CN110870916B - Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes - Google Patents

Abstract

The present invention relates to the application of FBXW7 or its up-regulator in the preparation of medicines for treating diabetes and preventing and treating the occurrence of tumors in individuals with diabetes. The present invention found that FBXW7 significantly reduced the high ROS levels and the excessive activation of PARP in diabetic large blood vessels, microvascular endothelium and retinal tissues; Repairing damaged DNA not only relieved the high glucose toxicity of vascular endothelium, but also exhibited retinal protection; FBXW7 could significantly enhance the catabolism and AMPK activity of large vascular endothelial cells, and reduce JNK activity. It is suggested that FBXW7 or its up-regulator can be used to prepare medicines for relieving the dysfunction of diabetic vascular endothelium, or strengthening DNA damage repair, or treating diabetes, or preventing and treating diabetic complications, or preventing and preventing the occurrence of tumors in diabetic individuals.

Description

Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of FBXW7 or an upper regulator thereof in preparing a medicine for treating diabetes and preventing and treating individual tumor of diabetes.

Background

Diabetes Mellitus (DM) is a lifelong metabolic disease characterized by chronic hyperglycemia. Diabetic vasculopathy is a characteristic pathological change of diabetes and is a main cause of disease and disability of patients. Long-term blood sugar elevation can damage large blood vessels and micro blood vessels, and further seriously endanger the heart, brain, kidney, peripheral nerves, eyes, feet and the like [1 ]. The American Diabetes Association (ADA) statistics show that diabetic patients who are more than 3 years have a probability of developing complications of more than 46%; the probability of the diabetic patients with more than 5 years of complications is more than 61%; the probability of the diabetic patients with more than 10 years of complications is as high as 98%. Among them, diabetic cardiovascular diseases (cardiovascular events such as coronary heart disease, atherosclerosis, myocardial infarction, etc.) and diabetic nephropathy (microangiopathy, early stage manifested as proteinuria, and later stage renal failure) are the main causes of more than 75% of patients with diabetes death, and diabetic retinopathy (microangiopathy) is the main cause of irreversible blindness among adults worldwide [2-4 ]. These three serious complications occur as a result of high sugar leading to damage and dysfunction of the endothelium of large vessels and microvessels. In addition, diabetic complications include diabetic cerebrovascular disease, neuropathy, diabetic foot disease, and the like, and have a lower incidence of fatal disability and less adverse consequences than the above complications.

Impairment of vascular endothelial function is the initiating factor and central link in vascular complications of diabetes. The famous "unified mechanistic theory" of diabetic complications [5], which has been acknowledged by the academia, has the core: high sugars cause excessive production of reactive oxygen species ROS in the mitochondria of target tissue cells. Excessive ROS chain-cleaves nuclear DNA, causing excessive activation of the endonuclear DNA repair enzyme poly- (ADP-ribose) polymerase (PARP), which generates large amounts of (ADP-ribose) Polymer (PAR) and nicotinic acid by cleaving substrate NAD +, and large amounts of PAR accumulate around glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other intranuclear proteins, causing ADP-ribosylation of GAPDH, resulting in inhibition of its activity [6 ]. Inhibition of GAPDH enzymatic activity causes accumulation of metabolic intermediates in the glycolytic pathway, i.e. glyceraldehyde 3-phosphate, fructose 6-phosphate or even glucose upstream of GAPDH, leading to the transfer of these metabolites to other glycolytic side branches, including activation of the polyol pathway (polyol pathway) [7], formation of non-enzymatic glycosylation end products [8], enhancement of the activity of protein kinase c (protein kinase c) [9] and activation of the hexosamine (hexosamine biosynthesis pathway) [10], ultimately triggering diabetic complications in target tissue cells. In short, it is: hyperglycemia → high ROS → PARP overactivation → GAPDH inactivation → activation of GAPDH upstream molecule, followed by transfer to activation of glycolytic bypass (polyol pathway activation, formation of non-enzymatic glycosylation end products, activation of protein kinase C signaling pathway, increase in activity of the hexosamine pathway) → diabetic complications. Although the consensus "unified mechanistic theory" in the industry has clearly revealed molecular mechanisms of high glycotoxicity, there is still a lack of direct and effective intervention.

Prevention and treatment of diabetic complications are key and difficult points in the field of diabetes. The methods for alleviating diabetic complications fall into two main categories. First, blood glucose is reduced. It is emphasized that the preference for hypoglycemic agents with cardiovascular protective effects is the latest trend to modify the therapeutic guidelines for diabetes. Diabetes is an imminent disease of cardiovascular disease, and the cardiovascular risk of diabetic patients is significantly increased. The T2DM milestone research result shows that the traditional treatment medicine in the diabetes hypoglycemic guide at present excessively emphasizes the hypoglycemic treatment, neglects the prevention and treatment of serious vascular complications and finally cannot be converted into a remarkable cardiovascular protection effect. Since the EMPA-RAG study 2015 and the LEADER study 2016 published to date, only 2-3 years, a cardiovascular outcome test (CVOT) with positive results brought obvious changes to the field of diabetes treatment, each of which was updated in succession, it was recommended that hypoglycemic drugs (e.g. SGLT2 inhibitor engeletin and GLP-1RA liraglutide) with clear cardiovascular benefits be preferentially selected for patients with type 2 diabetes (T2DM) who have combined cardiovascular disease or have a cardiovascular high risk factor. Second, other non-hypoglycemic therapeutic measures. Mainly intervene in the downstream links of the pathophysiological mechanism. The method comprises the following steps: aldose reductase inhibitors; a protein non-enzymatic glycosylation blocker; a growth factor antagonist; an angiotensin converting enzyme inhibitor; a neurotrophic agent. It is mainly used for treating diabetic nephropathy and retinopathy [4,11-12 ]. However, the current prevention and treatment of diabetic complications has not achieved satisfactory clinical results.

In addition, diabetic patients are more prone to develop tumors. Because it is known that the reduction of DNA repair capacity is a very important cause of tumorigenesis. Studies based on large samples of the population provide ample evidence for "a positive correlation between diabetes and cancer" [13-15 ]. The incidence rate of liver cancer, pancreatic cancer and endometrial tumor of a type 2 diabetes patient is 2 times of that of a common person, and the incidence risk of colorectal cancer, renal tumor, bladder cancer and breast cancer is 1.2-1.5 times of that of the common person [16 ].

E3 ubiquitin ligase FBXW7(F-box/WD40domain protein 7, FBXW7) is an important adipocyte differentiation regulatory protein and plays an important role in differentiation and maturation of adipocytes, and FBXW7 also degrades Sterol Regulatory Element Binding Protein (SREBP), thereby affecting synthesis and metabolism of fatty acids [17-19 ]. In addition, FBXW7 is also a broad-spectrum cancer suppressor protein, which can ubiquitinate and degrade the transcriptional activator protein C-myc, Cyclin-E, C-Jun, and has mutations in various tumors [30-34 ].

In 2015, it was found that the lipo-differentiation protein FBXW7 has a novel effect of regulating PGC-1 alpha in skeletal muscle and adipocytes [20 ]. In 2018, it was found that FBXW7 was significantly down-regulated in the livers of two obese mouse models and obese human subjects, that a functional low frequency human FBXW 7-encoded variant (p. ala204thr) in the chinese population was associated with elevated blood glucose and the risk of developing T2DM (i.e., type II diabetes), that mice with liver-specific knockout of FBXW7 developed hyperglycemia, glucose intolerance and insulin resistance even in normal diet, and that overexpression of FBXW7 in the liver not only prevented the development of high fat diet-induced insulin resistance, but also attenuated the disease profile in obese mice [35 ]. Although the research shows that the overexpression of FBXW7 in the liver of an obese mouse model prevents insulin resistance and reduces blood sugar, and plays a role in preventing obesity from developing into diabetes, the obese mouse model is not equal to hyperglycemia and is not equal to diabetes, and no document reports the role of FBXW7 in preventing and treating diabetic vascular complications and tumors.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of Fbxw7 or an upper regulator thereof in preparing a medicament for treating diabetes and preventing and treating individual tumor of diabetes.

In a first aspect of the invention there is provided the use of FBXW7 or a upregulation thereof in the manufacture of a medicament for the treatment of diabetes.

In another aspect of the invention, there is provided the use of FBXW7 or a upregulating agent thereof in the manufacture of a medicament for the prevention or treatment of a diabetic complication or for the prevention or treatment of tumorigenesis in a diabetic individual.

In a preferred embodiment, the diabetic complication is diabetic vascular complication or diabetic neuropathy.

In another preferred embodiment, the diabetic vascular complication is diabetic macroangiopathy or diabetic microangiopathy.

In another preferred embodiment, the diabetic macroangiopathy is selected from coronary heart disease, atherosclerosis, myocardial infarction and diabetic cerebrovascular disease.

In another preferred embodiment, the diabetic microangiopathy is selected from the group consisting of diabetic nephropathy and diabetic retinopathy.

In another preferred embodiment, the diabetic neuropathy becomes diabetic foot.

In another preferred embodiment, the up-regulator is selected from small molecule compounds or biological macromolecules.

In another aspect of the present invention, a pharmaceutical composition for treating diabetic complications is provided, which comprises FBXW7 or a upregulation thereof as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

In a preferred embodiment, the dosage form of the pharmaceutical composition is capsules, granules, tablets, pills, oral liquid or injection.

In another aspect of the invention, the application of FBXW7 as a target point in screening medicines for preventing and treating diabetes or diabetic complications or tumorigenesis of diabetic individuals is provided.

In another aspect of the present invention, there is provided a method for alleviating dysfunction of diabetic vascular endothelium, or enhancing DNA damage repair, or treating diabetes, or preventing diabetic complications, or preventing tumorigenesis in a diabetic individual, comprising administering FBXW7 or a upregulation thereof to a subject in need thereof.

The invention has the advantages that:

the fat differentiation protein FBXW7 is proved to be capable of obviously reducing the excessive activation of high ROS level and PARP in the great vessels, the microvascular endothelium and the retinal nerve tissues of diabetes for the first time; the FBXW7 protein repairs damaged DNA by activating the homologous recombination repair (HR) pathway and the non-homologous end joining repair (NHEJ) pathway; FBXW7 can improve AMPK activity of large blood vessel endothelial cell, reduce JNK activity, and is beneficial to reducing insulin resistance of diabetic blood vessel endothelium. Therefore, the application of FBXW7 or the up-regulation agent thereof in preparing the medicines for relieving the dysfunction of the vascular endothelium of diabetes, or strengthening the repair of DNA damage, or treating diabetes, or preventing and treating diabetic complications, or preventing and treating the tumorigenesis of diabetic individuals is provided.

Compared with the traditional PARP inhibitor, the FBXW7 not only inhibits the excessive activation of PARP, but also solves the DSB (namely DNA double strand break) threat remained by the PARP inhibitor, has obvious advantages, and is a new idea worth trying in the current treatment means of the diabetic complications. And the FBXW7 is used for effectively intervening in the upstream link of the pathophysiological mechanism of the complications of the diabetic damaged tissues, which is completely different from the existing treatment measures of the diabetic complications.

The invention provides a new direction for preventing and treating diabetes and related complications thereof as well as tumorigenesis of diabetic individuals.

Drawings

FIG. 1. the lipid-lowering drug, Fenofibrate, reduced the expression of EWS and Fbxw7 proteins in the retinal nerve tissue of DM rats. Set of vs Control, P<0.05，**P<0.01，***P<0.001; the set of vs DM's is set,^#P<0.05，^##P<0.01，^###P<0.001; the set of vs DM + Feno,^⊿P<0.05。

FIG. 2. in primary macroangio of human origin, the lipid-lowering drug Fenofibrate increased the expression of macroangio, microvascular endothelial EWS protein, FBXW7 protein and PGC-1 a.

FIG. 3. the lipid-lowering drug, Fenofibrate, increased the expression of macrovascular, microvascular endothelial EWS protein, FBXW7 protein and PGC-1a in microvascular endothelial cells.

FIG. 4. specific inhibitor of PFKFB 33 PO increased the expression of EWS protein, FBXW7 protein and PGC-1. alpha. protein.

FIG. 5 shows that the over-expression of the EWS protein promotes the proliferation and migration of the normal sugar NG group large vessel endothelium, and the knock-down of the EWS protein can resist the angiogenesis promoting effect of the recombinant protein VEGF growth factor.

FIG. 6. overexpression of the EWS protein did not promote endothelial proliferation in all high carbohydrate groups, HG + Met and HG + Feno.

Fig. 7 and 8. overexpression of FBXW7 protein inhibited vascular endothelial proliferation and migration in NG, HG, and NG + VEGF groups, and a number of vascular endothelial cells were killed after knockdown of FBXW 7.

FIG. 9. in human microvascular endothelium, overexpression of the EWS protein only promoted proliferation and migration of vascular endothelium in the NG group, with no significant effect on the HG group; knockdown of EWS protein severely inhibits vascular endothelial proliferation in NG and HG groups. The over-expression of the FBXW7 protein can inhibit the proliferation of vascular endothelium of the HG group, and the knock-down of the FBXW7 protein can inhibit the migration and proliferation of microvascular endothelial cells of the HG group, so that a large amount of cell death can be seen under an endoscope.

FIG. 10.30mM high sugar has strong toxic effect, inhibiting the luminal growth of HUVEC in the large vessel endothelium. Although the lumen forming ability of the endothelium of large blood vessels is enhanced after the EWS or Fbxw7 protein is over-expressed under high sugar, the statistical difference is not generated, and the high sugar toxicity effect of HUVEC is enhanced after the EWS protein or the Fbxw7 protein is knocked down, so that the lumen forming ability of the HUVEC is obviously weakened.

FIG. 11 shows that 30mM high sugar has obvious sugar toxicity in microvascular endothelial cells, and the over-expression of EWS can improve the toxic effect caused by 30mM high sugar, and knock down endogenously expressed EWS or Fbxw7 protein, thereby further weakening the endothelial lumen forming ability.

FIG. 12 EWS and Fbxw7 proteins dramatically reduced ROS levels in HUVEC cells and microvascular HREC endothelium at high sugar.

FIG. 13 high sugars promote PARP overexpression and overexpression of EWS and Fbxw7 dramatically reduced PARP expression at high sugars in the large and micro vascular endothelium HUVEC.

FIG. 14 shows that high sugar increases the PARP enzyme activity of the endothelial cells of large vessels and micro vessels, and both EWS and Fbxw7 can reduce the activity of the enzyme obviously.

Figure 15 high carbohydrate significantly increased PAR production in large vessel HUVEC endothelium, while EWS, Fbxw7 both dramatically reduced PAR production.

FIG. 16 DSB inducer triggers a large green fluorescent deposition in the endothelial nucleus, i.e., a large break in the DNA double strand. There was an increase in green punctate deposition of DSB in HUVEC nuclei after high sugar culture.

FIG. 17 shows that the HR and NHEJ pathway P-ATR kinase, CHK2 protein and P-P53 protein of large blood vessel endothelium HUVEC under high sugar are obviously increased. The expression level of P-ATM, P-ATR, CHK2 protein and P-P53 protein is reduced by the EWS protein, and the Fbxw7 protein is identical with the EWS protein.

FIG. 18 high sugars decrease the expression of the ATM and BRCA1 genes of the large vessel endothelial HUVEC. The EWS protein can greatly improve the expression of ATM, ATR, BRCA1, XRCC4 and MSH2 genes; the Fbxw7 protein also greatly improves the expression of ATM, ATR, XRCC4 and MSH2 genes.

FIG. 19. in microvascular endothelial cells, P-P53 protein decreased, and other proteins were unchanged. Under high sugar, the expression of CHK2 protein is reduced by the EWS, and the P-P53 protein is obviously reduced compared with the NG group; the Fbxw7 protein reduces the expression of P-ATR and CHK2, and still further reduces the expression of P-P53 protein.

FIG. 20. high sugar increases the expression of BRCA1 gene in microvascular endothelium at the transcriptional level. The Fbxw7 protein can greatly improve the expression of ATM, ATR, BRCA1 and MSH2 genes under high sugar.

FIG. 21 shows that HG promotes vascular endothelial cells to enter S phase, accelerating cell cycle operation; in large vessels, both EWS, Fbxw7 proteins block endothelial cells in G0/G1, providing sufficient time to repair damaged DNA.

FIG. 22 shows that Fbxw7 protein significantly enhances the expression of HIF-1 α, PGC-1 α, S6K1, ERR α, Cox5b, ATP5O, SCAD and VLCAD in large vessel endothelium under normal or high sugar; the EWS protein also exhibits similar effects to Fbxw7 at normal sugar (NG) concentrations, increasing HIF-1 α, PGC-1 α, S6K1 and ATP5O expression, but only HIF-1 α gene expression remains elevated at high sugar.

FIG. 23 high sugars promote JNK activity of large vessel endothelial HUVEC and expression of glycolytic key enzyme PFKFB 3. The protein EWS and Fbxw7 can promote energy metabolism of vascular endothelium, not only can reduce the activity of p-JNK and the expression of PFKFB3, but also can activate the activity of AMPK.

FIG. 24 early knockdown of endogenous EWS and Fbxw7 expression in DM rat retinal tissues increased the overall ROS levels in the retinal tissues, including RGC, inner and outer nuclear layers. The total ROS level and PAR product content of retina tissues of DM rats are obviously increased, and the over-expression of the EWS or Fbxw7 protein can obviously reduce the total ROS level of the retina tissues.

FIG. 25 early knockdown of expression of endogenous EWS and Fbxw7 in DM rat retinal tissues, aggravated retinal vascular leakage.

FIG. 26 overexpression of the EWS or Fbxw7 protein significantly reduced PAR production in retinal tissue in DM rats.

Figure 27. the degree of oxidation of diabetic retinal tissue proteins was significantly increased, and overexpression of Fbxw7 protein significantly reduced the level of oxidation of the protein, with a partial reduction in the overexpression of EWS protein.

Figure 28 overexpression of EWS significantly ameliorates retinal tissue glycosylation.

Detailed Description

The present inventors have extensively and intensively studied and found that the fat differentiation protein FBXW 7: the expression is extensive in large blood vessel, micro blood vessel and retina tissue, and participates in metabolism of sugar and lipid; ② the proliferation of the vascular endothelium is not promoted under high sugar, and the tube cavity forming experiment shows that the high sugar toxicity of the vascular endothelium can be relieved; ③ reducing the high ROS level of large blood vessels, micro blood vessels and retina tissues under the high glucose state of diabetes; the excessive activation of the diabetic great vessel and microvascular endothelial PARP is reduced, which is represented by the reduction of PARP expression quantity, the reduction of enzyme activity and the reduction of PAR products; FBXW7 can activate HR pathway or NHEJ pathway to repair DSB; cell cycle arrest at G0/G1 for DNA repair; the FBXW7 protein promotes the activity of the large vascular endothelium AMPK, reduces the activity of JNK and weakens the resistance of the JNK to insulin; the protection of the retinal tissue: FBXW7 reduces ROS levels and excessive activation of PARP in retinal neural tissue, reducing retinal tissue protein oxidation levels. Therefore, FBXW7 can be used as a target for relieving the dysfunction of the vascular endothelium of diabetes and strengthening DNA damage repair, and FBXW7 or an upper regulator thereof can be used for preparing a medicament for treating diabetes; ② preventing and treating diabetic complications; or ③ a medicine for preventing and treating individual tumor of diabetes.

FBXW7

E3 ubiquitin ligase FBXW7(F-box/WD40domain protein 7, FBXW7) is an important adipocyte differentiation regulatory protein and plays an important role in adipocyte differentiation and maturation.

In the present invention, the FBXW7 protein used may be naturally occurring, e.g., it may be isolated or purified from a mammal. In addition, the FBXW7 protein can also be artificially prepared, for example, the recombinant FBXW7 protein can be produced according to the conventional genetic engineering recombination technology. Preferably, the present invention may employ recombinant FBXW7 protein.

Any suitable FBXW7 protein may be used in the present invention. The FBXW7 protein comprises full-length FBXW7 protein or a biologically active fragment thereof. Preferably, the amino acid sequence of the FBXW7 protein may be substantially identical to the sequence shown in GenBank accession No. NM _018315, other variant sequences NM _001349798, NM _001013415, NM _00125706, NM _033632, NG _029466, XM _024454126, XM _011532088, XM _011532087, XM _024454125, XM _011532086, XM _011532085, XM _011532084, XM _024454124, XM _024454123, XM _024454122, XM _024454121, BC 944, BC037320, BC117246, BC117244, NM _032145, NM _001348092, XM _011536177, XM _017011353, AY049984, AY008274, NM _001330355, and NM _ 001304791.

The amino acid sequence of the FBXW7 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The FBXW7 protein or biologically active fragment thereof includes a portion of a conservative amino acid substitution sequence that does not affect its activity or retains some of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, molecular biology of the Gene, fourth edition, 1987, the Benjamin/Cummingspub. Co. P224.

Any biologically active fragment of the FBXW7 protein may be used in the present invention. Herein, the biologically active fragment of FBXW7 protein is meant to be a polypeptide that still retains all or part of the function of the full-length FBXW7 protein. Preferably, the biologically active fragment retains at least 50% of the activity of the full-length FBXW7 protein. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length FBXW7 protein.

The present invention may also employ modified or improved FBXW7 proteins, e.g., FBXW7 proteins modified or improved to promote half-life, efficacy, metabolism, and/or potency of the protein. The modified or improved FBXW7 protein may be a conjugate of FBXW7 protein, or it may comprise substituted or artificial amino acids. The modified or improved FBXW7 protein may be less common to the naturally occurring FBXW7 protein, but also may alleviate dysfunction of diabetic vascular endothelium or enhance DNA damage repair without other adverse effects or toxicity. That is, any variant that does not affect the biological activity of the FBXW7 protein may be used in the present invention.

The corresponding nucleotide coding sequence can be conveniently derived from the amino acid sequence of the FBXW7 protein.

Preferably, the nucleotide sequence of the FBXW7 protein may be substantially identical to the sequence shown in GenBank accession No. NM _018315, other variant sequences NM _001349798, NM _001013415, NM _00125706, NM _033632, NG _029466, XM _024454126, XM _011532088, XM _011532087, XM _024454125, XM _011532086, XM _011532085, XM _011532084, XM _024454124, XM _024454123, XM _024454122, XM _024454121, BC 944, BC037320, BC117246, BC117244, NM _032145, NM _001348092, XM _011536177, XM _017011353, AY049984, AY008274, NM _001330355, and NM _ 001304791.

Up regulator

As used herein, the up-regulation of FBXW7 includes promoters, agonists, and the like. Any substance that can increase the activity of FBXW7 protein, maintain the stability of FBXW7 protein, promote the expression of FBXW7 protein, promote the secretion of FBXW7 protein, prolong the effective duration of FBXW7 protein, or promote the transcription and translation of FBXW7 can be used in the present invention as an effective substance that can be used to alleviate the dysfunction of diabetic vascular endothelium or enhance the repair of DNA damage.

As a preferred mode of the invention, the up-regulator of FBXW7 protein includes (but is not limited to): an expression vector or expression construct that expresses (preferably overexpresses) FBXW7 after transfer into a cell. Typically, the expression vector comprises a gene cassette comprising a gene encoding FBXW7 operably linked to expression control sequences. The term "operably linked" or "operably linked" refers to the condition wherein certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In the present invention, the FBXW7 polynucleotide sequence may be inserted into a recombinant expression vector. Any plasmid and vector can be used in the present invention as long as they can replicate and are stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence of FBXW7 and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The transformation vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Use of

The invention provides an application of FBXW7 or a regulator thereof in preparing a medicament for relieving dysfunction of diabetic vascular endothelium or enhancing DNA damage repair.

The invention also provides FBXW7 or the upper regulator thereof for preparing medicines for treating firstly diabetes; ② preventing and treating diabetic complications; or the application of the medicine in preventing and treating diabetes individual tumor.

Diabetes is a lifelong metabolic disease characterized by chronic hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. Hyperglycemia existing in diabetes for a long time results in chronic damage and dysfunction of various tissues, particularly eyes, kidneys, brain, heart, blood vessels and nerves, and causes diabetic complications. The diabetic complication has a well-known "unified mechanistic theory", which is widely recognized in the industry, and the core of the theory is: hyperglycemia → high ROS → PARP overactivation → GAPDH inactivation → activation of glycolytic bypass (non-enzymatic glycosylation end product formation, activation of protein kinase C signaling pathway, activation of polyol pathway and increase of hexylamine pathway activity) → diabetic complications. Diabetic complications include, but are not limited to, diabetic vascular complications, diabetic neuropathy, and the like. Diabetic vascular complications involve diabetic macroangiopathy and diabetic microangiopathy. The diabetic macroangiopathy is especially prominent in cardiovascular and cerebrovascular events such as coronary heart disease, atherosclerosis, myocardial infarction, diabetic cerebrovascular diseases (such as cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, and brain atrophy). Diabetic microangiopathy is particularly prominent in diabetic nephropathy and diabetic retinopathy. Diabetic neuropathy is particularly prominent in diabetic feet (which refers to a disease state that the protective function of lower limbs of diabetic feet is reduced due to neuropathy, and microcirculation disturbance caused by insufficient arterial perfusion is caused by macrovascular and microvascular lesions to cause ulcer and gangrene). The above serious complications occur as a result of the damage and dysfunction of the endothelium of large vessels and micro vessels caused by high sugar. The essence of the large vessel and micro vessel pathological changes is that long-term hyperglycemia causes the decompensation of the vascular function homeostasis, and the vascular endothelial dysfunction caused by inflammation and oxidative stress is a shared core link. Impairment of vascular endothelial function is the initiating factor and central link in vascular complications of diabetes.

The invention finds that FBXW7 can: reducing ROS; inhibit PARP overactivation. The JNK activity is reduced by improving the AMPK activity of the large vessel endothelial cells. This is two well-recognized pathways for reducing cardiovascular events in diabetes. Recent research reports indicate that the pathological mechanism of cardiovascular events is still high carbohydrate → high ROS → overactivation of PARP → inhibition of GAPDH → activation of carbohydrate metabolism bypass, while high ROS high PARP → reduction of sirtuin, PGC1 α and AMPK activity → disruption of the synchronous rhythm of carbohydrate and lipid metabolism → induction of cardiovascular accidents.

Diabetic individuals are more prone to develop tumors and it is well known that a reduced DNA repair capacity is a very important cause of tumorigenesis. The invention discovers for the first time that DSB (namely DNA double-strand break) and DSB repair mechanism in cells and tissues of a diabetic patient are damaged, and FBXW7 has obvious evidence for strengthening DNA repair of diabetic cells, so that the invention can be applied to preventing and treating tumorigenesis of diabetic individuals.

The invention also provides an application of FBXW7 as a target spot in screening medicines for preventing and treating diabetes or diabetic complications or tumorigenesis of diabetic individuals, namely a method for screening potential substances for relieving dysfunction of diabetic vascular endothelium, enhancing DNA damage repair, treating diabetes, preventing and treating diabetic complications or preventing and treating tumorigenesis of diabetic individuals, wherein the method comprises the following steps: treating a system expressing FBXW7 with a candidate substance; and detecting expression of FBXW7 in said system; if the candidate substance increases expression of FBXW7, the candidate substance is an intended potential substance, and vice versa, the candidate substance is an undesired potential substance. The system for expressing FBXW7 can be a cell (or cell culture) system, and the cell can be a cell for endogenously expressing FBXW 7; or may be a cell recombinantly expressing FBXW 7. The system for expressing FBXW7 can also be a subcellular system, a solution system, a tissue system, an organ system, or an animal system (e.g., an animal model, preferably a non-human mammalian animal model, such as mouse, rabbit, sheep, monkey, etc.), etc.

In a preferred embodiment of the present invention, a control group may be provided in order to more easily observe the change in expression of FBXW7 during screening, and the control group may be a system expressing FBXW7 without the addition of the candidate substance.

Pharmaceutical composition

The pharmaceutical compositions of the invention may contain an active agent as described herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are generally safe, non-toxic, and may broadly include any material known in the pharmaceutical industry for preparing pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants, and the like. The mode of administration of the pharmaceutical composition is primarily a consideration in selecting excipients suitable for the delivery of the synthetic peptide, and is well known to those skilled in the art. The amount of the active agent in the medicament of the invention may be determined for different therapeutic uses. The above-mentioned Pharmaceutical compositions may be prepared according to known Pharmaceutical procedures, as described in detail in the Remington's Pharmaceutical Sciences, 17 th edition, Alfonoso R.Gennaro, Mac Publishing Company (Mack Publishing Company), Iston, Pa.1985. The medicament of the invention can be in various suitable dosage forms, including but not limited to capsules, granules, tablets, pills, oral liquid or injection and the like.

Method of treatment

The invention provides a method for relieving the dysfunction of diabetic vascular endothelium, or enhancing DNA damage repair, or treating diabetes, or preventing and treating diabetic complications, or preventing and treating tumorigenesis of a diabetic individual, which comprises administering FBXW7 or a upregulation thereof to a subject in need thereof. The amount administered is a therapeutically effective amount and can be determined according to the age, weight, sex, kind and severity of the disease of the individual. The subject may be a mammal, in particular a human, mouse, rabbit, pig, sheep, dog, etc. Methods of administration are conventional in the art, e.g., oral, injectable, etc., and may be adjusted for different agents. Such as by injection, and may be applied to a particular location of the individual, such as the eye, hip, thigh, knee, calf, foot, shoulder, arm, elbow, forearm, hand, head, etc., depending on the individual's condition.

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

Method and device

1. Construction of viral vectors overexpressing the EWS (nomenclature: E-OE) or Fbxw7 (nomenclature: F-OE) proteins

(1) Amplifying nucleotide sequences of the EWS and the Fbxw7 (the EWS sequence is shown as SEQ ID NO:1, the Fbxw7 sequence is shown as SEQ ID NO: 2) by a PCR reaction, and connecting EcoRI and BamHI enzyme cutting sites at two ends;

(2) performing double enzyme digestion on a PCR product and purifying;

(3) the H201pAdeno-MCMV-EGFP-3FLAG vector plasmid (Shanghai and Yuan biotechnology limited) is subjected to double digestion, a large fragment is recovered and purified, and the EWS or Fbxw7 gene fragment in the step (2) is inserted into the EcoRI and BamHI digestion sites of the shuttle plasmid by using a molecular cloning technology to replace EGFP;

(4) screening transformants by colony PCR, and carrying out sequencing verification on the screened positive clones;

(5) sequencing to verify correct cloning, and extracting high-purity plasmid;

(6) packaging, amplifying, purifying adenovirus, and performing virus titer determination and function identification.

2. Construction of viral vectors interfering with the EWS (nomenclature: E-sh or E-sh RNA), Fbxw7 protein (nomenclature: F-sh or F-sh RNA)

(1) Designing siRNA targets according to transcripts of the Human EWSR1 gene or Fbxw7 gene, and arranging primer synthesis;

(2) annealing the single-stranded primer into a double-stranded oligo sequence (the coding sequence of the EWS shRNA is shown as SEQ ID NO:3-4, and the coding sequence of the Fbxw7 shRNA is shown as SEQ ID NO: 5-6), connecting the double-stranded oligo sequence with a double-enzyme digestion linearized RNA interference vector pDKD-CMV-Puro-U6-shRNA (Shanghai and Biotechnology Co., Ltd.), and replacing the original ccdB toxic gene;

EWS knockdown sequence

Fbxw7 knockdown sequences

(3) Screening transformants by colony PCR, and carrying out sequencing verification on the screened positive clones;

(4) sequencing to verify correct cloning, and extracting high-purity plasmid;

(5) transfecting human HUVEC cells by using Lipofectamin2000 liposome, and identifying plasmid clones with the efficiency of knocking down the EWS or Fbxw7 protein being more than 70% according to western blot;

(6) co-transfecting the plasmid and adenovirus frame large plasmid to a packaging tool cell strain Ad 293;

(7) packaging, amplifying, purifying adenovirus, and performing virus titer determination and function identification.

3. Preparation of diabetic rat model

Male SD rats weighing about 200g were fasted overnight, injected intraperitoneally with 65mg/kg STZ, and tested by glucometer after 48 hours, and the model was successfully made with blood glucose >16.7 mM.

Intragastric treatment of DM rats with Fenfibrate

Randomly grouping after the molding is successful: control group, DM + Feno group. The stomach was gavaged with 35mg/kg of Fenofibrate daily for 8 weeks. The rats were sacrificed at 4 and 8 weeks of disease, the retinal whole tissue was dissected away, and appropriate amount of RIPA tissue protein lysate was added and disrupted by ultrasonic wave on ice to extract the protein. After protein quantification by the BCA method, western blot sample buffer is added into the protein extracting solution, boiled at 99 ℃ for 10min, ice-bathed for 10min, and stored at-30 ℃ for later use. The sample is to be subjected to Western Blot to detect the expression quantity of the proteins of EWS and Fbxw 7.

5. Effect of high sugar and glycolipid metabolism modulating Agents on human Primary vascular endothelial EWS and fbxw7 protein expression

Human primary large vessel endothelial HUVECs were cultured in ECM + ECGS + 5% FBS medium, dividing HUVECs in log-extended phase into 4 groups: NG (glucose concentration 5.5mM), HG (glucose concentration 30mM), HG + Met (1mM), HG + Feno (100. mu.M), were incubated for 48 hours under the 4 different treatment conditions described above. The culture medium is discarded, PBS is washed, a proper amount of RIPA protein lysate is added, and the samples are collected and subjected to ultrasonic wave disruption on ice to extract protein. The sample is subjected to Western Blot to detect the expression level of the EWS, Fbxw7 and PGC-1a protein.

6. Effect of high sugar and glycolipid metabolism-modulating Agents on human Primary retinal microvascular endothelial cell EWS and fbxw7 protein expression

Human primary retinal microvascular endothelial cells in log-extended phase were divided into 4 groups: NG, HG, HG + Met, HG + Feno, culturing for 48 hours, collecting cells, and extracting protein by RIPA lysate. The sample is to be subjected to Western Blot to detect the expression quantity of the proteins of EWS and Fbxw 7.

7. Effect of high-sugar and glycolytic regulatory drugs on human Primary vascular endothelial EWS and Fbxw7 protein expression

HUVECs in log-extended phase were divided into 4 groups: NG (glucose concentration 5.5mM), NG +3PO (20. mu.M), HG (glucose concentration 30mM), HG +3PO (20. mu.M), and after culturing for 48 hours, the cells were collected and the proteins were extracted from the cells using RIPA lysate. The sample is subjected to Western Blot to detect the expression level of the EWS, Fbxw7 and PGC-1a protein.

8. Western Blot detection of protein

A7.5% SDS-PAGE gel was prepared. 50 μ g of protein was added to each loading comb of SDS-PAGE gels, and electrophoresed at 90V for about 1.5 hours. Taking out the gel, attaching to nylon membrane with the same size, making into conventional sandwich (sponge-paper tower-gel-nylon membrane-paper tower-sponge), placing into a wet-transfer box, performing electrophoresis at 90V for 2 hr, and transferring the protein from the gel to the nylon membrane. After 5% skimmed milk is sealed and incubated, TBST is washed, the membrane is cut according to the molecular weight of protein, and is respectively incubated with antibodies such as EWS, Fbxw7, PGC-1a, actin and the like overnight at 4 ℃, after TBST is washed for three times, the membrane is respectively incubated with anti-rabbit or anti-mouse secondary antibody which is added with horseradish peroxidase and is marked for 1 hour at room temperature, TBST is washed for three times, and ECL exposure detection is carried out. Analyzing by using Image J software, calculating the ratio of each group of gray scales to actin gray scale, and analyzing the relative expression level of the protein.

Scoring healing Effect of EWS or Fbxw7 proteins on Large vascular endothelial HUVEC cells

HUVEC cells were seeded into 12-well plates and adenoviruses that over-expressed or interfered with EWS or Fbxw7 or Control were infected overnight at 100moi at a density of about 70-80%. After 200. mu.l pipette tips were cross-hatched and washed with PBS, fetal bovine serum-free medium was prepared and incubated with NG (5.5mM), HG (30mM), NG + VEGF (25NG/ml), HG + Met (1mM), HG + Feno (100. mu.M) for 48 hours. Pictures were taken under cross-hatch 0h, 12h, 24h, 48h microscope (100 ×), cell migration area was measured with NIH Image J software, and the healing rate of the cells was calculated as 1- (post-treatment blank area-0 hour blank area)/0 hour area.

Scoring healing effects of EWS or Fbxw7 proteins on microvascular endothelial HREC cells

In the same manner as above, HREC was first infected with adenovirus (100moi dose) that overexpresses or interferes with EWS or Fbxw7 or Control, and then culture medium without fetal calf serum was prepared after cross-scratching and cultured with NG (5.5mM) and HG (30mM) for 48 hours. Photographs were taken under a 0h, 12h, 24h, 48h microscope (100 ×), the cell migration area was counted with NIH Image J software, and the healing rate of the cells was calculated, the formula being as above.

11. Experiment for lumen formation of HUVEC cells and HREC cells of microvessels

50 mul/well of Matrigel gel was plated in a 96-well plate, after the gel was solidified, the same number of vascular endothelial cells were plated thereon, and cultured for each of different groups: NG (5.5mM), HG (30mM), NG + VEGF (25NG/ml), HG + Met (1mM), HG + Feno (100. mu.M). After 6 hours, the tube was observed under a microscope and photographed, and the total length of the lumen formation and the node were calculated using NIH Image J software. The NG group is taken as 1, and the ratio of each processing group/NG group is calculated.

Effect of the EWS or Fbxw7 proteins on the luminal Capacity of human Large vessel endothelium HUVEC or human microvascular endothelium HREC

As above, HUVEC or HREC cells were first infected overnight with adenovirus (100moi dose) that overexpresses or interferes with EWS or Fbxw7 or Control. Pancreatin each experiment consisted of a suspension of single cell fluid, inoculated with the same amount of cell suspension on a coagulated Matrigel gel, incubated for 6 hours with NG (5.5mM) or HG (30mM), observed under a microscope and photographed, and the total length of lumen formation and nodes calculated using NIH Image J software. Taking the NG group as 1, calculating the ratio of the HG group/the NG group under different processing conditions.

Effect of the EWS or Fbxw7 protein on ROS levels in human Large vessel endothelium HUVEC or human microvascular endothelium HREC

The active Oxygen detection Kit (Reactive Oxygen specifices Assay Kit) is a Kit for detecting active Oxygen by using a fluorescent probe DCFH-DA. Two kinds of vascular endothelial cells are planted in a six-well plate respectively, after the cells reach 70% fusion state, the cells are divided into 6 groups, Ad-Control, Ad E-OE or Ad F-OE are infected with 100moi dosage respectively overnight, and replaced by fresh NG or HG culture medium at 37 ℃ and 5% CO₂The cultivation was continued in the incubator for 48 hours. Removing the culture medium, diluting DCFH-DA with serum-free culture medium at a ratio of 1:1000 under the condition of keeping out of the sun, and incubating at 37 deg.C for 30 min. The cells were harvested, washed 3 times with PBS, resuspended in 300. mu.l of pre-cooled PBS, and examined by flow cytometry using 488nm excitation wavelength and 525nm emission wavelength to measure the mean fluorescence intensity of the cells.

Effect of EWS or Fbxw7 protein on the expression levels of HUVEC and HREC vascular endothelial PARP

Vascular endothelial cells were infected overnight with 100moi doses of Ad-Control, Ad E-OE or Ad F-OE, respectively, and cultured for an additional 48 hours in fresh NG or HG medium. And (3) removing the culture medium, washing with PBS, adding a proper amount of RIPA lysate, carrying out ultrasonic disruption in ice bath to extract protein lysate, and carrying out Western blot to detect the expression level of the PARP protein.

Effect of EWS or Fbxw7 proteins on vascular endothelial PARP Activity

The vascular endothelial cells were treated as above, 10000 cells were inoculated into a 96-well plate, Ad-Control, Ad E-OE or Ad F-OE (100moi) were infected overnight after adherence, and culture was continued for 48 hours with fresh NG, NG + mannitol (30mM), HG + Feno medium. The culture medium is discarded, washed with PBS 2 times, 100. mu.l of cell extract is added to each well, and the cells are lysed and incubated on ice for 30min, and the protein concentration is determined by BCA method for later use. The ELISA assay panels were removed and a series of PARP standard curves of known concentration were prepared. Add 50. mu.l of 1 XI-PAR Assay Buffer per well and incubate for 30min at room temperature; patting to dry 1 xI-PAR Assay Buffer, and adding 25 μ l of PARP standard substance or sample to be detected with different concentrations into each hole; a further 25. mu.l of PARP Substrate Cocktail was added and incubated at room temperature for 30 min. PBST washing 2 times, PBS additional washing 2 times; after patting to dry, 50. mu.l of anti-PAR monoclonal antibody dilution was added to each well and incubated at room temperature for 30 min. PBST washing 2 times, PBS additional washing 2 times; after patting to dryness, 50. mu.l of goat anti-mouse IgG-HRP conjugate antibody dilution was added to each well and incubated at room temperature for 30 min. PBST washing 2 times, PBS additional washing 2 times; after drying, adding 50 μ l of chromogenic substrate into each well, incubating for 15min at room temperature in the dark, stopping with 50 μ l/well of 0.2M HCl, and recording the OD value by reading a plate of a microplate reader with a wavelength of 450 nm. The PARP enzyme activity of each sample was calculated according to the plotted standard curve formula.

Effect of EWS or Fbxw7 proteins on vascular endothelial PAR production

The cell treatment is the same as in step 15. The number of cells in a six-well plate was washed 2 times with PBS, 300. mu.l of cell lysate was added to each well, the cells were hung on ice, and the ice bath was carried out for 15 min. Collecting the sample into a 1.5ml centrifuge tube, adding SDS to a final concentration of 1%; boiling at 100 deg.C for 5 min; cooling to room temperature; adding the same amount of Magnesium phosphate and DNase I, uniformly mixing, and incubating at 37 ℃ for 90 min; centrifuging at room temperature at 10000g for 10min, and taking the supernatant to determine the protein concentration by a BCA method for later use. Mu.l of the above sample or a PAR series standard of known concentration were added to the wells of the ELISA test plate strip and incubated overnight at 4 ℃ for 16 hours. After the bars were equilibrated to room temperature, PBST was washed 4 times; add 50 u l/hole PAR antibody dilution, incubate 2 hours at room temperature; PBST was washed 4 times, 50. mu.l/well of secondary antibody diluent was added, and incubated for 1 hour at room temperature; PBST was washed 4 times, 100. mu.l/well of color-developing solutions A and B were added, and the number of chemiluminescent photons was immediately detected with a Biotek Synergy 2. And calculating the PAR content of each sample according to a drawn standard curve formula.

DSB detection

HUVEC cells were seeded in 96-well plates and cultured for 48 hours in NG, NG + mannitol, HG + Feno. After the positive control wells were treated with DNA DSB inducer for 1 hour, all the medium was discarded. Adding 100 μ l paraformaldehyde to fix cells for 10min, and washing with PBS for 2 times; after patting to dry, adding 100 mul/hole frozen 90% methanol, standing at 4 deg.C for 10 min; washing with PBS for 1 time, drying, adding 200 μ l/hole sealing solution, and standing at room temperature for 30 min; discarding the confining liquid, adding 100 μ l/well of anti-phosphorylation-Histone antibody diluent, and incubating at room temperature for 1 hour; PBST is washed for 5 times, a second antibody diluent is added after the PBST is patted dry, and the PBST is incubated for 1 hour at room temperature and protected from light; PBST was washed 5 times and PBS was added and pictures were taken under the microscope on the FITC fluorescence channel.

Repair of high-sugar damaged vascular endothelium by EWS or Fbxw7 protein 1

The treatment of the large vessel endothelium and the micro vessel endothelium cells is the same as that of the prior treatment, and the treatment is divided into six groups: NG, NG + E-OE, NG + F-OE, HG, HG + E-OE, HG + F-OE. Collecting cell RIPA protein lysate, performing western blot technique to detect expression levels of P-ATM, P-ATR, CHK2, and P-P53 proteins, and referring to step 8.

Repair of high-sugar damaged vascular endothelium by EWS or Fbxw7 protein 2

The treatment of the large vessel endothelium and the micro vessel endothelium cells is the same as that of the prior treatment, and the treatment is divided into six groups: NG, NG + E-OE, NG + F-OE, HG, HG + E-OE, HG + F-OE. Extracting total RNA of cells by using a Trizol kit, measuring a D260/280 value by using an ultraviolet spectrophotometer, estimating the purity of the RNA, measuring the concentration of the RNA, calculating the content of the RNA, reversing the RNA into cDNA by using a TAKARA kit (RR047A), and detecting the gene transcription level conditions of ATM, ATR, BRCA1, XRCC4 and MSH2 by using the TAKARA kit (RR420A) on a fluorescence quantitative PCR instrument through real-time quantitative PCR.

20. Flow cytometry

The treatment of the large vessel endothelium and the micro vessel endothelium cells is the same as that of the prior treatment, and the treatment is divided into six groups: NG, NG + E-OE, NG + F-OE, HG, HG + E-OE, HG + F-OE. Carrying out pancreatin digestion, and preparing a sample to be detected as a single cell suspension; washing the cells with PBS, and centrifuging at 1500rpm/min to precipitate the cells; discarding PBS, adding 300. mu.l PBS, gently beating the cell pellet to loosen and resuspend the pellet; slowSlowly dripping 700 mu l of precooled absolute ethyl alcohol, uniformly mixing while dripping, and standing overnight; centrifuging, removing the stationary liquid, washing with PBS for 1 time, centrifuging to precipitate cells, and removing PBS; FxCycle in an amount of 0.5ml per sample^TMPI/RNase stabilizing solution, mixing well, incubating at room temperature in dark for 30 min; directly detecting by a computer, selecting 488nm and 532nm exciting light wavelengths, and collecting emitted light by an 585/42 band-pass filter. The results were simulated by Endofit software to calculate the cell number distribution of G0/G1, S, G2/M at each stage.

Effect of the protein EWS or Fbxw7 on the mitochondrial energy metabolism pathways of vascular endothelial cells 1

Vascular endothelial cells were treated as above, and after overexpressing virus-infected cells, cultured for 48 hours under different treatment conditions. Extracting total RNA of cells by using a Trizol kit, measuring a D260/280 value by using an ultraviolet spectrophotometer, estimating the purity of the RNA, measuring the concentration of the RNA, calculating the content of the RNA, reversing the RNA into cDNA by using a TAKARA kit (RR047A), and detecting the transcription level conditions of genes such as Cox5b, ATP50, SCAD, VLCAD, ERRa, S6K1, HIF-1a, PGC-1a and the like by using the TAKARA kit (RR420A) through real-time quantitative PCR on a fluorescence quantitative PCR instrument.

Effect of EWS or Fbxw7 proteins on mitochondrial energy metabolism pathways in vascular endothelial cells 2

Cell culture and treatment were as above. After a protein sample is collected, the phosphorylation levels of three energy pathways of AMPK, JNK and mTOR are detected by adopting a western blot technology, and the expression levels of PGC-1a and glycolytic key enzyme PFKFB3 are measured, and the steps are shown in step 8.

Intraocular injection of EWS or Fbxw7

(1) Interference EWS or Fbxw7

DM rats were divided into 4 groups. 5X 10 injections were administered on day 3 and week 4 after successful DM modeling⁷The EWS interfering adenovirus or Fbxw7 interfering adenovirus or control blank interfering adenovirus, the animals were sacrificed at 8 weeks of modeling and whole retinal tissue was removed to prepare samples for the vascular leakage experiment of step 25; or whole eye cryosections are removed and ROS stained (step 24). Intraocular NS injection was used as a control group.

(2) Overexpression of EWS or Fbxw7

DM rats were divided into 4 groups. Success of DM modelingInjection at 4th and 8 th weeks after the injection of 5X 10⁷The EWS overexpression virus or the Fbxw7 overexpression adenovirus or the blank control adenovirus, and the animals are sacrificed at the modeling period of 12 weeks to take out the whole retina tissue to prepare samples for three experiments of steps 25, 26 and 27; or whole eye cryosections are removed and ROS stained (step 24). Intraocular NS injection was used as a control group.

24. ROS staining of retinal tissue

Preparing DHE liquid: 5mg of DHE dry powder was added to 1.59ml of DMSO to prepare a 10mM DHE stock solution, which was stored in aliquots at-80 ℃. The solution was diluted with 0.1M PBS to 2. mu.M fresh DHE solution before the experiment. Immediately embedding the eyeball in OCT, slicing at constant temperature, and cutting eyeball tissue slices of about 10 μm near the optic nerve; adding 2 μ M DHE working solution, placing in an incubator at 37 ℃, keeping out of the sun, incubating for 30min, washing with PBS, observing under a fluorescence microscope, photographing (maximum excitation wavelength 300nm, emission wavelength 610nm), and analyzing fluorescence intensity by NIH Image J software.

25. Retinal vascular leakage assay

Anesthetizing a rat, injecting Evans Blue dye (30mg/kg) into the tail vein, and placing the rat on a heat-preservation blanket for 2 hours; the left ventricle was punctured by opening the thorax and pre-warmed sodium citrate buffer was perfused at 120mmHg for 2 min. After the perfusion is finished, the whole retina tissue is taken out, dried and weighed. Dissolving retinal tissue in 0.3ml formamide at 70 deg.c for 18 hr; 70000g high speed centrifugation for 45min, 4 deg.C; 60 μ l of the supernatant was used to determine the OD at 620 nm. Dissolving Evans Blue in formamide to prepare a standard curve concentration, and measuring the concentration of Evans Blue in a sample to be measured according to a formula.

26. Retinal tissue PAR quantification

Taking the whole retinal nerve tissue, adding 0.5ml of lysate, shearing, and then carrying out ice bath ultrasonic crushing; adding SDS to a final concentration of 1%, boiling at 100 deg.C for 5min, and immediately ice-cooling for 10 min; centrifuging at 10000g for 2min at 4 deg.C; taking the supernatant, and measuring the protein concentration by using a BCA method; the sample to be tested can be stored at-80 ℃. The subsequent ELISA detection procedure was as described in 16.

27. Retinal tissue protein oxidation level determination

And (3) cracking the protein lysate, carrying out ultrasonic disruption on retina tissues in ice bath, and taking the supernatant to perform BCA (burst amplification factor) method to determine the protein concentration. Will be ready forDiluting the sample to 10 μ g/ml, adding 100 μ l/well to OxiSelect^TMProtein Carbonyl ELISA plates were incubated overnight at 4 ℃. Washing with PBS for 3 times, beating to dry, adding DNPH working solution with 100 μ l/hole, incubating in dark for 45min, and shaking; 250 μ l/well PBS/ethanol (1:1) 5 times at 5min intervals; finally, the cells were washed 2 times with PBS. Adding 200 mul/hole sealing solution to incubate for 2 hours; wash buffer 3 times, beat dry, add 100 μ l/well anti-DNP antibody diluent, incubate 1 hour at room temperature; wash buffer 3 times, beat dry, add 100 u l/hole HRP conjugated secondary antibody dilution, incubate 1 hour at room temperature; wash buffer 5 times, add 100 μ L/well Substrate Solution after drying, terminate 100 μ L of Stop Solution after proper incubation, and detect OD 450. And calculating the content of the oxidized protein in the sample to be detected according to the standard curve.

28. Retinal tissue glycosylation level determination

And (3) cracking the protein lysate, carrying out ultrasonic disruption on retina tissues in ice bath, and taking the supernatant to perform BCA (burst amplification factor) method to determine the protein concentration. Selection of OxiSelect^TMMethoglyoxal (MG) Competitive ELISA Kit, 50. mu.l/well of the sample to be tested or the standard MG-BSA standard is added into the MG Conjugate coated ELISA plate, and the incubation is carried out for 10min at room temperature; adding 50 mu L/hole anti-MG antibody diluent, incubating for 1 hour at room temperature, and shaking up; washing the Wash Buffer for 3 times, beating to dry, adding 100 mu L Anti-HRP Conjugate secondary antibody diluent into each hole, and incubating for 1 hour at room temperature; wash Buffer was washed thoroughly 5 times, 100. mu.L of substrate Solution was added to each well, 2-20minutes were incubated at room temperature, 100. mu.L of Stop Solution was stopped, and OD450 was detected. And calculating the content of the glycosylated protein in the sample to be detected according to the standard curve.

Second, result in

1. The high sugar and glycolipid metabolism regulating medicine can change the expression level of the diabetic retina tissue and vascular endothelium EWS and fbxw7 protein obviously.

Western blot results show that the expression levels of the EWS and Fbxw7 proteins of the retinal tissues in the DM rat disease course at 4 weeks are obviously increased (vs Control group, P <0.001or P <0.05, recurrence) until the expression levels return to normal levels at 8 weeks (FIG. 1). The oral administration of the lipid-lowering drug of the diabetes mellitus, namely the Fenofibrate can continuously affect the expression of the EWS and Fbxw7 proteins in the retinal tissues of diabetic rats, so that the expression level of the EWS and Fbxw7 proteins in the retinal tissues of DM rats is continuously reduced and is obviously lower than the level of a normal group and a DM group (vs DM group, all P < 0.05).

In primary macrovessels of human origin, as shown in fig. 2, the Fenofibrate treated group significantly increased the expression levels of macrovessels, microvascular endothelial EWS protein, FBXW7 protein and PGC-1a (vs HG, all P < 0.05). In microvascular endothelial cells, the results are the same as shown in FIG. 3.

6-phosphofructose-2-kinase (6-phosphofructo-2-kinase/fructise-2, 6-biphosphatase 3, PFKFB3) is a key enzyme of the glycolytic pathway and plays an important role in sugar metabolism in endothelial cells. 3PO is a specific inhibitor of PFKFB3, which significantly increased the expression levels of EWS protein, FBXW7 protein (vs HG, all P <0.05) and PGC-1 alpha protein (vs HG, P <0.01) in the high sugar state, suggesting that both EWS and FBXW7 are also involved in the glycolytic process of endothelial cells (FIG. 4).

The above evidence suggests that the lipo-differentiation protein EWS/FBXW7 is present not only in the vascular endothelium and retinal tissue of large blood vessels, micro blood vessels; moreover, their expression is also significantly affected by high sugars, the lipid-lowering drug for diabetes, Fenofibrate, and the glycolytic regulator 3 PO.

The protein EWS and Fbxw7 can affect the proliferation and migration of vascular endothelium and relieve high-sugar toxicity injury of vascular endothelium.

We selected overexpressing EWS or FBXW7 genes, and knock-down (KD) plasmids carrying interfering shRNA sequences, and first observed their effect on the migration and proliferation of large and small microvascular endothelium.

(1) Two proteins affect proliferation and migration of vascular endothelium

The over-expression of the EWS protein, as shown in fig. 5 and fig. 6, significantly promoted the proliferation and migration of the large vessel endothelium of the NG group with normal sugars (vs NG, P <0.001), but did not promote the endothelial proliferation of all the high-sugar groups, such as the HG group, HG + Met group and HG + Feno group, indicating that the effect of the EWS on promoting the vessel migration and proliferation is only shown in the normal sugar concentration rather than the high-sugar state. Knockdown of the EWS protein was able to counteract the pro-angiogenic effect of the recombinant protein VEGF growth factor (P < 0.001).

As shown in fig. 7 and 8, overexpression of FBXW7 protein significantly inhibited vascular endothelial proliferation and migration in NG, HG, and NG + VEGF groups (all P <0.05), consistent with the function of FBXW7 as a cancer suppressor protein [20-21 ]. After knockdown of FBXW7 in all groups, a large number of vascular endothelial cells died (all P <0.001), suggesting that the physiological function of FBXW7 protein is very important.

In human microvascular endothelium, as shown in fig. 9, the over-expression of EWS protein only promoted proliferation and migration of vascular endothelium of NG group (vs NG, P <0.001), had no significant effect on HG group, and showed consistent with HUVEC of large vascular endothelium; knockdown of EWS protein severely inhibits vascular endothelial proliferation in NG and HG groups (all P < 0.001). The over-expression of FBXW7 protein can inhibit the proliferation of vascular endothelium of HG group (vs HG, P < 0.001); knocking down FBXW7 protein inhibits migration and proliferation of micro-vascular endothelial cells of HG group, and a large amount of cell death is seen under an endoscope (P < 0.001).

(2) Effect of two proteins on vascular endothelial lumen formation

High sugar impairs the homeostasis of vascular endothelial cells, and high sugar toxicity seriously compromises the ability to form vascular endothelial lumens.

As shown in FIG. 10, 30mM high sugar has a strong toxic effect, inhibiting the luminal growth of the large vessel endothelial HUVEC (vs NG, P < 0.001). When HG concentration is reduced to 15mM, the lumen forming capability of the large vessel endothelium is enhanced after the protein EWS or Fbxw7 is over-expressed under high sugar, but no statistical difference exists (vs NG, P > 0.05); knocking down the EWS protein or the Fbxw7 protein aggravates the high-sugar toxicity effect of HUVEC and obviously weakens the tube forming capability (vs NG, P <0.01, or P <0.05 respecively).

In microvascular endothelial cells, as shown in fig. 11, 30mM high sugar has more obvious sugar toxicity (vs NG, P <0.01), and over-expression of EWS can improve the toxic effect caused by 30mM high sugar (vs NG, P < 0.05); knocking down endogenously expressed EWS or Fbxw7 protein further weakens the endothelial lumen forming ability (vs NG, all P <0.001), and strengthens the high sugar toxicity of the microvascular endothelium.

In conclusion, the two proteins, namely the EWS and the Fbxw7, are used as a protective mechanism and show the effect of relieving the hyperglycemia toxicity of the vascular endothelium, and the EWS and the Fbxw7 do not promote the proliferation and migration of the hyperglycemia vascular endothelium.

The EWS and FBXW7 proteins substantially reduced the level of ROS in the hypo-glycal vascular endothelium, correcting the over-activation of the hyperglycogenic PARP.

As shown in fig. 12, EWS and Fbxw7 proteins dramatically reduced ROS levels in HUVEC cells at high sugar to 41.72% (vs HG, P <0.001) and 68.80% (vs HG, P < 0.01); reduced to 55.27% (vs HG, P <0.05), 65.23% (vs HG, P <0.05) levels in microvascular HREC endothelium, showing good antioxidant damage function.

Expression level of PARP: the high sugar promotes PARP overexpression, and the overexpression of EWS and Fbxw7 not only greatly reduces PARP expression of HUVEC (vs HG, P < 0.01; vs HG, P <0.05, respecively) of the large vascular endothelium under the high sugar, but also greatly reduces PARP expression of the microvascular endothelium (vs HG, all P <0.05), as shown in FIG. 13.

Activity of PARP protease: high sugar increases PARP enzyme activity in macrovascular, microvascular endothelial cells (vs NG, all P < 0.05); both EWS and Fbxw7 were able to significantly reduce the activity of the enzyme (vs HG, P <0.001), and EWS had the most potent effect of reducing PARP enzyme activity (vs HG + F-OE, P <0.01), as shown in fig. 14.

Content of polyADP-ribose (PAR): PARP over-activation leads to the production of a large amount of PAR product, and inhibition of downstream GAPDH activity is the source of a cascade amplification effect, so we measured the content of polyADP-ribose (PAR). As shown in FIG. 15, high carbohydrate significantly increased PAR production (vs NG, P <0.001) in the great vessel HUVEC endothelium, while both EWS, Fbxw7 significantly decreased PAR production (vs HG, all P <0.001), most notably Fbxw7 (vs HG + E-OE, P < 0.05). Similar results to HUVEC were not observed with very low PAR production in the 30mM hyperglycemic group after 48h of high-glucose culture of microvascular endothelial HREC cells.

In conclusion, the EWS, Fbxw7 protein and the Fenofibrate can reduce the expression level of high-sugar vascular endothelial PARP, PARP enzyme activity and PAR production.

The EWS and Fbxw7 proteins repair DSBs (DNA double-strand breaks, DSBs) by activating the HR pathway or the NHEJ pathway.

Using OxiSelect^TMDNA Double Strand Break (DSB) stabilizing Kit (Cell Biolabs), as shown in FIG. 16, DSB indicator triggers a large green fluorescent deposition in the endothelial nucleus, i.e., a large Break in the DNA Double Strand. After 48 hours of high-sugar culture, there was an increase in the green punctate deposition of DSB in the HUVEC nuclei.

DSB mainly activates Homologous recombination repair (HR) and Non-Homologous end joining (NHEJ) pathways of cells, and the two act synergistically to maintain genome stability.

As shown in FIG. 17, the HR and NHEJ pathways of HUVEC in the high-sugar large blood vessel were significantly changed, and P-ATR kinase (vs NG, P <0.01), CHK2 protein (vs NG, P <0.05) and P-P53 protein (vs NG, P <0.001) were significantly increased. Conversely, the EWS protein reduced the expression levels of P-ATM, P-ATR, CHK2 protein (vs HG, all P <0.01) and P-P53 protein (vs HG, P < 0.05); the Fbxw7 protein is identical to the EWS protein (vs HG, all P < 0.01).

At the gene transcription level, high sugars reduced the expression of the ATM and BRCA1 genes of the large vessel endothelial HUVEC (vs NG, P <0.05), as shown in fig. 18. The expression of ATM, ATR, BRCA1, XRCC4 and MSH2 genes (vs HG, all P <0.001) can be greatly improved by the EWS protein; the Fbxw7 protein also greatly improves the expression of ATM, ATR, XRCC4 and MSH2 genes (vs HG, all P < 0.001).

In microvascular endothelial cells, as shown in figure 19, P-P53 protein decreased (vs NG, P <0.001), with no changes in other proteins. Under high sugar, the EWS only reduces the expression of CHK2 protein (vs HG, P <0.01), but the P-P53 protein still has a remarkable reduction compared with the NG group (vs NG + E-OE, P < 0.001); the Fbxw7 protein reduced the expression of P-ATR and CHK2(vs HG, all P <0.01), and still further reduced the expression of P-P53 protein.

As shown in fig. 20, at the transcriptional level, high sugars increased the expression of BRCA1 gene in the microvascular endothelium (vs NG, P < 0.001). The Fbxw7 protein can greatly improve the expression of ATM, ATR, BRCA1 and MSH2 genes (vs HG, all P <0.001) under high sugar, and compared with the NG + F-OE group, the expression of the five genes is obviously improved (all P < 0.001).

The detection and repair of DNA damage depends not only on the role of the DNA repair system, but also on the role of checkpoint signaling pathways in the cell cycle. As shown in fig. 21, HG promotes vascular endothelial cells to enter S phase, accelerating cell cycle operation; in large vessels, both EWS, Fbxw7 proteins blocked endothelial cells in stage G0/G1, providing sufficient time to repair damaged DNA (vs HG, P < 0.05).

The effects of EWS and Fbxw7 proteins on the number, function and signaling pathways involved in the mitochondria of vascular endothelial cells.

The EWS and Fbxw7 can influence the expression level of PGC-1 alpha in cells, and further researches the influence of the EWS and Fbxw7 on the function of vascular endothelial mitochondria in diabetes.

HIF-1 alpha, PGC-1 alpha, S6K1, ERR alpha gene are the main regulatory genes of the upstream of energy metabolism of mitochondria, and the downstream target genes comprise glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, lipid synthesis and other related genes; cox5b and ATP5O reflect mitochondrial respiratory function; SCAD and VLCAD reflect Fatty Acid Oxidation (FAO) levels. In the large vessel endothelium, the Fbxw7 protein significantly enhanced the expression of all the above genes under normal or high sugar (FIG. 22, vs NG, P < 0.01; vs HG, P < 0.01); the EWS protein also showed similar effects as Fbxw7 at normal sugar (NG) concentrations, increasing HIF-1 α, PGC-1 α, S6K1 and ATP5O expression (vs NG, P <0.05), but only HIF-1 α gene expression remained elevated at high sugar (vs HG, P < 0.01).

Accordingly, we examined the activity of several key enzymes on the signaling pathway involved in energy metabolism. As shown in fig. 23, high sugars promoted JNK activity of large vessel endothelial HUVEC and expression of glycolytic key enzyme PFKFB3 (vs NG, P < 0.001); the EWS and Fbxw7 protein can promote energy metabolism of vascular endothelium, not only can reduce the activity of P-JNK (vs HG, P <0.001) and the expression of PFKFB3 (vs HG, P <0.05), but also can activate the activity of AMPK (vs HG, P <0.05, P <0.01 respecitvely).

Protection of DM retinal tissue by EWS and Fbxw7

In diabetic retinopathy, early knockdown of endogenous EWS and Fbxw7 expression in the retinal tissues of DM rats increased the overall ROS levels in the retinal tissues, including RGC, inner and outer nuclear layers, with interference with Fbxw7 being particularly pronounced (fig. 24, vs DM, P < 0.05); retinal vascular leakage also worsened to some extent (figure 25, vs DM, P > 0.05).

The overall ROS level and PAR product content of retina tissues of DM rats are both obviously increased (figure 24, vs Control, P < 0.01; figure 26, vs Control, P <0.05), and the overexpression of the EWS or Fbxw7 protein can not only obviously reduce the overall ROS level of retina tissues (figure 24, vs DM, P <0.05or P <0.01, respecitvey) but also obviously reduce the PAR yield in the tissues (figure 26, vs DM, P <0.05or P <0.001, respecitvey).

With the decrease of PAR production in diabetic retinal tissues, EWS and Fbxw7 proteins also show some resistance to oxidative stress damage of the retina. High levels of ROS can oxidize modified proteins, either directly or indirectly, using OxiSelect^TMProtein Carbonyl ELISAKit we examined the most common form of Protein oxidation, as shown in FIG. 27, with a significant increase in the degree of oxidation of diabetic retinal tissue Protein (vs Control, P)<0.001), overexpression of Fbxw7 protein significantly reduced the oxidation level of the protein (vs DM, P)<0.001) and over-expression of the EWS protein was partially reduced.

High levels of ROS activate PARP activity, resulting in PAR-ation of GAPDH, loss of its normal enzymatic activity, diverting upstream heavily trafficked glycolytic products to the bypass, including glycosylation of proteins, contributing to DM complications. We examined the level of glycosylation of DM retinal tissue, and no significant increase was seen at the disease course of 12week, but over-expression of EWS significantly reduced retinal tissue glycosylation (figure 28, vs DM, P < 0.05).

Third, discuss

The relationship between EWS/FBXW7 and diabetic vascular complications and tumorigenesis of diabetic individuals is confirmed for the first time in the study.

The research finds that the fat differentiation protein EWS/FBXW7 is universally present in vascular endothelium and retinal nerve tissues, and the protein EWS/FBXW7 is widely involved in a plurality of sugar and lipid metabolic processes of the vascular endothelium. Both the EWS and FBXW7 proteins overexpressed in the great vessel endothelium and the microvascular endothelium can obviously reduce the high ROS level and the excessive activation of PARP caused by high sugar, and relieve the high sugar toxicity of the vascular endothelium; the EWS and FBXW7 proteins affect the important regulatory molecules ATM, ATR, BRCA1, P53, CHK2, etc. in the homologous recombination repair (HR) pathway or the non-homologous end-joining repair (NHEJ) pathway and arrest damaged endothelial cells at G0/G1 for repair of damaged DNA. In diabetic rat retinal tissues, the over-expression of the proteins EWS and Fbxw7 shows a retinal protective function, and can reduce the high ROS level and PAR yield and reduce oxidative stress injury of the retinal tissues. These results indicate that the lipid-regulating protein EWS/FBXW7 has a good application prospect as a target molecule for resisting diabetic vascular complications, and the action mechanism of the lipid-regulating protein EWS/FBXW7 for reducing ROS and oxidative stress caused by high sugar is very consistent with the unified mechanism theory of diabetic complications recognized by the present academia [22-23 ].

The diabetes complications "unified mechanistic theory" (a unifing Mechanism) was proposed by professor hunter Brownlee 2005, who reviewed important research results in this field of research in recent 40 years. At the heart of this theory, high sugar causes excessive production of reactive oxygen species ROS in the mitochondria of target tissue cells. Excessive ROS strand breaks nuclear DNA, causing excessive activation of the endonuclear DNA repair enzyme poly- (ADP-ribose) polymerase (PARP), which generates a large amount of (ADP-ribose) Polymer (PAR) and nicotinic acid by cleaving the substrate NAD +, and a large amount of PAR accumulates around glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other intranuclear proteins, causing ADP-ribosylation of GAPDH, resulting in inhibition of its activity [6 ]. Inhibition of GAPDH enzymatic activity causes accumulation of metabolic intermediates in the glycolytic pathway, i.e. glyceraldehyde 3-phosphate, fructose 6-phosphate or even glucose upstream of GAPDH, leading to the transfer of these metabolites to other glycolytic side branches, including activation of the polyol pathway (polyol pathway) [7], formation of non-enzymatic glycosylation end products [8], enhancement of the activity of protein kinase c (protein kinase c) [9] and activation of the hexosamine (hexosamine biosynthesis pathway) [10], ultimately triggering diabetic complications in target tissue cells.

The excessive production of ROS in mitochondria caused by high sugar, undoubtedly, ROS is a key target spot for preventing and treating DR; secondly, blocking excessive activation of PARP to restore GAPDH activity is an important link to prevent ROS waterfall damage caused by signal cascade amplification. There is evidence that normalizing mitochondrial ROS levels can remedy both glyceraldehyde-3-phosphate dehydrogenase inactivation and glucose transport to the glycolytic alternative pathway [5, 21], and that inhibition of PARP can block activation of the hyperglycemia-induced multiple vascular injury pathways [22-23 ]. The research discovers for the first time that the fat differentiation proteins EWS and FBXW7 not only obviously reduce the high ROS level from the source, but also control the key upstream of the amplification of the cascade effect by inhibiting the over-activation of PARP, and the superposition of the two effects ensures the efficiency and the strength of resisting the vascular endothelial complications; these effects are widely manifested in a variety of cells or tissues, including large blood vessels, micro blood vessels, or even retinal nerve tissue, suggesting that the protective effects of EWS and Fbxw7 proteins may be prevalent in multiple tissues and multiple organs of diabetic patients, with broad tissue utility.

Therefore, two proteins, namely the EWS and the Fbxw7, are used as a protection mechanism and can relieve the high sugar toxicity of vascular endothelium; since neither of them promotes the proliferation and migration of vascular endothelium in a high-sugar environment, they can be used in diabetic patients in a large scale. Therefore, the two are expected to become novel target molecules for relieving diabetic vascular endothelial complications. It is noted that the EWS and FBXW7 proteins cannot be used in the normal population because they affect the proliferation and migration of normal vascular endothelium.

The activation of homologous recombination repair (HR) pathway or non-homologous end-joining repair (NHEJ) pathway by the EWS and the Fbxw7 also gives a new hint, and is a new idea worth trying in the current treatment means of diabetic complications. High-sugar high ROS leads to excessive activation of PARP to repair DNA damage and maintain stability of the genome of affected cells, and is a self-protection strategy adopted by higher eukaryotic cells under high sugar to avoid self-death. At present, the therapeutic approach for inhibiting excessive activation of PARP is mainly to use PARP inhibitor, which binds to PARP 1or PARP2 catalytic site, so that PARP protein cannot fall off from DNA damage site and loses the ability to repair DNA damage, therefore, it is mainly used as a hot anticancer drug successfully using Synthetic Lethality (Synthetic Lethality) concept in clinical application. Based on such mechanism of action, PARP inhibitors such as nicotinamide, 3-aminobenzamide and competitive specific inhibitor PJ34[22] only simply ensure the normal activity of GAPDH and to some extent relieve the pressure of glycolytic bypass such as PKC activation, AGEs formation, polyol accumulation in vitro when treating diabetic complications; in vivo, PJ34 can inhibit the death and pericyte loss of retinal capillary cells in diabetic rats and prevent the early damage of diabetic retinopathy [24 ]. However, the conventional PARP inhibitor treatment neglects a serious problem that the genome of the affected cells is damaged and not repaired, and the genome instability caused by high sugar and high ROS is still not solved effectively, and the affected cells are still exposed to death at any time. Therefore, the clinical treatment does not achieve good effect, the efficacy is low, and the effect is nonspecific.

Completely different from the action mechanism of the traditional PARP inhibitor, the lipid regulating proteins EWS and Fbxw7 have the DNA damage repair function and can play the repair role by influencing important regulatory molecules ATM, ATR, BRCA1, P53, CHK2, XRCC4 and MSH2 in homologous recombination repair (HR) or non-homologous end joining repair (NHEJ). DSB severely threatens cell survival as the most severe form of damage to genomic DNA, homologous recombination repair (HR) or non-homologous end joining repair (NHEJ) being the predominant form of repair of DSB damage by the body. Among them, ATM protein is responsible for initiating the response of cells to DNA double strand break damage, is the most core kinase regulating genome stability, and can directly phosphorylate over 1000 important substrates in cells, including p53 protein, cell cycle regulatory protein, etc. The Ku protein in NHEJ repair competes with PARP for binding to the ends of DSBs, and the high affinity of Ku protein for DNA and the competing effects of other types of damage limit the role of PARP in the DSB repair pathway; the function of PARP is of great importance for the repair of DSB only when the classical DNA-PK pathway is absent [25], which is why the EWS and Fbxw7 proteins can inhibit the excessive activation of PARP under high sugar. Therefore, the EWS and Fbxw7 proteins not only inhibit the excessive activation of PARP, but also solve the problem remained by the traditional PARP inhibitors, namely, the proteins can also repair DNA damage caused by high sugar and high ROS, and the two aspects are simultaneously considered and have consistent treatment thought, thereby having obvious advantages compared with the traditional PARP inhibitors which are commonly used at present.

The Fbxw7 protein and the EWS protein are found to show a DNA repair function in the diabetes vascular endothelial glycolipid toxicity for the first time in the research. Fbxw7 has been considered as a tumor suppressor gene, which can ubiquitinate Notch1, C-myc, Cyclin E, C-Jun, mTOR to play an anticancer role. The physiological function of EWS is still unclear, and it is notorious for the fact that it often undergoes chromosomal translocation with TET family, and the resulting chimeric protein has high transcriptional activity, resulting in various human malignancies. Therefore, there are very few reports on the research of EWS and DNA damage repair, and the mechanism is not known. The research firstly discovers that the EWS protein can activate HR and NHEJ to repair DNA damage of high-sugar vascular endothelium by activating ATM, ATR, CHK2 and P53.

In the large vessel endothelium, the protein EWS and Fbxw7 not only promote the increase of the copy number of mitochondria, but also activate the transcription of genes related to the biosynthesis of the mitochondria, and enhance the energy metabolism of the mitochondria by stimulating the activities of transcriptional regulatory factors such as PGC-1 alpha, HIF-1 alpha, estrogen related receptor (ERR alpha), S6K1 and the like and enhancing the functions of the respiratory chain of the mitochondria such as Cox5b and ATP 5O. At the protein expression level, the protein EWS and Fbxw7 improves the AMPK activity of endothelial cells under high sugar, reduces the JNK activity and the expression of glycolytic key enzyme PFKFB3, and enables the endothelial cells to be in a vigorous catabolic state. It is reported that AMPK activity is increased, JNK activity is reduced [26-27] to increase the sensitivity of hyperglycosylated vascular endothelium to insulin, and that reduction of the resistance of endothelial cells to insulin is beneficial to prevention and treatment of complications such as macroangiosclerosis and coronary heart disease which are accompanied with lipid deposition intracellularly [28 ]. Recent research reports indicate that the mechanism of increased atherosclerosis, coronary heart disease, and increased occurrence of cardiovascular accidents with diabetes is still high carbohydrate → high ROS → overactivation of PARP → inhibition of GAPDH → activation of carbohydrate metabolism bypass, while high ROS high PARP → reduction of sirtuin, PGC1 α and AMPK activity → disruption of the synchronous rhythms of carbohydrate and lipid metabolism → induction of cardiovascular accidents [29 ]. Therefore, the lipid regulating proteins EWS and Fbxw7 are beneficial in this respect to reducing the occurrence of cardiovascular accidents in diabetes.

PCR primer sequences:

reference documents:

[1] study progress on therapeutic targets of zheng, liu qiu shuang, jinjing, diabetes complications [ J ] strait pharmacology, 2014, 26 (1): 13-17.

[2] Centuries xiang American Diabetes Association (ADA) course [ J ] diabetes mellitus tiandi: clinical, 2015, 9 (7): 351-355.

[3]An Y,Zhang P,Wang J,Gong Q,Gregg EW,Yang W,Li H,Zhang B,Shuai Y,Chen Y,Engelgau MM,Cheng Y,Hu Y,Bennett PH,Li G.Cardiovascular and All-Cause Mortality Over a 23-Year Period Among Chinese With Newly Diagnosed Diabetes in the Da Qing IGT and Diabetes Study.Diabetes Care.2015 Jul；38(7):1365-71.

[4] Hedan, Zhengyi, Chen Chuang, Chenjian super, Sun Tiemin, research progress of medicine for treating diabetic complications, Zhongnan pharmacy, 2017, 15 (6): 714-721.

[5]Brownlee M.The pathobiology of diabetic complications:a unifying mechanism.Diabetes.2005；54:1615-1625.

[6]Pacher E,Szabo C.Role of poly(ADP-ribose)polymerase-1activation in the pathogenesis of diabetic complications：endothelial dysfunction，as a common underlying theme.Antioxid Redox Signal.2005,7：1568-1580.

[7]Lee AY,Chung SS.Contributions of polyol pathway to oxidative stress in diabetic cataract.FASEB J，1999，13：23-30.

[8]Du XL,Edelstein D，Rossetti L,et a1.Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1expression by increasing Spl glycosylation.Proc Natl Acad Sci U S A，2000，97：12222—12226.

[9]Koya D，King G L.Protein kinase C activation and the development of diabetic complications.Diabetes，1998，47：859—866.

[10]Kolm-Litty V,Sauer U,Nerlich A,et a1.High glucose-induced transforming growth factor betal production is mediated by the hexosamine pathway in porcine glomerular mesangial cells.J Clin Invest，1998，101：160-169.

[11] sheng, liu hongfei, xuximing, yunjiannan, research progress of medicine for treating diabetic complications, chinese drugstore, 2012, 26 (8): 906-910.

[12] Dingli, Dingyu, development and progress of drugs for treating diabetic complications, China New medicine journal, 1999, 8 (10): 1-5.

[13]Xu Y,Wang L,He J,et al.Prevalence and Control of Diabetes in Chinese Adults[J].JAMA,2013,310(9):948-959.

[14]Liu X,Hemminki K,Forsti A,et al.Cancer risk in patients with type 2 diabetes mellitus and their relatives.[J].International Journal of Cancer,2015,137(4):903-910.

[15]Brower V.Illuminating the Diabetes–Cancer Link.Journal of the National Cancer Institute,2012,104(14):1048-1050.

[16]Giovannucci E,Harlan D M,Archer M C,et al.Diabetes and Cancer:A Consensus Report.CA:A Cancer Journal for Clinicians,2010,60(4):207-221.

[17]Bengoechea-Alonso MT,Ericsson J.The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBPαfor degradation.PNAS.2010；107(26):11817–11822.

[18]Sundqvist A,Bengoechea-Alonso MT,Ye X,et al.Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF(Fbw7).Cell Metab.2005；1(6):379–391.

[19]Kim JB1,Spiegelman BM.ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism.Genes Dev.1996；10(9):1096-107.

[20]Jun Hong Park,Hong-Jun Kang,Yun Kyung Lee,et al.Inactivation of EWS reduces PGC-1αprotein stability and mitochondrial homeostasis.PNAS.2015；112(19):6074–6079.

[21]Nishikawa T,Edelstein D,Du XL,Yamagishi S,Matsumura T,Kaneda Y,Yorek MA,Beebe D,Oates PJ,Hammes HP,Giardino I,Brownlee M.Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.Nature.2000；404:787-790.

[22]Du X,Matsumura T,Edelstein D,Rossetti L,Zsengellér Z,SzabóC,Brownlee M.Inhibition of GAPDH activity by poly(ADP-ribose)polymerase activates three major pathways of hyperglycemic damage in endothelial cells.J Clin Invest.2003 Oct；112(7):1049-57.

[23]Garcia Soriano F,Virág L,Jagtap P,SzabóE,Mabley JG,Liaudet L,Marton A,Hoyt DG,Murthy KG,Salzman AL,Southan GJ,SzabóC.Diabetic endothelial dysfunction:the role of poly(ADP-ribose)polymerase activation.Nat Med.2001 Jan；7(1):108-13.

[24]Zheng L,SzabóC,Kern TS.Poly(ADP-ribose)polymerase is involved in the development of diabetic retinopathy via regulation of nuclear factor-kappaB.Diabetes.2004 Nov；53(11):2960-7.

[25]Wang M,Wu W,Wu W,Rosidi B,Zhang L,Wang H,Iliakis G.PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.Nucleic Acids Res.2006；34(21):6170-82.

[26]Hirosumi J,Tuncman G,Chang L,et al.A central role for JNK in obesity and insulin resistance.Nature.2002；420(6913):333–336.

[27]Kamata H,Honda S,Maeda S,et al.Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.Cell.2005；120(5):649–661.

[28]R Kosuru,Y Cai,V Kandula,et al.AMPK contributes to cardioprotective effects of pterostilbene against myocardial ischemia-reperfusion injury in diabetic rats by suppressing cardiac oxidative stress and apoptosis.Cell Physiol Biochem.2018,46(4):1381-1397.

[29]Manasi S.Shah,and Michael Brownlee.Molecular and Cellular Mechanisms of Cardiovascular Disorders in Diabetes.Circ Res.2016 May 27；118(11):1808–1829.

[30]Yada M,Hatakeyama S,Kamura T,Nishiyama M,Tsunematsu R,Imaki H,Ishida N,Okumura F,Nakayama K,Nakayama KI.Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7.EMBO J.2004；23(10):2116-25.

[31]Strohmaier H,Spruck CH,Kaiser P,Won KA,Sangfelt O,Reed SI.Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.Nature.2001；413(6853):316-22.

[32]Welcker M1,Clurman BE.FBW7ubiquitin ligase:a tumour suppressor at the crossroads of cell division,growth and differentiation.Nat Rev Cancer.2008；8(2):83-93.

[33]Jian-Hua Mao,Il-Jin Kim,Di Wu,et al.FBXW7Targets mTOR for Degradation and Genetically Cooperates with PTEN in Tumor Suppression.Science.2008；321(5895):1499–1502.

[34]Wang Z,Inuzuka H,Zhong J,Wan L,Fukushima H,Sarkar FH,Wei W.Tumor suppressor functions of FBW7in cancer development and progression.FEBS Lett.2012；586(10):1409-18.

[35]Zhao J,Xiong X,Li Y,et al.Hepatic F-Box protein FBXW7 maintains glucose homeostasis through degradation of Fetuin-A.Diabetes.2018；67(5):818-830.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> first-person hospital in Shanghai City

Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes

<130> /

<160> 32

<170> PatentIn version 3.3

<210> 1

<211> 1971

<212> DNA

<213> human

<400> 1

atggcgtcca cggattacag tacctatagc caagctgcag cgcagcaggg ctacagtgct 60

tacaccgccc agcccactca aggatatgca cagaccaccc aggcatatgg gcaacaaagc 120

tatggaacct atggacagcc cactgatgtc agctataccc aggctcagac cactgcaacc 180

tatgggcaga ccgcctatgc aacttcttat ggacagcctc ccactggtta tactactcca 240

actgcccccc aggcatacag ccagcctgtc caggggtatg gcactggtgc ttatgatacc 300

accactgcta cagtcaccac cacccaggcc tcctatgcag ctcagtctgc atatggcact 360

cagcctgctt atccagccta tgggcagcag ccagcagcca ctgcacctac aagaccgcag 420

gatggaaaca agcccactga gactagtcaa cctcaatcta gcacaggggg ttacaaccag 480

cccagcctag gatatggaca gagtaactac agttatcccc aggtacctgg gagctacccc 540

atgcagccag tcactgcacc tccatcctac cctcctacca gctattcctc tacacagccg 600

actagttatg atcagagcag ttactctcag cagaacacct atgggcaacc gagcagctat 660

ggacagcaga gtagctatgg tcaacaaagc agctatgggc agcagcctcc cactagttac 720

ccaccccaaa ctggatccta cagccaagct ccaagtcaat atagccaaca gagcagcagc 780

tacgggcagc agagttcatt ccgacaggac caccccagta gcatgggtgt ttatgggcag 840

gagtctggag gattttccgg accaggagag aaccggagca tgagtggccc tgataaccgg 900

ggcaggggaa gagggggatt tgatcgtgga ggcatgagca gaggtgggcg gggaggagga 960

cgcggtggaa tgggcagcgc tggagagcga ggtggcttca ataagcctgg tggacccatg 1020

gatgaaggac cagatcttga tctaggccca cctgtagatc cagatgaaga ctctgacaac 1080

agtgcaattt atgtacaagg attaaatgac agtgtgactc tagatgatct ggcagacttc 1140

tttaagcagt gtggggttgt taagatgaac aagagaactg ggcaacccat gatccacatc 1200

tacctggaca aggaaacagg aaagcccaaa ggcgatgcca cagtgtccta tgaagaccca 1260

cccactgcca aggctgccgt ggaatggttt gatgggaaag attttcaagg gagcaaactt 1320

aaagtctccc ttgctcggaa gaagcctcca atgaacagta tgcggggtgg tctgccaccc 1380

cgtgagggca gaggcatgcc accaccactc cgtggaggtc caggaggccc aggaggtcct 1440

gggggaccca tgggtcgcat gggaggccgt ggaggagata gaggaggctt ccctccaaga 1500

ggaccccggg gttcccgagg gaacccctct ggaggaggaa acgtccagca ccgagctgga 1560

gactggcagt gtcccaatcc gggttgtgga aaccagaact tcgcctggag aacagagtgc 1620

aaccagtgta aggccccaaa gcctgaaggc ttcctcccgc caccctttcc gcccccgggt 1680

ggtgatcgtg gcagaggtgg ccctggtggc atgcggggag gaagaggtgg cctcatggat 1740

cgtggtggtc ccggtggaat gttcagaggt ggccgtggtg gagacagagg tggcttccgt 1800

ggtggccggg gcatggaccg aggtggcttt ggtggaggaa gacgaggtgg ccctgggggg 1860

ccccctggac ctttgatgga acagatggga ggaagaagag gaggacgtgg aggacctgga 1920

aaaatggata aaggcgagca ccgtcaggag cgcagagatc ggccctacta g 1971

<210> 2

<211> 1884

<212> DNA

<213> human

<400> 2

atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactg tggagttttg 60

ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtc cacattagaa 120

tctgtgacat acctacctga aaaaggttta tattgtcaga gactgccaag cagccggaca 180

cacgggggca cagaatcact gaaggggaaa aatacagaaa atatgggttt ctacggcaca 240

ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctga ggtccgctct 300

ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccac tgggcttgta 360

ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatgg ccaagggcaa 420

caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatg gctaaaaatg 480

tttcagagct ggagtggacc agagaaattg cttgctttag atgaactcat tgatagttgt 540

gaaccaacac aagtaaaaca tatgatgcaa gtgatagaac cccagtttca acgagacttc 600

atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctgga acccaaagac 660

ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaaga caaccttctc 720

tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaa gagaagaaaa 780

gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcag acagcacaga 840

attgatacta actggaggcg aggagaactc aaatctccta aggtgctgaa aggacatgat 900

gatcatgtga tcacatgctt acagttttgt ggtaaccgaa tagttagtgg ttctgatgac 960

aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacatt agtgggacat 1020

acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtgg atctacagat 1080

cggacactca aagtgtggaa tgcagagact ggagaatgta tacacacctt atatgggcat 1140

acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcgg ttctcgagat 1200

gccactctta gggtttggga tattgagaca ggccagtgtt tacatgtttt gatgggtcat 1260

gttgcagcag tccgctgtgt tcaatatgat ggcaggaggg ttgttagtgg agcatatgat 1320

tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgtt gcaggggcat 1380

actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtgg atctcttgat 1440

acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgtt aacagggcac 1500

cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgg gaatgcagat 1560

tctacagtta aaatctggga tatcaaaaca ggacagtgtt tacaaacatt gcaaggtccc 1620

aacaagcatc agagtgctgt gacctgttta cagttcaaca agaactttgt aattaccagc 1680

tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttat tcgaaaccta 1740

gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagc ctcaaacaca 1800

aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagct gctggtgctg 1860

gactttgatg tggacatgaa gtga 1884

<210> 3

<211> 58

<212> DNA

<213> Artificial sequence

<400> 3

ccgggcatga gtggccctga taattcaaga gattatcagg gccactcatg cttttttg 58

<210> 4

<211> 58

<212> DNA

<213> Artificial sequence

<400> 4

aattcaaaaa agcatgagtg gccctgataa tctcttgaat tatcagggcc actcatgc 58

<210> 5

<211> 58

<212> DNA

<213> Artificial sequence

<400> 5

ccggcaacaa cgacgccgaa ttattcaaga gataattcgg cgtcgttgtt gttttttg 58

<210> 6

<211> 58

<212> DNA

<213> Artificial sequence

<400> 6

aattcaaaaa acaacaacga cgccgaatta tctcttgaat aattcggcgt cgttgttg 58

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

tttgcttgag gctgatcctt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

tgattgactc tgcagccaac 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

gtggtcatga gccgattttt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

gctggtagtc ctccaagctg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

tcatgccagc tcattacagc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<400> 12

taagccaggc tgtttgcttt 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

ttggacacca ttgcagaaaa 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

ctcggtcagc agtcatttca 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

ggtgttttgt gccatgtgag 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

ttccaacatt tcagccatga 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

ctgggttgga gagggagatc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

ttgttggaga tggaggggac 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> 19

ccaaagtggc tgcttctgtt 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

actgtgcaag gtacctctcc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<400> 21

gtgatactcc gggccttcat 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

tggcttctgg aaccttgaca 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

aatcagttct tgggacccgt 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

cagtgccagc caagatgatc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

ctactaaagg ccttggccct 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

cgagcatctc caagaacagc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<400> 27

ggacgctgga gaagttcaag 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<400> 28

cggatttttg gttcaaagga 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<400> 29

ccagatctcg gcgaagtaaa 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<400> 30

cctcacacgc aaatagctga 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

gcccaggtac agtgagtctt 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<400> 32

gtgaggactg aggacttgct 20

Claims (3)

1. The application of FBXW7 or the up-regulation agent thereof in preparing the medicine for preventing and treating diabetic retinopathy.
2. 2. The use of claim 1, wherein the upregulating agent is selected from the group consisting of small molecule compounds and biological macromolecules.
3. The application of FBXW7 as a target point in screening medicines for preventing and treating diabetic retinopathy.

Images