Application of asiatic acid in preparation of anti-atherosclerosis medicine

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of asiatic acid in preparation of an anti-atherosclerosis medicine. According to the application disclosed by the invention, an atherosclerosis animal induced by high-fat fed ApoE <- / -> mice and RAW264.7 cells induced by LPS and ox-LDL are adopted to establish a macrophage inflammation model or a macrophage-derived foam cell model, and the influence of asiatic acid on atherosclerosis lesion, macrophage inflammatory response and foaming is observed; researches find that the asiatic acid inhibits the formation of aortic atherosclerotic plaques of APOE- / -mice fed with high fat, and the mechanism is related to the fat regulating and anti-inflammatory effects of the asiatic acid; the asiatic acid is used for inhibiting RAW264.7 macrophage inflammatory response induced by LPS (Lipopolysaccharide); the asiatic acid is used for inhibiting foaming of RAW264.7 macrophages induced by ox-LDL (low density lipoprotein). Experimental results show that the asiatic acid has an obvious anti-atherosclerosis effect.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to application of asiatic acid in the preparation of anti-atherosclerosis drugs. Background Art

[0002] Atherosclerosis (AS) is a lipid-mediated inflammatory disease of the arterial intima. In atherosclerosis, the imbalance of pro-inflammatory and anti-inflammatory mechanisms ultimately leads to the occurrence of lesions. AS is the main pathological basis for adverse cardiovascular events such as coronary heart disease, myocardial infarction, and stroke, and exists in the entire stage of cardiovascular and cerebrovascular disease. Studies have shown that AS is the world's number one killer, with a high incidence, disability and mortality rate, and is gradually showing a trend of younger onset. Therefore, prevention and treatment of AS is crucial.

[0003] AS is a lipid-driven chronic inflammatory disease of the arterial intima. Inflammation and lipid metabolism disorders accompany and synergize with each other to promote the progression of AS lesions. Anti-inflammatory and regulation of lipid metabolism disorders have also become important strategies for the treatment of AS. At present, the treatment methods of AS mainly include drug therapy (Western medicine treatment, Chinese medicine treatment) and non-drug treatment (surgical therapy, interventional therapy). Among them, Western medicine treatment includes statins, antiplatelet drugs, antioxidants, etc. Statins and antiplatelet drugs have been considered by Western medicine as first-line drugs for the treatment of AS. However, their long-term use will cause bleeding and other side effects, and there is still a residual risk of cardiovascular events after use. Therefore, in order to improve the survival prognosis of AS patients, it is still necessary to actively seek new and more effective therapeutic drugs.

[0004] In recent years, with the continuous deepening of research on traditional Chinese medicine and its active ingredients, it has been found that traditional Chinese medicine has little toxic and side effects, has a wide range of biological activities, and has the characteristics of multi-link, multi-pathway, and multi-target effects. It has unique advantages in preventing and treating AS and related cardiovascular and cerebrovascular diseases, and has become the focus and hot spot of anti-AS drug research.

[0005] Centella asiatica Centella asiatica L Urban.) is also known as Gotu Kola and Beng Da Wan. It is widely distributed in various counties and cities in Guangxi. Its wild resources are extremely rich. It is a common folk medicine used by the Zhuang people in Guangxi. It is bitter, pungent and cold in nature. It enters the liver, spleen and kidney meridians and has the effects of "clearing heat and dampness, detoxifying and reducing swelling". Asiatic acid (AA) is a highly active ursane-type pentacyclic triterpene acid and is the main active ingredient of the Zhuang medicine Centella asiatica. Modern pharmacological studies have shown that AA has few toxic side effects and has multiple pharmacological effects such as antioxidant, anti-inflammatory, lipid regulation, blood pressure reduction, blood sugar reduction and anti-tumor. Studies have found that AA plays an important role in regulating blood lipids and can reduce liver damage induced by hyperlipidemia in golden hamsters through antioxidant effects (Lin Limin, Zhao Yun, Peng Lu, et al. Study on the lipid regulation and liver protection of asiatic acid on hyperlipidemia golden hamsters [J]. Chinese Journal of Biochemical Pharmaceutics, 2014, 34(04): 40-43+46.). Acute myocardial infarction is a cardiovascular disease with a high mortality rate, which is often based on coronary AS. Studies have shown that AA improves acute myocardial infarction in rats by inhibiting inflammatory response ([7] Rong Xia, Shi Yanxia, ​​Du Yu. Centella asiatica affects angiogenesis and ventricular remodeling in rats with acute myocardial infarction model through SIRT3 / β-catenin / PPARγ signaling pathway [J]. Chinese Journal of Arteriosclerosis, 2018, 26(06): 593-599.). Summary of the invention

[0006] The purpose of the present invention is to provide the use of asiatic acid (AA) in the preparation of anti-atherosclerosis drugs, so as to provide a new drug source for the prevention and treatment of atherosclerosis.

[0007] To achieve the above object, the present invention adopts the following technical solution: Application of asiatic acid in the preparation of anti-atherosclerotic drugs.

[0008] Furthermore, the active ingredient of the drug includes asiatic acid, and the drug includes one or more pharmaceutically acceptable carriers or excipients. The anti-atherosclerosis drug is a drug for preventing and treating atherosclerosis.

[0009] Furthermore, the above-mentioned Centella asiatica is used in the preparation of an anti-atherosclerotic drug, wherein the drug is an anti-atherosclerotic drug by inhibiting macrophage inflammatory response and / or inhibiting macrophage foaming.

[0010] Furthermore, the above-mentioned Centella asiatica is used in the preparation of an anti-atherosclerotic drug, and the drug can inhibit the formation of atherosclerotic plaques.

[0011] The present invention adopts high-fat feeding ApoE - / -The macrophage inflammation model or macrophage-derived foam cell model was established by using mice-induced atherosclerosis (AS) animals and LPS and ox-LDL-induced RAW264.7 cells to observe the effects of asiatic acid (AA) on AS lesions and macrophage inflammatory response, foaming, etc., and to deeply explore the anti-AS effect and mechanism of AA from the molecular, cellular and whole animal levels. The study found that: ① AA inhibits APOE in high-fat-fed - / - The formation of AS plaque in mouse aorta is related to its lipid-regulating and anti-inflammatory effects; ②AA inhibits LPS-induced inflammatory response of RAW264.7 macrophages; ③AA inhibits ox-LDL-induced foaming of RAW264.7 macrophages. The experimental results show that AA has a significant anti-atherosclerotic effect.

[0012] The beneficial effects of the present invention are: The present invention has shown through research that: asiatic acid has obvious anti-atherosclerotic effect, and can be used to prepare anti-atherosclerotic drugs. The present invention discloses the application of asiatic acid in the preparation of anti-atherosclerotic drugs, and provides a new drug source for the prevention and treatment of atherosclerosis. Moreover, asiatic acid is derived from the natural plant Centella asiatica, has little toxic and side effects, and has good application prospects. The present invention provides theoretical and experimental basis for the research and development of asiatic acid as a novel anti-atherosclerotic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effect of asiatic acid (AA) on ApoE in high-fat fed mice - / - Effects of four blood lipid items on mice ( , n=15, mmol / L), Note: Compared with the normal control group, ** P <0.01; compared with the AS model group, ## P <0.01; Figure 2 Effect of asiatic acid (AA) on ApoE - / - Oil red O staining of the whole aorta of mice (A and B) and HE staining (CE) and Masson staining (FG) of the aortic root sections ( , n=5), Note: Compared with the normal control group, ** P <0.01; compared with the AS model group, ## P <0.01; Figure 3 Effects of asiatic acid (AA) on the levels of TNF-α, IL-1β, MCP-1, NO and iNOS released by RAW264.7 macrophages induced by LPS , n=3), Note: Compared with the blank group, ＊＊ P <0.01; compared with the LPS group, # P <0.05, ## P <0.01; Figure 4 Effects of asiatic acid (AA) on the formation of RAW264.7 macrophage-derived foam cells and intracellular cholesterol content ( , n=3), Note: Compared with the normal control group, ＊＊ P <0.01; compared with the model group, ## P <0.01. DETAILED DESCRIPTION

[0014] The present invention is further described below in conjunction with the examples and drawings. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from chemical reagent companies.

[0015] Example 1 Study on the Anti-atherosclerotic Effect of Centella asiatica 1. Effect of asiatic acid (AA) on ApoE - / - Effects of atherosclerotic plaque formation on mice 1. Materials 1.1 Experimental animals and breeding environment 45 SPF 6-week-old male ApoE - / - The mice and the corresponding 15 wild-type C57BL / 6J male mice were all from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with the experimental animal qualification certificate number: 11400700133791. The animals required for the experiment were kept in the SPF-level experimental animal room of the Experimental Animal Center of Guangxi Medical University, with the experimental animal use license number: SYXK Gui 2020-0004. During the feeding period, the animals were allowed to drink water and eat freely, and 12 hours of lighting and 12 hours of dark room were alternated to simulate normal day and night, and the temperature and humidity were: 20-25℃, 40-70%, respectively.

[0016] 1.2 Methods 1.2.1 Preparation of experimental drugs and reagents

[0017] 1.2.2. Animal grouping and treatment After 2 weeks of adaptive feeding of 60 mice, 45 ApoE - / -Mice were fed a high-fat diet and randomly divided into three groups, namely the AS model group, the AA group (40 mg / kg / d, ig) and the atorvastatin group (10 mg / kg / d, ig), with 15 mice in each group; 15 wild-type C57BL / 6J mice served as the normal control group, which were fed with ordinary diet and gavaged with an equal volume of 0.5% sodium carboxymethylcellulose suspension every day. The body weight of all mice was measured and recorded once a week, and the dosage was adjusted with the change of body weight. After 18 weeks of feeding, blood and aorta samples were collected from 60 mice.

[0018] After 18 weeks, the four blood lipid items, namely serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), were detected by automatic serum biochemical analyzer; the whole-length aorta was stained with Oil Red O to detect the AS plaque area ratio (%); the aortic root sections were stained with HE and Masson staining to observe the AS plaque formation and collagen deposition, and the plaque area ratio (%) and intima-media thickness (IMT) were measured.

[0019] 1.2.3 Statistical analysis SPSS 26.0 and GraphPad Prism 8.0 were used for statistical analysis and drawing. The quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple samples and variance homogeneity test was performed. When the variance was equal (P>0.05), the LSD-t test was used to compare the two samples; when the variance was unequal (P<0.05), the Tamhane's T2 test was used to compare the two samples; P<0.05 was considered statistically significant.

[0020] 1.3 Experimental results The results of animal experiments showed that: ① After 18 weeks of high-fat feeding, the serum TC, TG and LDL-C levels of mice in the AS model group were significantly increased, while HDL-C was decreased (compared with the normal control group, P<0.01); the AA group and the Ator group significantly reduced serum TC, TG, LDL-C and increased HDL-C levels (compared with the AS model group, P<0.01); the experimental results are shown in Figure 1.

[0021] ② Obvious AS plaques appeared in the aorta of mice in the AS model group. Oral administration of 40 mg / kg AA for 18 weeks significantly reduced the ApoE expression induced by high-fat feeding. - / -Pathological changes of AS in mouse aorta reduce the positive plaque area ratio (%) of the whole aorta in Oil Red O staining, reduce the plaque area ratio (%) of the aorta root in HE staining and the intima-media thickness (IMT), and increase the content of collagen fibers in AS plaques. The experimental results are shown in Figure 2.

[0022] Experimental results show that asiatic acid (AA) inhibits APOE in high-fat diet - / - The mechanism of AS plaque formation in mouse aorta is related to its lipid-regulating and anti-inflammatory effects.

[0023] Effects of asiatic acid (AA) on LPS-induced inflammatory response in RAW264.7 macrophages In this experiment, we used LPS to induce a macrophage inflammation model established by mouse RAW 264.7 macrophages to study the effect of AA on this inflammatory model and further explore the effect of AA on the inflammatory response of AS at the cellular level.

[0024] 2.1 Materials 2.1.1 Cell line Mouse macrophage RAW 264.7 was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0025] 2.1.2 Reagents AA reference substance (Chengdu Purifa Technology Development Co., Ltd., batch number: PRF10102903); DMEM high glucose medium (Gibco, batch number: 8122132); high-quality fetal bovine serum (Lonsera, Uruguay, batch number: QT08773); lipopolysaccharide LPS (L2880) [Sigma, USA, batch number: 000114327]; nitric oxide (NO) kit (Nanjing Jiancheng Biotechnology Co., Ltd., batch number: 20210706); mouse TNF-α ELISA kit (Wuhan Yilai Ruite Biotechnology Co., Ltd., batch number: K9BVC5A3Y); mouse IL-1β, MCP-1 (Jiangsu Jingmei Biotechnology Co., Ltd., batch number: 2020-09); iNOS ELISA kit, etc.

[0026] 2.2 Reagent preparation and storage

[0027] 2.3 Methods Mouse RAW264.7 macrophages were divided into four groups: blank group, LPS group, AA group (12.5 μM) and AA group (25 μM). The cells in LPS group and AA group were pretreated with 0.1% DMSO or different concentrations of AA for 1 h, and then treated with 1 μg / mL LPS for 24 h; the cells in the blank group were treated with 0.1% DMSO for the same period.

[0028] The migration function of macrophages was detected by scratch and Transwell assays. The release of inflammatory factors such as TNF-α, IL-1β, MCP-1 and NO was detected by ELISA and Griess assays, respectively. The iNOS content of cells was detected by ELISA.

[0029] Statistical analysis: SPSS 26.0 software and GraphPad Prism 8.0 software were used for statistical analysis and drawing. The quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used to compare multiple samples and the variance homogeneity test was performed; when the variance was equal (P>0.05), the LSD-t test was used to compare between two samples; when the variance was unequal (P<0.05), the Tamhane's T2 test was used to compare between two samples; P<0.05 was considered statistically significant.

[0030] 2.4 Results The results of the cell experiment showed that after 24 hours of treatment with 1 μg / mL LPS, macrophages migrated significantly, released more TNF-α, IL-1β, MCP-1 and NO, and increased the content of cellular iNOS. AA (12.5 μM, 25 μM) intervention reduced the kinetic energy of macrophage migration, reduced the release of TNF-α, IL-1β, MCP-1 and NO, and reduced the content of cellular iNOS. The experimental results are shown in Figure 3 .

[0031] The experimental results showed that asiatic acid (AA) inhibited the inflammatory response of RAW264.7 macrophages induced by LPS. Effects of asiatic acid (AA) on ox-LDL-induced foaming of RAW264.7 macrophages In this experiment, ox-LDL was used to induce RAW264.7 cells to establish a macrophage-derived foam cell model and observe the effect of AA on macrophage foam cell formation.

[0032] 3.1 Methods RAW264.7 macrophages were randomly divided into normal control group, model group, and high-dose and low-dose AA groups (AA 15, 30 μM). After pre-incubation for 1 h, 80 μg / mL ox-LDL was added and incubated for 24 h to induce the formation of RAW264.7 macrophage-derived foam cells.

[0033] 3.2 Results The experimental results showed that AA concentration-dependently reduced intracellular lipid accumulation (Oil Red O staining), and reduced the total cholesterol (TC), cholesterol ester (CE) content and CE / TC ratio in foam cells. Figure 4 .

[0034] The experimental results showed that asiatic acid (AA) inhibited ox-LDL-induced foaming of RAW264.7 macrophages.

[0035] IV. Conclusion Asiatic acid (AA) can inhibit ApoE - / - The formation of AS plaques in mice may be through regulating blood lipids and anti-inflammation. AA inhibited the inflammatory response of RAW264.7 macrophages induced by LPS and inhibited the foaming of RAW264.7 macrophages induced by ox-LDL. It can be seen that Centella asiatica has a significant anti-atherosclerotic effect.

Claims

1. Application of Centella asiatica in the preparation of anti-atherosclerotic drugs.

2. The use according to claim 1, characterized in that: The active ingredient of the medicine includes asiatic acid, and the medicine includes one or more pharmaceutically acceptable carriers or excipients.

3. The use according to claim 1, characterized in that: The drug is a drug for resisting atherosclerosis by inhibiting macrophage inflammatory response and / or inhibiting macrophage foaming.

4. The use according to claim 1, characterized in that: The drug can inhibit the formation of atherosclerotic plaques.

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