Application of two-essence formula in preparation of medicine for treating hyperhomocysteinemia-derived atherosclerosis

Abstract

The invention provides application of a formula for treating hyperhomocysteinemia-derived atherosclerosis and relates to the technical field of application of formulas for treating hyperhomocysteinemia-derived atherosclerosis, and the formula has the unique advantages of multiple targets, multiple ways and low toxic and side effects, and is a medicine which is safe, effective and free of adverse reaction. The obvious effects are shown in the aspects of regulating Hcy metabolism, improving the vascular endothelial function, stabilizing the artery plaque and the like; at the same time, the Ersperm prescription can regulate lipid accumulation, inhibit inflammatory response, relieve oxidative stress injury and improve foaming of macrophages and VSMCs, thereby playing a role in relieving HHcy-derived atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of application technology of Erjingfang, specifically relating to the application of Erjingfang in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis. Background Technology

[0002] Atherosclerosis (AS), a chronic inflammatory vascular disease, is the core pathological basis for cardiovascular and cerebrovascular events such as coronary heart disease and cerebral infarction. Its incidence and mortality rates remain high globally, seriously threatening human health. With in-depth clinical research, hyperhomocysteinemia (HHcy) has been confirmed as an independent risk factor for the development of AS. When the level of homocysteine ​​(Hcy) in the blood exceeds 15 μmol / L, it can accelerate the formation and instability of atherosclerotic plaques through multiple pathways, including damaging vascular endothelial function, promoting lipid peroxidation, inducing inflammatory responses, and oxidative stress, significantly increasing the risk of cardiovascular and cerebrovascular diseases.

[0003] Currently, clinical interventions for hyperhomocysteinemia (HHcy) mainly rely on folic acid supplementation and statins. While these can lower Hcy levels and regulate blood lipids to some extent, long-term use can easily lead to adverse reactions such as liver damage and muscle pain. Therefore, it is necessary to explore a safe, effective, and side-effect-free drug for treating hyperhomocysteinemia-related atherosclerosis. Summary of the Invention

[0004] In view of this, the present invention provides a safe, effective, and adverse-reaction-free drug for treating hyperhomocysteinemia-related atherosclerosis.

[0005] Application of a certain formula in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis.

[0006] Preferably, the two-essence formula reduces the level of homocysteine ​​in serum.

[0007] Preferably, the two-essence formula can exert its anti-atherosclerotic effect by regulating lipids, reducing oxidative stress, and inhibiting inflammatory responses.

[0008] Preferably, the two-essence formula can improve foaming of macrophages and vascular smooth muscle cells.

[0009] Preferably, the effect of the two-essence formula in preventing atherosclerosis by regulating lipids is specifically as follows: it can improve the stability of atherosclerotic plaques in the aorta and reduce the levels of TC and TG in blood lipids.

[0010] Preferably, the two-essence formula exerts its anti-fibrotic effect by inhibiting macrophage infiltration and myofibroblast activation.

[0011] Preferably, the anti-atherosclerotic effect of the two-essence formula by reducing oxidative stress specifically involves: reducing MDA levels and restoring SOD and GSH-PX activities to normal levels.

[0012] Preferably, the anti-atherosclerotic effect of the Erjing formula by inhibiting the inflammatory response is specifically achieved by reducing TNF-α and IL-1β levels.

[0013] Preferably, the two-essence formula can reduce intracellular lipid accumulation.

[0014] Preferably, the dosage of the two-ingredient formula is 1.4g / kg-5.6g / kg.

[0015] The application of the composition containing Erjingfang in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The Erjing Formula provided by this invention has unique advantages in treating hyperhomocysteinemic atherosclerosis, namely "multi-target, multi-pathway, and low toxicity and side effects". It is a safe, effective drug with no adverse reactions.

[0018] This invention uses ApoE - / - Mice were used as experimental subjects. An HHcy-AS complex model was established by inducing a 1.7% methionine diet. The results showed that Erjingfang had significant effects on regulating Hcy metabolism, improving vascular endothelial function and stabilizing arterial plaques by intervening in the "Hcy-AS" pathological axis through multiple targets and pathways.

[0019] Meanwhile, Erjingfang can alleviate HHcy-derived atherosclerosis by regulating lipid accumulation, inhibiting inflammatory response, reducing oxidative stress damage, and improving foaming of macrophages and VSMCs. Attached Figure Description

[0020] Figure 1 The Erjing formula can alleviate methionine-induced ApoE. - / -Hyperhomocysteinemia and atherosclerosis in mice. A. Body weight change curves of mice in each group (n=10); B. Comparison of food intake in mice (n=10); C. Blood Hcy levels (n=6); D. Images of mouse aorta samples; E. Gross Oil Red O staining of mouse aorta; F. Quantitative analysis of gross Oil Red O staining of mouse aorta (n=3). Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001, #### indicates P<0.0001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001. p<0.05 was considered statistically significant.

[0021] Figure 2 The Erjing formula can alleviate hyperhomocysteinemia-related atherosclerosis and lipid accumulation. A. Total cholesterol level (n=6); B. Triglycerides (n=6); C. High-density lipoprotein level (n=6); D. Low-density lipoprotein level (n=6); E. Quantitative staining of mouse aortic sinus with Oil Red O; F. Pathological staining of mouse aortic sinus with Oil Red O, HE, and Masson's staining; G. Pathological staining of mouse aortic sinus with α-SMA and F4 / 80. Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001, #### indicates P<0.0001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001. p<0.05 is considered statistically significant.

[0022] Figure 3 The Erjing formula treatment can alleviate oxidative stress and inflammatory factor levels in hyperhomocysteinemia-related atherosclerosis. A. Malondialdehyde level (n=6); B. Superoxide dismutase activity (n=6); C. Glutathione peroxide activity (n=6); D. Tumor necrosis factor α level (n=6); E. Interleukin-1β level (n=6); F. Interleukin-1β level (n=6). Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001, #### indicates P<0.0001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001. p<0.05 is considered statistically significant.

[0023] Figure 4The effects of Erjingfang on the viability of RAW 264.7 macrophages and mouse vascular smooth muscle cells are as follows: A. Effect of Erjingfang on the viability of RAW 264.7 macrophages at 24 h (mg); BC. Effect of Erjingfang on the viability of RAW 264.7 macrophages at 24 h and 48 h (μg); DE. Effect of Erjingfang on the viability of mouse vascular smooth muscle cells at 24 h and 48 h (μg). Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, and ### indicates P<0.001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. p<0.05 is considered statistically significant.

[0024] Figure 5 The Erjing formula can improve foaming and lipid accumulation in macrophages and smooth muscle cells. A. Oil Red O staining of RAW 264.7 macrophages with Erjing formula; B. Oil Red O staining of mouse vascular smooth muscle cells with Erjing formula; C. TC level (n=3); D. FC level (n=3). Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, ### indicates P<0.001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. p<0.05 is considered statistically significant.

[0025] Figure 6 This study investigated the effects of Erjingfang on inflammatory factors and oxidative stress in a smooth muscle cell foaming model. The levels of tumor necrosis factor-α (n=3) were measured. A. Interleukin-1β (n=3) level; B. Interleukin-1β (n=3) level; C. Malondialdehyde (MDA) level (n=6); E. Superoxide dismutase (SOD) activity (n=3); F. Glutathione peroxide (GDP) activity (n=3). Note: Compared with the normal group, # indicates P<0.05, ## indicates P<0.01, and ### indicates P<0.001. Compared with the model group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. p<0.05 was considered statistically significant. Detailed Implementation

[0026] The technical solutions and effects of the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Example:

[0028] I. Animal Experiments

[0029] 1. Experimental Materials

[0030] 1.1. Laboratory animals

[0031] Eleven male SPF-grade C57 mice (weighing 18–22 grams, 6–8 weeks old) and male SPF-grade ApoE mice. − / − Sixty-six mice (weighing 18–22 grams, 6–8 weeks old) were purchased from Beijing Vital River Co., Ltd.

[0032] 1.2. Experimental Drugs

[0033] Erjing Formula: Composed of wolfberry and polygonatum. The wolfberry was purchased from Ningxia Ninganbao Specialty Products Co., Ltd., production license number: SC11464052101006; the polygonatum was purchased from Shandong Chixiang Chinese Medicine Pieces Co., Ltd., production license number: Lu20170360.

[0034] 1.3. Preparation of main solutions

[0035] 2% Sodium Pentobarbital Solution: Weigh 2g of sodium pentobarbital in a clean bench and place it in a 100mL beaker. Add physiological saline to a total volume of 100mL. After the sodium pentobarbital is fully dissolved, sterilize it through a 0.2μm sterile filter membrane and store it at 4°C for later use. For anesthetized mice, administer 0.1mL of 2% sodium pentobarbital solution intraperitoneally per 10g of body weight.

[0036] 1.4. Main Experimental Reagents

[0037] As shown in Table 1.

[0038] Table 1

[0039]

[0040] 2. Experimental Methods

[0041] 2.1. Preparation of Erjingfang Extract

[0042] Dry the Polygonatum and Lycium barbarum to constant weight. Chop the Lycium barbarum and pulverize the Polygonatum through a No. 2 sieve. Mix equal amounts of Lycium barbarum and Polygonatum, and extract three times by reflux with hot water, one hour each time, with a solid-liquid ratio of 1:10 each time. Filter the extract, combine the three filtrates, and concentrate. Dry to obtain the extract for later use.

[0043] 2.2. Animal Experiments

[0044] Mice were housed in an SPF-grade environment at the Laboratory Animal Center of Ningxia Medical University. The design and execution of the experimental protocol were reviewed and approved by the Institute for Laboratory Animal Use and Management (IACUC), with the approval number [IACUC-NYLAC-2024-161]. The environmental conditions for animal housing were: temperature: 22±1℃; humidity: 5-60%; 12 h / 12 ​​h light-dark cycle. These conditions met my country's standards for laboratory animal environments, and the design and operation of the animal experiments adhered to relevant animal experimental standards throughout. Eleven C57 mice were used as the non-ApoE gene knockout normal group (C57 Control group). - / - Sixty-six mice were randomly divided into six groups, namely the control group (ApoE) and the control group (ApoE). - / - The mice were divided into Control group, Model group, Positive drug folic acid group, low-dose Erjingfang group (EJP-L group), medium-dose Erjingfang group (EJP-M group), and high-dose Erjingfang group (EJP-H group). Each mouse's body weight and food intake were measured every three days. After one week of acclimatization feeding, C57 Control and ApoE... - / - The control group received a normal diet, while the other groups were fed a standard diet containing 1.7% methionine for 16 weeks, with drinking water changed every three days. HHcy was diagnosed when the blood Hcy level was above 15µM. Gross Oil Red O staining of the aorta indicated successful modeling, showing typical atherosclerotic plaques. The mice were then treated with drugs for 4 weeks. After 20 weeks of this intervention, the animals were sacrificed. Mice were fasted for 12 hours, and blood was collected by enucleation. Mice were then anesthetized with 2% sodium pentobarbital (0.1 mL per 10g body weight) via intraperitoneal injection. Once the corneal reflex disappeared, the mice were fixed on a dissecting board, and a small amount of 75% alcohol was sprayed onto their fur. The abdomen and chest cavity were then cut open, and a perfusion needle was inserted through the apex of the heart. Residual blood in the blood vessels was flushed away using PBS solution via reverse perfusion. The sternum and ribs of the mice were removed, the trachea was cut, and the lungs and digestive system were dissected from the side of the head to fully expose the heart and aorta. The aorta was carefully cut from the root of the heart, and then the heart was cut along the line connecting the two auricles to obtain the aortic sinus. The aortic sinus was placed in a 30% sucrose solution for dehydration and later use. The remaining heart, aorta, liver, kidneys, spleen, and lungs were rapidly frozen in liquid nitrogen and stored at -80°C for later use.

[0045] The adult clinical dose of Erjingfang is 24 g / 60 kg. According to the equivalent dose factor conversion method, the mouse dose of Erjingfang is 2.80 g / kg.

[0046] The mice were randomly divided into groups as follows:

[0047] (1) C57 normal group: fed with normal feed for 16 weeks, and then administered physiological saline by gavage for 4 weeks after successful modeling;

[0048] (2) ApoE - / - Normal group: fed with normal feed for 16 weeks, and then administered physiological saline by gavage for 4 weeks after successful model establishment;

[0049] (3) Model group: fed with 1.7% high methionine diet for 16 weeks, and then gavaged with physiological saline for 4 weeks after successful modeling;

[0050] (4) Positive group: fed with 1.7% high methionine diet for 16 weeks, and after successful modeling, administered 0.0275g / kg folic acid by gavage for 4 weeks;

[0051] (5) EJP low dose group: fed with 1.7% high methionine diet for 16 weeks, and after successful modeling, administered 1.40g / kg of Erjingfang extract solution by gavage for 4 weeks;

[0052] (6) EJP medium dose group: fed with 1.7% high methionine diet for 16 weeks, and after successful modeling, administered 2.80g / kg of Erjingfang extract solution by gavage for 4 weeks;

[0053] (7) High-dose EJP group: fed with 1.7% high-methionine diet for 16 weeks, and after successful modeling, administered 5.60g / kg of Erjingfang extract solution by gavage for 4 weeks;

[0054] The experimental animals were administered the above-listed dosage once daily in the morning, with each group receiving 10 mL / kg via gavage. The medication was administered for a total of 4 weeks. Note: The gavage dosage for the folic acid group was based on the clinically recommended daily oral dosage for a 60 kg adult, and converted to the mouse gavage dosage using an equivalent dose conversion factor between adults and mice.

[0055] 2.3. Serum Hcy and lipid level detection

[0056] The collected blood samples were allowed to stand at room temperature for 1 hour, then centrifuged at 3000 rpm for 15 minutes at room temperature. The supernatant was the serum. The supernatant was collected and aliquoted at -80 ℃ for later use. The concentrations of total Hcy, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) were determined using Hcy detection kits (enzyme cycling method), TC detection kits (microplate method), TG detection kits (microplate method), LDL-C detection kits (microplate method), and HDL-C detection kits (microplate method) provided by Jiangsu Jingmei, respectively.

[0057] 2.4. Detection of serum oxidative stress and inflammatory factor markers

[0058] The collected blood samples were allowed to stand at room temperature for 1 hour, then centrifuged at 3000 rpm for 15 minutes at room temperature. The supernatant was the serum. The supernatant was collected and aliquoted at -80 ℃ for later use. The concentrations of oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and inflammatory markers tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were obtained using MDA detection kits (microplate method), SOD detection kits (WST-8 method), GSH-PX detection kits (microplate method), TNF-α detection kits (double antibody one-step sandwich method), IL-6 detection kits (double antibody one-step sandwich method), and IL-1β detection kits (double antibody one-step sandwich method) provided by Jiangsu Jingmei, respectively.

[0059] 2.5. Preparation of frozen sections of the aorta

[0060] The removed aortic sinus was soaked in a 30% sucrose solution for one week to dehydrate it. The tissue surface was then blotted dry with filter paper. An appropriate amount of OCT embedding medium was added to the embedding cassette, and the tissue was placed with the cut side facing upwards within the embedding medium, adjusting the tissue to ensure the cut surface was horizontal. The embedding cassette was placed on the freezing stage of a cryostat, and the OCT embedding medium was frozen until it turned white and hardened. The tissue embedding block was fixed on the cryostat, and continuous transverse sections were made along the aortic root, marking the appearance of the three aortic valves. 6μm thick frozen sections were continuously collected. Two sections were mounted on clean, detachable glass slides, fixed in 4% paraformaldehyde for 30 minutes, and then stored at -20℃ for later use.

[0061] 2.6. HE staining

[0062] (1) Place the frozen sections at room temperature for 10 minutes, and then soak them in distilled water for 10 minutes to wash away the embedding agent.

[0063] (2) Soak the slices in Harris hematoxylin staining solution for 3 minutes, gently rinse the elution box containing the slices with tap water, observe under a microscope at any time, and differentiate the cells with 1% hydrochloric acid alcohol for a few seconds after the cell nuclei turn blue, and rinse again with running water.

[0064] (3) Place the slices into a staining vat containing eosin staining solution and soak for 3 minutes.

[0065] (4) Soak the sections in the following order and time: 95% ethanol I 5 min - 95% ethanol II 5 min - anhydrous ethanol I 5 min - anhydrous ethanol II 5 min - xylene I 5 min - xylene II 5 min. Finally, remove the sections from the last staining vat containing xylene and air dry them in a fume hood. Add neutral resin to the tissue sections and mount them with a glass slide of appropriate size.

[0066] (5) Scan the slide with a slide scanner.

[0067] 2.7. Oil Red O staining

[0068] (1) Take the frozen slices out of the refrigerator, let them warm to room temperature for 10 minutes, and then soak them in distilled water for 10 minutes.

[0069] (2) Soak in freshly prepared 60% isopropanol for 10 minutes.

[0070] (3) Soak the sections in freshly prepared Oil Red O working solution for 15 min.

[0071] (4) Rinse with 60% isopropanol for a few seconds to remove the surface color.

[0072] (5) Soak in Harris hematoxylin staining solution for 2 minutes, observe under a microscope until the cell nuclei turn blue, place the elution box containing the slices under a tap and rinse gently with running water, differentiate with 1% hydrochloric acid alcohol for a few seconds, and rinse again with running water.

[0073] (6) Rinse with distilled water.

[0074] (7) Quickly seal the tablets with warm liquid glycerin gelatin sealing tablets.

[0075] (8) Allow the glycerin gelatin to cool and solidify completely at room temperature.

[0076] (9) Scan the slide with a slide scanner.

[0077] 2.8. Masson staining

[0078] (1) Take the frozen sections out of the refrigerator and let them warm up to room temperature for 10 minutes. Soak them in distilled water for 10 minutes. Soak them in Harris hematoxylin staining solution for 2 minutes and observe them under a microscope until the cell nuclei turn blue. Place the elution box containing the sections under a tap and rinse gently with running water. Differentiate with 1% hydrochloric acid alcohol for a few seconds and rinse again with running water.

[0079] (2) Add the Ponceau S acid fuchsin solution from the kit to the tissue section, stain for 10 min, and rinse with freshly prepared 1% glacial acetic acid for 1 min.

[0080] (3) Treat with phosphomolybdic acid solution for 2 min.

[0081] (4) Remove the phosphomolybdic acid solution from the slide, do not rinse, add aniline blue solution for staining for 2 minutes, and rinse again with 1% glacial acetic acid for 1 minute.

[0082] (5) Dehydration and mounting with neutral resin were performed as before HE staining. The sections were then scanned using a slide scanner.

[0083] 2.9. Immunohistochemical detection of tissue-specific antigen expression in aortic sinus tissue sections

[0084] (1) Take the frozen sections out of the refrigerator and let them warm up at room temperature for 10 min. Place the sections in a destaining box containing PBS and shake the box on a shaker for 5 min each time.

[0085] (2) After the sections are slightly dried, use a PAP immunohistochemistry pen to draw a circle around the tissue section (to prevent the antibodies from flowing away), add goat serum working solution to the circle, and block at room temperature for 30 minutes.

[0086] (3) Gently shake off the blocking solution on the slide, add the primary antibody prepared with PBS in a certain proportion in the circle, place the slide flat in the humidified box, and incubate overnight at 4°C.

[0087] (4) Shake the sections three times on a shaker for 5 minutes each time. Shake off the water on the sections, then add the corresponding secondary antibody (HRP-labeled) to the tissue and incubate at room temperature for 50 minutes.

[0088] (5) Place the slide in PBS and wash it three times on a decolorizing shaker for 5 minutes each time. After shaking off the water on the slide, add freshly prepared DAB (solution A:solution B = 1:1) staining solution. Observe the first slide under a microscope and control the staining time. Rinse the slide with tap water to stop the staining. The DAB staining time for subsequent slides is the same as for the first slide.

[0089] (6) Place the slices into a hematoxylin staining jar, counterstain the cell nuclei for 3 minutes, rinse gently with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, and rinse with running water.

[0090] (7) The steps for dehydration, sealing, and reading section information are the same as before.

[0091] 3. Statistical Methods

[0092] The test results for all the above indicators have been processed using Graphpad Prism 9 and Excel software. One-way ANOVA (both nonparametric and mixed-mode) was used. Compared with the normal group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001. Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. A p < 0.05 was considered statistically significant.

[0093] 4. Test Results

[0094] C57 and ApoE were randomly assigned to groups respectively. - / - Standard diet for mice (normal group / C57 Control group, ApoE)- / - Control group), ApoE - / - Mice were fed a normal diet supplemented with methionine (1.7%, changed every three days; model group, positive drug group, low-medium-high dose group of Erjingfang / EJP-L, EJP-M, EJP-H group). Food intake and body weight changes were recorded for each cage of mice. The mice were fed continuously for 16 weeks. After successful model establishment, the mice were administered the drug for four weeks. Statistical analysis of mouse body weight and food intake revealed that the addition of methionine to the normal diet did not significantly affect the trend of body weight gain or food intake. Figure 1 (AB) indicates that the model establishment process did not cause nutritional metabolic disorders.

[0095] 4.1. Detection of plasma homocysteine ​​levels

[0096] After feeding, blood was collected from the eyes after enucleation, and the supernatant was analyzed. Results showed that adding 1.7% methionine to a normal diet significantly induced hyperhomocysteinemia. The Model group, C57 Control group, and ApoE group showed significantly higher levels of this symptom. - / - Compared with the control group, the level of homocysteine ​​(Hcy) in the blood was significantly higher (P < 0.0001), and the difference was statistically significant, with Hcy > 15 μM, reaching the diagnostic level for hemocytosis (HHcy). After drug intervention, the reduction in Hcy was statistically significant between the positive group and the model group (P < 0.05); the low, medium, and high dose EJP groups all showed stronger intervention effects compared with the model group (P < 0.0001), which was statistically significant. Figure 1 C). The Positive group and the low, medium and high dose EJP groups reduced blood Hcy levels to varying degrees, alleviating hyperhomocysteinemia.

[0097] 4.2. Pathological assessment of aortic atherosclerosis

[0098] The results of photographing the aorta of mice showed that the Model group, C57 Control group, and ApoE group... - / - Compared to the control group, the relative area of ​​aortic root plaques significantly increased, confirming the successful establishment of the aortic atherosclerosis model. However, after treatment with medium-to-high doses of EJP, the relative area of ​​aortic root plaques decreased. Figure 1 D). Then, the mouse aorta was grossly stained with Oil Red O and quantitatively analyzed. The results showed that the Model group, C57 Control group, and ApoE group... - / - Compared with the control group, typical atherosclerotic plaques were observed in the aorta. The difference was statistically significant compared to the C57 control group (P < 0.01). This was also consistent with the ApoE group. - / -Compared with the control group, (P<0.05) there was a statistically significant difference. After treatment with medium and high doses of EJP, the area of ​​typical atherosclerotic plaques in the aorta was reduced (P<0.05), which was also statistically significant. Erjingfang reduced plasma Hcy levels (P<0.0001), and the medium and high dose groups significantly reduced the formation of atherosclerotic plaques in the aorta (P<0.05), indicating that it has a clear ameliorative effect on HHcy-induced atherosclerosis.

[0099] Animal experiments have shown that the dosage of Erjingfang at 2.80 g / kg is the most effective in improving HDL-C and plaque stability. Erjingfang intervenes in the "HHcy-AS" pathological axis through multiple targets and pathways by regulating metabolism, protecting blood vessels, and reducing inflammation. It has shown significant effects in regulating Hcy metabolism, improving vascular endothelial function, and stabilizing arterial plaques.

[0100] II. Cell Experiments

[0101] 1. Experimental Materials

[0102] 1.1. Experimental Cells

[0103] The cells used in this experiment were RAW 264.7 macrophages provided by Wuhan Pronosei Biotechnology Co., Ltd.; and mouse vascular smooth muscle cells were derived from ATCCRAL-2797.

[0104] 1.2. Main Experimental Reagents

[0105] As shown in Table 2:

[0106] Table 2

[0107]

[0108] 1.3 Preparation of main solutions

[0109] (1) Complete culture medium: Add 50 mL of fetal bovine serum and 0.5 mL of penicillin-streptomycin solution to 500 mL of DMEM high glucose culture medium.

[0110] (2) Preparation of fetal bovine serum: The purchased fetal bovine serum was frozen. After purchase, it was placed in a 4°C freezer for slow thawing, with regular shaking during the process to avoid precipitation. After complete thawing, the packaging surface was disinfected with 75% alcohol and transferred to a clean bench. It was then dispensed into 50mL centrifuge tubes, the tube openings were sealed with sealing film, the date was marked, and the tubes were stored at -20°C for later use. Repeated freeze-thaw cycles should be avoided.

[0111] (3) Preparation of ox-LDL solution: Add ox-LDL to DMEM according to the original solution concentration, dilute to the concentration required for the experiment, store the original solution at 4℃, and prepare fresh each time.

[0112] (4) Erjingfang stock solution: Weigh 50mg of Erjingfang extract using a balance, add the extract to 5ml of DMEM, mix well and dissolve completely to prepare 10mg / mL Erjingfang stock solution, filter the drug solution with a filter membrane to sterilize, and store at -20℃ for one month. It is effective. Dilute to the required concentration according to the needs of subsequent experiments.

[0113] (2) Cell culture media containing different concentrations of Hcy: Weigh 6.8 mg of DL-homocysteine ​​powder using an analytical balance and place it in a 1.5 mL EP tube. Add 1 mL of DMEM high-glucose medium (free of FBS and antibiotics) to the EP tube and repeatedly pipette until the Hcy powder is completely dissolved. Draw the Hcy solution into a sterile 1 mL syringe, remove the needle, and filter it through a 0.22 μm microporous membrane for sterilization to obtain a 50 mM Hcy stock solution. The Hcy stock solution should be prepared fresh each time. When preparing Hcy cell culture media of different concentrations, add 20 μL of the freshly prepared Hcy stock solution to 1 mL of DMEM complete medium to obtain a 1000 μM Hcy cell culture medium. Dilute this concentration medium with DMEM complete medium at a 1:1 ratio to obtain a 500 μM Hcy cell culture medium, and then dilute the 500 μM Hcy cell culture medium to obtain a 200 μM solution.

[0114] 2. Experimental Methods

[0115] 2.1. Cell Culture and Processing

[0116] 2.1.1. Macrophage Culture Procedure

[0117] 2.1.1.1. Cell Culture

[0118] Mouse macrophages were uniformly seeded in T25 culture flasks and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C with 5% CO2. Experiments were conducted when cell confluence reached 80%. To simulate the in vivo atherosclerotic environment, different concentrations of homocysteine ​​(Hcy) and ox-LDL were simultaneously added to the culture medium.

[0119] 2.1.1.2. Cell resuscitation

[0120] (1) Prepare a pipette (1000μL), pipette tip (1000μL), culture dish, culture medium, and centrifuge tube (10mL), and irradiate with ultraviolet light on the clean bench for 30min;

[0121] (2) Take out the cryovial, put it in a 37°C water bath to thaw, and after thawing, transfer it to a sterile operating table;

[0122] (3) Aspirate the cell suspension from the cryopreservation tube into a centrifuge tube, add an appropriate amount of culture medium, and mix by pipetting.

[0123] (4) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and the lower layer is the cells;

[0124] (5) After adding an appropriate amount of culture medium, transfer the cells to a culture dish. The medium dish culture system is 4 mL. Incubate at 37℃ and observe the cell growth on the second day.

[0125] 2.1.1.3. Cell passage and plating

[0126] (1) Observe the cell morphology under a microscope to determine whether the cells need to be passaged. If passage or plating is required, proceed to the next step. If passage or plating is not required, put the culture dish back into the incubator to continue culturing.

[0127] (2) Prepare pipettes (1000μL, 200μL, 20μL, 10μL), pipette tips (1000μL, 200μL, 20μL, 10μL), appropriate amount of culture dishes (medium or large), 96-well plates, 6-well plates, centrifuge tubes (15mL), culture medium, PBS, etc. Irradiate with ultraviolet light on the clean bench for 30min;

[0128] (3) Take out the culture dish and open it in the clean bench. Aspirate the old culture medium into the waste liquid container and wash away the residual old culture medium with 2-3 mL of PBS.

[0129] (4) If the cells are adherent, add 1 mL of 0.25% trypsin to digest them. Once the cells are observed to become round under a microscope, immediately add culture medium to stop the digestion. Transfer them to a centrifuge tube and centrifuge at 1000 rpm for 10 min. Discard the supernatant and the cells that sink to the bottom are the cells.

[0130] (5) Add a small amount of fresh culture medium containing serum (complete culture), mix it with a pipette, add a certain amount of complete culture according to the number of passage plates, and then divide it into new culture dishes.

[0131] (6) If the cells are semi-adherent (e.g., RAW264.7 macrophages), trypsin is not needed. Just pipette them, centrifuge them and discard the supernatant. Add new complete culture according to the number of passage dishes, and then dispense them into each culture dish.

[0132] (7) The plate-laying operation requires cell counting after suspension. The volume of complete culture to be added is determined according to the number of cells, and then the corresponding volume is spread into each plate (100 μL per well of 96-well plate, 2 mL per well of 6-well plate, and 1 mL per well of 12-well plate).

[0133] 2.1.1.4. Cell cryopreservation

[0134] (1) Prepare pipettes (1000μL), pipette tips (1000μL), cryopreservation tubes, serum-free cell cryopreservation solution, centrifuge tubes (15mL), culture medium, PBS, etc., and irradiate with ultraviolet light in a clean bench for 30min.

[0135] (2) Remove the cell culture dish from the incubator and perform the following operations on the clean bench: aspirate the culture medium in the culture dish into the waste liquid tank, and wash the remaining old culture medium with 2-3 mL PBS.

[0136] (3) If the cells are adherent, add 1 mL of 0.25% trypsin to digest them. If the cells are semi-adherent (e.g., RAW264.7 macrophages), trypsin is not needed. Just pipette and centrifuge them and discard the supernatant. The lower layer is the cells we need to freeze. Add serum-free cell freezing solution and dispense into cryovials.

[0137] (4) When using serum-free cryopreservation solution, cryopreservation tubes can be directly placed in a -80°C freezer for storage.

[0138] 2.1.2. Procedures for culturing mouse vascular smooth muscle cells

[0139] 2.1.2.1. Preparation of culture medium and cryopreservation conditions:

[0140] (1) Prepare DMEM basal culture medium (iCell-0001) 89%

[0141] High-quality fetal bovine serum (iCell-0500) 10%

[0142] P / S Penicillin-Streptomycin (iCell-15140-122) 1%

[0143] G-418 (iCell-8160) 0.2mg / mL

[0144] (2) Cultivation conditions:

[0145] Gas phase: air, 95%; carbon dioxide, 5%.

[0146] Temperature: 37℃, humidity in incubator: 70%-80%.

[0147] (3) Cryopreservation of cells: When cryopreserving cells, resuspend the cells in FBS, then add a certain amount of DMSO, mix gently and transfer to cryopreservation tubes. The final concentration of DMSO is 10%.

[0148] 2.1.2.2. Cell treatment:

[0149] (1) Thawing of cryopreserved cells: Thaw cryovials containing 1 mL of cell suspension rapidly by shaking in a 37°C water bath, and add the cells to centrifuge tubes containing 4-6 mL of complete culture medium and mix well. Centrifuge at 1000 rpm for 3-5 min, discard the supernatant, and resuspend the cells in complete culture medium. Then add the cell suspension to a culture flask (or dish) containing 6-8 mL of complete culture medium and incubate overnight at 37°C. Observe cell growth and cell density under a microscope the next day.

[0150] (2) Cell passage: If the cell density reaches 80%-90%, passage culture can be performed. Discard the culture supernatant and wash the cells 1-2 times with PBS free of calcium and magnesium ions. Add 0.25% trypsin to the culture flask (1-2 mL for T25 flasks, 2-3 mL for T75 flasks), and incubate at 37℃ for 1-2 minutes (the digestion time can be appropriately extended for difficult-to-digest cells). Then observe the cell digestion under a microscope. If most of the cells become round and detach, quickly return the flask to the operating table, tap it a few times, and add 3-4 mL of medium containing 10% FBS to stop the digestion. Gently mix and aspirate, centrifuge at 1000 rpm for 3-5 minutes, discard the supernatant, add 1-2 mL of culture medium, and mix well. Divide the cell suspension into new T25 flasks at a ratio of 1:2, add 6-8 mL of fresh complete culture medium prepared according to the instructions to maintain cell growth viability, and perform subsequent passages at a ratio of 1:2 to 1:5 depending on the actual situation.

[0151] (3) Cell cryopreservation: During cell cryopreservation, digested cells are collected into centrifuge tubes according to the cell passage process. A hemocytometer can be used to count the cells to determine the cryopreservation density. The recommended cryopreservation density for cells is generally 1×10⁻⁶. 6 ~1×10 7 Cells / mL. Centrifuge at 1000 rpm for 3-5 min and discard the supernatant. Resuspend the cells in prepared cell cryopreservation medium at a concentration of 1×10⁻⁶ cells / mL. 6 ~1×10 7 Allocate cells per live cell / mL to a cryovial. Label the cryovials with their name, passage number, date, etc. Place the cells to be cryopreserved in a programmed cooling box and incubate overnight at -80°C. Afterward, transfer them to a liquid nitrogen container for storage. Record the position of the cryovials in the liquid nitrogen container for future reference and use.

[0152] 2.2. Cell Count

[0153] (1) Wipe the counting plate and coverslip clean with an alcohol swab, and then place the coverslip on the counting plate.

[0154] (2) Add 1 mL of complete culture medium to the cells collected after centrifugation, mix by pipetting, take 10 μL of the mixture and add it to 90 μL of complete culture medium (that is, dilute the cell suspension 10 times) and mix by pipetting.

[0155] (3) Take 10 μL of the cell suspension diluted 10 times and add it gently along the edge of the coverslip, being careful not to create air bubbles, so that the cell suspension fills the space between the coverslip and the counting chamber.

[0156] (4) Count the total number of cells in each of the four quadrants of the counting chamber. For cells pressed along the cross axis, only count the cells on the left and top sides. The calculation formula is: Total cells / mL = Total number of cells in four quadrants / 4 × 10⁻⁶ 4 × Dilution factor (10 times).

[0157] 2.3. Concentration Screening

[0158] 2.3.1. Screening the optimal concentration ratio of Erjing Formula using the CCK-8 method

[0159] (1) Screening of the intervention concentration of the two-precipitation formula

[0160] Cell seeding: RAW264.7 cells and mouse vascular smooth muscle cells were cultured to 90% confluence, then cell counts were performed, and the cells were diluted to 1x10⁻⁶. 5 Cells were seeded at 100 μL / well in a 96-well plate, with control wells, sample blanks, and a blank well for zeroing. The cells were incubated at 37°C and 5% CO2 for 24 hours. The following day, after observing the cells to ensure adhesion, subsequent experiments were performed.

[0161] Drug treatment: First, dilute the Erjingfang stock solution in the culture medium to different concentrations and discard the original cell culture medium. Then, add complete cell culture medium and Erjingfang containing different drug concentrations in sequence. Add complete culture medium to the blank wells. Each group has 3 replicates. Incubate in a 37℃, 5% CO2 incubator for 24 hours.

[0162] CCK-8 assay: Discard the cell culture medium in the 96-well plate, wash twice with PBS, then add 10 μL of CCK8 solution and 100 μL of culture medium to each well, incubate at 37°C and 5% CO2 for 1 h, and measure the OD value at 450 nm using a microplate reader; and assess cell viability according to the formula.

[0163]

[0164] 2.4. Establishment and Evaluation of Foam Cell Model

[0165] (1) Model construction: Log-phase RAW 264.7 macrophage cells or mouse vascular smooth muscle cells were collected and counted at a ratio of 5 x 10⁻⁶ cells. 4Macrophages were seeded at a density of 1 / mL onto sterile polystyrene plastic cell culture slides in 48-well plates and divided into 5 groups. After culturing in complete culture medium for 24 h, macrophages were treated with 50 μg / mL ox-LDL and 200 μM / mL Hcy culture medium, respectively. A blank control group was set up without ox-LDL. Macrophages were incubated in a 37℃, 5% CO2 incubator for 24 h, and mouse vascular smooth muscle cells were cultured for 48 h.

[0166] (2) Model evaluation: Discard the cell culture medium in the 48-well plate, rinse gently with PBS 3 times; add 4% paraformaldehyde for 20 min, discard the fixative, rinse once with distilled water; incubate with 60% isopropanol for 5 min; then discard the isopropanol, incubate with 0.5% Oil Red O solution at 37℃ for 15 min; discard the staining solution, rinse once with 60% isopropanol, then wash 3 times with distilled water; observe the formation of foam cells under a microscope.

[0167] 2.5 Macrophage experimental grouping and intervention are shown in Table 3:

[0168] Table 3

[0169]

[0170] 2.6. The grouping and intervention of vascular smooth muscle cells in the experiment are shown in Table 4:

[0171] Table 4

[0172]

[0173] 2.7. Detection of cell supernatant lipids, oxidative stress and inflammatory factors

[0174] The collected cell supernatant was centrifuged at 3000 rpm for 20 min at 4°C. The supernatant was carefully collected and aliquoted at -80°C for later use. Lipid markers TC and FC, oxidative stress markers MDA, SOD, and GSH-PX, and inflammatory markers TNF-α, IL-6, and IL-1β were all measured using kits provided by Jingmei.

[0175] 3. Results

[0176] 3.1. Effects of Erjingfang on the viability of RAW 264.7 macrophages and mouse vascular smooth muscle cells

[0177] Cell viability is a core indicator reflecting the effects of drugs on cell proliferation or toxicity. This study investigated the effects of Erjingfang on the viability of RAW264.7 macrophages and mouse vascular smooth muscle cells (MOVAS) through intervention at different time points (24h and 48h) and concentration gradients. Figure 4In RAW264.7 cells, at 24 h, concentrations above 0.25 μg / mL significantly increased macrophage proliferation, while concentrations of 16 mg / mL were toxic to macrophages, resulting in cell viability less than 50% compared to the normal group. Figure 4 A); a concentration of 100 μg / mL also showed proliferative activity (compared to the normal group, ###P<0.001), but at 48 h, none of the concentrations had a significant effect; finally, (12.5, 5, 50 μg / mL) were selected as the concentrations for the two-essence formula to intervene in macrophages. Figure 4 (BC); For MOVAS cells, a concentration of 100 μg / mL significantly reduced their viability at 24 h (compared to the normal group, ###P<0.001), while no significant effect was observed at any concentration at 48 h. The results suggest that the toxicity of the two-element formula to both cell types is characterized by "high concentration, short time (24 h) sensitivity," which disappears with prolonged intervention time (48 h), and the cell safety is good over a longer period.

[0178] 3.2. Erjingfang can improve foaming of macrophages and smooth muscle cells and lipid accumulation.

[0179] Depend on Figure 5 It can be seen that in the RAW264.7 macrophage Oil Red O staining experiment ( Figure 5 In the model group (-A), the number of intracellular lipid droplets (red stained area) was significantly higher than that in the normal control group, indicating successful model establishment. However, after intervention with Erjingfang (a traditional Chinese medicine), the number of intracellular lipid droplets in the 12.5, 25, and 50 μg / mL dose groups was significantly lower than that in the model group, showing a certain dose-dependent effect. In the Oil Red O staining experiment of mouse vascular smooth muscle cells (…),… Figure 5 In the model group (-B), the accumulation of lipid droplets was significantly higher than that in the normal control group. After treatment with Erjingfang at doses of 25, 50, and 100 μg / ml, the intracellular lipid droplet levels decreased to varying degrees compared to the model group. Combined with the results of intracellular total cholesterol (TC) and free cholesterol (FC) level detection, the TC level in the model group was significantly higher than that in the normal control group (###P<0.001), while each dose group of Erjingfang could downregulate intracellular TC and FC levels to varying degrees. The differences between the low, medium, and high dose groups and the model group were all statistically significant. Figure 5 CD). The above results indicate that Erjingfang can effectively improve the foaming process of macrophages and vascular smooth muscle cells, reduce intracellular lipid accumulation, and its effect shows a certain dose-related correlation.

[0180] 3.3. Effects of Erjing Formula on Improving Inflammatory Factors and Oxidative Stress in a Smooth Muscle Cell Foaming Model

[0181] Depend on Figure 6It was found that in the smooth muscle cell foaming model, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the model group were significantly higher than those in the normal control group (##P<0.01), indicating that the inflammatory response was significantly activated in the model group. After intervention with Erjingfang (EJP-L, EJP-M, EJP-H), the levels of each inflammatory factor decreased to varying degrees compared with the model group, and the differences were statistically significant. Figure 6 AC). In the detection of oxidative stress indicators, the malondialdehyde (MDA) level in the model group was significantly higher than that in the normal control group (#P<0.05), and the superoxide dismutase (SOD) level was significantly lower than that in the normal control group (##P<0.01), indicating that the model group suffered significant oxidative stress damage. After treatment with Erjingfang, the MDA level gradually decreased with increasing dosage, while the SOD level gradually increased. The differences between the medium- and high-dose groups and the model group were statistically significant (**P<0.01). The GSH-PX level showed some recovery ( Figure 6 (DF). The above results indicate that Erjingfang can effectively inhibit the release of inflammatory factors in the smooth muscle cell foaming model, reduce oxidative stress damage, and exert anti-inflammatory and antioxidant effects.

[0182] Cellular experiments revealed that Erjingfang exhibited "high-concentration, short-term (24h) sensitivity" to the toxicity of RAW264.7 macrophages and mouse vascular smooth muscle cells (MOVAS), with the toxicity disappearing after 48h. Therefore, 12.5, 25, and 50 μg / mL were selected as the intervention concentrations for macrophages, and 25, 50, and 100 μg / mL were selected as the intervention concentrations for MOVAS to ensure experimental safety and effectiveness. Oil Red O staining and TC and FC detection results showed that Erjingfang could dose-dependently reduce intracellular lipid droplet accumulation and decrease TC and FC levels in both types of cells. Therefore, it improves cell foaming by inhibiting ox-LDL receptor expression to reduce lipid uptake and activating cholesterol efflux-related proteins to promote cholesterol transport. Inflammation and oxidative stress index detection in the MOVAS foaming model showed that Erjingfang could downregulate the levels of inflammatory factors such as TNF-α, IL-6, and IL-1β, reduce MDA content, and increase SOD activity by inhibiting NF-κB. By blocking the inflammatory cascade and activating the antioxidant enzyme system to clear ROS, the vicious cycle of "inflammation-oxidative stress" is broken.

[0183] Through animal and cell experiments, it has been verified that Erjingfang can alleviate HHcy-related atherosclerosis by regulating lipid accumulation, inhibiting inflammatory responses, reducing oxidative stress damage, and improving macrophage and VSMCS foaming.

[0184] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. The application of a certain formula in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis.

2. The application of the Erjing formula as described in claim 1 in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis, characterized in that... The two-essence formula reduces serum homocysteine ​​levels.

3. The application of the Erjing formula as described in claim 2 in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis, characterized in that... The aforementioned Erjing formula can exert its anti-atherosclerotic effect by regulating lipids, reducing oxidative stress, and inhibiting inflammatory responses.

4. The application of the Erjing formula as described in claim 1 in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis, characterized in that... The aforementioned formula can improve foaming of macrophages and vascular smooth muscle cells.

5. The application of the Erjing formula as described in claim 1 in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis, characterized in that... The dosage of the two-ingredient formula is 1.4g / kg-5.6g / kg.

6. The application of the composition containing Erjingfang in the preparation of drugs for treating hyperhomocysteinemia-related atherosclerosis.

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