CN115998831A

CN115998831A - Traditional Chinese medicine composition for treating non-alcoholic steatohepatitis

Info

Publication number: CN115998831A
Application number: CN202310099104.1A
Authority: CN
Inventors: 姚凝; 王昕�
Original assignee: Gansu University of Chinese Medicine
Current assignee: Gansu University of Chinese Medicine
Priority date: 2023-02-11
Filing date: 2023-02-11
Publication date: 2023-04-25
Anticipated expiration: 2043-02-11
Also published as: CN115998831B

Abstract

The invention discloses a traditional Chinese medicine composition for treating nonalcoholic steatohepatitis, which consists of 21 traditional Chinese medicines such as coptis chinensis, rhizoma pinellinae praeparata, rhizoma atractylodis, peach kernel, coix seed stir-fried with bran, fingered citron, radix curcumae, ligusticum wallichii and the like. The invention has the effects of soothing liver, strengthening spleen, resolving phlegm, removing blood stasis, dehumidifying, resolving phlegm, promoting blood circulation, dispelling blood stasis, promoting qi circulation, promoting blood circulation, eliminating phlegm, regulating qi, dredging meridian passage, nourishing liver, strengthening spleen, clearing heat and tonifying spleen qi.

Description

Traditional Chinese medicine composition for treating non-alcoholic steatohepatitis

Technical Field

The invention belongs to the field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for treating nonalcoholic steatohepatitis.

Background

The medical guideline of Chinese non-alcoholic fatty liver disease in 2015 indicates that the prevalence of Chinese adult non-alcoholic fatty liver disease (NAFLD) is 12.5-35.4%, wherein the non-alcoholic fatty liver disease (NASH) accounts for about 10-30%, which is a key stage in the disease progress process. If NASH is not controlled and suppressed, it is still progressing, and the patient will undergo mild liver fibrosis, moderate liver fibrosis, and further develop severe liver fibrosis and cirrhosis. The incidence rate of cirrhosis in NASH patients reaches up to 15-25% within 10 years, which is one of the main causes of mortality in NAFLD.

NASH, once the disease progresses to the cirrhosis stage, means that a significant turn occurs in disease occurrence and progression. Cirrhosis will continue to deteriorate itself in a completely new pattern, and even if NASH primary causes are removed, it is difficult to block the progression of cirrhosis disease, with a final outcome being poor. The only globally accepted regimen for effective treatment of cirrhosis to date is simply liver transplantation. Limited by liver sources, the vast majority of patients will face severely fatal adverse outcomes.

For the whole disease development stage of NAFLD, the NASH stage is a key moment for blocking the disease progression deterioration, which is helpful for improving the life quality of patients and reducing the burden and pain of society, families and individuals. The development of drugs for treating NASH, through protecting the liver and reversing liver injury, actively blocks the occurrence of NASH-related liver fibrosis, and has positive scientific value, social value, economic value, reality and urgency when put into clinical application.

In modern medicine, no drugs are approved for NASH treatment worldwide.

Considering the pathogenesis of NASH, global advice is now being made: early encouragement of physical exercise by the patient and major measures to improve dietary structure, positive lifestyle interventions; patients who do not benefit from exercise and lifestyle interventions, or who have developed disease progression, can only be treated with drugs specific to liver inflammation, fibrosis, but no marketed drugs have been approved to date.

At present, several medicines are in the later development stage worldwide, and the medicines are respectively: (1) for liver fat accumulation: ilafebulin; (2) for oxidative stress, inflammation and apoptosis: an inhibitor of apoptosis signaling kinase 1 (ASK 1); (3) for intestinal microbiome and metabolic endotoxemia: TLR4 antagonists, solicomycin; (4) for anti-fibrotic drugs: cefrillo and CVC. No research results have been published so far.

The traditional Chinese medicine is characterized by multi-level, multi-target, multi-angle and integral regulation of organism dysfunction and disease state, thereby achieving and maintaining the health state of the organism. Under the guidance of Chinese medicine theory, each doctor clinically carries out Chinese medicine differentiation type theory treatment on the symptoms of the patient, wherein the following categories are adopted:

(1) Heat-clearing and dampness-resolving herbs

The interior syndrome of damp-heat is the common traditional Chinese medicine syndrome of NASH, and is mostly caused by improper diet, spleen failing to transport, stomach qi failing to descend and damp turbidity accumulated in middle energizer, and long-lasting depression transforming into heat. The disease is repeated due to the accumulation of damp-heat caused by the accumulation of damp-heat in the body due to the intermingling of heat and damp-evil. The traditional Chinese medicines for clearing heat and eliminating dampness are adopted, so that dampness is eliminated without assisting heat, heat is removed without generating dampness, and both damp and heat can be achieved. Guo Yideng by using Gegenqinlian decoction to intervene in a high-fat diet NASH rat model, the result shows that the expression of NLRP3 inflammation corpuscles in the model group is increased, and the expression of NLRP3 inflammation corpuscles is obviously reduced after the medicine intervention, which indicates that Gegenqinlian decoction has an inhibiting effect on NLRP3 inflammation corpuscles. Cell experiments show that emodin can inhibit activation of NLRP3 inflammatory small body signaling pathway induced by LPS.

(2) Phlegm-resolving and stasis-resolving herbs

The syndrome of phlegm-blood stasis is often caused by excessive eating of the food, which results in impairment of spleen qi, adverse transportation and transformation, and generation of dampness, accumulation of dampness into phlegm, obstruction of collaterals by phlegm, unsmooth blood circulation and generation of blood stasis. The phlegm and blood stasis are caused by the cause and effect of the phlegm and blood stasis, and the disease is repeated. Wu Liu et al induced mice NASH with MCD diet, found that NASH mice had significantly reduced NLRP6 protein expression; intervention of the decoction for removing blood stasis can improve the expression of NLRP6, obviously improve liver functions and obviously reduce liver inflammation, and shows that a possible action mechanism of the decoction for removing blood stasis for improving NASH is to lower the expression of NLRP 6. In the pinellia tuber magnolia bark decoction intervention experiment, the prescription is found to have no obvious effect on serum IL-1 beta level, but can down regulate the activation of NLRP3 inflammatory corpuscles in liver.

(3) Tonifying liver and kidney

The traditional Chinese medicine considers that kidney yang is the root of yang qi of a human body, and kidney yang deficiency, poor transportation and distribution of essence and micro-transportation, accumulation of fat into turbidity and stagnation in liver. The research shows that the NLRP3 expression in the liver of the NASH mice induced by high-fat diet is increased, and the liver cells have obvious lipid deposition, and the dry prognosis of the compound Zhenzhu lipid regulating granule (FTZ) not only obviously reduces the formation of NLRP3 inflammatory corpuscles, but also reduces liver steatosis and fibrosis. At the same time, in FTZ treated cells, inflammatory body formation and activation were also significantly reduced.

The essence of traditional medicine is that "treating no disease in the upper part, treating the desired disease in the middle part, treating the disease in the lower part", aiming at the characteristics of chronic disease course of NASH, the traditional medicine is required to focus on the transmission of disease symptoms rather than the current situation of disease symptoms in the treatment strategy and method.

The existing traditional Chinese medicine treatment scheme is deficient from the treatment angle, the internal correlation between the life characteristics of people and diseases in the current society cannot be deeply analyzed, the disease pathogenesis of NASH cannot be treated, the disease transmission and development cannot be blocked, and more clinical common symptoms cannot be interfered. The disease treatment and the root elimination are comprehensively analyzed based on factors such as causes, transmission characteristics and the like, and the phlegm-blood stasis internationis always penetrated in the NASH and is the root of pathogenesis.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for treating non-alcoholic steatohepatitis.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a traditional Chinese medicine composition for treating non-alcoholic steatohepatitis is prepared from the following raw materials in parts by weight: 20-30 parts of coptis chinensis, 12-24 parts of rhizoma pinellinae praeparata, 15-25 parts of rhizoma atractylodis, 18-30 parts of peach kernel, 20-30 parts of semen coicis stir-fried with bran, 15-30 parts of fingered citron, 15-30 parts of radix curcumae, 9-18 parts of rhizoma ligustici wallichii, 18-30 parts of herba artemisiae capillaris, 20-30 parts of radix salviae miltiorrhizae, 18-30 parts of radix paeoniae rubra, 12-30 parts of rhizoma curcumae, 12-30 parts of concha arcae, 10-24 parts of trogopterus dung, 10-26 parts of caulis spatholobi, 10-18 parts of safflower, 15-30 parts of white paeony root, 15-30 parts of white hyacinth bean, 9-18 parts of lotus leaf, 9-15 parts of jujube and 9-12 parts of raw liquorice.

The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight as a preferred scheme: 24 parts of coptis chinensis, 18 parts of rhizoma pinellinae praeparata, 18 parts of rhizoma atractylodis, 24 parts of peach kernel, 25 parts of semen coicis stir-fried with bran, 20 parts of fingered citron, 20 parts of radix curcumae, 18 parts of ligusticum wallichii, 25 parts of herba artemisiae capillaries, 27 parts of radix salviae miltiorrhizae, 24 parts of radix paeoniae rubra, 24 parts of rhizoma curcumae, 30 parts of concha arcae, 18 parts of trogopterus dung, 18 parts of caulis spatholobi, 12 parts of safflower, 30 parts of radix paeoniae alba, 24 parts of white hyacinth bean, 15 parts of lotus leaf, 9 parts of jujube and 9 parts of raw liquorice.

As a preferred scheme, the traditional Chinese medicine composition can be prepared into decoction, pills, tea, oral liquid, tablets, capsules, granules, paste or powder.

The invention relates to a principle of etiology and pathogenesis analysis and treatment of non-alcoholic steatohepatitis:

according to the theory of "state target differentiation" under the guidance of "state and target combination" theory, NASH mainly uses "phlegm" and "blood stasis", while core "target" is alanine Aminotransferase (ALT), imaging and liver biopsy based on non-alcoholic simple fatty liver disease; the external appearance is serum ALT elevation, gastric cavity fullness, pain in both hypochondriac areas, bitter taste in mouth, loose stool and the like, the imaging accords with diffuse fatty liver diagnosis, the liver histological appearance accords with fatty hepatitis diagnosis, and the internal essence is inflammatory injury of the liver.

According to the theory of "syndrome differentiation of state target", the recipe is based on the "phlegm" state, and is combined with "rhizoma Pinelliae Preparata, rhizoma Atractylodis, coptidis rhizoma, coicis semen parched with bran, curcumae rhizoma, and Concha arcae"; for the "blood stasis" state, the Chinese medicinal materials of peach kernel, salvia root, red peony root, spatholobus stem, carthamus flower, ligusticum root and curcuma root are combined by adopting the method of activating blood and dissolving stasis; the recipe is aimed at "target of" distention and fullness in the stomach and pain in the hypochondrium ", compatibility of" white peony root, trogopterus dung and fingered citron ", compatibility of" target of "bitter taste in the mouth and loose stool", compatibility of "target of" ALT rising "and compatibility of" raw licorice ".

The formula principle and the compatibility rule for treating the nonalcoholic steatohepatitis provided by the invention are as follows:

according to the theory of monarch, minister, assistant and guide under the theoretical guidance of the dialectical treatment and the nature and taste of traditional Chinese medicine, the recipe takes coptis chinensis, rhizoma pinellinae praeparata, rhizoma atractylodis, peach kernel, semen coicis stir-fried with bran, fingered citron, radix curcumae, rhizoma ligustici wallichii, herba artemisiae, radix salviae miltiorrhizae, radix paeoniae rubrathe, rhizoma curcumae zedoariae, concha arcae, trogopterus dung, caulis spatholobi, safflower, radix paeoniae alba, white hyacinth bean, lotus leaf, jujube and raw liquorice as the compatibility to play the role of soothing liver and strengthening spleen, reducing phlegm and removing blood stasis together. In the formula, "rhizoma Pinelliae Preparata, coptidis rhizoma, rhizoma Atractylodis and semen Persicae" are used as monarch agents, and have the effects of removing dampness and resolving phlegm, and promoting blood circulation and removing blood stasis; the bran-fried coix seed, fingered citron, radix curcumae and ligusticum wallichii are used for strengthening spleen and resolving depression and promoting qi circulation, and the virgate wormwood herb, the root of red-rooted salvia, the root of common peony, the curcuma zedoary and the concha arcae are used as ministerial agents for promoting diuresis, activating blood circulation and dissolving phlegm; "Oletum Trogopterori, caulis Spatholobi, carthami flos" has effects of regulating qi-flowing, dredging meridian passage, and "radix Paeoniae alba, semen lablab album, folium Nelumbinis" has effects of softening liver, invigorating spleen, clearing heat, and can be used as adjuvant, and "fructus Jujubae, glycyrrhrizae radix" has effects of invigorating spleen qi, and can be used as medicinal preparation.

Among the monarch agents, the 'prepared pinellia tuber' has pungent taste and is warm in nature and subsidence of temperature, warms and eliminates cold dampness, and is good at clearing and resolving phlegm, eliminating turbid and spleen dampness, lowering adverse qi, relieving vomiting and eliminating masses; with "Huang Lian", the latter is bitter in flavor, cold in nature, low in cost, clear heat and dry dampness, good in removing dampness-heat and fire toxin in middle-jiao, and free in dispersing qi-separating heat accumulation in middle-jiao; the two are compatible, one cold and one heat, and the cold and the heat are mutually used for yin and yang; one bitter and one pungent, pungent and bitter, lowering the qi flow; clear heat and not disturb dampness, dry dampness but not disturb heat; it has the actions of removing dampness, resolving phlegm, relieving stuffiness and stopping vomiting and complement each other. Rhizoma Pinelliae Preparata is combined with rhizoma Atractylodis, which is pungent, warm and dispersed, and is free from being left, and is good at drying dampness and strengthening spleen, and relieving stuffiness and resolving accumulation; the two are combined together to play the roles of drying dampness, resolving phlegm, resolving accumulation, relieving stuffiness, lowering adverse qi and preventing vomiting. The Chinese atractylodes is compatible with coptis, so that the Chinese atractylodes has the effects of warming one cold, bitter one pungent, clearing heat and drying dampness without damaging yang qi, and regulating the ascending, descending, floating and sinking properties and complement each other. The three medicines of rhizoma pinellinae praeparata, coptis chinensis and rhizoma atractylodis are combined, so that the medicine can dry dampness and resolve phlegm, soothe liver and invigorate spleen, regulate qi movement and relieve distension and fullness and accumulation. The theory of traditional Chinese medicine holds that qi, blood and body fluids are homologous, so-called "homologous phlegm and blood stasis" and "homologous phlegm and blood stasis" disease. Phlegm can be generated and can be generated, phlegm and blood stasis can be generated mutually, phlegm is the initial of the blood stasis, and the blood stasis is the deep transformation of the phlegm, and the phlegm and the blood stasis can be generated mutually in a homologous mode. Among the monarch agents, the peach kernel is sweet and bitter in nature, has the effects of relieving stagnant blood, sweet in nature, promoting blood production, moistening body, nourishing intestine and dryness, and is good for dredging channels, removing blood stasis, astringing, breaking blood and eliminating mass; it is combined with "rhizoma Pinelliae Preparata", "Coptidis rhizoma", "rhizoma Atractylodis" to treat phlegm and blood stasis, and has the effects of eliminating dampness and promoting blood circulation, resolving phlegm and removing blood stasis, and also has the effects of dispersing stagnated liver qi, invigorating spleen, resolving phlegm and removing blood stasis.

Among ministerial agents, "bran-fried coix seed" is matched with "herba artemisiae scopariae", the ministerial agents are used for strengthening spleen, eliminating dampness, clearing heat and resolving masses, and the auxiliary monarch agents are used for exerting the effects of drying dampness and strengthening spleen; the curcuma zedoary is matched with concha arcae, so that the effects of resolving food stagnation, resolving masses, promoting qi circulation and resolving phlegm are achieved, and the auxiliary monarch agent plays the roles of relieving stuffiness and resolving phlegm; the red sage root is matched with red paeony root, so as to promote blood circulation to remove blood stasis, promote menstruation and relieve menalgia, and the auxiliary monarch agent plays the role of promoting blood circulation to remove blood stasis; the fingered citron is matched with the curcuma aromatica and the ligusticum wallichii to relieve depression and promote blood circulation, dry dampness and phlegm, and assist the monarch agent in playing the role of soothing liver and regulating qi. The ministerial drugs are compatible and applied to supplement each other, and the multi-layer multidimensional synergy strengthens the root of the monarch drug 'phlegm stasis and treatment'.

In the adjuvant, trogopterus dung is matched with white paeony root, suberect spatholobus stem and safflower, and has the effects of dredging channels, removing blood stasis, softening liver and nourishing blood, and is used for adjuvant treatment of hypochondriac pain due to blood stasis and deficiency of liver yin and liver blood; the white hyacinth bean is matched with lotus leaf, and has the effects of strengthening spleen and regulating stomach, raising clear yang to strengthen yang, raising yang to dispel fire, raising yang to dehumidify, raising yang to relieve depression, and assisting in treating heart lower fullness and spleen deficiency and dampness obstruction of middle energizer. The adjuvant has synergistic effect, and can strengthen the curative effects of the monarch agent and ministerial agent on strengthening spleen and removing dampness, dredging channels and removing blood stasis, and relieve the syndrome of liver-yin blood deficiency and diarrhea.

In the preparation, the Chinese date is matched with the raw licorice to play the roles of introducing the medicines into the channels, tonifying qi and nourishing blood, reinforcing spleen and regulating stomach and harmonizing the medicines.

Drawings

FIG. 1 shows the effect of the formulation of the present invention on the expression of key molecules of TLR4 and TLR9 signaling pathways in PBMCs of patients with NASH;

FIG. 2 is a graph showing the effect of the formulation of the present invention on the secretion of TLR4 signaling pathway effectors in PBMCs of patients with NASH;

FIG. 3 is a graph showing the effect of the formulation of the present invention on the secretion of TLR9 signaling pathway effector molecules in PBMCs of patients with NASH;

FIG. 4 shows the protective effect of the traditional Chinese medicine formulation on liver injury of NASH mice;

FIG. 5 shows the effect of the Chinese medicinal composition on the Th17 cell differentiation immune inducing molecule of NASH mouse;

FIG. 6 shows the effect of the traditional Chinese medicine formulation on the Th17 cell differentiation immunosuppressive molecule of NASH mice;

FIG. 7 shows the effect of the traditional Chinese medicine formulation on the Th17 cell differentiation transcription factor of NASH mice;

FIG. 8 shows the effect of the traditional Chinese medicine composition on the expression of the functional molecules of the Th17 cells of the NASH mice, wherein, the A diagram shows the effect of the traditional Chinese medicine composition on the expression of the functional molecules of the Th17 cells of the NASH mice, and the B diagram shows the effect of the traditional Chinese medicine composition on the secretion of the functional molecules of the Th17 cells of the NASH mice;

FIG. 9 shows the effect of a traditional Chinese medicine formula on the expression of NASH mouse hepatocyte IL-17R signaling pathway molecules, wherein A shows the effect of a traditional Chinese medicine formula on the expression of NASH mouse hepatocyte IL-17R signaling pathway, B shows the effect of a traditional Chinese medicine formula on the expression of a chemokine gene downstream of the NASH mouse hepatocyte IL-17R signaling pathway, C shows the effect of a traditional Chinese medicine formula on the secretion level of NASH mouse chemokines, and D shows the effect of a traditional Chinese medicine formula on IL-17R in vitro induced NASH mouse hepatocyte chemokines;

FIG. 10 shows the effect of a traditional Chinese medicine formulation on the expression of key constitutive molecules of the signaling pathways of TLR2, TLR4, TLR7 and TLR9 of the liver of a NASH mouse, wherein A shows the effect of a traditional Chinese medicine formulation on the TLR of the liver of a NASH mouse _S Influence of receptor expression, panel B shows that the traditional Chinese medicine formula has no effect on TLR (toll-like receptor) of liver of NASH mouse _S The influence of the expression of signal path joint molecule on TLR of liver withered cell of NASH mouse by Chinese medicine composition _S Influence of expression of transcription factors of the signal pathway;

FIG. 11 is a graph showing the effect of a traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver, wherein A is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the expression of interleukin-like effector molecules, B is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the secretion level of interleukin-like effector molecules, C is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the expression of TNF-alpha, TGF-beta 1 and interferon-like effector molecules, D is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the secretion level of TNF-alpha, TGF-beta 1 and interferon-like effector molecules, E is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the secretion level of matrix metalloproteinase effector molecules, F is the effect of the traditional Chinese medicine formula on the TLR2 signaling pathway of NASH mouse liver to mediate the secretion level of matrix metalloproteinase-like effector molecules;

FIG. 12 is a graph showing the effect of a traditional Chinese medicine formulation on the expression of NASH mouse liver-up or not, wherein A is the effect of a traditional Chinese medicine formulation on the expression of NASH mouse liver-up or not, B is the effect of a traditional Chinese medicine formulation on the secretion level of NASH mouse liver-up or not, TLR4 is the effect of a traditional Chinese medicine formulation on the secretion level of NASH mouse liver-up or not, C is the effect of a traditional Chinese medicine formulation on the expression of NASH mouse liver-up or not, TLR4 is the effect of a traditional Chinese medicine formulation on the expression of TNF-alpha, TGF-beta 1 and interferon effector molecules, D is the effect of a traditional Chinese medicine formulation on the secretion level of NASH mouse liver-up or not, E is the effect of a traditional Chinese medicine formulation on the expression of NASH mouse liver-up or not, F is the effect of a traditional Chinese medicine formulation on the secretion level of a NASH mouse liver-up or not, G is the effect of a traditional Chinese medicine formulation on the secretion level of a mouse liver-up or not, and D is the effect of a mouse liver-up or not;

FIG. 13 is a graph showing the effect of a traditional Chinese medicine formula on the expression of NASH mouse liver-drying cell HGF, wherein graph A shows the effect of a traditional Chinese medicine formula on the expression of NASH mouse liver-drying cell HGF gene, and graph B shows the effect of a traditional Chinese medicine formula on the level of NASH mouse HGF protein;

fig. 14 shows the effect of the traditional Chinese medicine composition on the expression of the ALR in the liver cells of the NASH mice, wherein the A diagram shows the effect of the traditional Chinese medicine composition on the expression of the ALR gene in the liver cells of the NASH mice, and the B diagram shows the effect of the traditional Chinese medicine composition on the ALR protein level of the NASH mice.

Detailed Description

The invention is further described below with reference to the drawings and the detailed description.

1. Examples:

example 1:

20g of coptis chinensis, 12g of rhizoma pinellinae praeparata, 15g of rhizoma atractylodis, 18g of peach kernel, 20g of semen coicis stir-baked with bran, 15g of fingered citron, 15g of radix curcumae, 9g of ligusticum wallichii, 18g of herba artemisiae capillaris, 20g of radix salviae miltiorrhizae, 18g of radix paeoniae rubra, 12g of rhizoma curcumae, 12g of concha arcae, 10g of trogopterus dung, 10g of caulis spatholobi, 10g of safflower, 15g of radix paeoniae alba, 15g of white hyacinth bean, 9g of lotus leaf, 9g of jujube and 9g of raw liquorice.

Example 2:

24g of coptis chinensis, 18g of rhizoma pinellinae praeparata, 18g of rhizoma atractylodis, 24g of peach kernel, 25g of coix seed fried with bran, 20g of fingered citron, 20g of radix curcumae, 18g of ligusticum wallichii, 25g of herba artemisiae capillaries, 27g of radix salviae miltiorrhizae, 24g of radix paeoniae rubra, 24g of rhizoma curcumae, 30g of concha arcae, 18g of trogopterus dung, 18g of caulis spatholobi, 12g of safflower, 30g of radix paeoniae alba, 24g of white hyacinth bean, 15g of lotus leaf, 9g of jujube and 9g of raw liquorice.

Example 3:

30g of coptis chinensis, 24g of rhizoma pinellinae praeparata, 25g of rhizoma atractylodis, 30g of peach kernel, 30g of semen coicis stir-fried with bran, 30g of fingered citron, 30g of radix curcumae, 18g of ligusticum wallichii, 30g of herba artemisiae capillaris, 30g of radix salviae miltiorrhizae, 18-30g of radix paeoniae rubra, 30g of rhizoma curcumae, 30g of concha arcae, 24g of trogopterus dung, 26g of caulis spatholobi, 18g of safflower, 30g of radix paeoniae alba, 30g of white lablab seed, 18g of lotus leaf, 15g of jujube and 12g of raw liquorice.

Example 4:

20g of coptis chinensis, 18g of rhizoma pinellinae praeparata, 20g of rhizoma atractylodis, 24g of peach kernel, 20g of semen coicis stir-fried with bran, 20g of fingered citron, 20g of radix curcumae, 12g of ligusticum wallichii, 18g of herba artemisiae capillaris, 20g of radix salviae miltiorrhizae, 18g of radix paeoniae rubra, 18g of rhizoma curcumae, 20g of concha arcae, 20g of trogopterus dung, 18g of caulis spatholobi, 12g of safflower, 30g of radix paeoniae alba, 20g of white hyacinth bean, 18g of lotus leaf, 9g of jujube and 9g of raw liquorice.

The Chinese medicinal composition can be made into decoction, pill, tea, oral liquid, tablet, capsule, granule, unguent or powder.

2. Clinical experiments:

1. general data and study design:

according to the clinical guidelines of the related non-alcoholic fatty liver disease, which were set by the American society of liver disease, the American society of gastroenteropathy, the European society of liver disease, the Chinese society of medical science, the fatty liver and alcoholic liver disease groups, the Chinese society of fatty liver disease expert committee, the clinical study was designed by parallel randomized controlled study, the treatment group and the control group of the formula of the invention were included in a 1:1 ratio, and 104 NASH patients were collected altogether, wherein the lipid-lowering chemical soup group was 52, and the control group was 52. Patients all come from the affiliated hospitals of Gansu traditional Chinese medicine university, and the study is approved by the ethical committee of the affiliated hospitals of Gansu traditional Chinese medicine university, and the patients agree with the knowledge.

2. Inclusion criteria and exclusion criteria:

patient inclusion criteria were strictly followed by the clinical guidelines described above, with color ultrasound examination showing the presence of fatty liver and blood examination showing the presence of liver inflammatory lesions. Exclusion criteria included patients with diabetes, pregnant and lactating mothers, viral hepatitis, autoimmune liver disease, alcohol consumption, cirrhosis, hepatocellular carcinoma or any other cause of liver disease.

3. Treatment protocol: the formula treatment group comprises: giving a healthy lifestyle change; meanwhile, the decoction of the formula example 2 is continuously taken for 8 weeks at 3/day; the study period prohibits the use of any other liver disease-related drugs; control group: giving a healthy lifestyle change for 8 consecutive weeks; the study period was prohibited from using any other liver disease related drugs. Taking blood before and after treatment on an empty stomach, separating serum, and detecting relevant enzymology indexes and inflammatory cytokine indexes of the serum; peripheral blood PBMCs were isolated, cellular mRNA was extracted, and the levels of key molecular gene expression were detected using real-time quantitative polymerase chain reaction (real-time PCR).

4. Study and observation indexes:

(1) Clinical efficacy study:

detecting serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), triglyceride (TG), total Cholesterol (CHOL), albumin (ALB), total bilirubin (T-Bil), blood glucose (Glu); calculating Body Mass Index (BMI) to evaluate the fat and thin degree of the patient; the fibriscan test controls the attenuation index (Cap) to assess the liver fat content of the patient, calculates the steady state model insulin resistance index (HOMA-IR) to assess the insulin resistance level of the patient.

(2) Inflammatory mechanism study:

(1) peripheral blood inflammatory cytokine levels: detection of serum Interleukin 6 (IL-6), IL-1 beta, IL-12, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), C-X-C motif chemokine 10 (CXCL 10), MMP9

(2) TLR4 and TLR9 signaling pathway expression:

detecting the expression level of TLR4 and TLR9 signaling pathway key molecules TLR4, TLR9, myeloid differentiation factor 88 (MyD 88), tumor necrosis factor receptor-related factor 6 (TRAF 6) and nuclear factor κB (NF- κB) in Peripheral Blood Mononuclear Cells (PBMCs), and interferon regulatory factor 7 (IRF 7) mRNA.

(3) TLR4 and TLR9 signaling pathway functions:

PBMCs are extracted, and stimulated by TLR4 specific ligand Lipopolysaccharide (LPS) and TLR9 specific ligand (CpG OND 2216) respectively, and an enzyme-linked immunosorbent assay (ELISA) is used for detecting secretion levels of inflammatory cytokines IL-6, IL-1 beta, IL-12, TNF-alpha, IFN-gamma, CXCL10 and MMP 9.

5. Statistical analysis:

statistics were performed using SPSS16.0 analysis software. The metering data are expressed in terms of mean ± standard deviation, or in terms of median (25% quartile, 75% quartile). Analyzing normal distribution by inter-group variation, if the normal distribution is met, dividing by adopting independent sample t test; if the normal distribution is not met, a non-parametric test is used. A two-sided assay standard was used with P <0.05 as the statistical difference.

6. Results of the study

(1) Clinical study of liver protection and insulin resistance of NASH patient by the formula of the invention

(1) Patient baseline data:

note that: data are expressed in mean ± standard deviation, or median (25% quartile, 75% quartile)

(2) Patient group baseline data:

(3) liver function, blood lipid, HOMA-IR and Cap of the patient after treatment:

(4) comparison of the extent of change in serum ALT and AST of patients after treatment:

the data show that the formula of the invention has remarkable liver protection effect on NASH patients, and effectively improves insulin resistance level, liver fat deposition degree and blood lipid level of the patients, and is characterized in that ALT, AST, TG, HOMA-IR and Cap of the patients are remarkably reduced before and after treatment, and compared with a control group, the formula has statistical difference (P < 0.01). After healthy lifestyle changes, the control group had statistical differences in serum ALT compared to AST before change (P < 0.01); the degree of ALT and AST changes before and after treatment was analyzed in the control and drug groups, and the data showed that the drug group promoted recovery of liver function significantly higher than the control group and had a statistical difference (P < 0.01).

(2) Clinical study of the formulation of the invention on the level of inflammatory cytokine Release in NASH patients

(1) General data

Patient baseline profile related data are as above.

(2) Experimental method

The control group and the drug group patients with the formula of the invention collect blood from veins at baseline and 8 weeks of intervention, extract plasma, and analyze the content of IL-6, IL-1 beta, IL-12, TNF-alpha, IFN-gamma, CXCL10 and MMP9 in the plasma by ELISA, and analyze the change level of inflammatory cytokines before and after the intervention of the two groups.

(3) Experimental results

The data show that the formula has remarkable effect on downregulating the inflammatory cytokines of the organism of the NASH patient, and is characterized in that the serum IL-6, IL-1 beta, IL-12, TNF-alpha, IFN-gamma, CXCL10 and MMP9 of the patient are obviously reduced before and after treatment; meanwhile, the pharmaceutical group with the formula has statistical difference (P < 0.01) compared with the reduction amplitude of inflammatory cytokine indexes before and after intervention of a control group.

The prescription of the invention can reduce the liver inflammatory injury of the NASH patient by down regulating the release of the organism inflammatory cytokines, thereby exerting the liver protection effect, and possibly being one of the action mechanisms.

(3) Clinical study of the Effect of Lipidation chemical fiber decoction on TLR4 and TLR9 Signal paths in peripheral blood PBMCs of NASH patients

(1) General data: patient baseline profile related data are as above.

(2) Experimental method

Control and drug group patients were bled at baseline and 8 weeks of intervention, PBMCs were extracted, and real-time quantitative polymerase chain reaction (real-time PCR) techniques were used to detect TLR4 and TLR9 signaling pathway key molecule expression, including myeloid differentiation factor 88 (MyD 88), tumor necrosis factor receptor associated factor 6 (TRAF 6), nuclear factor κb (NF- κb), interferon regulatory factor 7 (IRF 7) gene expression levels;

patients in the control group and the drug group are subjected to baseline and intervention for 8 weeks, blood is collected by veins, PBMCs are extracted for in vitro cell culture, and TLR4 specific ligand LPS is respectively addedEscherichia coli055:B5) and TLR9 specific ligand CpG ODN2216, stimulation for 24h, ELISA analysis to detect the content of IL-6, IL-1 beta, IL-12p40, TNF-alpha, IFN-gamma and CXCL10 in the cell culture supernatant.

(3) Experimental results

As shown in the data of fig. 1, compared with baseline 0w, the inventive formula pharmaceutical group 8w significantly down-regulates gene expression of TLR4 and TLR9 signaling pathway key molecules, with statistical difference (P < 0.01); the gene expression of TLR4 and TLR9 signaling pathway key molecules did not show significant changes in control 8w compared to baseline 0 w.

As the data in fig. 2 show, the inventive formulation drug group 8w significantly down-regulates TLR4 signaling pathway inflammatory cytokine secretion compared to baseline 0w, with statistical differences (P < 0.01); control 8w showed no significant change in TLR4 signaling pathway inflammatory cytokine secretion compared to baseline 0 w.

As the data in fig. 3 show, the inventive formulation drug group 8w significantly down-regulates TLR9 signaling pathway inflammatory cytokine secretion compared to baseline 0w, with statistical differences (P < 0.01); control 8w showed no significant change in TLR9 signaling pathway inflammatory cytokine secretion compared to baseline 0 w.

Researches show that the formula of the invention can down regulate the expression and the function of TLR4 and TLR9 signal channels of organisms and can effectively improve the injury of the NASH liver.

3. Animal experiment:

study method

1. Experimental animal and experimental place

SPF grade C57BL/6 mice, male, 6-8 weeks old, 18-22 g, provided by the university of Lanzhou laboratory animal center (license number: SCXK (Glycine) 2018-0002; quality detection unit: national laboratory animal quality detection center); all experiments were done at the university of Lanzhou GLP medical laboratory center.

2. Animal model preparation:

preparing a quasi-induced phlegm-blood stasis interengaged non-alcoholic steatohepatitis mouse model according to an animal model: taking healthy C57BL/6 mice, feeding the mice with high-fat feed, and freely drinking water; the squirrel cage is paved with wet broken wood shavings and is placed in a molding box, the bottom Cheng Manshui of the box is provided with vent holes, the temperature of the molding box is kept at (33+/-1) DEG C, and the relative humidity is kept at (95+/-3)%; unpredictable random noise stimulation, blinking glare stimulation, light and dark stimulation were applied 1 time each week. The molding was continued for 8w.

3. Grouping and intervening animals:

(1) Blank control (kbdz): normal feeding of healthy C57BL/6 mice was standardized; mice were sacrificed at 12w end and relevant indicators were detected. (2) animal model group (DWMXZ): strictly preparing an animal model; mice were sacrificed at end 8w and the relevant index was detected. (3) animal model control group (mxdz): strictly preparing an animal model; mice recovered from standardized normal feeding after 8 w; lavage with normal saline for 2 times/d for 4w; mice were sacrificed at 12w end and relevant indicators were detected. (4) The pharmaceutical intervention group (YWGYZ) strictly performs animal model preparation; mice recovered from standardized normal feeding after 8 w; the drug concentrate for gastric lavage experiment is 2 times/d for 4w; mice were sacrificed at 12w end and relevant indicators were detected.

4. Drug formulation, preparation and conversion:

the experiment adopts the traditional Chinese medicine composition of the embodiment 2, and adopts a water decoction method, wherein the concha arcae is decocted for 30min to obtain the standard water decoction.

According to the dose conversion coefficient table of each kilogram of body weight of animals and human bodies and the calculation formula: dosage of animals B (mg/kg) =dosage of animals w×a (mg/kg), the mice lavage drug dosage was calculated as: 0.90g/20g mice.

5. Sample collection:

at the end of 12w, mice were fasted for 12h, were free to drink water, and were given general anesthesia by intraperitoneal injection of 10% chloral hydrate. After the mice were anesthetized, blood was collected from the orbital venous plexus, and serum was prepared. Fully exposing the liver of the mouse under the aseptic low-temperature condition, and taking the right-leaf tissue of the liver to prepare liver tissue homogenate; placing left leaf of liver in ice PBS, low-temperature mechanical milling, and performing Percoll density gradient centrifugation to separate liver lymphocyte; taking the middle leaf of the liver of the mouse, placing the middle leaf in ice PBS for flushing, completely digesting the digestive juice, sieving, collecting and purifying to prepare the primary cells of the liver.

6. Statistical analysis:

analysis is carried out by adopting SPSS.22 statistical software, and the data of normal distribution of experimental data is obtained

To show, the comparison between groups uses one-way analysis of variance and LSD to make the comparison between groups, and the data of non-normal distribution is represented by M (P ₂₅ ，P ₇₅ ) Representing, inter-group non-parametric test with independent samples, P<The 0.05 difference is statistically significant.

(II) experimental project:

experiment one, chinese medicinal composition for protecting liver injury of NASH mice

1. Detection reagent, sample, method and instrument

ALT, AST, ALP, GGT, T-BIL, ALB, CHOL, TG, BS; the kit is produced in Nanjing to build a bioengineering institute; the detection sample is mouse serum; the detection method is a colorimetric analysis method; the detection instrument was an Olinbas full-automatic biochemical analyzer (AU 640, japan). All assays were performed strictly according to the kit instructions.

2. Conclusion of the experiment

The experimental study shows that the traditional Chinese medicine composition can effectively protect liver parenchymal cells and biliary tract system injury, promote liver biotransformation function, promote recovery and balance of liver substance metabolism function, and promote repair of NASH liver tissue cell injury in multiple targets, multiple angles and multiple layers, and promote restoration of liver function injury.

Experiment II, revealing the protection mechanism of traditional Chinese medicine formula on liver of NASH mice based on Th17 cell differentiation and functions

1. Detection reagent, sample, method and instrument

Rorγt, IL-17, IL-21, IL-22, TNF- α, IL-17RA, act1, TRAF6, NF- κ B, CXCL9, CXCL10, CXCL11 mRNA, detected by real-time PCR technology, and the kit is derived from TaKaRa Bio Inc, japan; the detection instrument is a PCR instrument (Bio-Rad cycler IQ5, USA); the detection sample is mouse liver lymphocytes; primers were synthesized in Beijing Bomaide Biotechnology Co.

TGF-beta 1, IL-6, IL-23, IL-1 beta, IL-2, IFN-gamma, IL-4, RORgamma t, IL-22, IL-17, IL-21, TNF-alpha, CXCL9, CXCL10 and CXCL11 proteins are detected by ELISA detection methods. All assays were performed strictly according to the kit instructions.

2. Conclusion of the experiment

Th17 cell infiltration is one of the pathological features of NASH liver. Th17 cells infiltrate the liver and secrete a special functional cytokine IL-17, and through an IL-17R mediated cell signaling pathway, the cascade secretion effect of inflammatory factors of the liver is amplified, and pericyte infiltration is recruited, so that the generation and development of complex inflammatory lesions of the liver are induced and promoted. According to the experimental data shown in fig. 5-9, the traditional Chinese medicine composition can effectively regulate the secretion of Th17 cell differentiation induction factors and Th17 cell differentiation inhibition factors, down regulate the expression of nuclear transcription factors RORγt, inhibit the differentiation of Th17 cells and reduce the release of functional effector molecules; meanwhile, the traditional Chinese medicine formula can down regulate the expression of IL-17R mediated cell signal pathway key molecules, inhibit the cell signal pathway function, and reveal the mechanism of the traditional Chinese medicine formula for playing the role of protecting the liver of the NASH through multiple targets, angles and planes.

Experiment III, study of Chinese medicinal formula for inhibiting expression and function of Toll-like receptor (TLRs) signaling pathway of liver of NASH mouse

1. Detection reagent, sample, method and instrument

TLR2, TLR4, TLR7, TLR9, myD88, TRAF6, NF-. Kappa. B, c-Jun, IRF3, IRF7 mRNA, and detecting by real-time PCR detection method.

IL-6, IL-1 beta, IL-12p40, TNF-alpha, TGF-beta 1, IFN-beta, IFN-gamma, MMP2, MMP3, MMP9 mRNA, using real-time PCR detection method.

IL-6, IL-1 beta, IL-12p40, TNF-alpha, TGF-beta 1, IFN-beta, IFN-gamma, MMP2, MMP3, MMP9, CXCL10, CXCL11 proteins, and detection by ELISA detection method.

2. Conclusion of the experiment

NASH is closely related to intestinal microbial disorders and hepatic cell injury stimulation, and can activate cellular signaling pathways mediated by hepatic stem cells TLRs to promote NASH progression and exacerbation. According to the experimental data shown in FIGS. 10-12, the traditional Chinese medicine composition can effectively improve the expression and abnormal secretion of TLR2, TLR4, TLR7 and TLR9 mediated cell signaling pathways, effectively improve the abnormal secretion of L-6, IL-1 beta, IL-12p40, TNF-alpha, TGF-beta 1, IFN-beta, IFN-gamma, MMP2, MMP3, MMP9 and CXCL9, CXCL10 and CXCL11, protect liver cells and promote liver function restoration by multi-target, multi-angle and multi-layer inhibition of NASH liver inflammation injury, and the traditional Chinese medicine composition disclosed by the invention takes TLRs signaling pathways as the center and plays a role in liver protection (because the statistical characteristics of the data results of TLR7 and TLR9 are similar to that of TLR2 and TLR4, the data diagram is not listed)

Research on influence of traditional Chinese medicine formula on improving liver cell regeneration capability of NASH mice

1. Detection reagent, sample, method and instrument

HGF and ALR mRNA expression are detected by adopting a real-time PCR detection method.

HGF and ALR protein expression are detected by ELISA detection method. All assays were performed strictly according to the kit instructions.

2. Conclusion of the experiment

Chronic liver inflammation impairs the normal regenerative capacity of liver cells, induces the occurrence and accumulation of liver fibrosis processes, and eventually causes adverse consequences such as cirrhosis. According to the experimental data shown in fig. 13-14, the traditional Chinese medicine formula disclosed by the invention can play a role in regenerating and repairing liver cells through multiple targets, multiple angles and multiple planes by improving abnormal expression and secretion of HGF molecules of liver dead cells and ALR molecules of liver cells of NASH mice, and can promote liver function recovery, and one of the mechanisms of the traditional Chinese medicine formula disclosed by the invention for playing a role in protecting liver is disclosed.

Claims

1. The traditional Chinese medicine composition for treating the nonalcoholic steatohepatitis is characterized by being prepared from the following raw materials in parts by weight: 20-30 parts of coptis chinensis, 12-24 parts of rhizoma pinellinae praeparata, 15-25 parts of rhizoma atractylodis, 18-30 parts of peach kernel, 20-30 parts of semen coicis stir-fried with bran, 15-30 parts of fingered citron, 15-30 parts of radix curcumae, 9-18 parts of rhizoma ligustici wallichii, 18-30 parts of herba artemisiae capillaris, 20-30 parts of radix salviae miltiorrhizae, 18-30 parts of radix paeoniae rubra, 12-30 parts of rhizoma curcumae, 12-30 parts of concha arcae, 10-24 parts of trogopterus dung, 10-26 parts of caulis spatholobi, 10-18 parts of safflower, 15-30 parts of white paeony root, 15-30 parts of white hyacinth bean, 9-18 parts of lotus leaf, 9-15 parts of jujube and 9-12 parts of raw liquorice.

2. The traditional Chinese medicine composition for treating non-alcoholic steatohepatitis according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 24 parts of coptis chinensis, 18 parts of rhizoma pinellinae praeparata, 18 parts of rhizoma atractylodis, 24 parts of peach kernel, 25 parts of semen coicis stir-fried with bran, 20 parts of fingered citron, 20 parts of radix curcumae, 18 parts of ligusticum wallichii, 25 parts of herba artemisiae capillaries, 27 parts of radix salviae miltiorrhizae, 24 parts of radix paeoniae rubra, 24 parts of rhizoma curcumae, 30 parts of concha arcae, 18 parts of trogopterus dung, 18 parts of caulis spatholobi, 12 parts of safflower, 30 parts of radix paeoniae alba, 24 parts of white hyacinth bean, 15 parts of lotus leaf, 9 parts of jujube and 9 parts of raw liquorice.

3. The traditional Chinese medicine composition for treating non-alcoholic steatohepatitis according to claim 1 or 2, wherein: it can be made into decoction, pill, tea, oral liquid, tablet, capsule, granule, unguent or powder.