Traditional Chinese medicine compound with novel methylation effect for treating AS and application of traditional Chinese medicine compound

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and discloses a traditional Chinese medicine compound with a novel methylation effect for treating AS and application of the traditional Chinese medicine compound, and the traditional Chinese medicine compound is characterized by being prepared from the following raw medicinal materials in parts by weight: 10-20g of trichosanthes kirilowii maxim, 5-15g of allium macrostemon, 5-15g of ligusticum wallichii, 5-15g of radix paeoniae rubra, 5-15g of cape jasmine and 5-15g of pericarpium citri reticulatae. The traditional Chinese medicine compound can be used for preparing a medicine for treating AS. The traditional Chinese medicine compound disclosed by the invention has a bidirectional methylation regulation effect mainly based on gene methylation promotion, can be used for preparing medicines for treating atherosclerosis, has small side effects, and can fill the blank of medicines for AS abnormal gene methylation.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine compound for treating AS, and more specifically, to a traditional Chinese medicine compound with a novel methylation effect on AS and application thereof. Background Art

[0002] Atherosclerosis (AS) is the most common disease of the cardiovascular and cerebrovascular systems. There is no specific treatment for it. Approximately 20 million people die from atherosclerotic diseases every year worldwide.

[0003] In recent years, people have gradually realized that abnormal DNA methylation is of great significance to the occurrence and development of AS. DNA methylation belongs to the category of epigenetics. It refers to the chemical modification process of 5-cytosine in CpG sequence selectively adding methyl groups to form 5-methylcytosine under the catalysis of DNA methyltransferase (mainly including DNMT3a, DNMT3b and DNMT1). Since DNA methylation can affect the downstream expression of genes, it is of great significance in life activities and is considered to be an important therapeutic target for diseases. Studies have found that AS presents a situation where low methylation of the whole genome and abnormal local high methylation coexist. Many studies have shown that the methylation of the genome of AS patients is in a disordered state. For example, Einari et al. detected the methylation of 22 femoral artery atherosclerotic plaques in the whole genome. After comparing with 9 normal groups, it was found that the AS genome had a large number of abnormal low methylation sites in the gene promoter region, accompanied by relatively few abnormal high methylation sites (3997VS 782). Therefore, treatment of abnormal gene methylation in AS is an important direction to break through the dilemma of AS treatment.

[0004] Elevated low-density lipoprotein is the main pathogenic factor of AS. Over the past 30 years, lipid-lowering drugs represented by statins have made significant research progress in the treatment of AS-related diseases. However, it has also been found in clinical use that even after standardized statin treatment, a considerable number of patients still inevitably suffer from acute cardiovascular and cerebrovascular events.

[0005] Abnormal inflammatory response is considered to be another key pathogenic factor of AS. The recent landmark event in the field of AS treatment is the success of the CANTOS study, which is a multicenter, randomized, double-blind, placebo-controlled clinical trial involving 10,061 patients after myocardial infarction in nearly 40 countries. The trial showed that canakinumab, a drug targeting the inflammatory marker IL-1β, can reduce the incidence of cardiovascular events (non-fatal myocardial infarction, non-fatal stroke, or death caused by cardiovascular disease), with statistical differences (HR=0.85, P=0.01). This study proved for the first time that simple anti-inflammatory rather than lipid-lowering treatment can significantly reduce cardiovascular events in patients with coronary heart disease, which has elevated the "inflammatory hypothesis" of AS to the "inflammatory theory." However, the study also found that compared with the placebo group, canakinumab treatment increased the incidence of adverse reactions such as pseudomembranous colitis, severe infection, and sepsis, and the cost of a single canakinumab infusion was about $16,000, and its application prospects are unclear.

[0006] At present, a large amount of evidence shows that abnormal gene methylation is also one of the important causes of AS disease progression, and there is currently a lack of drugs that target abnormal gene methylation in AS. Therefore, the development of a traditional Chinese medicine with the effect of regulating methylation is of great significance for the treatment of AS. Summary of the invention

[0007] The purpose of the present invention is to provide a traditional Chinese medicine compound with a novel methylation effect for treating AS.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A traditional Chinese medicine compound with a novel methylation effect for treating AS, the traditional Chinese medicine compound is composed of the following raw medicinal materials: trichosanthes, allium macrostemon, ligusticum chuanxiong, red peony root, gardenia, and tangerine peel.

[0010] Preferably, the Chinese medicine compound is made of the following raw medicinal materials in parts by weight: 10-20g of Trichosanthes kirilowii, 5-15g of Allium macrostemon, 5-15g of Ligusticum chuanxiong, 5-15g of Paeonia lactiflora, 5-15g of Gardenia jasminoides, and 5-15g of Citrus reticulatae.

[0011] Preferably, the Chinese medicine compound is composed of the following raw medicinal materials in parts by weight: 12-18g of Trichosanthes kirilowii, 8-12g of Allium macrostemon, 8-12g of Ligusticum chuanxiong, 8-12g of Paeonia lactiflora, 8-12g of Gardenia jasminoides, and 8-12g of Citrus reticulatae.

[0012] Preferably, the Chinese medicine compound is composed of the following raw medicinal materials in parts by weight: 15g of Trichosanthes kirilowii, 10g of Allium macrostemon, 10g of Ligusticum chuanxiong, 10g of Paeonia lactiflora, 10g of Gardenia jasminoides, and 10g of Citrus reticulatae.

[0013] Another object of the present invention is to provide the use of the above-mentioned Chinese medicine compound in preparing a drug for treating atherosclerosis (AS).

[0014] Furthermore, the Chinese herbal compound can be used to lower the levels of total cholesterol and low-density lipoprotein.

[0015] Furthermore, the Chinese medicine compound can be used to reduce the levels of serum inflammatory factors tumor necrosis factor-α (IL-6), interleukin-6 (TNF-α) and intercellular adhesion molecule-1 (ICAM-1).

[0016] Furthermore, the Chinese herbal compound can reduce the expression level of the monocyte / macrophage immune marker CD68.

[0017] Furthermore, the Chinese herbal compound has a bidirectional methylation regulatory effect mainly promoting gene methylation.

[0018] Furthermore, the dosage form of the Chinese medicine compound is decoction, capsule, tablet, granule, powder or pill.

[0019] Furthermore, the preparation method of the decoction is to weigh the following raw medicinal materials according to the prescription amount in the above-mentioned Chinese medicine compound: Trichosanthes kirilowii, Allium macrostemon, Ligusticum chuanxiong, Paeonia lactiflora, Gardenia jasminoides, and Tangerine peel, soak them in water for 1 hour, boil the water over high heat, then turn to low heat and simmer for 30-40 minutes, filter out the residue, and the filtrate is the decoction.

[0020] According to traditional Chinese medicine theory, "phlegm, blood stasis and toxins" are the core pathogenesis elements of atherosclerosis (AS). The present invention is based on the classic prescriptions such as Gualou Xiebai Banxia Decoction by Zhang Zhongjing in the Han Dynasty, Yueju Pills by Zhu Danxi in the Yuan Dynasty and Xuefu Zhuyu Decoction by Wang Qingren in the Qing Dynasty, to create a Chinese herbal compound prescription with a novel methylation effect for the treatment of AS, namely Gualou Huoxue Jiedu Decoction (GHJD). Among them, Gua Lou Xie Bai Ban Xia Tang comes from "Jingui Yaolue", which is Zhang Zhongjing's main prescription for treating chest pain. It is composed of Gua Lou, Xie Bai, Ban Xia, and white wine, which can resolve phlegm, promote yang and eliminate numbness; Yue Ju Wan comes from "Dan Xi Xin Fa" by Zhu Danxi in the Yuan Dynasty, and is composed of Cyperus rotundus, Ligusticum chuanxiong, Atractylodes macrocephala, Gardenia jasminoides, and Shenqu, which can treat six kinds of depression such as qi, blood, phlegm, fire, dampness, and food; Xuefu Zhuyu Tang comes from "Yi Lin Gaicuo" by Wang Qingren in the Qing Dynasty. The prescription is composed of Rehmannia root, peach kernel, safflower, Citrus aurantium, red peony root, Bupleurum, Ligusticum chuanxiong, Platycodon grandiflorum, Cyathula officinalis, and Angelica sinensis, which has the effects of promoting blood circulation, removing blood stasis, promoting qi and relieving pain. According to the pathogenesis theory of AS "phlegm, blood stasis, and poison", the Gualou Xiebai Banxia Decoction is composed of Gualou Huoxue Jiedu Decoction, including Gualou Xiebai Banxia Decoction, Gardenia in Yueju Pills, Chuanxiong and Red Peony Root in Xuefu Zhuyu Decoction, and Tangerine Peel. The Gualou Huoxue Jiedu Decoction in the present invention is composed of the following raw materials: Gualou Xiebai, Chuanxiong, Red Peony Root, Gardenia, and Tangerine Peel. Gualou Xiebai and Tangerine Peel in the Chinese medicine compound can resolve phlegm, promote qi and eliminate dampness, targeting "stubborn phlegm"; Chuanxiong and Red Peony Root can promote blood circulation and remove blood stasis, and eliminate "dead blood"; Gardenia can clear away heat and detoxify to transform "toxic evil".

[0021] It should be noted that the raw medicinal materials and pharmacological effects used in the Chinese medicinal compound of the present invention are as follows:

[0022] Trichosanthes kirilowii Maxim. or Trichosanthes rosthornii Harms, a plant of the Cucurbitaceae family, is a dried and mature fruit. Nature and flavor and meridians: sweet, slightly bitter, cold. It enters the lung, stomach, and large intestine meridians. Functions and indications: clearing away heat and removing phlegm, relieving chest congestion and dispersing stagnation, moistening dryness and lubricating intestines. It is used for cough due to lung heat, turbid yellow and thick phlegm, chest obstruction and heart pain, chest congestion and fullness, breast abscess, lung abscess, intestinal abscess, and constipation.

[0023] Xiebai: the dried bulb of Allium macrostemon Bge. or Alliumchinense G.Don, a plant of the Liliaceae family. Nature and flavor and meridians: pungent, bitter, warm. It enters the heart, lung, stomach, and large intestine meridians. Functions and indications: promote yang and dissipate stagnation, promote qi and relieve stagnation. It is used for chest pain, abdominal distension and pain, and heaviness after diarrhea.

[0024] Chuanxiong: The dried rhizome of the plant Ligusticum chuanxiong Hort. of the Umbelliferae family. Nature and flavor and meridians: pungent, warm. Enters the liver, gallbladder, and pericardium meridians. Functions and indications: promotes blood circulation and qi, dispels wind and relieves pain. Used for chest pain, heart pain, stabbing pain in the chest and ribs, swelling and pain due to falls, irregular menstruation, amenorrhea, dysmenorrhea, abdominal pain due to lumps, headache, and rheumatic pain.

[0025] Red peony root: the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, Ranunculaceae. Nature and flavor and meridians: bitter, slightly cold. Enters the liver meridian. Functions and indications: clearing heat and cooling blood, dispersing blood stasis and relieving pain. Used for heat entering the blood, fever toxicity causing rash, vomiting blood and epistaxis, red and swollen eyes, liver depression and flank pain, amenorrhea and dysmenorrhea, abdominal pain due to lumps and masses, injuries from falls, carbuncles and ulcers.

[0026] Gardenia: The dried mature fruit of Gardeniajasminoides Ellis, a plant of the Rubiaceae family. Nature and flavor and meridians: bitter, cold. It enters the heart, lung, and triple burner meridians. Functions and indications: It can clear away heat and relieve restlessness, clear away heat and dampness, cool blood and detoxify; it can be used externally to reduce swelling and relieve pain. It is used for restlessness caused by fever, jaundice caused by dampness and heat, astringent pain caused by stranguria, vomiting and bleeding caused by blood heat, red and swollen eyes, and fire poison sores; it can also be used externally to treat sprains and contusions.

[0027] Tangerine peel: The dried mature peel of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties. Nature and flavor and meridians: bitter, pungent, warm. Enters the lung and spleen meridians. Functions and indications: Regulates qi and strengthens the spleen, dries dampness and resolves phlegm. Used for abdominal distension, loss of appetite, vomiting and diarrhea, and cough with excessive phlegm.

[0028] The present invention has the following beneficial effects:

[0029] 1. GHJD is created based on the core pathogenesis of AS, phlegm, blood stasis, and toxicity. In the formula, Trichosanthes and Allium macrostemon are the main drugs. Trichosanthes is sweet and cold in nature, and can dispel phlegm and dissipate stagnation. Allium macrostemon is pungent and bitter in nature, warm in nature, and can dissipate phlegm and dissipate cold. The two drugs are warm and cool, and the yin and yang match to dissipate phlegm turbidity; Chuanxiong is pungent in nature and warm in nature, and can activate blood circulation and promote qi. Red peony root is bitter in nature and slightly cold in nature, and can activate blood circulation and cool blood. The two drugs are ministerial drugs that activate blood circulation and dissipate blood stasis; phlegm and blood stasis are stagnant for a long time and turn into heat and toxicity. Gardenia is bitter in taste and cold in nature, and it enters the heart, lung, and triple burner meridians, purging fire and detoxifying. Tangerine peel is bitter and pungent in taste and warm in nature, and can regulate qi and strengthen the spleen, dry dampness and resolve phlegm. It assists the main drug to strengthen the power of expectoration. The above two drugs are adjuvant drugs. The combination of these drugs strives to remove phlegm and dampness, resolve blood stasis, and detoxify evil, which can comprehensively target the core pathogenesis of AS, phlegm, blood stasis, and toxicity.

[0030] 3. The Chinese medicine compound of the present invention can reduce the levels of total cholesterol and low-density lipoprotein, reduce the levels of serum inflammatory factors tumor necrosis factor-α (IL-6), interleukin-6 (TNF-α) and intercellular adhesion molecule-1 (ICAM-1); reduce the expression level of monocyte / macrophage immune marker CD68, and has a bidirectional methylation regulatory effect mainly promoting gene methylation. It can be used to prepare drugs for the treatment of atherosclerosis with small side effects, filling the gap in drugs for abnormal gene methylation of AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The anti-AS effect of GHJD in the experimental case is observed by pathology (A is ApoE fed with high-fat diet - / - Mice were 16 weeks old and were given compound GHJD by gavage for 8 weeks according to the groups; B is the weight of mice in each group during the experiment; C is the pathological staining photos of mice in each group; D shows that GHJD can reduce the area of ​​AS plaques);

[0032] Figure 2 The results of the effect of GHJD on blood lipids and inflammatory response of AS mice in the test example (A is the change of TC in each group in the biochemical test; B is the change of TG in each group in the biochemical test; C is the change of LDL in each group in the biochemical test; D is the change of HDL in each group in the biochemical test);

[0033] Figure 3 This is the heat map of differentially methylated sites between the AS model group and the normal group (Control) in the experimental example (Control represents the normal mouse group, and Model represents the AS model mouse);

[0034] Figure 4The volcano map of differentially methylated sites between the AS model group and the normal group (Control) in the test case (DMS: differentially methylated sites; hyper: high; hypo: low; red represents statistically significant high methylated sites; green represents statistically significant low methylated sites);

[0035] Figure 5 This is a volcano map of differentially methylated sites between the high-dose GHJD group and the AS model group. (DMS: differentially methylated sites; hyper: high; hypo: low; red represents statistically significant high methylated sites; green represents statistically significant low methylated sites). DETAILED DESCRIPTION

[0036] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0037] The term "atherosclerosis (AS)" in this article refers to a chronic inflammatory disease that occurs in the blood vessel wall. It is the pathological basis of many cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral infarction. Its pathological process involves dyslipidemia, chronic inflammation, endothelial damage, etc. About 19.8 million people die from cardiovascular and cerebrovascular diseases with AS as the pathological basis every year worldwide.

[0038] The term "DNA methylation" herein refers to DNA methylation, which belongs to the category of epigenetics and refers to the chemical modification process in which, under the action of DNA methyltransferase, S-adenosylmethionine provides a methyl donor and a methyl group is selectively added to the cytosine of the CpG sequence to form 5-methylcytosine.

[0039] The term "demethylation" herein refers to the biological process in which 5-methylcytosine loses its methyl group under the mediation of various DNA methylases.

[0040] The term "gene methylation" herein refers to the biological process in which 5-cytosine acquires a methyl group under the mediation of various DNA methylases.

[0041] The term "ApoE knockout mouse model" herein refers to the AS model induced by feeding ApoE- / - mice with a high-fat diet, which is the currently recognized AS model.

[0042] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0043] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0044] Embodiment 1:

[0045] A method for preparing a traditional Chinese medicine compound with a novel methylation effect for treating AS, the method specifically comprising: weighing the following raw medicinal materials according to the prescribed amount: 15g of Trichosanthes kirilowii, 10g of Allium macrostemon, 10g of Ligusticum chuanxiong, 10g of Paeonia lactiflora, 10g of Gardenia jasminoides, and 10g of Citrus reticulatae; soaking them in 10 times the volume of water for 1 hour, boiling the water over high heat, then simmering over low heat for 30 minutes, filtering to remove the residue, and obtaining the filtrate as a decoction.

[0046] Embodiment 2:

[0047] A method for preparing a traditional Chinese medicine compound with a novel methylation effect for treating AS, the method specifically comprising: weighing the following raw medicinal materials according to the prescribed amount: 10g of Trichosanthes kirilowii, 5g of Allium macrostemon, 5g of Ligusticum chuanxiong, 5g of Paeonia lactiflora, 5g of Gardenia jasminoides, and 5g of Citrus reticulatae; soaking the mixture in 10 times the volume of water for 1 hour, boiling the water over high heat, then simmering over low heat for 40 minutes, filtering to remove the residue, and obtaining a decoction using the filtrate.

[0048] Embodiment 3:

[0049] A method for preparing a traditional Chinese medicine compound with a novel methylation effect for treating AS, the method specifically comprising: weighing the following raw medicinal materials according to the prescribed amount: 20g of Trichosanthes kirilowii, 12g of Allium macrostemon, 12g of Ligusticum chuanxiong, 12g of Paeonia lactiflora, 12g of Gardenia jasminoides, and 12g of Citrus reticulatae; soaking them in 10 times the volume of water for 1 hour, boiling the water over high heat, then simmering over low heat for 35 minutes, filtering to remove the residue, and obtaining the filtrate as a decoction.

[0050] Embodiment 4:

[0051] A method for preparing a Chinese medicine compound with a novel methylation effect for treating AS, the method specifically comprising: weighing the following raw materials according to the prescribed amount: 18g of Trichosanthes kirilowii, 8g of Allium macrostemon, 8g of Ligusticum chuanxiong, 8g of Paeonia lactiflora, 8g of Gardenia jasminoides, and 8g of Citrus reticulatae; soaking in 10 times the volume of water for 1 hour, boiling the water over high heat, then simmering over low heat for 30 minutes, filtering to remove the residue, and the filtrate is a decoction. .

[0052] Embodiment 5:

[0053] A method for preparing a traditional Chinese medicine compound with a novel methylation effect for treating AS, the method specifically comprising: weighing the following raw medicinal materials according to the prescribed amount: 12g of Trichosanthes kirilowii, 12g of Allium macrostemon, 12g of Ligusticum chuanxiong, 12g of Paeonia lactiflora, 12g of Gardenia jasminoides, and 12g of Citrus reticulatae; soaking them in 10 times the volume of water for 1 hour, boiling the water over high heat, then simmering over low heat for 40 minutes, filtering to remove the residue, and obtaining the filtrate as a decoction.

[0054] Test example:

[0055] In order to illustrate that the Chinese herbal compound of the present invention has the effect of regulating methylation in treating AS, the following experiment was conducted using Example 1:

[0056] 1 Experimental methods

[0057] 1.1 Preparation of experimental decoction drugs

[0058] The Chinese medicinal pieces of Trichosanthes kirilowii, Allium macrostemon, dried orange peel, red peony root, Ligusticum chuanxiong and Gardenia jasminoides in the Trichosanthes kirilowii decoction (GHJD) were purchased from the Pharmacy Department of Xiyuan Hospital, China Academy of Chinese Medical Sciences. The same batch of pieces were selected. The decoction in Example 1 was the concentration of the low-dose group, and further concentrated to half the volume of the decoction in Example 1 was the concentration of the high-dose group.

[0059] 1.2 Preparation of AS animal model

[0060] 50 male ApoE - / - After one week of adaptive feeding, the mice were fed with a high-fat diet (basic diet 78.85%, lard 21%, cholesterol 0.15%) for 8 weeks to induce the establishment of the AS model.

[0061] 1.3 Grouping and drug treatment

[0062] Ten SPF C57BL / 6J mice were used as a normal control group. ApoE - / - Mice were randomly divided into model group, GHJD low-dose and high-dose groups and positive control drug atorvastatin (Ator), with 10 mice in each group. The model group continued to be fed with high-fat diet for 8 weeks, and the modeling period was 16 weeks; the Chinese medicine compound dose groups and atorvastatin group were gavaged with corresponding concentrations of drugs, once a day, 0.1mL / 10g each time, for 8 consecutive weeks. The specific grouping is shown in Table 1.

[0063] Table 1 Grouping and dosing regimen

[0064]

[0065]

[0066] 1.4 Collection of materials

[0067] Fast for 12-14 hours before sampling, free access to water. Intraperitoneal injection of 1.5% sodium pentobarbital anesthesia (0.06mL / 10g), remove the eyeball to collect blood. Dissect and fully expose the chest cavity, cut a small hole in the right atrial appendage, draw 1-2mL of pre-cooled (4℃) deionized water with a disposable syringe, inject from the left ventricle, and push slowly until the liver and spleen turn white, then stop the perfusion. Completely remove the heart and aorta and its branches (to the left and right common iliac arteries). One part of the sample is placed in 4% paraformaldehyde fixative at room temperature, and one part is placed in a dry ice box, and then transferred to a -80℃ low-temperature refrigerator for storage.

[0068] 1.5 Index detection

[0069] 1.5.1 Pathological examination to observe the effect of GHJD in improving AS

[0070] a. The mouse aorta was removed from 4% paraformaldehyde, rinsed with PBS, peeled, cut, and flattened under a stereomicroscope, washed with staining and washing solution at room temperature, and placed in a culture dish. It was stained with Oil Red O staining solution for 10 to 20 minutes, washed with staining and washing solution, placed on a black background, wiped dry with absorbent paper, observed and photographed under a microscope, and the Oil Red O positive area was selected by Image-Pro Plus and the ratio of its area to the area of ​​the entire aorta was calculated; b. The heart fixative was rinsed clean, embedded, sliced, and the mouse heart was fixed with a slicer. The heart was cut along the transverse axis of the heart, and the lesions at the aortic valve were mainly observed. The cutting distance was changed to 5 mm, and the slices were collected and spread. After hematoxylin-eosin (H&E) staining, the pathological changes such as the thickness of the intima, atherosclerotic plaques, cholesterol crystals and necrotic cores, and fibrous caps were observed; c. At a certain interval (several slices), the slices were collected for Masson staining to observe the amount of collagen fiber production and the thickness of the fibrous cap in the lesion site.

[0071] 1.5.2 GHJD lipid-lowering and anti-inflammatory effects

[0072] The whole blood of mice was kept at room temperature for 1-2 hours and then centrifuged at 4°C, 3000 rpm, for 15 minutes. The four serum lipids of mice were detected by automatic biochemical analyzer: total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein cholesterol (HDL). The levels of serum inflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and intercellular adhesion factor 1 (ICAM-1) were detected by ELISA.

[0073] 1.5.3 Effect of GHJD on genome-wide gene methylation in AS mice

[0074] The aorta specimens were ground and genomic DNA was extracted using a DNA extraction kit (Tiangen Company). The methylation capture combined with sequencing method was used to detect the methylation status of the above-mentioned gene groups. The main steps were: a: sample preparation, mainly including DNA fragmentation and purification; b: library hybridization and hybridization capture; c: DNA sample sulfite treatment; d: machine; PCR amplification and sequencing; e: data quality control; f: analysis of differential methylation sites and corresponding genes.

[0075] 2 Test results

[0076] 2.1 Pathological staining showed that GHJD could reduce the area of ​​AS plaques

[0077] Figure 1 A is ApoE fed with high-fat diet - / - Mice aged 16 weeks were given compound GHJD by gavage for 8 weeks according to the group; Figure 1 B is the weight of mice in each group; Figure 1 C is the pathological staining photos of mice in each group; Figure 1 D shows that GHJD can reduce the area of ​​AS plaques.

[0078] 2.2.1GHJD has a significant lipid-lowering effect

[0079] according to Figure 2 A. Figure 2 The biochemical test results in C showed that, like atorvastatin, GHJD could significantly reduce the levels of total cholesterol (TC) and low-density lipoprotein (LDL) in AS mice. Figure 2 B. Figure 2 The results of D showed that GHJD could not reduce triglycerides (TG) and high-density lipoprotein (HDL).

[0080] 2.2.2GHJD has significant anti-inflammatory effects

[0081] according to Figure 2 E. Figure 2 F and Figure 2 ELISA test in G showed that, like atorvastatin, GHJD could significantly reduce the levels of serum inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) in AS mice. Figure 2 H and Figure 2 Immunofluorescence detection in I showed that GHJD could significantly reduce the expression level of monocyte / macrophage immune marker CD68.

[0082] 2.3.1 Whole genome methylation detection shows that AS has a large number of abnormally high-methylated genes and low-methylated genes

[0083] After the normal group and AS model group mice were treated accordingly, the aortas of the two groups were collected for methylation immunoprecipitation combined with sequencing technology detection. The results showed that compared with the normal group (Control), the whole genome methylation of the two groups was significantly different ( Figure 4 ), there were 61492 differentially methylated sites (DMS) in the AS model group, including 41281 hypermethylated sites and 20211 hypomethylated sites ( Figure 5 ), these sites corresponded to 8056 hypermethylated genes and 5183 hypomethylated genes, respectively, suggesting that the whole genome methylation of AS is in an unbalanced state, that is, abnormal hypermethylation and hypomethylation coexist.

[0084] 2.3.2 Whole genome methylation detection showed that GHJD has a significant methylation regulatory effect

[0085] After AS mice were treated with high-dose GHJD, the aortas of the two groups were collected for methylation immunoprecipitation combined with sequencing technology. The results showed that after GHJD treatment, there were 61,492 differentially methylated sites (DMS), including 41,281 high-methylated sites and 20,211 low-methylated sites ( Figure 5 ), these sites correspond to 8056 hypermethylated genes and 5183 hypomethylated genes, respectively, suggesting that GHJD has a bidirectional methylation regulatory effect mainly promoting gene methylation. Table 2 lists some of the hypermethylated genes induced by GHJD. Table 3 lists some of the hypomethylated genes induced by GHJD.

[0086] Table 2 Some hypermethylated genes induced by GHJD (TOP 30)

[0087] Hypermethylated genes Differential methylation value Hypermethylated genes Differential methylation value Bcar3 0.391109233 Cnbd2 0.34333506 Nrarp 0.386634885 Cpa6 0.341234048 Plcg2 0.383539959 Mypn 0.341141312 Gm6921 0.377022785 En2 0.34113371 Arhgap27 0.366881959 Gm24096 0.340192792 Fam129a 0.360012591 Dlc1 0.33841579 C530008M17Rik 0.35802397 Kank2 0.33656511 Lasp1 0.354034857 Scarf1 0.329102011 Rnf43 0.351028326 1500017E21Rik 0.32905427 Gm4773 0.349291774 Ccdc81 0.328452682 HkD 0.34665511 Myo9b 0.327936394 Dpyd 0.34638076 Nxpe2 0.325222486 Mmgt2 0.34402435 Gm4773 0.324207838 Nupr1 0.343772411 Map7d1 0.32347681 Rgp1 0.343592252 Vim 0.322517816 Bcar3 0.391109233 Cnbd2 0.34333506 Nrarp 0.386634885 Cpa6 0.341234048

[0088] Table 3 Part of hypomethylated genes induced by GHJD (TOP 30)

[0089] Hypomethylated genes Differential methylation value Hypomethylated genes Differential methylation value Gm8334 -0.61387276 Tcf7l1 -0.322081728 Arhgef3 -0.436893512 Elf4 -0.321545423 Fam129c -0.391400799 Nav2 -0.319973307 Ptpn6 -0.370813478 Chrna3 -0.319282417 Fendrr -0.369337583 St8sia4 -0.315993072 Iqce -0.346464454 Gm11945 -0.313950335 Frmd4b -0.344653509 Axdnd1 -0.313687804 Ccdc60 -0.3408866 Epb4.1l4b -0.30993333 Espn -0.337014785 Atoh8 -0.308362002 Gm14165 -0.331282541 Sec14l2 -0.30789146 Zp1 -0.330239332 Zscan10 -0.307411951 Arhgef1 -0.330200678 Olfr647-ps1 -0.306203468 Milr1 -0.32825452 Gm25155 -0.306097978 Tead1 -0.325086013 Gm14232 -0.305880389 Pigx -0.323450513 Adamtsl5 -0.30432402

[0090] In summary, the results in the test examples show that the Chinese medicine compound of the present invention can reduce the levels of total cholesterol and low-density lipoprotein, reduce the levels of serum inflammatory factors tumor necrosis factor-α (IL-6), interleukin-6 (TNF-α) and intercellular adhesion molecule-1 (ICAM-1); reduce the expression level of monocyte / macrophage immune marker CD68, and has a bidirectional methylation regulatory effect mainly promoting gene methylation. It can be used to prepare drugs for the treatment of atherosclerosis with small side effects, and can fill the gap in drugs for abnormal gene methylation of AS.

[0091] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A Chinese medicinal compound with a novel methylation effect for treating AS, characterized in that: The traditional Chinese medicine compound is prepared from the following raw medicinal materials in parts by weight: 10-20g of Trichosanthes kirilowii, 5-15g of Allium macrostemon, 5-15g of Ligusticum chuanxiong, 5-15g of Paeonia lactiflora, 5-15g of Gardenia jasminoides, and 5-15g of Citrus reticulatae.

2. A Chinese medicinal compound with a novel methylation effect for treating AS according to claim 1, characterized in that: The traditional Chinese medicine compound is composed of the following raw medicinal materials in parts by weight: 12-18g of Trichosanthes kirilowii, 8-12g of Allium macrostemon, 8-12g of Ligusticum chuanxiong, 8-12g of Paeonia lactiflora, 8-12g of Gardenia jasminoides, and 8-12g of Citrus reticulatae.

3. A Chinese medicinal compound with a novel methylation effect for treating AS according to claim 1, characterized in that: The traditional Chinese medicine compound is composed of the following raw medicinal materials in parts by weight: 15g of Trichosanthes kirilowii, 10g of Allium macrostemon, 10g of Ligusticum chuanxiong, 10g of Paeonia lactiflora, 10g of Gardenia jasminoides, and 10g of Citrus reticulatae.

4. Use of the Chinese medicine compound according to any one of claims 1 to 3 in preparing a drug for treating AS.

5. The use according to claim 4, characterized in that: The Chinese medicinal compound is used in medicines for reducing total cholesterol and low-density lipoprotein levels.

6. The use of the Chinese medicine compound according to claim 4, characterized in that: The Chinese medicinal compound is used in a medicine for reducing the levels of serum inflammatory factors tumor necrosis factor-α, interleukin-6 and intercellular adhesion molecule-1.

7. The use of the Chinese medicine compound according to claim 4, characterized in that: The Chinese medicinal compound is used in a drug for reducing the expression level of monocyte / macrophage immune marker CD68.

8. The use of the Chinese medicine compound according to claim 4, characterized in that: The application of the Chinese medicine compound in drugs for regulating methylation effects.

9. The use of the Chinese medicine compound according to any one of claims 5 to 8, characterized in that: The dosage form of the Chinese medicine compound is decoction, capsule, tablet, granule, powder or pill.

10. The use according to claim 9, characterized in that: The preparation method of the decoction is as follows: the following raw medicinal materials are weighed respectively according to the prescription amount of the traditional Chinese medicine compound described in any one of claims 1 to 3: trichosanthes, allium macrostemon, ligusticum chuanxiong, red peony root, gardenia jasmine, and tangerine peel, soaked in water for 1 hour, boiled, boiled over high heat, then boiled over low heat for 30-40 minutes, filtered to remove the residue, and the filtrate is the decoction.

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