CN102258771A - Application of recombinant hirudin in preventing and treating diabetic cataract - Google Patents

Abstract

The invention discloses new application of recombinant hirudin, and in particular relates to application of recombinant hirudin in preventing and treating diabetic cataract.

Description

Application of Recombinant Hirudin in Prevention and Treatment of Diabetic Cataract

technical field

The invention belongs to the field of bioengineering pharmacy or protein polypeptide medicine, and relates to the application of hirudin in preventing and treating diabetic cataract. the

Background technique

Diabetes seriously endangers human health, and diabetic cataract (DC) is a major diabetic complication and blinding eye disease. With the continuous improvement of living standards and population aging, the number of diabetic patients worldwide is increasing day by day, and the number of diabetic cataract patients is also increasing dramatically. According to statistics, the incidence of cataract among type II diabetic patients in my country is as high as 62.37%. the

Although cataracts can be removed surgically, complications such as clouding of the posterior capsule of the lens (post-cataract) often occur after surgery. What's more noteworthy is that cataracts in diabetic patients are often not suitable for this kind of surgical treatment, because many patients will have severe complications such as opacity of the posterior lens capsule (post-cataract) after surgery. Therefore, the drug treatment of diabetic cataract has always been concerned by medical experts, especially in the early stage of cataract, doctors tend to prefer drug treatment to relieve symptoms and control the development of the disease. Therefore, finding effective preventive and treatment drugs for diabetic cataract has important practical application value. the

Hirudin is a natural anticoagulant protein polypeptide, composed of 65-66 amino acids, and its molecule is like a tadpole. The hirudin molecule consists of two structural domains, which respectively interact with two different surface sites on the surface of the thrombin molecule. Its N-terminal domain is a spherical dense core structure formed by amino acids 1-47, which binds to the active site of thrombin and inhibits its catalytic activity; while its negatively charged carboxy-terminal domain Amino acids (49-65/66) bind to the anion binding exosite (or fibrinogen recognition site) of thrombin, thereby inhibiting the interaction between thrombin and fibrinogen. Between these two domains there is a finger like structure that protrudes outward from the complex and does not come into contact with thrombin. This finger-like structure is based on the amino acid residues 27-31 and 36-40 of hirudin forming an antiparallel β-sheet (antiparallel β-sheet) chain, and the finger end is formed by amino acid residues 32-35 to form a flexible β-turn. It shows that the finger structure is not involved in the inhibition of thrombin, and belongs to the non-functional essential region. the

Hirudin is the most powerful thrombin inhibitor found so far, it can effectively inhibit the formation of arterial and venous thrombosis and disseminated intravascular coagulation (DIC), and has a very high role in the prevention and treatment of cardiovascular and cerebrovascular thrombotic diseases. Medicinal value. Because hirudin can inhibit both free thrombin and thrombin bound to thrombus (heparin can only act on free thrombin), and there is no side effect of thrombocytopenia caused by heparin allergy, so the effect of hirudin better than heparin. In addition, hirudin has great potential clinical application value in the prevention and treatment of unstable angina, disseminated intravascular coagulation, cerebral coagulation, thrombophlebitis and coronary artery thrombosis. the

Wu Wutong et al. (Chinese Invention Patent, Patent No.: 03132224.7) pharmacological tests have proved that recombinant hirudin has a significant preventive effect on animal cataract induced by D-galactose and sodium selenite in vivo and in vitro, but no experimental evidence has been found. Prevention and treatment effect of recombinant hirudin on streptozotocin (streptozotocin, STZ)-induced diabetic cataract model. the

Contents of the invention

The invention discloses a new application of recombinant hirudin, especially the application in preventing and treating diabetic cataract. the

1. Experimental materials

1. Drug under test: Recombinant Hirudin (III) Eye Drops, a colorless transparent liquid, the preparation method of which can be found in the Chinese Invention Patent (Patent No.: 03132224.7). The bases of hirudin eye drops were made into 2000U/ml, 1000U/ml and 500U/ml respectively, and refrigerated at 4°C. the

2. Positive control drug: pirenoxine sodium eye drops (Bainaiting), product of Wuhan Yuanda Pharmaceutical Group Co., Ltd. (batch number: 101015), specification: each tablet contains pirenoxine sodium 0.8 mg, stored at 4°C Refrigerator chilled. the

3. Experimental animals: Male SPF grade SD rats, weighing 130±20g, a total of 120 were purchased from Shanghai Xipuer-Bikay Laboratory Animal Co., Ltd. (certificate number: SCXK (Shanghai) 2008-0016). Rats were fed pelleted diet and had free access to water. the

4. Other reagents: STZ (provided by Sigma Company of the United States, China CAS NO.18883-66-4 Pcode: 1000830091.); citric acid (Shanghai Chemical Reagent Company, China Pharmaceutical Group, batch number: 20000801); sodium citrate (Shanghai Reagent 4 factory, batch number: 080330); compound tropicamide eye drops (Shenyang Xingqi Pharmaceutical Co., Ltd., batch number: 110102); total superoxide dismutase (T-SOD) test kit, malondialdehyde (MDA) test kit , Coomassie Brilliant Blue Protein Assay Kit was purchased from Nanjing Jiancheng Bioengineering Institute. the

5. Main experimental instruments: YZ5E slit lamp microscope (Suzhou Liuliu Vision Co., Ltd.)

Two, some pharmacological test data of the present invention. the

1. STZ preparation: take 0.21g citric acid and add 10ml distilled water to make citric acid solution, take 0.29g sodium citrate and add 10ml distilled water to make sodium citrate solution. Take 1.1ml of the above-mentioned citric acid solution and 0.86ml of the sodium citrate solution to prepare 100ml buffer solution with pH=4.5. Take 70ml of buffer solution, add 1.4g of STZ to fully dissolve, and prepare 2% STZ solution [1]. the

2. Preparation of diabetic cataract model and therapeutic effect of recombinant hirudin (III) eye drops

Rats were fasted for 24 hours without water, and were divided into two groups: normal control group and model group. Rats in the normal control group were intraperitoneally injected (65 mg/kg) the above buffer solution, and rats in the model group were intraperitoneally injected with 2% STZ (65 mg/kg, 0.325 ml/100 g) solution. After the injection, the rats were fed normally. After 72 hours, fasting blood glucose > 11mmol L ^-1 in rats was the criterion for success of the diabetes model. After 2 weeks of modeling, the rats were dilated with compound tropicamide eye drops, and the degree of lens turbidity in the rats was observed with a slit lamp microscope. Record the result ^[1] . According to the degree of turbidity of the lens, the model rats were randomly divided into 5 groups, so that the rats with different degrees of turbidity could be evenly distributed to the model group, the positive drug group, the high-dose group, the middle-dose group, and the low-dose group; As the normal control group, the rats were divided into 6 groups, with 16 rats in each group. The high-dose group, the middle-dose group, and the low-dose group were given 2000U/ml, 1000U/ml, and 500U/ml recombinant hirudin III eye drops respectively, and the positive drug group was given Bai Nei Ting eye drops, all 3 times a day, each time 1 drop. After 4 weeks of administration, compound tropicamide eye drops were used to dilate the pupils, and the turbidity of the lens of the rats was regularly observed with a slit lamp microscope, and the results were recorded after observation. The degree of lens opacity was assigned different scores according to the standards in Table 1, and a t-test between groups was performed ^[2,3] .

Table 1. Scoring criteria for rat lens opacity

Two weeks after modeling, the degree of lens turbidity in the model group, the recombinant hirudin III high, low, and high dose groups, and the positive drug group was significantly different from that in the normal control group (p<0.01). The lens of the normal control group was transparent, and the lenses of other groups showed obvious vacuoles, indicating that the rat diabetic cataract model was successfully prepared (see Table 2 and Figure 1). the

n＝16)  Table 2. Rat lens opacity degree after 2 weeks of modeling (

n=16)

Note: ^* p<0.05, ^** p<0.01vs normal control group

After 4 weeks of administration, there was a very significant difference (p＜0.01) between the high, medium and low dose groups of recombinant hirudin III and the model group in terms of lens turbidity, and there was a very significant difference between the high, high and low dose groups and the positive drug group (p <0.01) show that each dose group can significantly relieve lens opacity, and is better than the curative effect of the active drug (see Table 3). the

Table 3. The effect of recombinant hirudin III on the degree of lens opacity in rats after 4 weeks of administration

Note: ^* p<0.05, ^** p<0.01vs model group; ^# p<0.05, ^## p<0.01vs positive drug group

The lens of the normal control group was transparent and clear; the peripheral cortex and nucleus of the lens in the model group were opaque, and had reached the stage of deep cataract; the cortical opacity around the lens of the low-dose group had a certain curative effect compared with the model group, and was similar to the positive drug; the lens of the medium-dose group There are medium vacuoles, and the high-dose group has fine vacuoles. Compared with the model group, the curative effect is significant, and it is better than the positive drug group. (See Figure 2)

After administration of recombinant hirudin III for 4 weeks, the rats were sacrificed to take the eyeballs and separate the lens. Superoxide dismutase (SOD) kit and malondialdehyde (MDA) kit were used to detect SOD activity and MDA content in rat lens. Compared with the model group, the high-dose group, the middle-dose group and the low-dose group could significantly increase the SOD activity (p<0.01); the high-dose group and the middle-dose group could significantly reduce the MDA content (p<0.01), and the low-dose group The dose group can significantly reduce the content of MDA (p<0.05), indicating that recombinant hirudin has a better therapeutic effect on diabetic cataract. Compared with the positive control group, the high-dose group can increase the SOD activity (p<0.05), indicating that the effect of high-dose recombinant hirudin is better than that of Baineiting (see Table 4). the

Table 4. Effect of recombinant hirudin (III) on SOD activity and MDA content in rat lens

Note: *p<0.05, **p<0.01 compared with the model group; ##p<0.05, ##p<0.01 compared with the positive group

3. Experimental conclusions: The experimental results of diabetic cataract in rats showed that after giving recombinant hirudin high (2000U/ml), medium (1000U/ml) and low (500U/ml) doses of eye drops, after four weeks, the degree of lens turbidity was observed and determined by slit lamp. SOD activity and MDA content, the results show that recombinant hirudin can significantly reduce lens turbidity, alleviate the development of diabetic cataract symptoms, increase SOD activity, reduce MDA content, significantly improve the severity of diabetic cataract in rats, and the effect is better than positive Medicine (Bai Nei Ting). the

Figure 1 Rat cataract lens two weeks after modeling: 1 is the normal control group, 2 is the model group, 3 is the positive drug group, 4 is the high-dose group, 5 is the middle-dose group, 6 is the low-dose group

Figure 2 Cataract lens of rats after 4 weeks of administration: 1 is the normal control group, 2 is the model group, 3 is the positive drug group, 4 is the high dose group, 5 is the middle dose group, 6 is the low dose group

references

[1] Ding Zhenghua, Yan Hong, Guo Yong, Ha Wenjing, Wang Jianwei. Pathogenesis of streptozotocin-induced diabetic cataract in rats [J]. J Fourth Mil Med Univ, 2006, 27 (13): 1208-1210. 

[2] Bian Xiaoyun, Zhang Yachao, Song Xiujun. Experimental study on the prevention and treatment of diabetic cataract with taurine [J]. Ophthalmology Research, 2000, 18(4): 302-304.

[3] Leng Fei, Guan Linan, Liu Ping, Gao Weiqi, Li Zhijian, Ge Hongyan, Zhang Jiaying. The effect of salvianolic acid B eye drops on diabetic cataract in rats. Journal of Harbin Medical University, 2009, 43(3): 249-251.

Claims (1)

1. The application of recombinant hirudin in the prevention and treatment of diabetic cataract.

Images