Application of fresh bamboo juice in preparation of medicine for preventing or treating atherosclerosis

Abstract

The invention discloses application of fresh bamboo juice in preparation of a medicine for preventing or treating atherosclerosis, and relates to the technical field of atherosclerosis treatment. The invention finds that the fresh bamboo juice can achieve the effects of lowering lipid and resisting atherosclerosis by regulating glycolipid metabolism, improving liver injury, inhibiting inflammation, activating brown fat, promoting browning of white fat and improving the stability of vulnerable plaques; the effect is related to the regulation effect of the medicine on a steroid hormone biosynthesis signal channel and the remodeling effect of intestinal flora. It is found for the first time that the fresh bamboo juice can effectively improve dyslipidemia and atherosclerosis, has good safety and can serve as a candidate medicine for lowering lipid and resisting atherosclerosis, and a new thought is provided for traditional Chinese medicine lipid regulation and atherosclerosis resistance.

Description

Technical Field

[0001] The invention relates to the technical field of atherosclerosis treatment, and in particular to application of fresh bamboo sap in preparing medicine for preventing or treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a lipid-driven chronic inflammatory disease. Its main clinical manifestations include many serious cardiovascular and cerebrovascular events, such as cerebral infarction, cerebral hemorrhage, stroke, arrhythmia, myocardial infarction, and coronary heart disease [1]. It seriously threatens the health and life of people all over the world and has become a serious public health problem worldwide. The etiology of AS is very complex, with many risk factors, including extracellular lipid accumulation, chronic inflammation, oxidative stress damage, and foam cell formation and death. Lipid metabolism disorders and abnormal inflammatory responses are widely considered to be the main risk factors for AS [2,3]. A large amount of evidence-based medical data has shown that lipid regulation and anti-dyslipidemia can significantly reduce the disability and mortality of myocardial infarction and coronary heart disease. Active lipid regulation has become one of the important measures to prevent atherosclerosis. To date, statins and a healthy lifestyle have become the preferred initial intervention measures for treating dyslipidemia and reducing the risk of CVD. However, despite the success of statins in effectively lowering cholesterol levels and reducing cardiovascular causes of death, two-thirds of patients receiving statin treatment still experience adverse coronary events. Therefore, new treatment strategies are very necessary. With the development of modern Chinese medicine, Chinese medicine has certain advantages in preventing and treating AS. A series of studies have been conducted on the molecular mechanisms of Chinese medicine in preventing and treating AS, mainly focusing on the classic theories of lipid infiltration, injury response, thrombosis and inflammation [4]. Chinese medicine believes that the occurrence and development of dyslipidemia and atherosclerosis are closely related to "phlegm", and lipid metabolism is the material basis of "phlegm". Professor Liu Xinyu proposed that the basic principle of regulating dyslipidemia should be to resolve phlegm and reduce turbidity [5]. Chinese medicine has achieved good clinical results in treating dyslipidemia and atherosclerosis by starting with the pathogenesis of phlegm and turbidity [6]. Based on this, we are looking for possible Chinese medicines that are effective and safe for improving dyslipidemia and atherosclerosis from the perspective of "resolving phlegm and reducing lipids".

[0003] Fresh bamboo juice (Succus Bambusae, SB) is the liquid that naturally drips from the fresh stems of Phyllostachys glaucaMcClure, Phyllostachys nuda McClure, and several other plants of the same genus after heating.[7] It is sweet and cold in nature and has the effects of clearing away heat and resolving phlegm, calming the nerves and relieving constipation. Fresh bamboo juice has a significant effect in resolving phlegm and is praised by traditional Chinese medicine as the "holy remedy for phlegm"[8]. Since ancient times, there have been many cases where fresh bamboo juice has been used to resolve "tangible phlegm" with significant effects, which has been widely recognized. However, there are few studies on its use in "intangible phlegm". Previous studies have shown that bamboo leaves (Lophatherum gracileBrongn) have a positive intervention effect on obesity and blood lipid regulation. They can alleviate obesity induced by high-fat diet in mice by reducing weight gain, total adipose tissue mass and adipocyte size, lowering leptin and adiponectin levels, and improving plasma lipid profiles [9,10]. They can also improve in vitro digestibility and exert liver protection potential

[11] .

[0004] When mice have a defective Apoe gene, lipid metabolism disorders occur, leading to lipid deposition in the liver and elevated plasma and liver lipid levels. It is worth noting that both elevated blood lipids and elevated liver lipids are lipid metabolism disorders, but there is a correlation rather than a causal relationship between the two. Relevant studies have shown that some methods of reducing liver steatosis can cause elevated blood lipids, so drugs that simultaneously improve hyperlipidemia and liver steatosis would be ideal.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide application of fresh bamboo sap in preparing medicine for preventing or treating atherosclerosis.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a use of fresh bamboo sap in preparing a medicine for preventing or treating atherosclerosis.

[0009] In a second aspect, the present invention also provides the use of fecal microbiota transplantation solution or fecal microbiota transplantation capsule after administration of fresh bamboo sap in the preparation of a drug for preventing or treating atherosclerosis.

[0010] The present invention has the following beneficial effects:

[0011] The present invention found that fresh bamboo juice can play a lipid-lowering and anti-atherosclerotic role by regulating sugar and lipid metabolism, improving liver damage, inhibiting inflammation, activating brown fat and promoting white fat browning, and improving the stability of vulnerable plaques. This effect is related to the drug's regulatory effect on the steroid hormone biosynthesis signaling pathway and the remodeling effect of intestinal flora. The present invention first discovered that fresh bamboo juice can effectively improve dyslipidemia and atherosclerosis and has good safety. It can be used as a candidate drug for lipid-lowering and anti-atherosclerosis, providing a new idea for traditional Chinese medicine lipid regulation and anti-atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 This is the experimental flow chart;

[0014] Figure 2 Effects of fresh bamboo juice on body weight, food intake and related organ indexes of mice (A) Weight change curve, final body weight and total body weight gain of WT mice; (B) Apoe - / - Mouse weight change curve, final weight and total weight gain; (C) WT mouse food intake change curve, average daily food intake and food efficiency ratio; (D) Apoe - / - Mouse food intake change curve, daily average food intake and food efficiency ratio; (E) Apoe - / - Representative images of mice and related organ indexes; (F) Organ indexes of WT mice; Data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0015] Figure 3 The results of the steady-state regulation of fresh bamboo juice on glucose and lipid metabolism disorders; (A) Plasma TG, TC, LDL-C, HDL-C, AST and ALT levels in WT mice; (B) Apoe - / - Plasma TG, TC, LDL-C and HDL-C levels in mice; (C) Apoe - / - Mouse liver TG, TC, LDL-C and HDL-C levels; (D) OGTT curve and area under the curve of WT mice; (E) Apoe - / - Mouse OGTT curve and area under the curve; (F) Apoe - / - Oral glucose tolerance curve and area under the curve of mice in the FMT group; data are expressed as mean ± standard deviation,* P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0016] Figure 4 Fresh bamboo juice improves hepatic steatosis and protects the liver. (A) Representative images of H&E staining of WT mouse liver (B) Apoe - / - Representative images of H&E staining of mouse liver; (C) Plasma AST and ALT levels of WT mice; (D) AST and ALT levels of WT mouse liver; (E) Apoe - / - (F) CAT and SOD levels in the liver of WT mice. Data are expressed as mean ± standard deviation. * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0017] Figure 5 Fresh bamboo sap reduces the risk of Apoe - / - Results of the mouse atherosclerosis and stabilization of vulnerable plaque experiments; (A) Morphological images of aortic arch atherosclerotic lesions under a stereomicroscope; (B) Representative images of H&E staining of the aortic root; (C) Representative images of Oil Red O staining of the aortic root; (D) Representative images of Masson staining of the aortic root; (E) Representative images of immunohistochemistry of Mac-3 of the aortic root; (F) Representative images of immunohistochemistry of α-SMA of the aortic root; (G) Lipid content in the aortic root (Oil Red O positive rate area); (H) Collagen content in the aortic root (collagen fiber positive rate area); (I) α-SMA content (α-SMA positive rate area); (J) Mac-3 content (macrophage positive rate area; (K) Plaque vulnerability index (vulnerability index = (Mac-3 + lipid) area positive percentage / (α-SMA + collagen fiber) area positive percentage). The images were obtained under an optical microscope at 40× magnification, scale bar = 50 μm; data are expressed as mean ± standard deviation. * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0018] Figure 6Statistical graph showing that fresh bamboo juice inhibits the expression of pro-inflammatory cytokines and promotes the expression of anti-inflammatory factors in HFD-fed mice; (A) Protein levels of IL-6, IL-1β, TNF-a, MCP-1, IL-10, and TGF-β in the liver of WT mice; (B) Protein levels of IL-6, IL-1β, TNF-a, MCP-1, IL-10, and TGF-β in the plasma of WT mice; (C) Protein levels of Apoe - / - Protein levels of IL-6, IL-1β, TNF-a, MCP-1, IL-10 and TGF-β in mouse liver; (D) Apoe - / - Protein levels of IL-6, IL-1β, TNF-a, MCP-1, IL-10, and TGF-β in mouse liver; data are expressed as mean ± SD. * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0019] Figure 7 Fresh bamboo juice can reduce the size of lipid droplets in white adipose tissue and the amount of white fat in brown adipose tissue; (A) Apoe - / - Representative images of H&E staining of epididymal white fat and interscapular brown fat tissue in mice (200×); (B) Representative images of H&E staining of epididymal white fat and interscapular brown fat tissue in WT mice (200×); (C) Apoe - / - The positive area of ​​lipid droplets in the interscapular brown adipose tissue of mice (D) The positive area of ​​lipid droplets in the interscapular brown adipose tissue of WT mice. The images were obtained under an optical microscope at a magnification of 200×, scale bar = 50 μm, and the data are expressed as mean ± standard deviation. * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0020] Figure 8 Fresh bamboo juice activates thermogenesis in brown adipose tissue of mice. (A) Apoe - / - Representative images of Ucp-1 immunohistochemistry in interscapular BAT of mice; (B) Representative images of Ucp-1 immunohistochemistry in interscapular BAT of WT mice; (C) Ucp-1 content in interscapular BAT of WT mice; (D) Apoe - / - Ucp-1 content in interscapular BAT of mice; (E) Apoe - / -mRNA levels of the thermogenic genes Ucp1, Pgc1α, and Prdm16, fatty acid oxidation-related genes Ppar-γ and Cpt1, and mitochondrial biological genes Nrf1 and Nrf2 in interscapular brown adipose tissue of mice. Images were obtained under an optical microscope at 200× magnification, scale bar = 50 μm, and data are expressed as mean ± standard deviation. * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001;

[0021] Fig. 9 Alpha diversity and species composition analysis. (A) Rarefaction curve; (B) Alpha diversity analysis; (C) OUT_Venn diagram; (D) Bar graph of community composition at the phylum and genus level;

[0022] Fig.10 Beta diversity and species difference analysis. (A) PCoA and its box plot; (B) Lefse multi-level species difference discriminant analysis from phylum to genus level; (C) Relative abundance of intestinal microbiota at phylum and genus level. DETAILED DESCRIPTION

[0023] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0024] In a first aspect, the present invention provides a use of fresh bamboo sap in preparing a medicine for preventing or treating atherosclerosis.

[0025] The inventors found that fresh bamboo juice may play a lipid-lowering and anti-atherosclerotic role by regulating glycolipid homeostasis, activating brown fat to promote browning, regulating intestinal microorganisms, inhibiting inflammation and improving the stability of vulnerable plaques, and has application prospects in preventing and treating atherosclerosis.

[0026] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses:

[0027] (1) Reduce the subject's body weight;

[0028] (2) reduce the subjects' daily food intake;

[0029] (3) Improve the glucose and lipid metabolism disorders of the subjects;

[0030] (4) improving hepatic steatosis in subjects;

[0031] (5) Prevent liver damage;

[0032] (6) Reduce atherosclerotic lesions;

[0033] (7) Stabilize vulnerable plaques;

[0034] (8) Inhibit inflammation;

[0035] (9) Inducing the activation of brown fat and promoting browning;

[0036] (10) Regulate intestinal microorganisms.

[0037] The subject in the present invention includes but is not limited to mammals, such as humans, monkeys, mice, dogs, rabbits, sheep or cows.

[0038] The aforementioned reduction in the subject's weight includes but is not limited to, during the test period, compared with before drug administration, the subject's weight after drug administration significantly decreases, shows a decreasing trend, or slightly decreases.

[0039] The aforementioned reduction in the daily food intake of the subject includes but is not limited to: during the test period, compared with before drug administration, the total daily food intake of the subject decreases or shows a decreasing trend after drug administration, or the amount of food consumed each time decreases, etc.

[0040] In a preferred embodiment of the present invention, the above-mentioned improvement of the subject's glucose and lipid metabolism disorder includes but is not limited to at least one of the following methods: reducing the level of any one of the following substances in the subject's plasma and / or liver: TG, TC (total cholesterol) and LDL-C (low-density lipoprotein cholesterol) levels, improving the subject's glucose tolerance and increasing the subject's HDL-C (high-density lipoprotein cholesterol) level in the subject's plasma and / or liver.

[0041] Glucose tolerance refers to the body's ability to regulate blood sugar concentration, and generally refers to testing the body's ability to regulate blood sugar concentration through an oral glucose tolerance test. The higher the glucose tolerance, the higher the body's ability to regulate blood sugar concentration. The present invention's research shows that fresh bamboo juice can improve the disorder of sugar metabolism in mice and has a good effect of lowering blood sugar and lipids.

[0042] In a preferred embodiment of the present invention, the above-mentioned improvement of hepatic steatosis in the subject includes but is not limited to at least one of the following methods: reducing the ALT (alanine aminotransferase) level and / or AST (aspartate aminotransferase) level in the subject's liver and / or plasma and improving the subject's liver antioxidant capacity.

[0043] In a preferred embodiment of the present invention, improving the antioxidant capacity of the subject's liver includes but is not limited to: improving the subject's SOD (superoxide dismutase) and / or CAT (catalase) levels.

[0044] In a preferred embodiment of the present invention, the above-mentioned prevention of liver damage includes at least one of the following ways: reducing the size of the subject's liver fat droplets and reducing the content of liver lipids in the subject. After being treated with fresh bamboo juice, the liver fat droplets become smaller and the content is significantly reduced, which can significantly reduce lipid accumulation and has a similar effect to simvastatin. This suggests that fresh bamboo juice can improve the adverse effects of high fat on the liver, has a lipid-lowering and liver-protecting effect, and this effect is related to changes in intestinal flora.

[0045] In a preferred embodiment of the present invention, the above-mentioned reduction of atherosclerotic lesions includes but is not limited to at least one of the following methods:

[0046] (1) Reduce the area of ​​arterial plaque in subjects;

[0047] (2) reduce the degree of arterial stenosis in subjects;

[0048] (3) reduce the thickness of the aortic fibrous cap in subjects;

[0049] (4) reducing lipid deposition in the aortic root region of the subjects;

[0050] (5) reducing aortic foam cell formation in subjects;

[0051] (6) Increase the content of smooth muscle cells in plaques;

[0052] (7) Reduce the infiltration of macrophages in plaques.

[0053] Reducing aortic foam cell formation in a subject includes, but is not limited to: a decreasing trend in foam cell formation.

[0054] Studies have shown that a high-fat diet (HFD) can induce the migration of inflammatory macrophages, accelerate vascular inflammation and the formation of atherosclerotic plaques, participate in the development of AS and increase the instability of plaques. Treatment with fresh bamboo juice and fresh bamboo juice-fecal microbiota transplantation alleviated the infiltration of Mac-3 positive macrophages in AS plaques.

[0055] In a preferred embodiment of the present invention, the above-mentioned stabilization of vulnerable plaques includes at least one of the following methods: reducing the collagen content of aortic root plaques and lowering the plaque vulnerability index.

[0056] The research of the present invention shows that the pharmacological effect of fresh bamboo juice is better than that of simvastatin and can play a role in stabilizing plaques.

[0057] In a preferred embodiment of the present invention, the above-mentioned inhibition of inflammation includes at least one of the following methods: reducing the level of pro-inflammatory factors in the subject's liver tissue and / or plasma, and increasing the level of anti-inflammatory factors in the subject's liver tissue and / or plasma. In addition, fecal microbiota transplantation experiments also show that fresh bamboo juice has a significant effect of inhibiting pro-inflammatory factors and increasing pro-inflammatory factors.

[0058] In a preferred embodiment of the present invention, the liver tissue proinflammatory factors include but are not limited to at least one of IL-1β, IL-6, TNF-α and MCP-1.

[0059] In a preferred embodiment of the present invention, the anti-inflammatory factors in liver tissue and / or plasma include but are not limited to at least one of IL-10 and TGF-β.

[0060] Fresh bamboo juice can increase mitochondrial function and fatty acid oxidation, effectively activate intBAT, enhance energy consumption, and promote the browning of adipose tissue. In a preferred embodiment of the present invention, the above-mentioned induction of brown fat activation to promote browning includes at least one of the following methods:

[0061] (1) Reduce the epididymal white fat (epiWAT) index of the subjects;

[0062] (2) reduce the interscapular brown adipose tissue (intBAT) organ index of the subjects;

[0063] (3) reduce the subjects' perirenal white adipose tissue (prWAT) index;

[0064] (4) reducing the size of lipid droplets in the subjects’ white adipose tissue;

[0065] (5) Increase mitochondrial function;

[0066] (6) Increase fatty acid oxidation;

[0067] (7) Enhanced energy expenditure;

[0068] In a preferred embodiment of the present invention, increasing mitochondrial function includes the following method: reversing the decrease in Nrf1 and / or Nrf2 gene expression in the subject induced by a high-fat diet.

[0069] In a preferred embodiment of the present invention, increasing fatty acid oxidation includes the following methods: increasing the expression of Cpt1 gene and reversing the decrease of Ppar-γ gene expression in the subject induced by a high-fat diet.

[0070] In a preferred embodiment of the present invention, enhancing energy expenditure comprises at least one of the following methods: reversing the decrease in Ucp-1, Pgc-1α and Prdm-16 gene expression in the subject induced by a high-fat diet.

[0071] The present invention shows that after administration of SB, the decrease of ACE and Shannon index caused by HFD can be significantly reversed, and the diversity of intestinal microorganisms almost returns to normal levels, while simvastatin and FMT transplantation have no significant effect on the diversity of intestinal microorganisms. The above results show that the administration of fresh bamboo juice can increase the richness and diversity of intestinal flora in mice. In summary, the above-mentioned regulation of intestinal microorganisms includes at least one of the following ways: increasing the richness and diversity of intestinal flora.

[0072] In a preferred embodiment of the present invention, the richness and diversity of intestinal flora is improved in at least one of the following ways:

[0073] (1) Reduce the abundance ratio of Firmicutes to Bacteroidetes (F / B);

[0074] (2) regulating the intestinal microbial community structure to move toward the intestinal microbial community structure of normal subjects;

[0075] (3) Regulate the levels of Bifidobacterium and Ileibacterium;

[0076] (4) Improve the level of Rikenellaceae_RC9_gut_group and unclassified_fOscillospiraceae.

[0077] Studies have shown that high levels of Firmicutes are associated with inflammation, and can promote the uptake of fatty acids and accelerate the deposition of lipid droplets, while Bacteroidetes can esterify cholesterol into coprostanol (a type of non-absorbable cholesterol), thereby lowering blood cholesterol levels. This suggests that fresh bamboo juice may play a lipid-lowering and anti-AS role by reducing F / B.

[0078] The medicine also includes a combination of medicines, and the combination of medicines is selected from at least one of dried tangerine peel, pinellia tuber and tangerine peel. The medicine includes the combination of fresh bamboo sap and dried tangerine peel, or the medicine includes the combination of fresh bamboo sap and tangerine peel.

[0079] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0080] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a lubricant, a flavoring agent, a binder, a suspending agent and a flavor.

[0081] Pharmaceutically acceptable excipients include but are not limited to: pharmaceutically acceptable carriers, auxiliary substances or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and / or auxiliary materials because they are commonly used for pharmaceutical purposes, especially for solid pharmaceutical preparations. Examples include: excipients, such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders, such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropyl pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants, such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, ethylene glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants, such as magnesium stearate, talc, sodium lauryl sulfate; flavors, such as citric acid, menthol, glycine , orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers, such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0082] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0083] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as solution, suspension and gel usually contain liquid carrier, such as water and / or pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include pH adjusting agents, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (such as methylcellulose), dyes and / or flavoring agents, such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be regulated by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be regulated by pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0084] In a preferred embodiment of the present invention, the drug is formulated for oral administration or injection administration. Injection administration includes but is not limited to subcutaneous injection, intramuscular injection, intravenous injection, intradermal injection, and enteral injection.

[0085] In a preferred embodiment of the present invention, the fresh bamboo sap is obtained by dry distillation of bamboo.

[0086] In a second aspect, the present invention also provides the use of fecal microbiota transplantation solution or fecal microbiota transplantation capsules after administration of fresh bamboo sap in the preparation of a drug for preventing or treating atherosclerosis.

[0087] The fecal microbiota transplantation liquid is prepared by the following method: feces of the subject after administration of fresh bamboo sap is taken to prepare a suspension. The fecal microbiota transplantation capsule can be prepared by a conventional fecal microbiota transplantation capsule method reported in the prior art.

[0088] The drug can be directly delivered to the subject's intestines through a medical device by enema.

[0089] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0090] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0091] The following is a study on the lipid-regulating and anti-atherosclerotic effects of fresh bamboo juice from the perspective of resolving phlegm and reducing turbidity. - / - The hyperlipidemia and atherosclerosis models were constructed by using C57BL / 6J wild-type (WT) mice, and the drug efficacy experiments were conducted for 7 and 14 weeks. Simvastatin (SV) was used as a positive control drug to compare and analyze the effects of fresh bamboo juice on Apoe - / - The anti-atherosclerotic effect of fresh bamboo juice on rats and their wild-type WT mice was studied, with a focus on glycolipid disorders, glucose tolerance, inflammation levels, liver damage, aortic lesions and plaque stability. The levels of genes related to brown fat activation were detected by real-time fluorescence quantitative PCR to evaluate the contribution of brown fat to the anti-atherosclerotic effect of fresh bamboo juice. In addition, based on the important role of intestinal flora in obesity, dyslipidemia and atherosclerosis [12,13], the present invention added a fecal transplantation experiment to the 14-week experiment to further explore the causal role of intestinal bacteria in the development of atherosclerosis, and explored the effect of fresh bamboo juice on the structural changes of intestinal microbiota through microbial 16SrRNA diversity analysis. At the same time, the network pharmacology and transcriptomics analysis methods were combined to clarify the lipid-lowering and anti-atherosclerotic mechanism of fresh bamboo juice.

[0092] 1. The medicinal materials and reagents involved in the examples are as follows:

[0093] The main materials and reagents required are listed in Table 1.

[0094] Table 1 Materials and reagents

[0095]

[0096]

[0097] 2. The instruments and equipment involved in the embodiment are shown in Table 2:

[0098] Table 2 Instruments and equipment

[0099]

[0100] 3. Experimental Animals

[0101] 4-week-old SPF C57BL / 6J wild-type (WT) mice and Apoe knockout (Apoe - / - ) mice, weighing 16-18 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd., license number SCXK (Beijing) 2019-0010. All mice were raised under SPF conditions, with room temperature controlled at (22±2)℃, humidity (50±10)%, and a light-dark cycle of 12h. During this period, the animals were allowed to freely access food and water, and were fed adaptively.

[0102] After 1 week of feeding, the animals were randomly assigned to different groups for experiment. Except for the normal control group, which was given a normal diet, the rest were fed a high-fat diet. - / - Mice received an antibiotic cocktail as antibiotic-sterilized mice for fecal microbiota transplantation experiments. All animal experiments were approved by the Ethics Committee of Jiangxi University of Traditional Chinese Medicine (No. JXZLLSC20220004).

[0103] 4. Statistical analysis methods

[0104] GraphPad Prism 9.3.1 and SPSS 21.0 software were used for statistical analysis of the data, and the data were expressed as mean ± standard deviation (±s). One-way analysis of variance (ANOVA) and post hoc test (LSD) were used to analyze the significant differences between the groups. When P < 0.05, the difference was statistically significant.

[0105] Example 1

[0106] Systematic toxicological evaluation of fresh bamboo sap slices prepared by different processing methods.

[0107] Acute toxicity test: 80 KM mice were randomly divided into a normal control group, a fire roasting process group (5860 mL·kg -1 ·day), reflux extraction process group (12610mL·kg-1 ·day) and dry distillation process group (2113mL·kg -1 ·day).

[0108] Fire roasting method: Take fresh bamboo and saw it into bamboo strips about 40 cm in length. Remove the bamboo nodes at both ends, heat the middle part with an alcohol lamp, collect the light yellow juice dripping from both ends, and stop heating when no liquid flows out. Store at 4°C.

[0109] Reflux extraction method: split fresh bamboo into pieces of 2cm in length and 1cm in width, take a certain amount of bamboo pieces and place them in a round-bottom flask, add 7 times the amount of pure water, make the water cover the bamboo pieces, soak for 1 hour, reflux extraction for 1 hour, filter; add 5 times the amount of pure water for the second time, extract for 1 hour in the same operation, filter. Mix the two extracts and store them at 4℃.

[0110] Dry distillation method: Take fresh bamboo and saw it into bamboo pieces with a length of about 40 cm, remove the bamboo nodes at both ends, and place it in a dry distillation device with a fixed temperature of 370℃ and a pressure of about 0.4MPa. After 0.5h, the distillate is taken as a sample and stored at 4℃.

[0111] The maximum dosage test method was used, and the mice were gavaged three times within 24 hours. The weight changes, diet, appearance, behavior, secretions, excretions, and death of the mice were observed for 14 consecutive days after administration. The subacute toxicity test focused on the dry distillation of fresh bamboo sap slices prepared by the innovative process of the research team to carry out further toxicological evaluation. 80 SD rats were randomly divided into 4 groups, namely normal control group (NC), dry distillation low-dose group (188 mL·kg -1 ), medium dose group (625 mL·kg -1 ) and the high-dose group (938 mL·kg -1 The mice were given the drug by oral gavage once a day for 30 consecutive days. The daily body weight changes, diet and water intake, and mental state were recorded. After the experiment, blood routine and blood biochemistry tests were performed, and pathological histological analysis was performed on the heart, liver, spleen, lung, kidney, testis, and gastrointestinal organ tissues. The pathological changes of organ tissues and changes in indicators such as blood routine, blood biochemistry, and organ index were observed.

[0112] The results of the acute toxicity test showed that the dry distillation of fresh bamboo sap slices had adverse effects on the body weight and gastrointestinal tract of mice. The results of the subacute toxicity test were consistent with the results of the acute toxicity test. The high-dose group of dry distillation of fresh bamboo sap had adverse effects on gastrointestinal tissues, and the organ indexes of the heart, liver, lung and kidney were also significantly increased. However, the dose used in the subacute toxicity test covered 100 times the clinical equivalent dose of adults, and no obvious adverse effects were observed, indicating that the overall safety of fresh bamboo sap slices produced by different processes was good.

[0113] Example 2

[0114] This example tests the effect of fresh bamboo juice on the body weight, food intake and organ index of mice. Figure 1 shown.

[0115] 1. Sample Preparation

[0116] Take fresh bamboo and saw it into bamboo pieces with a length of about 40cm, remove the bamboo joints at both ends, and place it in a dry distillation device with a fixed temperature of 370℃ and a pressure of about 0.4MPa. After 0.5h, the distillate is taken as a sample and stored at 4℃. The obtained stock solution is concentrated 15 times by a rotary evaporator to a drug concentration of 15mL / mL, and stored at -80℃ for later use.

[0117] 2. Animal Experiments

[0118] (1) A 14-week intervention study of fresh bamboo juice on wild-type WT mice

[0119] After 1 week of adaptive feeding, WT mice were randomly divided into three groups (n=8): normal control group (WT+CD), model group (WT+HFD), fresh bamboo sap group (WT+SB, 45 mL·kg -1 ·d -1 , calculated based on the daily oral dose of 60 mL / 60 kg for adults in the market, which is equivalent to 5 times the clinical dose). WT mice in the normal control group were given a normal diet, and WT mice in the model group and fresh bamboo sap group were given a high-fat diet to induce dyslipidemia. Among them, the fresh bamboo sap group was given fresh bamboo sap by gavage every day, and the normal control group and model group were given an equal amount of purified water according to body weight, and the administration was continued for 14 weeks. After the experiment, the mice were fasted for 12 hours but not water, and then anesthetized with isoflurane. Blood samples were collected and centrifuged to obtain plasma and stored at -80°C; kidneys, livers, spleens, perirenal white adipose tissue (prWAT), epididymal white adipose tissue (epiWAT) and interscapular brown adipose tissue (intBAT) were collected, weighed and preserved in time, one part of which was fixed in 10% formalin, and the other part was immediately immersed in liquid nitrogen and stored at -80°C for further analysis.

[0120] (2) Effect of fresh bamboo juice on Apoe - / - 14-week intervention study in mice

[0121] Apoe - / - The mice were randomly divided into six groups (n=8): normal control group (CD, the rest were high-fat diet groups), model group (HFD), simvastatin group (SV, 5 mg·kg -1 ·d-1 ), fresh bamboo juice group (SB, 90 mg kg -1 ·d -1 , calculated based on the daily oral dose of 60mL / 60kg for adults in the market, which is equivalent to 10 times the clinical dose) and fecal microbiota transplantation group (FMT group, divided into fecal microbiota transplantation model group (HDF-FMT) and fecal microbiota transplantation fresh bamboo sap group (SB-FMT) after continuous HFD for 5 weeks). A high-fat diet was used to induce dyslipidemia and atherosclerosis in mice, and the intervention effect of fresh bamboo sap for 14 consecutive weeks was observed. Simvastatin was selected as a positive drug as a reference, and at the 8th week, a fecal microbiota transplantation experiment was added to observe the intervention effect of fresh bamboo sap fecal microbiota transplantation. During the experiment, the weight changes and dietary conditions of each group of mice were carefully observed and recorded. The diet and weight of the mice were recorded every week, and the weight and food intake curves of the mice were drawn. The weight gain, average diet and food efficiency ratio (FER) of each group of mice were calculated. After the experiment, blood was collected from the eyeballs after isoflurane anesthesia, and the heart, liver, spleen, perirenal fat, epididymal white fat (epiWAT) and brown adipose tissue (intBAT) were collected after cardiac perfusion with 20 ml of PBS. The aorta was isolated and obtained under a stereomicroscope, and the tissue was weighed and divided into two parts for storage. One part was fixed in 10% formalin, and the other part was immediately immersed in liquid nitrogen and stored at -80°C for subsequent detection and analysis.

[0122] (3) Fecal microbiota transplantation experiment

[0123] In the 14-week treatment experiment, feces were collected daily from the 8th to the 14th week in the HFD group and the SB group. The collected feces were used to prepare fecal microbial supernatant for fecal microbiota transplantation experiments. After the mice in the fecal microbiota transplantation group were given a high-fat diet for 5 weeks, they drank an antibiotic cocktail for 2 weeks to eliminate the original intestinal flora and construct pseudo-germ-free mice. Pre-treatment of the intestinal flora of the recipient mice with antibiotics before fecal microbiota transplantation can eliminate pathogenic bacteria such as Fusobacterium, Sutera, Escherichia coli and Streptococcus, thereby promoting the implantation of beneficial microorganisms and promoting clinical remission. The antibiotic cocktail experiment was based on a previous study with slight modifications (Yu L, Wang L, Yi H, Wu X. Beneficial effects of LRP6-CRISPR on prevention of alcohol-related liver injury surpassed fecal microbiota transplant in a rat model. Gut Microbes. 2020; 11(4): 1015-1029. doi: 10.1080 / 19490976.2020.1736457). In the previous study, the FMT experiment included the following steps: rats with alcohol-related liver injury received antibiotics (ampicillin, 1 mg / ml; neomycin, 1 mg / ml; metronidazole, 1 mg / ml; and vancomycin, 0.5 mg / ml) once a day for one week to deplete the intestinal microbiota. After antibiotic treatment, each rat in the FMT group was orally gavaged with 200 μl of healthy rat fecal filtrate once a day for 5 consecutive days (other groups were given sterile saline).

[0124] The FMT experimental steps of this example are as follows: depletion of Apoe by antibiotic cocktail method - / - The original intestinal microflora of mice was affected by the addition of broad-spectrum antibiotics (ampicillin 1 g L -1 , Sigma; neomycin sulfate 1 g·L -1 , Sigma; Metronidazole 1 g·L -1 , Sanofi-Aventis; vancomycin 0.5 g·L-1, Sandoz) for 2 consecutive weeks. After removing the native intestinal flora, antibiotics were stopped for 2 days and fecal microbiota transplantation was performed for 7 consecutive weeks. - / -Mice served as fecal donors, and feces were collected every day. The feces of donor mice were mixed together, and each 100 mg was suspended in 1 mL of sterile saline. The solution was vortexed and homogenized for 1 min, then centrifuged at 800 × g for 3 min, and the supernatant was immediately used for further transplantation. While receiving high-fat diet, 200 μL of fresh fecal supernatant was gavaged to the corresponding group every day for 7 consecutive weeks. Donor feces were freshly prepared within 2 hours before gavage on the day of transplantation to prevent changes in bacterial composition. In addition, the additional supplementation of antibiotic cocktail was terminated during the fecal microflora transplantation.

[0125] The body weight results showed that the body weight of WT-HFD mice and HFD mice gradually increased with the extension of feeding time; however, there was no significant difference in body weight between WT-HFD mice and WT-CD mice, and between HFD mice and CD mice ( Figure 2 It is worth noting that Apoe - / - Compared with the CD and HFD groups, the total body weight of the mice in the SV group increased significantly (13.09% and 8.14% respectively compared with the CD and HFD groups). - / - The total weight gain of mice decreased (compared with the model group, it decreased by 25.49% and 4.76%, respectively). To explore whether the weight change of mice is related to dietary factors, the food intake of each group of mice was further compared and analyzed. It can be seen that the average daily food intake of mice in the WT-HFD and HFD groups, WT-SB and SB groups decreased significantly (compared with the WT-CD and CD groups, it decreased by 14.08% and 17.87%, 23.73% and 18.93%, respectively), while the average daily food intake of mice in the SV group increased significantly by 19.82% compared with the HFD group ( Figure 2 Figures C and D in the figure suggest that weight changes may be related to the amount of food intake of mice. - / - The food efficiency ratio (FER) of the mouse model group was significantly higher than that of the CD group (24.20%), and the FER of the WT mouse model group was also increased compared with the WT-CD group (26.07%), which was related to the high-fat diet of the mice.

[0126] In addition, after 14 weeks of high-fat diet feeding, Apoe - / - There was no obvious difference in the gross appearance of each group of mice, but there was a certain difference in the size of epididymal adipose tissue ( Figure 2Figure E in the figure). The epididymal white fat (epiWAT) index of the HFD group was higher than that of the CD group. After intervention with fresh bamboo juice and simvastatin, the epiWAT index of mice decreased (compared with HFD, the SB and SV groups decreased by 27.21% and 21.77%, respectively); at the same time, SB-FMT also decreased by 8.43% compared with HFD-FMT, and the interscapular brown adipose tissue (intBAT) organ index of mice in the SB-FMT group was significantly reduced by 34.59% compared with that in the CD group ( Figure 2 Similarly, in WT mice, the perinephric white adipose tissue (prWAT) and epiWAT indexes of the WT-HFD group were significantly increased by 1.64 and 0.68 times compared with those of the WT-CD group, and this phenomenon was reversed by administration of fresh bamboo juice ( Figure 2 Figure F in the figure).

[0127] Example 3

[0128] This example explores the steady-state regulation of fresh bamboo juice on glucose and lipid metabolism disorders.

[0129] 1. Oral Glucose Tolerance Test

[0130] Oral glucose tolerance test (OGTT) was performed 3 days before the end point experiment. - / - After the mice were fasted for 16 h but not water,

[14] 0.01 mL·g -1 The dosage was 2 g·kg. The 20% glucose solution (5 g of anhydrous glucose was added to 25 mL of sterile water and filtered through a 0.22 microporous membrane for sterilization) was administered by gavage. -1

[15] Fasting blood glucose levels in tail vein blood were measured before drug administration (0 min) and 15 min, 30 min, 60 min, 90 min, and 120 min after sugar administration

[16] (Roche Active blood glucose meter) was used to draw the blood glucose change curve using GraphPad Prism 9.0.0 software, and the area under the curve (Area Under Curve, AUC) was calculated.

[0131] 2. Determination of blood lipid related indicators

[0132] The TC, TG, LDL-C and HDL-C in mouse plasma were determined using a fully automatic biochemical analyzer. The TC, TG, LDL-C and HDL-C in liver tissue were detected using a kit strictly in accordance with the instructions.

[0133] After high-fat diet feeding, plasma TC levels and Apoe - / - Plasma TG, TC and LDL-C levels in mice were significantly increased, and Apoe- / - The mice showed significantly higher blood lipid levels than WT mice. Fresh bamboo juice and simvastatin had no significant effect on the blood lipid levels of WT mice, but improved the expression of Apoe - / - The dyslipidemia of mice was significantly reduced after SV administration compared with the HFD group. - / - The plasma TG level of mice was significantly lowered and the plasma HDL-C level was increased (P<0.0001, P<0.0001). SB (fresh bamboo sap) had a lipid-lowering effect comparable to SV (simvastatin) ( Figure 3 A, B). For Apoe - / - Changes in liver lipid levels in mice. The experimental results of this example show that after HFD feeding, the TC level in the liver of mice increased significantly

[0134] (P<0.001, Figure 3 In the C), the levels of liver TG and LDL-C increased by 12.29% and 65.22% respectively compared with the CD group, and the level of liver HDL-C decreased by 63.98%. Both SB and SV treatments significantly reduced the level of liver TG and increased the level of liver HDL-C in mice. At the same time, SV was able to significantly reduce the level of liver TC in mice (P<0.05).

[0135] The fecal microbiota experiment results showed that the fecal microbiota of the transplanted feeding model group made Apoe - / - The plasma LDL-C level of mice was significantly increased, while after transplantation of fecal microbiota in the fresh bamboo sap group, the plasma TG level of mice was significantly decreased compared with that in the HFD-FMT group (P<0.05), and the plasma TC and LDL-C levels were decreased by 6.79% and 10.39%, respectively. At the same time, the liver TC, TG and LDL-C levels were decreased by 0.71%, 11.33% and 1.19%, respectively, and the liver HDL-C level was increased by 3.7 times.

[0136] In addition, blood sugar disorders may affect the conversion of glucose and lipid metabolism, so this example also conducted an oral glucose tolerance test (OGTT). After sugar administration, mice fed with HFD showed a decrease in glucose tolerance compared with mice fed with normal feed ( Figure 3 (D, E) AUC analysis showed that compared with the other groups, WT and Apoe - / - The mice in the model group had the highest AUC. Both SB and SV treatments could effectively reduce AUC and improve glucose tolerance. There was no significant difference in the changes in blood glucose curves and AUC results between HFD-FMT and SB-FMT in the fecal microbiota transplantation experiment, indicating that fecal microbiota transplantation had little effect on glucose tolerance ( Figure 3 (F) This experiment supplemented the lipid-lowering mechanism of fresh bamboo sap from the perspective of glucose metabolism, and confirmed that fresh bamboo sap can improve glucose metabolism disorders in mice and has a good effect of lowering blood sugar and lipids.

[0137] Example 4

[0138] This example explores the improvement of hepatic fatty degeneration and the liver-protecting effect of fresh bamboo juice.

[0139] 1. Use an automatic biochemical analyzer to determine the levels of AST and ALT, liver injury markers in mouse plasma; strictly follow the instructions and use kits to detect the levels of ALT, AST, and antioxidant markers superoxide dismutase (SOD) and catalase (CAT) in liver tissue.

[0140] 2. Pathological histological analysis and immunohistochemistry.

[0141] After the experiment, the liver tissue was fixed, embedded, sliced ​​and stained with H&E, and observed and photographed under a microscope to evaluate the fat deposition in the mouse liver tissue. The specific operation is as follows:

[0142] A. Liver tissue embedding and sectioning

[0143] (1) Fixation and sampling: After fresh tissue is collected, place it in the fixative immediately and fix it at room temperature for more than 24 hours, then transport and store it at room temperature. Take the tissue out of the fixative in a fume hood, trim the target area of ​​the tissue with a scalpel, and place the trimmed tissue and the corresponding label paper into the embedding frame.

[0144] (2) Dehydration and wax dipping: Place the embedding frame neatly into the dehydration box of the dehydrator, set the dehydration program, and dehydrate the dehydration box in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol ① for 30 minutes, anhydrous ethanol ② for 30 minutes, alcohol benzene for 5-10 minutes, xylene ① for 5-10 minutes, xylene ② for 5-10 minutes, 65℃ melted paraffin ① for 1 hour, 65℃ melted paraffin ② for 1 hour, 65℃ melted paraffin ③ for 1 hour.

[0145] (3) Paraffin embedding: Place the wax-soaked tissue in an embedding machine for embedding. Inject melted wax into the embedding frame mold, quickly place the cut surface of the tissue facing down into the embedding frame mold, and attach a label. After cooling to room temperature, trim the wax block.

[0146] (4) Paraffin section: Place the wax block in a -20℃ freezer for several minutes, and place it in a paraffin slicer for sectioning, with a thickness of 2-5 microns. Flatten the slices in 40℃ warm water on the slicer, and then use a glass slide to pick up the slices, place them in a 60℃ oven, and take them out for staining after the wax is melted.

[0147] BH&E staining

[0148] (1) Dewaxing: Place paraffin sections in xylene① for 20 minutes, xylene② for 20 minutes, anhydrous ethanol① for 5 minutes, anhydrous ethanol② for 5 minutes, and anhydrous ethanol③ for 5 minutes, and rinse with tap water. (Frozen sections should be rinsed directly with tap water).

[0149] (2) Hematoxylin staining: Stain the sections with hematoxylin for 3-5 minutes, wash off the excess stain with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, then blue with hematoxylin bluing solution for a few seconds, and rinse with running water for a few seconds.

[0150] (3) Eosin staining: Dehydrate the sections with graded alcohol (85% ethanol → 95% ethanol) and stain them in eosin solution for 5 minutes.

[0151] (5) Dehydration and sealing: The sections were placed in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, anhydrous ethanol ③ for 5 minutes, n-butanol for 5 minutes, xylene ① for 5 minutes, and xylene ② for 5 minutes in sequence until transparent. The sections were taken out of xylene and slightly dried, and then sealed with neutral gum.

[0152] (6) Interpretation of results: The cell nucleus is blue and the cytoplasm is red.

[0153] After HFD feeding and HFD-FMT fecal microbiota transplantation, significant liver damage was detected in mice. The liver AST and ALT levels of WT-HFD mice were significantly increased (P<0.05, P<0.001), and the plasma AST level increased by 39.16% ( Figure 4 Medium CD); Apoe - / - The plasma AST and ALT levels of mice in the HFD group increased by 81.23% and 29.93% respectively compared with those in the CD group. Meanwhile, the plasma AST levels of mice in the HFD-FMT group were significantly higher than those in the CD group (P<0.0001, Figure 4 Middle E). SB administration significantly improved the expression of WT and Apoe - / - The liver ALT and plasma ALT levels of mice were increased (P<0.05, P<0.05). Similarly, SB-FMT treatment significantly reduced the expression of Apoe - / - In addition, the liver antioxidant indexes were measured, and it was found that the administration of fresh bamboo juice increased the SOD and CAT levels of WT mice to a certain extent (compared with the WT-CD group, they increased by 30.69% and 4.82%, respectively, Figure 4 Middle F).

[0154] Further analysis of mouse liver tissue pathology showed that WT and Apoe - / - A large number of spherical vacuoles appeared in the liver of the mouse model group, showing obvious lipid accumulation and fatty degeneration ( Figure 4 A, B), and Apoe- / - Mice showed more obvious lipid accumulation than WT mice. However, after simvastatin treatment, the lipid droplets in the liver became smaller and the content decreased significantly, which could significantly reduce lipid accumulation. The therapeutic effects of fresh bamboo sap and SB-FMT were the same as simvastatin. This suggests that fresh bamboo sap can improve the adverse effects of high fat on the liver, has lipid-lowering and liver-protecting effects, and this effect is related to changes in intestinal flora.

[0155] Example 5

[0156] This example studies the effect of fresh bamboo sap on improving atherosclerosis.

[0157] 1. Histopathological analysis and immunohistochemistry.

[0158] After the experiment, Apoe was isolated under a stereo microscope. - / - The aortic tree of mice (from the aortic arch to the iliac bifurcation and brachiocephalic trunk) was analyzed and atherosclerotic lesions were evaluated. - / - Atherosclerotic lesions in the aortic root of mice. Heart slices containing the aortic root were embedded in tissue freezing OCT medium. The aortic root was cross-sectioned at intervals of 8 μm from the appearance of the aortic valve to the aortic valve cusp. The aortic sinus sections were stained with hematoxylin-eosin (H&E) to evaluate the thickness of the aortic fibrous cap, plaque area, and degree of lumen stenosis; the cross-sections were stained with Oil Red O to detect lipid deposition (red staining area) and quantify lipid content; the collagen content was quantified by Masson trichrome staining, and the blue staining area of ​​the sections represented collagen fibers. In addition, immunohistochemical analysis was performed on paraffin sections of the mouse aortic root using primary antibodies against monocyte / macrophage antibody-3 (Mac-3) and α-smooth muscle actin (α-SMA), and diaminobenzidine (DAB) was used to develop and stain to identify macrophages and smooth muscle cells (brown staining area) and quantify the analysis. Images were captured by light microscopy and then quantitatively analyzed using ImageJ software. The extent of the lesion area was expressed as the percentage of the total aortic area covered by the lesion.

[0159] In order to further determine the intervention effect of fresh bamboo juice on AS, the morphological images of the aortic arch and H&E staining of the aortic root samples showed that the thickness of the aortic fiber cap, plaque area, and luminal stenosis in the HFD and HFD-FMT groups were significantly aggravated compared with the CD group due to the formation of foam cells, and endothelial shedding was observed in severe lesions. - / - Plaque area and luminal narrowing in mice ( Figure 5At the same time, Oil Red O staining showed that the lipid-positive area of ​​the aortic root plaque in the HFD and HFD-FMT groups increased to a certain extent (compared with the CD group, it increased by 98.69% and 98.96%, respectively, Figure 5 C, G), simvastatin significantly reduced lipid content (P < 0.05), SB and SB-FMT had no significant effect on the lipid content of arterial root plaques, but lipid deposition and foam cell formation in the aortic root area showed a downward trend, which further indicated that fresh bamboo juice could inhibit Apoe - / - AS lesions in mice.

[0160] Masson staining showed that the collagen content of aortic root plaques in each high-fat diet group increased to varying degrees compared with the CD group ( Figure 5 D, H), the collagen fiber content in the aortic root plaque of SB-FMT mice was significantly increased compared with that of HFD-FMT and CD groups (P<0.05, P<0.01), indicating that the stability of the plaque increased. In addition, immunohistochemical staining was used to evaluate the content of macrophages and smooth muscle cells (SMC) in the plaque. By measuring and analyzing the positive expression of α-SMA, the positive expression of α-SMA in the aortic plaque of mice in HFD and HFD-FMT groups was reduced compared with that in CD group. Simvastatin and fresh bamboo juice increased the number of SMCs on the surface of AS plaques (P<0.01, P<0.05, Figure 5 According to the MAC-3 macrophage positive area, the infiltration of macrophages in the plaques of the HFD-FMT group was significantly aggravated (P<0.05, Figure 5 In the middle (F, J), the HFD group also increased relative to the CD group, proving that a high-fat diet (HFD) can induce the migration of inflammatory macrophages, accelerate vascular inflammation and the formation of atherosclerotic plaques, participate in the development of AS and increase the instability of plaques. SB and SB-FMT treatment alleviated the infiltration of Mac-3 positive macrophages in AS plaques.

[0161] In addition, the effect of drug intervention on plaque stability was further evaluated by calculating the plaque stability index. The results showed that compared with the CD group, the plaque vulnerability index of the HFD and HDF-FMT groups was significantly increased (P<0.01, P<0.001, Figure 5In the middle K, AS plaques showed obvious instability, and the severity of HFD-FMT was worse than that of the HFD group. Compared with the HFD group, SB and SV could significantly reduce the vulnerability index (P<0.05, P<0.01). Similarly, SB-FMT could significantly reduce the vulnerability index increased by HFD-FMT (P<0.01), playing a role in stabilizing plaques, and the efficacy of fresh bamboo sap was better than that of simvastatin. These data show that fresh bamboo sap and fresh bamboo sap fecal microbiota transplantation can improve high-fat diet-induced atherosclerosis and stabilize vulnerable plaques. The fecal microbiota of mice in the fecal microbiota transplantation model group aggravated HFD-induced AS, and fresh bamboo sap fecal microbiota transplantation could reverse the disease outcome.

[0162] Example 6

[0163] Experiment on the effect of fresh bamboo juice on the inflammatory state of mice.

[0164] 1. Determination of inflammatory factors:

[0165] The pro-inflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 and the anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in liver tissue and plasma were measured by ELISA kits.

[0166] After 14 weeks of high-fat feeding, the levels of pro-inflammatory factors in liver tissue and plasma of mice in the WT-HFD group increased significantly, while the levels of anti-inflammatory factors decreased significantly ( Figure 6 Fresh bamboo juice treatment effectively reversed this phenomenon, significantly reducing the concentrations of proinflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1 in liver tissue and significantly increasing the concentrations of anti-inflammatory factors IL-10 and TGF-β. It also significantly reduced the concentrations of plasma proinflammatory cytokines IL-1β, IL-6, and MCP-1 and significantly increased the concentrations of anti-inflammatory factors IL-10 and TGF-β. - / - In mice, after feeding a high-fat diet, the trend of inflammation levels in HFD mice was consistent with that in WT-HFD mice, with significantly increased concentrations of IL-1β, IL-6, TNF-α, and MCP-1 and reduced concentrations of anti-inflammatory factors IL-10 and TGF-β ( Figure 6C, D). After administration of fresh bamboo juice, compared with the HFD group, SB significantly reduced the plasma TNF-α level (P<0.0001) and the levels of IL-6 and TNF-α in liver tissue (P<0.01, P<0.001), and significantly increased the plasma IL-10 and liver tissue TNF-α levels (P<0.05, P<0.05). In addition, the results of the fecal microbiota transplantation experiment showed that after transplantation of fecal microbiota in the SB group, the plasma and liver tissue TNF-α levels and the liver tissue IL-6 levels of the SB-FMT mice were significantly decreased compared with the HFD-FMT group.

[0167] It is worth noting that the administration of SV did not reverse the inflammatory state caused by the high-fat diet. Previous related studies have shown that the therapeutic effect of statins was only observed when a high-fat, high-cholesterol diet with relatively low cholesterol levels (HFHC < 0.15%) was used

[17] , which may explain this phenomenon. In addition, when comparing WT-HFD mice with Apoe - / - -HFD mice, as well as WT-CD mice and Apoe - / - -CD mice showed a significant decrease in inflammation compared with WT mice. - / - This is consistent with previous studies on Apoe - / - This is consistent with the results of studies that compared to WT mice, which showed spontaneously increased inflammation and high cholesterol levels. Wild-type mice are highly resistant to atherosclerosis due to their high levels of anti-atherogenic HDL and low levels of pro-atherogenic LDL and VLDL, and are therefore not susceptible to atherosclerosis. However, the deletion of the Apoe gene in mice predisposes to obesity, hypercholesterolemia, and the development of atherosclerotic changes

[18] . Apoe mice fed a high-fat diet are more susceptible to atherosclerosis. - / - Mice have been shown to be an ideal model for the study of atherosclerosis

[19] . - / - One of the reasons why mice are used as a model.

[0168] Example 7

[0169] This example conducts an experiment on inducing browning of white fat and activating brown adipose tissue using fresh bamboo juice.

[0170] 1. The immunohistochemical experimental method of BAT is as follows:

[0171] WT mice and Apoe - / -The interscapular BAT, epididymal WAT, and liver specimens of mice were fixed, embedded, sectioned, and stained with H&E. Immunohistochemistry was performed on interscapular BAT. Immunohistochemistry kits were used to detect intBAT sections incubated with Ucp-1. ImageJ software was used to quantify the percentage of lipid droplet-positive area in intBAT

[20] and the Ucp-1-positive area.

[0172] Based on the fact that white fat in the body can be transformed into brown fat with anti-atherosclerotic effects under certain conditions

[21] , this example observed the morphology of epididymal white fat tissue in mice. The results showed that the size of lipid droplets in white fat cells of mice in the HFD and HFD-FMT groups was larger than that of CD mice ( Figure 7 After 14 weeks of SV, SB and SB-FMT administration, the epiWAT weight of mice decreased ( Figure 2 E, F), and at the same time, the large fat droplets in the white adipose tissue were transformed into multiple small fat droplets, among which SB could basically restore the size of adipocytes to the level of CD mice, which is a typical feature of the browning of white adipose tissue. In addition, along with the significant activation of intBAT, the size of intracellular fat droplets in the brown adipose tissue of mice in the high-fat diet and various drug-treated groups was significantly reduced and appeared reddish brown ( Figure 7 In the AD), the changes in intBAT organ index showed the same trend ( Figure 2 (E, F) The lipid droplet-positive area in intBAT of SB-FMT mice was the smallest, suggesting that fecal microbiota transplantation can improve AS by inducing brown adipose tissue activity.

[0173] Immunohistochemistry and fluorescence quantitative PCR analysis of BAT further revealed the mechanism of action of fresh bamboo juice in lowering lipids and preventing atherosclerosis. The results of immunohistochemistry showed that the Ucp-1 protein content per unit area of ​​intBAT of mice fed a high-fat diet increased to varying degrees compared with mice fed a normal diet ( Figure 8 AD), in which the Ucp-1 protein content of SB-FMT mice was significantly increased compared with that of the CD group, which was consistent with the change in the intBAT lipid droplet-positive area, indicating an increase in energy expenditure.

[0174] 2. Fluorescence quantitative PCR analysis method is as follows:

[0175] (1) Total RNA extraction from interscapular brown adipose tissue

[0176] 1) Take Apoe - / - About 50 mg of mouse tissue sample was placed in a mortar pre-cooled with liquid nitrogen. The tissue was ground with a pestle while adding liquid nitrogen continuously until it was thoroughly ground into powder. The sample was transferred to an enzyme-free centrifuge tube, the final weight was determined, and 1.5 mL of RNAisoPlus was added.

[0177] 2) Mix thoroughly and let stand at room temperature (15-30℃) for 5 minutes.

[0178] 3) Centrifuge at 12,000×g at 4°C for 5 min and carefully pipette the supernatant into a new centrifuge tube.

[0179] 4) Add 300 μL of chloroform, invert the centrifuge tube to mix until the solution is emulsified and turns milky white, and let it stand at room temperature for 5 minutes.

[0180] 5) Centrifuge at 12,000×g and 4°C for 15 min. The homogenate is separated into three layers. The colorless supernatant is aspirated and transferred to another new centrifuge tube.

[0181] 6) Add 750 μL of pre-cooled isopropanol, mix thoroughly by inverting the tube, and let stand at room temperature.

[0182] 10 minutes.

[0183] 7) Centrifuge at 12,000×g at 4℃ for 10 minutes and carefully discard the supernatant without touching the precipitate.

[0184] 8) Add 1.5 ml of pre-cooled 75% ethanol, gently invert, centrifuge at 7,500 × g at 4°C for 5 min, and carefully discard the supernatant without touching the precipitate. Repeat this operation 3 times.

[0185] 9) Carefully remove the supernatant and place the centrifuge tube on a clean bench to air dry.

[0186] 10) After the precipitate is blown dry, add 20 μl RNase-free water to dissolve the RNA.

[0187] (2) RNA purity analysis and quantification

[0188] Use Biodrop2000 to detect RNA concentration and purity: After the instrument is zeroed with RNase-free water, take 1 μl of the RNA solution to be tested into the detection well to measure its concentration and keep a record. It is best to control the A260 / A280 ratio between 1.8 and 2.1.

[0189] (3) Reverse transcription

[0190] Strictly follow the instructions of the BIOG cDNA Synthesis Kit reverse transcription kit, and prepare the reaction system according to the concentration of the extracted RNA according to Table 3. The above reaction system was gently mixed and centrifuged, and reverse transcription was completed on a PCR instrument (reaction conditions: 25°C, 5 min; 50°C, 45 min; 85°C, 5 min).

[0191] Table 3 Reverse transcription reaction system preparation

[0192]

[0193] (4) Fluorescence quantitative PCR

[0194] The cDNA obtained by reverse transcription was used as a template, and 10 μL of DEPC water was added to each tube to dilute it 1.5 times for later use. A PCR tube without RNAse was taken and the reaction system was prepared according to Table 4. The reaction was carried out using a Biorad qPCR instrument (the program settings were: step 1: 94°C, 2 min; step 2: 94°C 10 sec, 58°C 30 sec (40 cycles); step 3: MeltCurve). The β-Actin gene was selected as the internal reference gene, and the relative expression of the gene was measured using 2 -△△Ct Primer synthesis was provided by Changzhou EMI Biotechnology Co., Ltd., see Table 5 for details.

[0195] △△Ct=(CT 实验组目的基因 -CT 实验组内参基因 )-(CT 对照组目的基因 -CT 对照组内参基因 )

[0196] Table 4 PCR reaction system

[0197]

[0199] Table 5 PCR primers

[0200] qRT-PCR further confirmed that after HFD treatment, Apoe - / - The expression of the thermogenic gene Ppar-γ, fatty acid oxidation-related genes (Ppar-γ and Cpt1), and mitochondrial biology genes (nrf2) in mouse intBAT was significantly downregulated ( Figure 8E), after HFD-FMT treatment, the expression level of the thermogenic gene Prdm16 in mice was significantly decreased (compared with the HFD group, P<0.001), and the expression of the fatty acid oxidation gene Ppar-γ and the mitochondrial biobase Nrf2 was significantly downregulated (compared with the CD group, P<0.001, P<0.001. S) B treatment can reverse the HFD-induced decrease in the expression of Ucp-1, Pgc-1α, Prdm-16, Ppar-γ and Nrf1 genes to a certain extent, and significantly increase the expression of the Cpt1 gene (P<0.01). SV can increase the expression levels of Cpt1 and Ppar-γ genes but without significant effect. However, SB-FMT was able to significantly upregulate the HFD-FMT-induced decrease in the mRNA levels of Ucp1, Pgc1α, Prdm16, Ppar-γ, Nrf1, and Nrf2 (P<0.01, P<0.0001, P<0.05, P<0.001, P<0.0001), inducing the activation of brown adipose tissue.

[0201] In summary, the above data results overall prove that Apoe - / - Under atherosclerotic conditions in mice, fresh bamboo juice can increase mitochondrial function and fatty acid oxidation, effectively activate intBAT, enhance energy consumption, and promote the browning of WAT.

[0202] Example 8

[0203] In this example, 16S rRNA gene sequencing was performed on the intestinal flora in mouse feces.

[0204] In order to evaluate the effect of fresh bamboo sap on intestinal flora, in this example, Apoe - / - Mouse fecal samples, collection process: First, prepare sterile cryopreservation tubes, alcohol, gloves, disposable culture dishes, autoclaved tweezers, etc. In the SPF animal room environment, use a 75% alcohol spray bottle to treat a relatively clean platform area to create a relatively sterile operating environment. Wear sterile disposable gloves, use 75% alcohol to treat gloves and tweezers before each operation, catch mice, wipe the mouse anus with an alcohol cotton swab, and gently massage the mouse's lower abdomen with your fingers. When there is a defecation reaction, immediately prepare tweezers and cryopreservation tubes to collect them. Collect 3 or more fresh feces in sterile cryopreservation tubes for subsequent 16S RNA sequencing.

[0205] 1. Alpha Diversity Analysis

[0206] From the diversity dilution curve ( Fig. 9As shown in Figure A, the number of samples and sequencing depth of this experiment were sufficient to reflect the diversity of intestinal flora in each group of mice and meet the requirements of subsequent analysis. Compared with CD, the aces, obs and shannon index of the HFD group were significantly reduced (P<0.05, P<0.01, P<0.01, Fig. 9 B), indicating that a high-fat diet leads to a decrease in intestinal microbial diversity. After administration of SB, the decrease in ACE and Shannon index caused by HFD was significantly reversed (P<0.05, P<0.05), and the intestinal microbial diversity almost returned to normal levels, while simvastatin and FMT transplantation had no significant effect on intestinal microbial diversity. The above results show that the administration of fresh bamboo sap can increase the richness and diversity of the intestinal flora in mice.

[0207] 2. OUT composition and species composition analysis

[0208] There were 264 OUTs among the six groups of mice, 126 in the CD group, 36 in the HFD group, 110 in the SV group, 236 in the SB group, 96 in the HFD-FMT group, and 54 in the SB-FMT group ( Fig. 9 Middle C).

[0209] To explore the differences in intestinal microbial composition, the relative abundance of bacterial communities at the phylum and genus levels was analyzed. Among the top 10 bacterial communities, the dominant bacterial communities with a relatively large proportion are Bacteroidota, Firmicutes, Actinobacteriota, Deferribacterota, Verrucomicrobiota, Desulfobacterota, Protebacteria; norank_f_Muribaculacea e; Bifidobacterium; Lactobacillus, Dubosiella, Faecalibaculum, Lachnospiraceae_NK4A136_group (Lachnospiraceae) ( Fig. 9(D). Compared with the CD group, the ratio of Firmicutes / Bacteroidetes (F / B) in the HFD group was significantly increased (from 0.45 to 6.32). Fresh bamboo juice and simvastatin intervention can reduce the abundance of Firmicutes and increase the abundance of Bacteroidetes. Studies have shown that high levels of Firmicutes are associated with inflammation

[22] , and can promote the uptake of fatty acids and accelerate lipid droplet deposition

[23] . Bacteroidetes can esterify cholesterol into coprostanol (a type of non-absorbable cholesterol), thereby reducing blood cholesterol levels

[24] . This suggests that fresh bamboo juice may play a lipid-lowering and anti-AS role by reducing F / B.

[0210] 3. Beta Diversity

[0211] In this study, principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) were used to calculate beta diversity. Gut microbial beta diversity was assessed by principal coordinates analysis (PCoA) of the euclidean distance matrix. Fig.10 Results in A show that CD overlapped with other groups but was significantly separated from the HFD group, with the longest distance, indicating that a high-fat diet had a significant impact on the structure of the intestinal flora. SV, SB, and SB-FMT interventions caused the intestinal microbial flora to move toward CD, with the SV and SB groups completely separated from the HFD group and basically restored to a level similar to that of mice on a normal diet, suggesting that SB and SV interventions can significantly affect the intestinal microbial community structure of Apoe- / - mice on a high-fat diet.

[0212] 4. Analysis of Differential Microflora

[0213] The linear discriminant analysis effect size (LEfSe) method was used to find the representative microorganisms with different differences between the groups ( Fig.10The effect sizes of the features with abundant changes were calculated based on linear discriminant analysis (LDA). The results were screened with LDA>1 and P-value<0.05, and 82 OTUs were found to be significantly different from the other groups, with 20, 10, 7, 34, 7, and 2 OTUs in each group in the CD, HFD, SV, SB, HFD-FMT, and SB-FMT groups, respectively. In the CD group, the Bacteroidota phylum, Rikenellaceae family, Bacteroidia class, Muribaculaceae family, Muribaculaceae genus, Muribaculaceae family and Turicibacter genus were relatively abundant; in the HFD group, the differential microorganisms between the HFD group and other groups were the Firmicutes phylum, Ileibacterium genus, Bifidobacterium genus, Erysipelotrichales order, Erysipelotrichaceae family, Bacilli class, Actinobacteriota phylum, Bifidobacteriales order, Bifidobacteriaceae family and Actinobacteria class; in the SV group, the Lactobacillus genus, Lactobacillaceae family, Lactobacillales order, Erysipelotrichaceae family, Bacteroidaceae family, Bacteroides genus and Muribaculum genus were the dominant microorganisms. In the SB group, Dubosiella, Lachnospiraceae_NK4A136_group, norank_fLachnospiraceae, Clostridia, Lachnospirales, Lachnospiraceae, Marvinbryantia, norank_fEggerthellaceae, and unclassified_pFirmicutes were abundant. Faecalibaculum, Family_XIII_UCG-001, Acetatifactor, UCG-010, norank_fUCG-010, Butyricicoccus, and Butyricicoccaceae were abundant in the HFD-FMT group. Atopobiaceae and Coriobacteriaceae_UCG-002 were abundant in the SB-FMT group. These results suggest that SB modulates the composition of the intestinal flora.

[0214] Reference Fig.10 The Kruskal-Wallis rank sum test was used to compare the differences among multiple groups. It can be seen that HFD significantly increased the relative abundance of Firmicutes, Bifidobacterium, and Ileibacterium, and decreased the levels of Bacteroidota, norank_f_Muribaculaceae, and Rikenellaceae_RC9_gut_group; SV and Firmicutes, Bifidobacterium, and Ileibacterium were significantly decreased. ium(Bifidobacterium) and Ileibacterium(Ileobacterium) levels and significantly increased the levels of Bacteroidota(Bacteroidetes) and norank_f_Muribaculaceae. Consistently, SB had the same regulatory effect on the levels of Bifidobacterium(Bifidobacterium) and Ileibacterium(Ileobacterium) and could significantly increase the levels of Rikenellaceae_RC9_gut_group and unclassified_f_Oscillospiraceae, and reduce the F / B ratio to a certain extent. It is worth noting that HFD-FMT may aggravate AS by significantly increasing the relative abundance of Family_XIII_UCG-001.

[0215] In summary, the present invention focuses on evaluating the therapeutic effect of fresh bamboo juice intervention on the onset of related metabolic disorders, inflammation and atherosclerosis. In the hyperlipidemia and atherosclerosis mouse model induced by high fat and high cholesterol, fresh bamboo juice intervention treatment can improve lipid metabolism disorders, reduce oxidative stress and inflammation levels, and regulate intestinal microorganisms, thereby reducing dyslipidemia and hepatic fatty degeneration in mice; through further pathological histological analysis, fresh bamboo juice can significantly reduce aortic lesions and stabilize vulnerable plaques to improve high-fat diet-induced atherosclerosis; at the same time, relevant immunohistochemistry and qRT-PCR analysis prove that fresh bamboo juice can promote white fat browning and induce the activation of brown adipose tissue, increase energy consumption, and improve hyperlipidemia and atherosclerosis in mice.

[0216] When the Apoe gene is defective, lipid metabolism disorders will occur, leading to liver lipid deposition and increased plasma and liver lipid levels. It is worth noting that both increased blood lipids and increased liver lipids are lipid metabolism disorders, but there is a correlation rather than a causal relationship between the two. Relevant studies have shown that some methods of reducing liver fatty degeneration can cause increased blood lipids [25,26]. Therefore, drugs that can improve both hyperlipidemia and liver fatty degeneration would be ideal. The present invention found that fresh bamboo juice has the same lipid-lowering and glucose tolerance-improving effects as simvastatin, and fresh bamboo juice has a more consistent regulatory effect on liver lipids. It also has the ability to significantly reduce blood lipids and liver lipid TG levels and increase HDL-C levels, and can even reduce liver TC and LDL-C levels to a certain extent and improve liver fatty degeneration. In addition, WT and Apoe were fed the same high-fat diet. - / - Both mouse species lasted the same amount of time, with Apoe - / - The mice were given twice the dose of WT mice. It can be seen that under the same conditions, the Apoe - / - -HFD mice had higher blood lipid levels and liver lipid deposition than WT-HFD mice, indicating that Apoe gene deficiency leads to impaired circulation and liver lipid metabolism. - / - The changes in blood lipid parameters of mice are not completely consistent. Fresh bamboo juice can reduce liver fatty degeneration of WT mice to a certain extent, but has no significant effect on the changes in blood lipid levels and glucose tolerance caused by their high-fat diet. This suggests that it may be related to the dosage of fresh bamboo juice. The lipid-lowering effect of 10 times the clinical dosage may be better, which also provides a basis for the later dosage selection. Similarly, the present invention also observed that Apoe - / - -CD group had higher inflammation level than WT-CD group, and Apoe - / - No significant difference was observed in the inflammatory level between the -HFD group and the WT-HFD group. It is worth noting that fresh bamboo juice has a more significant anti-inflammatory effect on WT mice. It can effectively reduce the inflammatory state of the body by significantly reducing the concentrations of proinflammatory cytokines IL-1β, IL-6, TNF-α and MCP-1 in the liver and plasma of WT mice and increasing the concentrations of anti-inflammatory factors IL-10 and TGF-β. A significant decrease in liver ALT levels can be seen. - / - In mice, the inflammatory state of the liver was improved mainly by reducing the levels of IL-6 and TNF-α in the liver and increasing the level of TGF-β. It could also significantly reduce the level of plasma TNF-α and increase the level of IL-10, and a significant decrease in the level of plasma ALT was observed.

[0217] In addition, in the 7th week of the experiment, the present invention conducted a fecal microbiota transplantation experiment, and the fecal microbiota transplantation in the HFD group significantly increased the Apoe - / --HFD mice plasma LDL-C level, and to a certain extent increased liver LDL-C level and aggravated liver fatty degeneration. It can be seen that HFD-FMT fecal bacteria treatment can aggravate hyperlipidemia to a certain extent, suggesting that dyslipidemia caused by a high-fat diet is related to changes in intestinal flora. It is worth noting that ELISA results showed that FMT (including HFD-FMT and SB-FMT groups) had lower inflammation levels than the HFD group, but the degree of atherosclerotic lesions was not significantly different from that of the HFD group, and no significant aggravation was observed. After transplantation of fecal bacteria liquid treatment in the SB group, consistent with SB treatment, it was also able to significantly reduce Apoe - / - The SB-FMT intervention had no significant effect on the anti-inflammatory factor IL-10, TGF-β, plasma ALT and liver lipid levels, but significantly reduced the increase in liver function AST levels caused by HFD-FMT transplantation. In addition, pathological results showed that SB-FMT had the same ability as SB to reduce atherosclerotic lesions and stabilize vulnerable plaques. In addition, it is worth noting that SB mainly induced the activation of brown fat by upregulating the expression level of Cpt1 gene, while SB-FMT could significantly upregulate the decrease in mRNA levels of Ucp1, Pgc1α, Prdm16, Ppar-γ, Nrf1 and Nrf2 induced by HFD-FMT, inducing the activation of brown fat and the browning of white fat. Further 16SrRNA sequencing analysis showed that the protective effect of SB on atherosclerosis was accompanied by the remodeling of intestinal microbial composition. In conclusion, the fecal microbiota transplantation experiment and 16SrRNA sequencing results demonstrated that fresh bamboo juice can improve high-fat diet-induced dyslipidemia atherosclerosis by regulating intestinal microbiota.

[0218] In summary, these results suggest that fresh bamboo juice may play a lipid-lowering and anti-atherosclerotic role by regulating glycolipid homeostasis, activating brown fat to promote browning, regulating intestinal microorganisms, inhibiting inflammation and improving the stability of vulnerable plaques.

[0219] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

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Claims

1. Application of fresh bamboo sap in the preparation of medicines for preventing or treating atherosclerosis.

2. The use according to claim 1, characterized in that: The drug has at least one of the following uses: (1) Reduce the subject's body weight; (2) reduce the subjects' daily food intake; (3) Improve the glucose and lipid metabolism disorders of the subjects; (4) improving hepatic steatosis in subjects; (5) Prevent liver damage; (6) Reduce atherosclerotic lesions; (7) Stabilize vulnerable plaques; (8) Inhibit inflammation; (9) Inducing the activation of brown fat and promoting browning; (10) Regulate intestinal microorganisms.

3. The use according to claim 2, characterized in that: The improvement of the subject's glucose and lipid metabolism disorder includes at least one of the following ways: reducing the level of any one of the following substances in the subject's plasma and / or liver: TG, TC and LDL-C levels, improving the subject's glucose tolerance and increasing the subject's plasma and / or liver HDL-C levels.

4. The use according to claim 2, characterized in that: The improving of the subject's hepatic steatosis comprises at least one of the following ways: reducing the ALT level and / or AST level in the subject's liver and / or plasma and improving the subject's liver antioxidant capacity; Preferably, improving the antioxidant capacity of the subject's liver includes: improving the SOD and / or CAT levels of the subject.

5. The use according to claim 2, characterized in that: The prevention of liver damage includes at least one of the following ways: reducing the size of lipid droplets in the subject's liver and reducing the content of lipids in the subject's liver.

6. The use according to claim 2, characterized in that: The mitigation of atherosclerotic lesions includes at least one of the following methods: (1) Reduce the area of ​​arterial plaque in subjects; (2) reduce the degree of arterial stenosis in subjects; (3) reduce the thickness of the aortic fibrous cap in subjects; (4) reducing lipid deposition in the aortic root region of the subjects; (5) reducing aortic foam cell formation in subjects; (6) Increase the content of smooth muscle cells in plaques; (7) Reduce the infiltration of macrophages in plaques; Preferably, the stabilization of vulnerable plaques includes at least one of the following methods: reducing the collagen content of aortic root plaques and lowering the plaque vulnerability index.

7. The use according to claim 2, characterized in that: The inhibition of inflammation includes at least one of the following ways: reducing the level of pro-inflammatory factors in the subject's liver tissue and / or plasma, and increasing the level of anti-inflammatory factors in the subject's liver tissue and / or plasma; Preferably, the liver tissue proinflammatory factor is selected from at least one of IL-1β, IL-6, TNF-α and MCP-1; Preferably, the liver tissue and / or plasma anti-inflammatory factor is selected from at least one of IL-10 and TGF-β.

8. The use according to claim 2, characterized in that: The inducing activation of brown fat to promote browning includes at least one of the following methods: (1) Reduce the epididymal white fat (epiWAT) index of the subjects; (2) reduce the interscapular brown adipose tissue (intBAT) organ index of the subjects; (3) reduce the subjects' perirenal white adipose tissue (prWAT) index; (4) reducing the size of lipid droplets in the subjects’ white adipose tissue; (5) Increase mitochondrial function; (6) Increase fatty acid oxidation; (7) Enhanced energy expenditure; Preferably, the increasing mitochondrial function comprises the following manner: reversing the decrease in Nrf1 and / or Nrf2 gene expression in the subject induced by a high-fat diet; Preferably, the increasing fatty acid oxidation comprises the following manners: increasing the expression of Cpt1 gene and reversing the decrease of Ppar-γ gene expression of the subject induced by a high-fat diet; Preferably, the enhancing energy expenditure comprises at least one of the following ways: reversing the decrease in the expression of Ucp-1, Pgc-1α and Prdm-16 genes in the subject induced by a high-fat diet.

9. The use according to claim 2, characterized in that: The intestinal microorganism regulation includes at least one of the following methods: increasing the richness and diversity of intestinal flora; Preferably, the richness and diversity of intestinal flora is improved by at least one of the following methods: (1) Reduce the abundance ratio of Firmicutes to Bacteroidetes (F / B); (2) regulating the intestinal microbial community structure to move toward the intestinal microbial community structure of normal subjects; (3) Regulate the levels of Bifidobacterium and Ileibacterium; (4) Improve the level of Rikenellaceae_RC9_gut_group and unclassified_f Oscillospiraceae; Preferably, the drug further comprises a combined drug, and the combined drug is selected from at least one of dried tangerine peel, pinellia tuber and tangerine peel; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the fresh bamboo sap is obtained by dry distillation of bamboo.

10. Use of fecal microbiota transplantation solution or fecal microbiota transplantation capsule after administration of fresh bamboo sap in the preparation of drugs for preventing or treating atherosclerosis.

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