CN115252622B - Aldose reductase inhibitor and its application in the preparation of medicines for treating lung cancer - Google Patents

Abstract

The invention provides an aldose reductase inhibitor, the active ingredient of which includes one or more of olanzapine, quercetin and baicalin; Drug application in lung cancer. The present invention finds for the first time that olanzapine, as an aldose reductase inhibitor, can significantly inhibit the activity of lung cancer cells and cause cell apoptosis. Due to insufficient supply. Combined use of natural product aldose reductase inhibitors (quercetin or baicalin) can significantly inhibit the activity of aldose reductase, and the effect is better. More importantly, olanzapine among the aldose reductase inhibitors has been marketed as a drug with high safety and is mainly used clinically for the treatment of schizophrenia and manic episodes. The present invention finds for the first time that olanzapine alone or in combination with a natural product aldose reductase inhibitor can be used to treat lung cancer, which will benefit more lung cancer patients.

Description

Aldose reductase inhibitor and its application in the preparation of medicines for treating lung cancer

technical field

The invention relates to the technical field of medical applications, in particular to an aldose reductase inhibitor and its application in the preparation of medicines for treating lung cancer.

Background technique

Lung cancer is mainly divided into two types: small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer is the main subtype of lung cancer, accounting for about 80%-85% of all lung cancers. For patients with early and locally advanced non-small cell lung cancer, surgical resection or combined radiotherapy and chemotherapy is the current first-line treatment, but the efficiency is low. So far, in the treatment of lung cancer, only immunotherapy is better. In view of the high price, many lung cancer patients still choose chemotherapy as the first-line treatment. The survival prognosis of lung cancer is still not optimistic, and it is urgent to try and develop new drugs.

Aldose reductase (aldose reductase, AR) is a member of the aldoketone reductase superfamily, and it is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductase, which is a polyol metabolizer. The key rate-limiting enzyme of the pathway. This type of enzyme is expressed in a variety of cells and tissues of different species, and widely exists in animal tissues and organs such as nerves, muscles, lenses, brains, kidneys, and lungs. AR catalyzes the reduction of saturated and unsaturated aldehydes, including aldoses and monosaccharides, as well as a host of other substrates, such as the reduction of glucose to sorbitol. Because sorbitol itself has a strong polarity and is difficult to pass through the cell membrane, it will accumulate in the cell, change the permeability of the cell, and reduce the activity of Na ⁺ -K ⁺ -ATPase in the cell, resulting in the loss of inositol, resulting in the loss of cell metabolism and Impairment of function. Taken together, AR plays an important role in cancer, diabetic complications, ischemia/reperfusion-induced liver injury, and cardiac lipid accumulation. Finding economical, efficient and low-toxic AR inhibitors is of great significance for the treatment of diseases such as cancer, diabetes, myocardial infarction and ischemic injury, asthma, transplantation and harmful inflammatory responses.

AR inhibitors are divided into three categories according to their sources: natural products from plants, antibiotics from microorganisms, and synthetic compounds. Most of the artificially synthesized AR inhibitors have not passed clinical trials due to their low curative effect and high toxicity. Currently, there are very few AR inhibitors available for clinical use. Therefore, it is imminent to find new high-efficiency and low-toxicity AR inhibitors.

Contents of the invention

Aiming at the above-mentioned technical limitations, the present invention proposes an aldose reductase inhibitor and its application in the preparation of a drug for treating lung cancer, which overcomes the deficiencies and defects mentioned in the background technology.

To achieve the above object, the present invention adopts the following technical solutions:

The inventive point of the present invention is to provide an aldose reductase inhibitor, the active ingredient of which includes one or more of olanzapine, quercetin and baicalin.

Olanzapine has been on the market for more than 20 years. It is mainly used clinically to treat schizophrenia and manic episodes, with few adverse reactions. The invention finds for the first time that the drug can effectively inhibit the activity of aldose reductase AR, and can be used as a candidate drug for treating lung cancer. In addition, plants are a rich source of AR inhibitors, and natural products rarely cause serious adverse reactions. At present, natural product AR inhibitors are more and more widely used in disease treatment, and become the best choice besides synthetic drugs.

Further, the above-mentioned aldose reductase inhibitor, the active ingredients of the inhibitor are olanzapine, olanzapine compounded with sylcetin, olanzapine compounded with baicalin, olanzapine compounded with sylcetin and baicalin.

In the embodiments of the present invention, relevant cell experiments and animal experiments have been carried out for the effective dose of olanzapine. 175μM, 200μM), the effective dose of olanzapine in animal experiments is 3-6 mg/kg; the effective doses of quercetin and baicalin in cell experiments are 25-150μM quercetin (25μM, 50μM, 75μM, 100μM , 125 μM, 150 μM), baicalin 50-200 μM (50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM), and the best effective doses were selected as quercetin 150 μM and baicalin 200 μM.

The effective doses of olanzapine, quercetin and baicalin meet the requirements of clinical drug safety and efficacy.

The second invention point of the present invention is to provide the application of the above-mentioned aldose reductase inhibitor in the preparation of a drug for treating lung cancer.

The combination of olanzapine and natural product AR inhibitors to treat lung cancer is a new method, which has great sociological and economic significance.

Further, in the above application, the lung cancer includes one or more of small cell lung cancer, non-small cell squamous cell lung cancer, non-small cell lung adenocarcinoma, non-small cell adenosquamous cell carcinoma and large cell lung cancer.

Further, in the above application, the aldose reductase inhibitor in the drug can inhibit the glutamine metabolism of lung cancer cells.

Further, in the above application, the aldose reductase inhibitor in the drug can inhibit the glycolysis of lung cancer cells.

Further, in the above application, the aldose reductase inhibitor in the drug can inhibit the TCA cycle of lung cancer cells.

Further, in the above application, the drug for treating lung cancer containing an aldose reductase inhibitor also includes a pharmaceutically acceptable carrier and auxiliary material, and the drug is administered orally.

Compared with prior art, technical effect of the present invention is as follows:

1. Olanzapine and its drug combination can significantly inhibit the activity of aldose reductase;

2. Olanzapine can hinder the glutamine metabolism of lung cancer cells at the cellular level, thereby inhibiting the proliferation of lung cancer cells and promoting their apoptosis;

3. Olanzapine can hinder the glycolysis process at the cellular level and reduce the supply of ATP energy, thereby inhibiting the proliferation of lung cancer cells and promoting their apoptosis;

4. Olanzapine can hinder the TCA cycle of lung cancer cells at the cellular level, reduce the supply of ATP energy, thereby inhibiting the proliferation of lung cancer cells and promoting their apoptosis.

Description of drawings

Figure 1 shows the inhibition of olanzapine on AKR1B10 and AKRAB1 at the mRNA level; among them, A in Figure 1 is the inhibition of AKR1B10 mRNA level in H520 cells by olanzapine; B in Figure 1 is olanzapine Inhibition of AKR1B10 mRNA level in H520 tumor-bearing mice; C in Figure 1 is the inhibition of AKR1B1 mRNA level in H520 cells by olanzapine; D in Figure 1 is olanzapine in H520 tumor-bearing mice Inhibition of AKR1B1 mRNA levels.

Figure 2 shows the effect of olanzapine on the viability of BASE-2B, H520 and H226 cells detected by CCK8 assay, wherein, A in Figure 2 is the cell viability detection results of 24h and 48h after H520 was treated with different concentrations of olanzapine, Fig. B in Figure 2 is the test results of cell viability at 24h and 48h after treating H226 with different concentrations of olanzapine, and C in Figure 2 is the test results of cell viability at 24h and 48h after treating BASE-2B with different concentrations of olanzapine.

Figure 3 shows the effect of olanzapine on the proliferation ability of lung squamous cell carcinoma H520 detected by EDU experiment, wherein, A in Figure 3 is the staining result of EDU cell proliferation experiment on lung squamous carcinoma cell H520, and B in Figure 3 It is the statistical result of EDU cell proliferation experiment on lung squamous cell carcinoma cell H520.

Figure 4 shows the effect of olanzapine on the migration ability of lung squamous cell carcinoma cells H520 detected by the cell scratch test, wherein, A in Figure 4 is the detection result of the cell scratch test, and B in Figure 4 is the cell scratch Statistical graph of experimental data.

Figure 5 shows the impact of olanzapine on the migration ability of lung squamous cell carcinoma cells H520 detected by Transwell experiment, wherein, A in Figure 5 is the detection result diagram of Transwell experiment, and B in Figure 5 is the detection data statistical diagram of Transwell experiment .

Figure 6 shows the degree of apoptosis of olanzapine on lung squamous cell carcinoma cell H520 detected by the Annexin-7AAD double staining experiment, wherein, A in Figure 6 is the flow cytometric detection result of the Annexin-7AAD double staining experiment, Figure 6 B in Annexin-7AAD double-staining experiment of flow cytometry data statistics.

Figure 7 shows the detection of olanzapine on lung squamous cell carcinoma H520 cell apoptosis protein by Western Blot experiment, wherein, A in Figure 7 is the detection result figure of Western Blot experiment, and B in Figure 7 is the detection data of Western Blot experiment summary graph.

Figure 8 shows the anti-tumor effect of olanzapine in H520 tumor-bearing mice, wherein, A in Figure 8 is a statistical chart of the weight data of nude mice in which olanzapine acts on H520 tumor-bearing mice, and B in Figure 8 is olanzapine Statistical chart of tumor volume data of H520 tumor-bearing mice treated with Olanzapine, C in Figure 8 is the tumor tissue measurement results of Olanzapine treated with H520 tumor-bearing mice, D in Figure 8 is Olanzapine treated with H520 tumor-bearing mice Statistical chart of tumor weight data in mice.

Figure 9. The effects of quercetin and baicalin on the activity of BASE-2B and H520 cells were detected by CCK8 assay.

Figure 10 The effects of olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the viability of BASE-2B and H520 cells were detected by CCK8 assay.

Figure 11. The effect of olanzapine + baicalin on the proliferation ability of lung squamous cell carcinoma cell line H520 was detected by EDU experiment.

Figure 12. The effect of olanzapine + quercetin on the proliferation ability of lung squamous cell carcinoma cell H520 was detected by EDU experiment.

Figure 13. The effect of olanzapine + quercetin + baicalin on the proliferation of lung squamous cell carcinoma cells H520 was detected by EDU experiment.

Figure 14 was used to detect the effect of olanzapine + baicalin on the migration ability of lung squamous cell carcinoma cell H520 by cell scratch test.

Fig. 15 The effect of olanzapine + quercetin on the migration ability of lung squamous cell carcinoma cell H520 was detected by cell scratch test.

Figure 16 Detects the effect of olanzapine + quercetin + baicalin on the migration ability of lung squamous cell carcinoma cell H520 by cell scratch test.

Figure 17 was used to detect the effect of olanzapine + baicalin on the degree of apoptosis of lung squamous cell carcinoma cell H520 by Annexin-7AAD double staining experiment.

Figure 18 The effect of olanzapine + quercetin on the apoptosis of lung squamous cell carcinoma cell H520 was detected by Annexin-7AAD double staining experiment.

Figure 19 Detected the effect of olanzapine + quercetin + baicalin on the degree of apoptosis of lung squamous cell carcinoma cell H520 by Annexin-7AAD double staining experiment.

Fig. 20 PCA score plot of intracellular compounds of control group-drug group.

Fig. 21 PLS-DA model diagram of the intracellular compound in the control group-drug group.

Fig. 22 Analysis diagram of intracellular compound metabolism channel of control group-drug group.

Fig. 23 Effect of olanzapine on mRNA expression of key enzymes in glutamate metabolism, glycolysis and TCA cycle pathways.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below. However, it should be understood that the description herein is only used to explain the present invention, and is not intended to limit the scope of the present invention.

Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention, and the terms used in the description of the present invention herein are only to describe specific implementations The purpose of the example is not intended to limit the present invention. The reagents and instruments used in this article are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, and will not be repeated here.

In order to further understand the present invention, the present invention will be further described in detail below in conjunction with the best embodiments.

Example 1

An aldose reductase inhibitor, the active ingredient of which includes one or more of olanzapine, quercetin and baicalin.

The active ingredients of the inhibitor are olanzapine, olanzapine compounded with sylcetin, olanzapine compounded with baicalin, olanzapine compounded with sylcetin and baicalin.

The effective dose of olanzapine in cell experiments is 25-200 μM (25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM), and the effective dose of olanzapine in animal experiments is 3-6 mg/kg; cell experiments The effective doses of quercetin and baicalin are quercetin 25-150 μM (25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM), baicalin 50-200 μM (50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM), the best effective doses were selected as quercetin 150 μM and baicalin 200 μM.

The optimal effective doses of olanzapine, quercetin and baicalin meet the requirements of clinical drug safety and efficacy.

Also provided is the use of the aldose reductase inhibitor in the preparation of medicines for treating lung cancer.

Lung cancer includes one or more of small cell lung cancer, non-small cell squamous cell lung cancer, non-small cell lung adenocarcinoma, non-small cell adenosquamous cell carcinoma, and large cell lung cancer.

The aldose reductase inhibitor in the drug can inhibit the glutamine metabolism of lung cancer cells.

The aldose reductase inhibitor in the drug can inhibit the glycolysis of lung cancer cells.

The aldose reductase inhibitor in the drug can inhibit the TCA cycle of lung cancer cells.

The oral medicine for treating lung cancer also includes pharmaceutically acceptable carrier and auxiliary materials.

The present invention finds that the mRNA expression level of aldose reductase in lung cancer cells is significantly increased. In the present invention, lung cancer cells (H520 cell line) are used as cell models and H520 tumor-bearing mice are used as animal models, and olanzapine is used for in vitro cell and animal in vivo experiments. The experimental results show that olanzapine can significantly inhibit lung squamous cell carcinoma cells H520 and The mRNA expression level of aldose reductase in H520 tumor-bearing mice.

The CCK8 experiment showed that olanzapine could significantly inhibit the proliferation of lung squamous cell carcinoma cell line H520 in a dose-dependent manner. The cell scratch test and Transwell test showed that olanzapine could significantly inhibit the migration of H520 lung squamous cell carcinoma cells, and showed a dose-dependent effect. Flow cytometry experiments and detection of apoptosis pathway regulatory protein levels showed that olanzapine could significantly promote the apoptosis of lung squamous cell carcinoma cell H520. Experiments in H520 tumor-bearing mice showed that olanzapine significantly inhibited the growth of H520 tumors in vivo.

The present invention finds that olanzapine, as an aldose reductase inhibitor, can inhibit glutamine metabolism by inhibiting genes of c-Myc, GLUD, GLS and GS in lung cancer cells.

The present invention finds that olanzapine, as an aldose reductase inhibitor, can inhibit the glycolysis process by inhibiting the genes of HKII, GAPDHS, PK, PDH and LDH in lung cancer cells.

The present invention finds that olanzapine, as an aldose reductase inhibitor, can inhibit TCA cycle by inhibiting SDH, MDH, CS and IDH genes in lung cancer cells.

Therefore, olanzapine can be used as an aldose reductase inhibitor in the treatment of lung cancer.

The compounds of the present invention can be formulated into various suitable pharmaceutical preparation forms. It can be used alone, or mixed with pharmaceutical excipients (such as excipients, diluents, etc.), and formulated into tablets, capsules, granules, syrups, etc. for oral administration or powder injections, solutions for injection agent.

Example 2

Olanzapine on the expression of aldose reductase mRNA in lung squamous cell carcinoma cells H520 and H520 tumor-bearing mice:

Main reagents: reverse transcription kit KR116-02 (Tiangen Biochemical Technology Co., Ltd.); fluorescence quantitative kit P205-02 (Tiangen Biochemical Technology Co., Ltd.); primer sequences (BGI Co., Ltd.), as shown in Table 1 shown.

Table 1

experimental method:

Cell culture:

(1) Cell preparation: Take the cells that are in a good state of logarithmic growth phase, digest them with 0.25% trypsin, and blow them into single cells at the same time, and then suspend the single cells in the medium containing 10% fetal bovine serum , waiting to be used.

(2) Cell inoculation: Inoculate in a 96-well plate at a cell density of 20,000/well, 2,000 μL per well, and set 3 replicate wells for each group of cells.

(3) Cell dosing: After the cells adhered to the wall for 12 hours, 0.1% DMSO in the control group and olanzapine at different gradient concentrations were added respectively, and cultured in a cell incubator at 37°C, 5% CO ₂ and saturated humidity for 72 hours.

(4) Cell collection: Scrape the cells of each group with a cell scraper. Centrifuge at 1000rpm for 5min, discard the culture medium, wash the cells once with PBS and centrifuge at 1000rpm for 5min, discard the supernatant.

Animal experiment:

H520 cells (1×10 ⁶ /0.2 ml PBS per mouse) were injected subcutaneously into the right side of the mice. Seven days after inoculation, tumors grew to a volume of 80-100 mm ³ . Mice were randomly divided into three groups (3 mice per group) and administered daily with PBS (vehicle group) or olanzapine 3 mg/kg, 6 mg/kg by intragastric injection for 21 days. Tumor volume was measured every 3-4 days after tumor appearance and was calculated by the formula V=a*b2/2 (a=longest axis; b=shortest axis). Mice were sacrificed on day 21 after treatment, tumors were isolated, volumes were measured and weighed.

Total RNA extraction:

Transfer the collected cells and tumor tissues to 1.5mL enzyme-free centrifuge tubes, and add 1mL Tirzol to each tube to fully lyse. Add 200 μL of chloroform to each 1 mL of TRIzol, shake the centrifuge tube vigorously for 15 s, incubate at room temperature for 5 min, and then centrifuge at 12000 rpm for 15 min at 4°C. After centrifugation, it was separated into a lower red organic phase, a middle white phase and an upper clear aqueous phase. Transfer the aqueous phase containing RNA to a new centrifuge tube. Add 600ul of isopropanol to mix up and down, incubate at room temperature for 10min, then centrifuge at 4°C, 12000rpm, for 10min. The supernatant was discarded, and a white precipitate was visible. Add 1ml of 75% ethanol, reverse centrifugation at 4°C, 7500rpm, 5min. As far as possible all the supernatant in the pipette was dried at room temperature for 10 minutes, dissolved in 20 μL DEPC water, and stored at -80°C.

Synthesis:

(1) 5×gDNA Buffer 2 μL; Total RNA 2 μg; RNase-Free ddH ₂ O make up to 10 μL, mix thoroughly. Briefly centrifuge and incubate at 42°C for 3 min, then place on ice;

(2) 10×King RT Buffer 2 μL; FastKing RT Enzyme Mix 1 μL; FQ-RT PrimerMix 2 μL; RNase-Free ddH ₂ O to make up to 10 μL. Add it to the product obtained in step (1), and mix well.

(3) Incubate at 42°C for 15 minutes, and at 95°C for 3 minutes.

Real-time fluorescent quantitative PCR:

Using the above cDNA as a template, quantify according to the following reaction system and procedure. Reaction system: 10uL 2×SuperReal PreMix Plus, 1μL Primer F, 1μL Primer R, 2μL cDNA, 18μL ddH ₂ O. A three-step reaction procedure was adopted: 95°C for 15 min; 95°C for 10 s, 60°C for 20 s, and 72°C for 32 s to collect fluorescence signals, 40 cycles.

Experimental results:

The results shown in Figure 1 show that olanzapine can inhibit the expression of aldose reductase mRNA in lung squamous cell carcinoma cells H520 and H520 tumor-bearing mice.

Example 3:

Olanzapine on the proliferation and migration of lung squamous cell carcinoma cells H520:

experimental method:

1.1 Cell viability was measured by CCK8 assay.

H520 cells in good growth state were taken to prepare a certain concentration of cell suspension, and 100ul per well was added to a 96-well cell culture plate. Incubate overnight. Add 0.1% DMSO and different gradient concentrations of olanzapine, respectively, and incubate for 24 and 48 hours. Take 10ul of CCK-8 solution and add it to a 96-well cell culture plate, and continue to incubate for 2 hours in a 37°C incubator. Obtain the absorbance of each well using an automated fluorescent microplate reader with a wavelength of 450 nm.

1.2 Evaluation of cell proliferation ability by EDU cell proliferation assay

H520 cells were treated with different concentrations of olanzapine for 24 h and 48 h, the medium was replaced with fresh medium containing 10 μM EdU and incubated for another 2 h. Cells were fixed with 4% neutral paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with reaction mix and Hoechst 33342 according to the manufacturer's instructions. Cells were imaged with a fluorescent inverted microscope.

1.3 The cell scratch test was used to detect the effect of olanzapine on the migration ability of lung squamous cell carcinoma H520 cells.

When the H520 cells reached 90% confluency in the six-well plate, injure the cells on the cell monolayer with a sterile 200 μl pipette tip and wash with serum-free medium to remove detached cells, photograph the gap between the scratches using a microscope image. Next, cells were treated with different concentrations of olanzapine for 24 and 48 hours. Finally, images of the wound gap are taken using a microscope.

1.4 The effect of olanzapine on the migration ability of lung squamous cell carcinoma cell H520 was detected by Transwell experiment.

H520 cells were starved overnight and then cultured with serum-free medium in the upper chamber (24-well transwell chamber, 8 μm), while high-serum medium (10% FBS with different concentrations of olanzapine) was added to the lower chamber. After incubation for 48 hours at 37°C, cells were fixed with methanol and stained with 0.1% crystal violet. Finally, images are taken using a microscope.

Experimental results:

In order to study whether olanzapine can effectively inhibit the proliferation of human lung squamous cell carcinoma cells, the CCK8 method was used to detect the inhibition rate of cell proliferation. Olanzapine was used to treat BASE-2B (human normal lung cells), H520 (lung squamous cell carcinoma cells), and H226 (lung squamous cell carcinoma cells) at different concentrations. As shown in Figure 2, the data of 24h and 48h showed that compared with the blank control, the inhibition rate of cell proliferation in the treatment group was significantly increased, and the effect was dose-dependent. Moreover, olanzapine inhibited the proliferation of lung squamous cell carcinoma cells significantly higher than that of normal lung cells. 24 hours later. The IC50 of BASE-2B is 211.7μM; the IC50 of H226 is 155.0μM; the IC50 of H520 is 162.9μM. In order to further study the effect of olanzapine on the viability of lung squamous cell carcinoma cells, EDU cell proliferation experiment was carried out on lung squamous cell carcinoma H520 cells. As shown in Figure 3, compared with the control group, olanzapine can significantly reduce the growth rate of lung squamous cell carcinoma H520 cells, the number of cell proliferation is significantly smaller, the positive rate of EDU is reduced, and the proliferation ability of cells can be inhibited in a concentration-dependent manner. sex (P<0.05). This result further supports that olanzapine is able to inhibit the proliferation of H520 lung squamous cell carcinoma cells. In order to study the effect of olanzapine on inhibiting the ability of cell migration, the cell scratch test was used, as shown in Figure 4, the cell migration and ability were significantly inhibited after adding olanzapine (P<0.05). In order to further verify the effect of olanzapine on the ability to inhibit cell migration, Transwell experiments were used, as shown in Figure 5, after adding olanzapine, cell migration and ability were significantly inhibited (P<0.05). This result further supports that olanzapine is able to inhibit the migration of H520 lung squamous cell carcinoma cells.

Example 4:

Olanzapine on the degree of apoptosis of lung squamous cell carcinoma cell line H520:

experimental method:

1.1 Annexin-7AAD double-staining assay to detect the degree of olanzapine-promoted apoptosis in lung squamous cell carcinoma H520 cells

H520 cells were grown in 6-well plates and incubated overnight, and H520 cells were treated with different concentrations of olanzapine for 24 and 48 hours. Cells were harvested and resuspended in 200 μL binding buffer, then incubated with 5 μL Annexin V-FITC and 10 μL 7AAD for 15 min at room temperature in the dark. A total of 10,000 cells were collected per tube for data analysis.

1.2 Western Blot test to detect the degree of olanzapine-promoted apoptosis of lung squamous cell carcinoma cells H520

H520 cells were grown in 6-well plates and incubated overnight, and cells were treated with different concentrations of olanzapine for 24 and 48 hours. Cells were harvested and washed with PBS, then lysed with cold lysis buffer containing protease inhibitor cocktail. After centrifugation at 12 000 rpm for 15 min, the protein supernatant was extracted and the protein concentration was measured by BCA protein detection kit. Equal amounts of protein from each sample were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PDVF membrane. The PDVF membrane was blocked with 5% skimmed milk for 4 hours at room temperature, washed and incubated with the primary antibody overnight at 4°C. The next day, the PDVF membrane was washed and incubated with secondary antibody for 1 hour. After the membrane was washed, the blot was detected with the Odyssey infrared imaging system.

Experimental results: In order to study the mechanism of olanzapine inhibiting the proliferation of lung squamous cell carcinoma cells, Annexin-7AAD double staining was performed on lung squamous cell carcinoma H520, and analyzed by flow cytometry, as shown in Figure 6, with the increase of drug concentration , the apoptosis rate of H520 cells increased significantly (P<0.05). In order to further study the mechanism of olanzapine-induced cell apoptosis, the apoptosis pathway regulatory proteins were detected by WB. As shown in Figure 7, the expression ratio of BCL-2/BAX was significantly reduced (P<0.05).

Example 5:

Study on the antitumor effect of olanzapine in H520 tumor-bearing mice:

Experimental method: with embodiment 2.

Experimental Results: We evaluated the effect of olanzapine on the growth of lung squamous cell carcinoma H520 cells in vivo. As shown in Figure 8, the treatment basically did not affect the average body weight of mice, but could significantly reduce the weight and volume of H520 tumor-bearing tumors (P<0.05).

Embodiment 6:

Effects of quercetin, baicalin, olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the viability of BASE-2B and H520 cells.

Experimental method: with embodiment 3.

Experimental results: In order to study whether quercetin and baicalin can effectively increase the effect of olanzapine on inhibiting the proliferation of human lung squamous cell carcinoma cells, we used the CCK8 method to detect the inhibition rate of cell proliferation. Quercetin and baicalin were used to treat BASE-2B (human normal lung cells), H520 (lung squamous cell carcinoma cells), and H226 (lung squamous cell carcinoma cells) at different concentrations. As shown in Figure 9, the data at 24h and 48h showed that compared with the blank control, the inhibition rate of cell proliferation in the treatment group was significantly increased, and the effect was dose-dependent. Moreover, we used the CCK8 method to detect the inhibition rate of cell proliferation. The degree of inhibition of the proliferation of lung squamous cell carcinoma cells was significantly higher than that of normal lung cells. We chose quercetin 150 μM and baicalin 200 μM as the concentration for the next experiment. In order to study the effects of olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the activity of lung squamous cell carcinoma cells, we used the CCK8 method to detect the inhibition rate of cell proliferation. As shown in Figure 10, after 48 hours of combined administration of olanzapine, quercetin, and baicalin, the effect on the proliferation inhibition rate of lung squamous cell carcinoma H520 was significant. In order to further study the effects of olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the activity of lung squamous cell carcinoma cells, we conducted EDU cell proliferation experiments on lung squamous cell carcinoma H520 cells. As shown in Figures 11, 12, and 13, compared with the control group, olanzapine, quercetin, and baicalin combined for 48 hours can significantly reduce the growth rate of lung squamous cell carcinoma H520 cells, the number of cell proliferation is significantly less, and EDU is positive The reduction of the rate can inhibit the proliferation ability of cells, and it is concentration-dependent (P<0.05).

Embodiment 7:

Effects of olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the proliferation and migration of lung squamous cell carcinoma cells H520

Experimental method: with embodiment 3.

Experimental results: In order to study the effects of olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on the ability to inhibit cell migration, cell scratch experiments were used, as shown in Figures 14 and 15 , Olanzapine, quercetin, and baicalin were used together for 48 hours, and the cell migration and ability were significantly inhibited (P<0.05). In Figure 16, we can find that after the combined use of olanzapine + quercetin + baicalin, the cells were directly suspended in a large area. It may be that in the case of low serum, olanzapine + quercetin + baicalin intensified the inhibition of cell activity. cause the cells to no longer adhere to the wall.

Embodiment 8:

Olanzapine + quercetin, olanzapine + baicalin, olanzapine + quercetin + baicalin on apoptosis of lung squamous cell carcinoma cell line H520

Experimental method: with embodiment 4.

Experimental results: In order to study the mechanism of olanzapine inhibiting the proliferation of lung squamous cell carcinoma cells, we performed Annexin-7AAD double staining on lung squamous cell carcinoma H520, and analyzed by flow cytometry, as shown in Figures 17, 18, and 19. Olanzapine The apoptotic rate of H520 cells was significantly increased (P<0.05) after combined use of quercetin and baicalin.

Embodiment 9:

Olanzapine on cell metabolomics of lung squamous cell carcinoma cells:

Experimental method: H520 cells were grown in 6-well plates and incubated overnight, and the cells were treated with different concentrations of olanzapine for 72 hours. Then, wash the cells twice with ice-cold PBS and fix with 1 mL of ice-cold aqueous solution of 10% methanol + 1% formic acid. Cells were scraped off the plate, sonicated for 30 min, and centrifuged at 10,000 rpm for 1 min. Take 50 μL of the supernatant and add 450 μL of sample pretreatment solution, mix well and centrifuge at 12000 rpm for 10 min. Take 5 µL of the supernatant for LC-MS/MS-based metabolome assays.

Experimental results: Before the formal analysis by MetaboAnalyst5.0 software, Paretoscaling was performed on the data set to obtain more intuitive and reliable results. In order to determine whether there is a difference between the two groups, the PCA modeling method was used to analyze the samples. Perform principal component analysis on it, as shown in Figure 20. In order to obtain the metabolite information leading to this significant difference, the partial least squares discriminant analysis (PLS-DA) was further used to analyze the 4 groups of samples (see Figure 21). VIP is the main parameter to evaluate the importance of each variable in the PLS-DA model, the larger the VIP value, the greater the contribution to the classification model. With VIP>1, metabolites were screened out, defined as differential metabolites, and used to distinguish the control group from the experimental group. The changes in endogenous substances in cells between the control group and the drug group (VIP>1) are shown in Table 2. MetaboAnalyst5.0 software was used to analyze the main differential endogenous metabolites with a VIP value greater than 1, and those with a P value greater than 0.1 were selected as the main metabolic pathways affected, see Table 3 and Figure 22. Table 3 shows the analysis results of metabolic pathways of intracellular compounds in the control group-drug group.

Table 2

no Metabolites VIP 15um/0um(%) 50um/0um(%) 150um/0um(%) 1 Phenylethylamine 12.248 85.31±35.36 76.15±31.21 46.8±17.49 2 spermine 5.5181 164.15±11.48 159.67±8.05 148.21±7.78 3 creatine 5.4879 94.3±5.29 79.56±3.1 51.01±2.08 4 Phosphocholine 4.6051 71.27±9.14 47.74±4.04 20.32±1.31 5 lactic acid 3.8248 85.26±15.17 107.07±20.59 192.8±7.82 6 histamine 3.2534 108.65±39.82 126.92±41.22 166.69±49.27 7 Valine 2.6627 86.48±36.93 91.33±31.15 67.69±35.28 8 L-proline 2.2792 113.79±11.47 143.56±12.12 200.97±9.67 9 L-Arginine 2.2743 17.7±1.66 17.8±1.83 2.08±1.46 10 Spermidine 2.0203 104.25±2.93 94.03±3.16 85.85±5.03 11 PC(14:0/22:1(13Z)) 1.8592 121.1±38.13 205.97±133.35 254.11±192.41 12 PC(14:0/18:1(9Z)) 1.8446 69.7±32.73 636.88±490.98 263.03±405.94 13 L-carnitine 1.7773 151.98±10.66 48.09±2.34 15.7±2.43 14 caffeine 1.6564 35.28±16.83 19.36±9.39 20.71±7.67 15 citric acid 1.5398 271.18±50.97 286.98±64.55 192.46±18.21 16 Propionylcarnitine 1.5244 55.43±27.92 128.8±58.57 234.79±72.19 17 N-acetyl-L-aspartic acid 1.4791 165.63±14.27 201.76±34.47 122.79±11.96 18 PC(o-16:1(9Z)/18:0) 1.4153 169.72±131.74 163.71±104.02 230.32±162.07 19 PC(O-16:0/18:2(9Z, 12Z)) 1.4086 96.04±49.8 219.44±220.49 204.24±250.83 20 Propionylcarnitine 1.3934 59.55±25.75 151.51±82.07 268.97±114.34 twenty one Oleamide 1.3673 96.18±15.04 109.66±18.5 93.93±41.46 twenty two SM(d18:0/24:1(15Z)) 1.3301 327.77±149.1 622.42±227.82 1108.34±422.02 twenty three PC(o-18:1(9Z)/18:2(9Z, 12Z)) 1.3255 134.92±188.12 270.37±283.8 222.4±266.7 twenty four 7-ketolithocholic acid 1.3135 106.28±8.64 122.72±17.24 113.67±23.65 25 4-Methylbenzotriazole 1.2014 122.8±50.77 140.93±41.17 166.85±51.91 26 PC(14:0/P-18:0) 1.1994 142.65±195.68 261.11±230.23 230.84±212.5 27 stearic acid 1.186 107.75±10.19 95.28±11.24 104.16±13.93 28 2,4-Diaminobutyric acid 1.1764 87.6±51.48 125.71±73.78 129.09±22.18 29 L-acetylcarnitine 1.1518 192.74±13.64 106.55±15.75 40.34±2.85 30 PC(o-18:0/18:2(9Z, 12Z)) 1.1509 114.45±59.02 165.03±103.7 259.43±218.27 31 L-histidine 1.1503 287.32±124.65 334.71±137.36 260.13±251.88 32 L-acetylcarnitine 1.1489 192.98±13.7 107±15.33 40.04±2.22 33 Nicotinamide 1.1292 153.81±12.64 141.02±13.48 131.31±12.47 34 L-leucine 1.1243 84.96±9.09 86.53±7.42 103.93±5.85 35 creatinine 1.1001 89.3±5.72 85.95±8.12 45.62±3.84 36 D-glucose 1.0878 12.47±3.87 9.1±3.71 6.63±2.16 37 adenine 1.0434 143.22±30.24 108.78±18.58 81.85±29.57 38 3-Hydroxy-3-methyl-2-oxobutanoic acid 1.0161 90.06±16.61 147.36±72.56 193.03±87.74 39 PC(16:1(9Z)/22:2(13Z, 16Z)) 1.004 92.75±66.76 101.84±73.86 105.85±65.94

table 3

NO metabolic pathway Total Hits FDR Impact 1 Histidine metabolism 16 2 0.5070 0.4098 2 Phenylalanine metabolism 10 1 1.0000 0.2381 3 Niacin and Niacinamide Metabolism 15 1 1.0000 0.1943 4 Arginine and Proline Metabolism 38 5 0.0310 0.1818 5 Glycerophospholipid metabolism 36 2 1.0000 0.1038

Example 10:

Effect of olanzapine on mRNA expression of key enzymes in lung squamous cell carcinoma cells:

Main reagents: reverse transcription kit KR116-02 (Tiangen Biochemical Technology Co., Ltd.); fluorescence quantitative kit P205-02 (Tiangen Biochemical Technology Co., Ltd.); primers (Hua Da Genomics Co., Ltd.). The details are shown in Table 4.

Table 4

name Primer sequence c-MYC F:5-GTGCCACGTCTCCACACATCAG-3 R: 5-CCTTGGGGGCCTTTTCATTGTTTTC-3 GS F:5- AAAATGTCCCTCCGTTCTTATGG -3 R:5-CTGAAGTTGAGCGTAATACCAGT-3 GLS F:5-AGGGTCTGTTACCTAGCTTGG-3 R: 5-ACGTTCGCAATCCTGTAGATTT-3 R:5-AGTCATCCGTGCGATATGCTC-3 GLUD F:5-GGGATTCTAACTACCACTTGCTCA-3 R:5-AACTCTGCCGTGGGTACAAT-3 HKII F: 5-GAGCCACCACTCACCCTACT-3 R: CCAGGCATTCGGCAATGTG-3 GAPDHS F: 5-CTCACCGGATGCACCAATGTT-3 R: CGCGTTGCTCACAATGTTCAT-3 PK F:5-TCAAGGCCGGGATGAACATTG-3 R: CTGAGTGGGGAACCTGCAAAG-3 PDH F:5-AAGAGGCGCTTTCACTGGAC-3 R: ACTAACCTTGTATGCCCCATCA-3 CS F:5-AACTGCTACCCAAGGCTAAGG-3 R: CTTTTGAGAGCCAAGATACCTGT-3 IDH F:5-TGTGGTAGAGATGCAAGGAGA-3 R: TTGGTGACTTGGTCGTTGGTG-3 SDH F:5-CAAACAGGAACCCGAGGTTTT-3 R: CAGCTTGGTAACACATGCTGTAT-3 Mdh F:5-GGTGCAGCCTTAGATAAATACGC-3 R:AGTCAAGCAACTGAAGTTCTCC-3 LDH F:5-ATGGCAACTCTAAAGGATCAGC-3 R: CCAACCCCAACAACTGTAATCT-3 β-actin F:5-C TGTGATGGTGGGAATGGGTCAG-3 R:5-TTTGATGTCACGCACGATTTCC-3

Experimental method: with embodiment 1.

Experimental results:

The mRNA expression levels of key enzymes in the glutamine metabolic pathway: c-Myc, GLUD, GLS, and glutamine synthetase (Glutamine Synthetase, GS) were detected. As shown in Figure 23, lung squamous cell carcinoma cells H520 and The expressions of C-MYC, GLUD, GLS and GS decreased significantly in a dose-dependent manner after co-cultured with olanzapine for 72 hours.

Key enzymes for glycolysis and TCA cycle pathways: hexokinase (Hexokinase, HKII), 3-phosphate glyceraldehyde dehydrogenase (Glyceraldehyde 3-Phosphate Dehydrogenase, GAPDHS), pyruvate kinase (PyruvateKinase, PK), pyruvate Dehydrogenase (Pyruvate Dehydrogenase, PDH), citrate synthetase (CitroylSynthetase, CS), isocitrate dehydrogenase (Isocitrate Dehydrogenase, IDH), succinate dehydrogenase (Succinodehydrogenase, SDH), malate dehydrogenase ( Malic Dehydrogenase , MDH ), lactate dehydrogenase (Lactic Dehydrogenase , LDH ), the detection of mRNA expression levels, after co-cultured lung squamous cell carcinoma cell H520 and olanzapine for 72 hours, the expression of related genes decreased significantly and had Dose dependent. Finally, olanzapine induces apoptosis by inhibiting glutamate metabolism, glycolysis and TCA cycle pathways.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement or improvement made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (5)

1. The application of the aldose reductase stopping agent in preparing the medicine for treating the lung cancer is characterized in that the effective components of the aldose reductase stopping agent are olanzapine compounded with quercetin and baicalin, and the lung cancer is non-small cell lung squamous carcinoma.

2. The use according to claim 1, wherein the aldose reductase inhibitor in the medicament is capable of inhibiting glutamine metabolism in lung cancer cells.

3. The use according to claim 1, wherein the aldose reductase inhibitor in the medicament is capable of inhibiting glycolysis of a lung cancer cell.

4. The use as claimed in claim 1, wherein the aldose reductase inhibitor in the medicament is capable of inhibiting TCA cycle TCA in lung cancer cells.

5. The use according to any one of claims 1 to 4, wherein the medicament for treating lung cancer further comprises a pharmaceutically acceptable carrier and adjuvant, and the medicament is administered orally.

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