Application of gemcitabine in preparation of medicine for treating atherosclerosis

Abstract

The invention discloses an application of gemcitabine in preparation of a medicine for treating atherosclerosis. The low-dose gemcitabine can inhibit the formation of foam cells, reduce the uptake of lipid by macrophages and inhibit the aging effect and inflammatory response of oxLDL on the macrophages, and can be used as a medicine for treating atherosclerosis.

Description

Technical Field

[0001] The invention belongs to the technical field of cardiovascular diseases, and specifically relates to the application of gemcitabine in preparing a drug for treating atherosclerosis. Background Art

[0002] Coronary heart disease and other cardiovascular diseases kill millions of people every year. In 2019, the number of deaths caused by cardiovascular disease worldwide was about 18 million, and by 2024, this number will continue to remain high, although some regions in Eastern Europe and Central Asia still have high mortality rates despite the overall downward trend in cardiovascular disease mortality worldwide. In China, cardiovascular disease is one of the diseases with the highest morbidity and mortality rates among middle-aged and elderly people in my country, accounting for almost half of all causes of death in middle-aged and elderly patients. According to the 2023 "China Cardiovascular Health and Disease Report", there are currently 330 million cardiovascular disease patients in China. Coronary heart disease is the second leading cause of death among cardiovascular diseases. The proportion of deaths due to coronary heart disease in urban areas is about 22%, while in rural areas it is 13%. In China, the burden of coronary heart disease is increasing due to the increasing number of risk factors such as hypertension, hyperlipidemia, obesity, diabetes, and the aging of the population.

[0003] Atherosclerosis is an important pathological basis of cardiovascular disease and the main cause of high mortality worldwide. The development of atherosclerosis begins with impaired endothelial cell function, which can be caused by a variety of factors, including high blood pressure, high cholesterol, smoking, and inflammatory response. After endothelial damage, low-density lipoprotein (LDL) cholesterol invades the arterial wall and forms atherosclerotic plaques through oxidation and accumulation. These plaques cause the blood vessel wall to lose elasticity and block blood flow, leading to myocardial ischemia. When the plaque becomes unstable and eventually ruptures, it leads to platelet aggregation and thrombosis, causing myocardial infarction. Among them, the formation of foam cells is a key link in the process of atherosclerosis. Phagocytic cells (such as macrophages) under the endothelium recognize and engulf oxidized low-density lipoprotein (oxLDL) particles and transform into foam cells. The accumulation and death of foam cells release more lipids and cell fragments. These substances further accumulate in the plaque, causing the plaque to enlarge and may eventually cause the plaque to rupture, causing serious vascular events.

[0004] Common drug treatments for atherosclerosis include statins, ACE inhibitors, beta-blockers, and anticoagulants and antiplatelet drugs. These treatments are designed to manage symptoms and prevent disease progression by lowering cholesterol levels, improving blood pressure, and preventing thrombosis, but these drugs often have side effects such as myalgia, liver damage, and hyperkalemia. For this reason, the development of new drugs and new therapies is essential to further improve treatment outcomes and patients' quality of life. Gemcitabine is a nucleoside analog chemotherapy drug. Due to this structural similarity, gemcitabine can be recognized by enzymes in cells and inserted into DNA chains, thereby interfering with the normal DNA replication process. This property is used to treat a variety of cancers, including pancreatic cancer, breast cancer, ovarian cancer, and non-small cell lung cancer. There are currently no studies on the use of gemcitabine as a treatment for atherosclerosis. Summary of the invention

[0005] The purpose of the present invention is to provide the use of gemcitabine in preparing a drug for treating atherosclerosis.

[0006] Application of gemcitabine in the preparation of a drug for treating atherosclerosis.

[0007] Furthermore, gemcitabine inhibited the formation of foam cells.

[0008] Furthermore, gemcitabine inhibited the uptake of oxLDL by macrophages.

[0009] Furthermore, gemcitabine inhibited the senescence effect of oxLDL on macrophages and the inflammatory response.

[0010] A drug for treating atherosclerosis, the active ingredient of which is gemcitabine.

[0011] Furthermore, it also includes one or more pharmaceutically acceptable excipients or carriers.

[0012] The auxiliary materials or carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

[0013] The pharmaceutical composition can be prepared into the dosage form of tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral liquids, pills or injections.

[0014] Beneficial effects of the present invention: The present invention finds that low-dose gemcitabine can inhibit the formation of foam cells, reduce the uptake of lipids by macrophages, inhibit the aging effect of oxLDL on macrophages and the inflammatory response, and can be used as a therapeutic drug for atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Results of the cytotoxicity experiment of low concentration gemcitabine on THP-1 cells.

[0016] Figure 2 Gemcitabine inhibits the formation of foam cells.

[0017] Figure 3 Gemcitabine inhibits the uptake of oxLDL by macrophages.

[0018] Figure 4 Changes in the expression levels of SR-A1 and CD36 genes in macrophages after treatment with low concentrations of gemcitabine.

[0019] Figure 5 Gemcitabine delays macrophage senescence.

[0020] Figure 6 Gemcitabine modulates the expression of senescence-associated secretory phenotype (SASP) factors IL-6, IL-8, MMP3, and MCP1.

[0021] Figure 7 Gemcitabine inhibits atherosclerosis in a mouse model. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Example 1 Low concentration of gemcitabine has no obvious cytotoxicity to THP-1 cells Human mononuclear THP-1 cells were selected as the research object. The culture medium was prepared in the following proportions: 90% RPMI1640 culture medium (Cat. No. PYG0006, Wuhan Boster Biological Co., Ltd.) + 10% serum (Cat. No. FSP500, Suzhou Yikesai Biotechnology Co., Ltd.) + 1% double antibody (Cat. No. 2240831, Thermo Fisher, USA). The cells were cultured at 37°C and 5% carbon dioxide concentration. When the cells were in good condition, the appropriate cell density was selected, and the inducer PMA (Phorbol 12-myristate 13-acetate) was added to plate the 96-well plates. After 24 hours, THP-1 cells were induced to differentiate into macrophages by PMA. Different concentrations of gemcitabine were added. After 48 hours of action, cell count kit-8 (CCK-8) cell proliferation and toxicity were detected. The cell group without gemcitabine was selected as the control. The CCK-8 test data was normalized and analyzed using GraphPad prism 9 was used for drawing and statistical analysis of differences. One-way analysis of variance was used for statistical analysis. The experimental results showed that low-dose gemcitabine (≤1 μM) had no obvious cytotoxicity to THP-1 cells ( Figure 1 ).

[0024] Experimental methods: (1) THP-1 cells were plated into 96-well plates and induced with PMA (20 ng / ml) for 24 hours.

[0025] (2) Different concentrations of Gemcitabine (0, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM) were added and treated for 24 hours.

[0026] (3) Add 0.5 ml of CCK-8 reagent to 9.5 ml of culture medium and mix well for later use.

[0027] (4) Aspirate the culture medium in the 96-well plate and add 100 ml of CCK-8 reagent working solution to each well. Place the plate in the incubator for 2 hours and then use an M5 microplate reader to measure the absorbance at 450 nm.

[0028] Example 2 Gemcitabine inhibits the formation of foam cells PMA was added to THP-1 cells to induce differentiation into macrophages. After 24 hours, low-dose gemcitabine was added to the experimental group. After 24 hours, oxLDL was added to the experimental group and the positive control group, and the negative control group was not treated. After 24 hours, each sample was stained with oil red. The experimental results are shown in the table. Figure 2The results showed that compared with the negative control group, the staining intensity of the positive control group was significantly enhanced. After adding low-dose gemcitabine before the action of oxLDL, the oil red staining intensity of macrophages was significantly lower than that of the positive control group. The above results suggest that low-dose gemcitabine may inhibit the formation of foam cells.

[0029] Experimental methods: (1) THP-1 cells were plated in 12-well plates with cell culture slides and induced with PMA (20 ng / ml) for 24 hours. (2) After treatment with different concentrations of Gemcitabine (0, 0.5 μM, 1 μM) for 24 hours, oxLDL (20 μg / ml) was added and incubated for 24 hours; (3) Aspirate the culture medium and wash once with PBS; (4) Fixation with 10% neutral formaldehyde for 10 min; (5) Add 60% isopropanol (diluted with PBS) and wash briefly; (6) Dilute 0.5% Oil Red O solution (Sigma) with water in a ratio of 3:2, let stand for 10 min, and then filter with medium-speed qualitative filter paper; (7) Stain for 30 min, keeping away from light; (8) Add 60% isopropanol to wash briefly; (9) Stain the nucleus with hematoxylin and rinse with tap water; (10) Take out the slide, seal it with glycerol gelatin, and then take a photo under a microscope.

[0030] Example 3 Gemcitabine inhibits macrophage uptake of oxLDL PMA was added to THP-1 cells to induce differentiation into macrophages for 24 hours. After treatment with different doses of gemcitabine (0, 0.5μM, 1μM) for 24 hours, fluorescent lipoprotein (Dil-oxLDL) was added and incubated for 4 hours to detect the uptake of lipoprotein. The results showed that after gemcitabine treatment, the uptake of Dil-oxLDL by macrophages was significantly reduced ( Figure 3 ). The above results suggest that low-dose gemcitabine inhibits the uptake of oxLDL by macrophages.

[0031] Experimental methods (1) THP-1 cells were plated in 12-well plates with cell culture slides and induced with PMA (20 ng / ml) for 24 hours. (2) After treatment with different concentrations of Gemcitabine (0, 0.5 μM, 1 μM) for 24 hours, Dil-oxLDL (20 μg / ml) was added and incubated for 4 hours; (3) Wash once with PBS solution and fix with paraformaldehyde for 10 min; (4) After fixation, wash with PBS three times and stain with DAPI solution for 10 min; (5) Wash twice with PBS solution. Add 1 ml of PBS to each dish and take photos under a fluorescence microscope.

[0032] Example 4 Gemcitabine regulates the expression of scavenger receptor SR-A1 and CD36 PMA was added to THP-1 cells to induce differentiation into macrophages for 24 hours. After being treated with different doses of gemcitabine (0, 0.5μM, 1μM) for 24 hours, oxLDL (20μg / ml) was added for 24 hours to induce foam cells. RNA was extracted from each sample group, and the expression levels of SR-A1 and CD36 in each group of samples were detected by real-time fluorescence quantitative PCR. The results showed that the expression levels of SR-A1 and CD36 in the group not treated with gemcitabine after the addition of oxLDL were significantly higher than those in the control group. After gemcitabine treatment and the addition of oxLDL, the expression levels of SR-A1 and CD36 were significantly lower than those in the group not treated with gemcitabine. One-way analysis of variance was used to calculate the p value for statistical analysis, and the data were expressed as the mean value with positive and negative annotated errors. See the experimental schematic diagram. Figure 4 The above results suggest that low-dose gemcitabine may inhibit the expression of SR-A1 and CD36 genes in macrophages.

[0033] Example 5 Gemcitabine delays senescence of macrophages PMA was added to THP-1 cells to induce differentiation into macrophages for 24 hours. Different doses of Gemcitabine (0, 0.5μM, 1μM) were added for 24 hours, and then oxLDL (20μg / ml) was added for 24 hours to induce foam cells. Cells in each group were stained for cell senescence β-galactosidase. The results showed that the intensity of cell senescence β-galactosidase staining of macrophages treated with gemcitabine was significantly lower than that of the control group ( Figure 5 ). The above results suggest that low-dose gemcitabine may delay macrophage senescence.

[0034] Experimental methods: (1) THP-1 cells were plated in 12-well plates with cell culture slides and induced with PMA (20 ng / ml) for 24 hours. (2) After treatment with different concentrations of Gemcitabine (0, 0.5 μM, 1 μM) for 24 hours, oxLDL (20 μg / ml) was added and incubated for 24 hours; (3) Use the cell senescence β-galactosidase staining kit (Beyotime; C0602) for staining, and follow the instructions of the kit; (4) After staining overnight, aspirate the staining solution, add PBS, and take photos under a NIKON microscope.

[0035] Example 6 Gemcitabine regulates the expression of senescence-associated secretory phenotype (SASP) factors IL-6, IL-8, MMP3 and MCP1 PMA was added to THP-1 cells to induce differentiation into macrophages for 24 hours. After treatment with different doses of gemcitabine (0, 0.5μM, 1μM) for 24 hours, oxLDL (20μg / ml) was added for 24 hours to induce foam cells. RNA was extracted from each sample group, and the expression levels of IL-6, IL-8, MMP3, and MCP1 in each group of samples were detected by real-time fluorescence quantitative PCR. The results showed that the expression levels of IL-6, IL-8, MMP3, and MCP1 in the group not treated with gemcitabine after the addition of oxLDL were significantly higher than those in the control group. After gemcitabine treatment and the addition of oxLDL, the expression levels of IL-6, IL-8, MMP3, and MCP1 were significantly lower than those in the group not treated with gemcitabine. One-way analysis of variance was used to calculate the p value for statistical analysis, and the data were expressed in the form of positive and negative errors of the mean. See the experimental schematic diagram. Figure 6 The above results suggest that low-dose gemcitabine can inhibit the expression of IL-6, IL-8, MMP3, and MCP1 genes in THP-1 cells after the action of oxLDL.

[0036] Example 7 Gemcitabine inhibits atherosclerosis in a mouse model In order to further study the function of gemcitabine in the process of atherosclerosis, we selected ApoE, a mouse model currently recognized as an ideal model for studying atherosclerosis. - / - We isolated eight-week-old ApoE - / - The mice were randomly divided into two groups. The experimental group was injected with 10 mg / kg gemcitabine, and the control group was injected with 200 μL / kg PBS (twice a week for eight consecutive weeks). They were also fed a high-fat diet to establish an atherosclerosis model.

[0037] Two groups of mice were phenotyped: A: The aorta of mice was stained with Oil Red O and the plaque area was calculated. The aortic plaque area of ​​mice in the gemcitabine group was significantly lower than that of mice in the control group. This indicates that gemcitabine can inhibit the formation of atherosclerotic plaques. Statistical analysis was performed using one-way ANOVA to calculate the p value, and the data were expressed in the form of mean plus or minus standard error.

[0038] Experimental methods: (1) Mice were induced with a high-fat diet and then anesthetized and killed, and their organs were removed; (2) Remove the entire aorta and the fat tissue around the aorta; (3) Cut the mouse aorta longitudinally and lay it flat with the intima facing down; (4) Add 0.5 ml 4% paraformaldehyde and fix at 4°C overnight; (5) Rinse with 5 ml PBS for 2 hours; (6) Remove the aortic adventitia and rinse with PBS; (7) Dehydrate in 5 ml propylene glycol at room temperature for 2 min; (8) Stain in 5 ml 0.5% Oil Red O at room temperature for 2 hours; (9) Wash four times in 85% propylene glycol (diluted with PBS); (10) PBS washing; (11) Scan and photograph the aorta after fixation.

[0039] (12) The ratio of the Oil Red O-stained plaque area to the total aorta area was calculated. Image J software was used for quantitative analysis.

[0040] B: The plaque sections of the aorta root of mice were taken for HE staining and the absolute area of ​​the plaque was calculated. The plaque area of ​​the mice in the gemcitabine experimental group was significantly lower than that of the mice in the control group, and the difference between the two was statistically significant. This shows that gemcitabine can significantly inhibit the occurrence of atherosclerosis. The statistical analysis used one-way analysis of variance to calculate the p value, and the data were expressed in the form of mean positive and negative standard errors.

[0041] Experimental methods: (1) Mice were anesthetized and killed after high-fat diet induction, and the root tissue of aortic plaques was isolated and fixed in 10% formalin for 48 hours; (2) Place the fixed tissue blocks into an embedding box; (3) Dehydrate the tissue in an alcohol gradient: 70%, 80%, 90% I, 90% II, 95% I, 95% II, 100% I, 100% II for 10 min each; (4) Transparent the tissue in xylene: xylene I and xylene II for 5 min each; (5) Immerse the tissue in wax: wax tank I and wax tank II for 40 min each; (6) Embedding: making the tissue into wax blocks; (7) Sections, 4 μm thick; (8) Bake in oven overnight.

[0042] (9) Dewaxing of sections: xylene I and xylene II for 10 min each; rehydration: 100% I, 100% II alcohol for 5 min each, 95%, 90%, 80%, 75% alcohol for 3 min each; (10) Rinse with water 3 times, 2 min each time; (11) Stain the nucleus with hematoxylin for 10 minutes; (12) Wash with tap water for 2 min; (13) Eosin staining for 2 min; (14) Wash with tap water for 2 min; (15) 90% and 100% alcohol for 2 min each; xylene I and xylene II for 4 min each; (16) Neutral resin sealing; (17) Observation was performed using a Nikon Bx60 microscope connected to a Nikon DP70 camera and photography was performed using Cell-F imaging software. Quantitative analysis was performed using Image J software.

[0043] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of gemcitabine in the preparation of drugs for treating atherosclerosis.

2. The use of gemcitabine in the preparation of a drug for treating atherosclerosis according to claim 1, characterized in that: Gemcitabine inhibits the formation of foam cells.

3. The use of gemcitabine in the preparation of a drug for treating atherosclerosis according to claim 1, characterized in that: Gemcitabine inhibits the uptake of oxLDL by macrophages.

4. The use of gemcitabine in the preparation of a drug for treating atherosclerosis according to claim 1, characterized in that: Gemcitabine inhibits the senescence effect of oxLDL on macrophages and the inflammatory response.

5. A drug for treating atherosclerosis, characterized in that: Its active ingredient is gemcitabine.

6. The drug for treating atherosclerosis according to claim 5, characterized in that: It also includes one or more pharmaceutically acceptable excipients or carriers.

7. The drug for treating atherosclerosis according to claim 6, characterized in that: The auxiliary materials or carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

8. The drug for treating atherosclerosis according to claim 5, characterized in that: The drug can be prepared into the dosage form of tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral liquids, pills or injections.

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