A pharmaceutical composition for treating non-alcoholic fatty liver disease, liver fibrosis and / or liver cirrhosis, a preparation method thereof, and uses thereof

Abstract

The present invention discloses a pharmaceutical composition for treating non-alcoholic steatohepatitis, liver fibrosis and / or liver cirrhosis, which is prepared from the following raw materials in the following weight ratios: 15-50 parts of Astragalus membranaceus, 5-25 parts of turtle shell, 5-25 parts of Angelica sinensis, 10-30 parts of safflower, 10-30 parts of peach kernel, and 1-10 parts of liquorice. The pharmaceutical composition of the present invention for treating non-alcoholic steatohepatitis can reduce serum transaminase, and significantly reduce lipid deposition, liver tissue inflammation and collagen fiber deposition; the composition of the present invention for treating liver fibrosis can significantly reduce liver tissue pathological inflammation and collagen fiber deposition, as well as the numerical values of related indexes such as serum liver function ALT and AST, and at the same time significantly down-regulate the numerical values of related indexes such as serum PⅢP, PCⅣ, HA and LN, significantly inhibit the activation of hepatic stellate cells (HSCs), the central link of liver fibrosis, significantly reduce the activation of NLRP3 inflammasome in liver tissue, reduce HSC autophagy and activation, and has a good therapeutic effect on both liver fibrosis and liver cirrhosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a pharmaceutical composition for treating liver fibrosis, a preparation method thereof and uses thereof. Background Art

[0002] Non-alcoholic fatty liver disease is a common liver disease, which can evolve from simple steatosis to non-alcoholic steatohepatitis (NASH), increasing the risk of serious chronic diseases such as type 2 diabetes and cardiovascular diseases. In recent years, the incidence of non-alcoholic fatty liver disease in China has been gradually increasing, and it can further develop into liver fibrosis. Data shows that about 20% of NASH patients will develop into cirrhosis or even liver cancer.

[0003] Liver fibrosis is a common pathological change process in most chronic liver diseases. Without timely intervention, it may progress to cirrhosis and hepatocellular carcinoma. Cirrhosis caused by liver fibrosis affects nearly 2% of the global population. Globally, 70%-90% of hepatocellular carcinoma cases are based on cirrhosis. China is one of the countries with a high incidence of chronic liver diseases, and the incidence of hepatitis B ranks first in the world. The occurrence of liver fibrosis and its related diseases has greatly increased the economic and medical burden and has become one of the major public health events that cannot be ignored. Actively intervening and reversing liver fibrosis and controlling the progression of cirrhosis are urgent. The treatment of liver fibrosis mainly aims to reduce or even reverse fibrosis according to its pathogenesis, mainly from aspects such as anti-inflammatory, inhibiting the proliferation and activation of HSCs, and immunomodulation. Currently, the anti-liver fibrosis candidate drugs at the research forefront mainly include OCA, resmetirom, CVC, selonsertib and elafibranor. Although effective in experimental animal models, they are limited in clinical trials. Currently, there is no approved specific drug for liver fibrosis. Liver transplantation is the only option for advanced liver diseases.

[0004] Traditional Chinese medicine does not have the names of non-alcoholic fatty liver disease, liver fibrosis and cirrhosis. According to its main symptom manifestations, it can be classified into the categories of diseases such as "abdominal mass", "abdominal distension", "hypochondriac pain", "accumulation", "liver mass", etc. In recent years, traditional Chinese medicine has accumulated rich experience in treating non-alcoholic fatty liver disease, liver fibrosis and cirrhosis by virtue of its advantages of multi-target, good curative effect, low price and high safety, providing another option for the treatment of chronic liver diseases. For example, patent CN113262283A discloses a traditional Chinese medicine composition for improving the liver function and liver fibrosis of patients with cirrhosis, but it only plays the roles of softening hard masses, replenishing qi and strengthening the spleen, promoting blood circulation to remove stasis, nourishing yin to promote fluid production, clearing heat and detoxifying, promoting diuresis to remove jaundice, strengthening healthy qi to eliminate pathogenic factors, and treating both the liver and the spleen, so as to improve the quality of life of patients with liver fibrosis or cirrhosis, and does not have a substantial therapeutic effect on the treatment of chronic liver diseases such as non-alcoholic fatty liver disease and liver fibrosis. Summary of the Invention

[0005] To solve the above problems, the present invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, liver fibrosis and / or liver cirrhosis, which is prepared from the following raw materials in the following weight ratios:

[0006] Astragalus membranaceus 15 - 50 parts, turtle shell 5 - 25 parts, angelica sinensis 5 - 25 parts, safflower 10 - 30 parts, peach kernel 10 - 30 parts, licorice 1 - 10 parts.

[0007] Furthermore, it is prepared from the following raw materials in the following weight ratios:

[0008] Astragalus membranaceus 30 parts, turtle shell 15 parts, angelica sinensis 9 parts, safflower 18 parts, peach kernel 18 parts, licorice 5 parts.

[0009] The present invention also provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, liver fibrosis and / or liver cirrhosis, which is prepared from the following raw materials in the following weight ratios:

[0010] Astragalus membranaceus 15 - 50 parts, turtle shell 5 - 25 parts, angelica sinensis 5 - 25 parts, safflower 10 - 30 parts, peach kernel 10 - 30 parts, spina gleditsiae 5 - 25 parts, zedoary 5 - 25 parts, ground beetle 5 - 25 parts, licorice 1 - 10 parts.

[0011] Furthermore, it is prepared from the following raw materials in the following weight ratios:

[0012] Astragalus membranaceus 25 - 35 parts, turtle shell 10 - 20 parts, angelica sinensis 7 - 13 parts, safflower 15 - 25 parts, peach kernel 15 - 25 parts, spina gleditsiae 10 - 20 parts, zedoary 10 - 20 parts, ground beetle 8 - 12 parts, licorice 2 - 7 parts.

[0013] Furthermore, it is prepared from the following raw materials in the following weight ratios:

[0014] Astragalus membranaceus 30 parts, turtle shell 15 parts, angelica sinensis 9 parts, safflower 18 parts, peach kernel 18 parts, spina gleditsiae 15 parts, zedoary 15 parts, ground beetle 10 parts, licorice 5 parts.

[0015] The present invention also provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, liver fibrosis and / or liver cirrhosis, which is prepared from the following raw materials in the following weight ratios:

[0016] Astragalus membranaceus 20 - 50 parts, turtle shell 5 - 25 parts, angelica sinensis 5 - 25 parts, safflower 10 - 30 parts, peach kernel 10 - 30 parts, salvia miltiorrhiza 3 - 20 parts, leech 3 - 20 parts, ground beetle 5 - 25 parts, licorice 1 - 10 parts.

[0017] Furthermore, it is prepared from the following raw materials in the following weight ratios:

[0018] 30 parts of Astragalus membranaceus, 15 parts of turtle shell, 9 parts of Angelica sinensis, 18 parts of safflower, 18 parts of peach kernel, 10 parts of Salvia miltiorrhiza, 5 parts of Hirudo, 10 parts of Eupolyphaga seu Steleophaga, 5 parts of Glycyrrhiza uralensis Fisch.

[0019] The present invention also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the following steps:

[0020] S1: Weigh the raw materials according to the ratio, mix them, add 8 - 10 times the amount (g / g) of water, soak for 0.5 - 1 hour, decoct twice, each decoction for 1 - 1.5 hours, filter, combine the filtrates, concentrate into an extract, dry in vacuum, and pulverize to obtain a dry extract powder;

[0021] S2: Add the dry extract powder obtained in step S2 to pharmaceutically acceptable excipients to prepare a preparation.

[0022] The present invention also provides the use of the aforementioned pharmaceutical composition in the preparation of a medicament for treating non - alcoholic fatty liver disease.

[0023] The present invention also provides the use of the aforementioned pharmaceutical composition in the preparation of a medicament for treating liver fibrosis.

[0024] Finally, the present invention provides the use of the aforementioned pharmaceutical composition in the preparation of a medicament for treating liver cirrhosis.

[0025] The pharmaceutical composition of the present invention is based on the pathogenesis of non - alcoholic fatty liver disease and liver fibrosis, which involves the basic mechanism of the disease due to deficiency of healthy qi and retention of pathogenic factors, long - term retention of pathogenic toxins, qi stagnation and blood stasis, and damage to the liver collaterals. It particularly emphasizes stasis and deficiency. "Blood stasis is the body of accumulation, and deficiency is the root of accumulation". And it puts forward the theory of collateral disease, believing that the pathogenesis is caused by the invasion of pathogenic toxins, deficiency of healthy qi, retention of pathogenic toxins in the liver meridian, deepening into the blood collaterals over time, obstruction of collaterals, spasm of meridians, resulting in obstruction of liver collaterals, thus forming non - alcoholic fatty liver disease and liver fibrosis. The major clinical treatment principle is to strengthen healthy qi and dredge collaterals, and the pharmaceutical composition of the present invention is optimized and formed accordingly.

[0026] In the formula of the present invention, Astragalus membranaceus is used in large amounts as the monarch drug. Because it tastes sweet and is slightly warm in nature, and is "good at replenishing qi", it is an important drug for replenishing qi and tonifying deficiency, and is used for qi deficiency or qi and blood deficiency syndromes. The minister drug is Trionyx sinensis carapace, which tastes salty and is flat in nature. Saltiness is used to soften hardness and disperse nodules. It is combined with peach kernels and safflower. Peach kernels are bitter and flat in taste, enter the blood aspect and have a strong effect on promoting blood circulation and removing blood stasis. They can also lead the pathogenic factors in the jueyin blood aspect downward. Safflower has a pungent and warm smell, enters the liver meridian blood aspect, can promote blood circulation and break stasis, activate blood circulation and dredge the meridian. It was anciently called safflower. Angelica sinensis is used as an assistant drug. It tastes sweet and is warm in nature. It can both generate blood and promote blood circulation. It is a holy drug for nourishing and activating blood circulation, and runs along the jueyin liver meridian, enters the jueyin blood aspect, and can nourish blood, activate blood circulation and dredge collaterals. Sparganium stoloniferum tastes light, and Curcuma zedoaria is slightly bitter. Both have a pungent taste and are slightly warm in nature. They are often used together as important drugs for removing stasis. Sparganium stoloniferum is good at removing stasis, and Curcuma zedoaria is good at promoting qi movement. Their combination has a strong effect on promoting qi movement, removing stasis and softening hardness, and can "slowly eliminate even the stasis as hard as iron stone". When used together with Trionyx sinensis carapace, they can help the effect of dispersing nodules and dredging collaterals. Eupolyphaga seu Steleophaga can enter the collaterals to search for pathogenic factors. Licorice tastes sweet and is flat in nature. Its nature is slow and it is stronger in relieving various emergencies and harmonizing various drugs. The above-mentioned drugs are combined to achieve the effects of supplementing qi and promoting blood circulation, strengthening the healthy qi and dredging collaterals.

[0027] In order to better realize different dosage forms, pharmaceutically acceptable excipients need to be added during the preparation of these dosage forms, such as: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc. Disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked carboxymethyl cellulose sodium, etc. Lubricants include: magnesium stearate, sodium lauryl sulfate, talc powder, silicon dioxide, etc. Suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc. Binders include, starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc. Sweeteners include: sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc. Flavoring agents include: sweeteners and various flavors. Preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, etc.

[0028] The beneficial effects of the present invention are as follows: Experimental studies have shown that when the pharmaceutical composition of the present invention is used to treat non-alcoholic steatohepatitis, it can reduce serum transaminases, significantly reduce lipid deposition, liver tissue inflammation and collagen fiber deposition; when the composition of the present invention is used to treat liver fibrosis, it can significantly reduce liver tissue inflammation, reduce collagen fiber deposition in liver tissue, significantly reduce the liver index of rats, significantly reduce the numerical values of related indicators such as serum liver function ALT and AST in rats, significantly down-regulate the numerical values of related indicators such as serum PⅢP, PCⅣ, HA and LN, significantly inhibit the NLRP3 inflammasome pathway in liver tissue, inhibit the activation of hepatic stellate cells, which is the core link of liver fibrosis, and reduce the autophagy and activation of hepatic stellate cells. These results indicate that the pharmaceutical composition of the present invention has good therapeutic effects on non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis. The formula of the present invention is novel, the composition is simple, the prices of all main drugs are relatively cheap, and the medication cost is lower. The preparation method of the present invention is simple to operate and can be applied to industrial production. Description of the Drawings

[0029] Figure 1 Liver index, liver function and concentration of serum liver fibrosis markers in each group of rats: (a) Liver index, (b) Alanine aminotransferase (ALT); (c) Aspartate aminotransferase (AST); (d) Albumin (ALB); (e) Procollagen type III peptide (PⅢP); (f) Collagen type IV (PCⅣ); (g) Laminin (LN); (h) Hyaluronic acid (HA).

[0030] Figure 2 Inflammatory and collagen fiber deposition in liver tissue of each group of rats: (a) HE staining (×200) and Masson staining (×200) of rat liver tissue; (b) Appearance of liver tissue in each group of rats; (c) Percentage of collagen fiber area in Masson staining.

[0031] Figure 3 Expression of miR-23a-3p and results of GO function and KEGG pathway analysis of target genes in each group of rats: (a) Expression of miR-23a-3p in each group; (b) Results of GO function analysis of target genes; (c) KEGG signaling pathway analysis of miR-23a-3p target genes (Top 20).

[0032] Figure 4 Expression of CXCL12 / CXCR4 mRNA and protein in each group of rats: (a) Relative expression of CXCL12 mRNA; (b) Relative expression of CXCR4 mRNA; (c) Protein expression of CXCL12; (d) Protein expression of CXCR4; (e) Band grayscale map of CXCL12, CXCR4 and β-actin.

[0033] Figure 5Expression of α-SMA, CollagenⅠmRNA and TGF-β1 in liver tissues of rats in each group: (a) Expression level of α-SMA mRNA in rat liver tissue; (b) Expression level of Collagen mRNA in rat liver tissue; (c)(d)(e) Protein expression of α-SMA and CollagenⅠin rat liver tissue; (f)(g)(h) Immunohistochemical results of α-SMA and CollagenⅠin liver tissues of rats in each group; (i) Expression level of TGF-β1 in rats.

[0034] Figure 6 mRNA and protein expression of key factors in the TXNIP / NLRP3 inflammasome pathway in liver tissues of rats in each group: (a) Relative expression level of TXNIP mRNA; (b) Relative expression level of NLRP3 mRNA; (c) Relative expression level of ASC mRNA; (d) Relative expression level of caspase 1mRNA; (e) TXNIP protein expression; (f) NLRP3 protein expression; (g) ASC protein expression; (h) pro-caspase1 protein expression; (i) Western blot protein band diagram; (j) Expression of IL-1β.

[0035] Figure 7 Effects of Qijia Fuzheng Tongluo formula-containing serum on autophagy and activation of HSC cells: (a) Cell survival rate at each serum concentration; (b) The formula-containing serum reduces the number of autophagosomes (yellow arrows indicate autophagosomes); (c) The formula-containing serum reduces the fusion process of autophagosomes into autophagolysosomes (yellow spots, autophagosomes; red spots, autophagolysosomes); (d) The formula-containing serum reduces the fusion process of autophagosomes into autophagolysosomes.

[0036] Figure 8 Effects of pathway inhibitors on the regulation of autophagy by Qijia Fuzheng Tongluo formula-containing serum: (a) Pathway inhibitors partially inhibit the effect of Qijia Fuzheng Tongluo formula-containing serum on autophagosomes (yellow arrows indicate autophagosomes; Rapa, Rapamycin); (b) Pathway inhibitors partially inhibit the effect of the formula-containing serum on the autophagy activation process; (c) Pathway inhibitors partially inhibit the effect of the formula-containing serum on the autophagy activation process.

[0037] Figure 9 Regulatory effects of formula-containing serum on α-SMA protein expression, LC3II / LC3I ratio and PI3K / AKT / mTOR protein phosphorylation: (a) Effects of formula-containing serum on α-SMA protein expression and LC3II / LC3I ratio; (b) Regulatory effects of formula-containing serum on PI3K / AKT / mTOR protein phosphorylation and α-SMA expression.

[0038] Figure 10Effect of Qijia Fuzheng Tongluo Prescription on Serum ALT, AST, and ALP in NASH Rats: (a) Effect of Qijia Fuzheng Tongluo Prescription on Serum ALT in NASH Rats; (b) Effect of Qijia Fuzheng Tongluo Prescription on Serum AST in NASH Rats; (c) Effect of Qijia Fuzheng Tongluo Prescription on Serum ALP in NASH Rats.

[0039] Figure 11 Regulatory Effect of Qijia Fuzheng Tongluo Prescription on Morphology, Histopathological HE Staining, Oil Red O Staining, and Masson Staining in NASH Rats (200×, Scale bar = 100μm): (a) Effect of Qijia Fuzheng Tongluo Prescription on the Morphology of NASH Rats; (b) Effect of Qijia Fuzheng Tongluo Prescription on HE Staining of Liver Tissue in NASH Rats; (c) Effect of Qijia Fuzheng Tongluo Prescription on Oil Red O Staining of Liver Tissue in NASH Rats; (d) Effect of Qijia Fuzheng Tongluo Prescription on Masson Staining of Liver Tissue in NASH Rats. Detailed Implementation Modes

[0040] According to the above content of the present invention, and in accordance with the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made. The technical solutions of the present invention will be further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited to the following description.

[0041] Example 1 Preparation of the Composition of the Present Invention

[0042] Astragalus membranaceus 15g, Trionyx sinensis carapax 5g, Angelica sinensis 5g, Carthamus tinctorius 10g, Prunus persica 10g, Sparganium stoloniferum 5g, Curcuma zedoaria 5g, Eupolyphaga seu Steleophaga 5g, Glycyrrhiza uralensis 1g.

[0043] The specific preparation method includes: S1: Weigh Astragalus membranaceus, Trionyx sinensis carapax, Angelica sinensis, Carthamus tinctorius, Prunus persica, Sparganium stoloniferum, Curcuma zedoaria, Eupolyphaga seu Steleophaga, and Glycyrrhiza uralensis according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in Step S1, add 8 times the weight of the medicinal materials of water, soak for 0.5 hours, decoct twice, each time for 1 hour, filter the two decoction liquids, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in Step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in Step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0044] Example 2 Preparation of the Composition of the Present Invention

[0045] Astragalus membranaceus 50g, Trionyx sinensis carapax 25g, Angelica sinensis 25g, Carthamus tinctorius 30g, Prunus persica 30g, Sparganium stoloniferum 25g, Curcuma zedoaria 25g, Eupolyphaga seu Steleophaga 25g, Glycyrrhiza uralensis 10g.

[0046] The specific preparation method includes: S1: Weigh astragalus membranaceus, turtle shell, angelica sinensis, safflower, peach kernel, rhizoma sparganii, rhizoma zedoariae, ground beetle and liquorice according to the weight ratio for later use; S2: Mix the Chinese medicinal materials in step S1, add water 10 times the weight of the medicinal materials and soak for 1 hour, decoct twice, each time for 1.5 hours, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Concentrate the medicinal liquid obtained in step S2 by heating to obtain an extract, then vacuum-dry the extract to obtain a dry extract, and crush the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0047] Example 3 Preparation of the Composition of the Invention

[0048] Astragalus membranaceus 25g, turtle shell 10g, angelica sinensis 7g, safflower 15g, peach kernel 15g, rhizoma sparganii 10g, rhizoma zedoariae 10g, ground beetle 8g, liquorice 2g.

[0049] The specific preparation method includes: S1: Weigh astragalus membranaceus, turtle shell, angelica sinensis, safflower, peach kernel, rhizoma sparganii, rhizoma zedoariae, ground beetle and liquorice according to the weight ratio for later use; S2: Mix the Chinese medicinal materials in step S1, add water 8 times the weight of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1.5 hours, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Concentrate the medicinal liquid obtained in step S2 by heating to obtain an extract, then vacuum-dry the extract to obtain a dry extract, and crush the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0050] Example 4 Preparation of the Composition of the Invention

[0051] Astragalus membranaceus 35g, turtle shell 20g, angelica sinensis 13g, safflower 25g, peach kernel 25g, rhizoma sparganii 20g, rhizoma zedoariae 20g, ground beetle 12g, liquorice 7g.

[0052] The specific preparation method includes: S1: Weigh astragalus membranaceus, turtle shell, angelica sinensis, safflower, peach kernel, rhizoma sparganii, rhizoma zedoariae, ground beetle and liquorice according to the weight ratio for later use; S2: Mix the Chinese medicinal materials in step S1, add water 10 times the weight of the medicinal materials and soak for 1 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Concentrate the medicinal liquid obtained in step S2 by heating to obtain an extract, then vacuum-dry the extract to obtain a dry extract, and crush the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0053] Example 5 Preparation of the Composition of the Invention

[0054] Astragalus membranaceus 30g, Trionyx sinensis carapax 15g, Angelica sinensis 9g, Carthamus tinctorius 18g, Prunus persica 18g, Sparganium stoloniferum 15g, Curcuma zedoaria 15g, Eupolyphaga sinensis 10g, Glycyrrhiza uralensis 5g.

[0055] The specific preparation method includes: S1: Weigh Astragalus membranaceus, Trionyx sinensis carapax, Angelica sinensis, Carthamus tinctorius, Prunus persica, Sparganium stoloniferum, Curcuma zedoaria, Eupolyphaga sinensis and Glycyrrhiza uralensis according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add 8 - 10 times the weight of the medicinal materials of water to soak for 0.5 - 1 hour, decoct twice, each time decocting for 1 - 1.5 hours, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0056] Example 6 Preparation of the composition of the present invention

[0057] Astragalus membranaceus 15g, Trionyx sinensis carapax 5g, Angelica sinensis 5g, Carthamus tinctorius 10g, Prunus persica 10g, Sparganium stoloniferum 5g, Curcuma zedoaria 5g, Glycyrrhiza uralensis 1g.

[0058] The specific preparation method includes: S1: Weigh Astragalus membranaceus, Trionyx sinensis carapax, Angelica sinensis, Carthamus tinctorius, Prunus persica, Sparganium stoloniferum, Curcuma zedoaria and Glycyrrhiza uralensis according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add 8 times the weight of the medicinal materials of water to soak for 0.5 hour, decoct twice, each time decocting for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0059] Example 7 Preparation of the composition of the present invention

[0060] Astragalus membranaceus 15g, Trionyx sinensis carapax 5g, Angelica sinensis 5g, Carthamus tinctorius 10g, Prunus persica 10g, Glycyrrhiza uralensis 1g.

[0061] The specific preparation method includes: S1: Weigh Astragalus membranaceus, Trionyx sinensis carapax, Angelica sinensis, Carthamus tinctorius, Prunus persica and Glycyrrhiza uralensis according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add 8 times the weight of the medicinal materials of water to soak for 0.5 hour, decoct twice, each time decocting for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add the dry extract powder obtained in step S3 to pharmaceutically acceptable excipients to prepare a pharmaceutically common pharmaceutical preparation.

[0062] Example 8 Preparation of the composition of the present invention

[0063] 50 g of Astragalus membranaceus, 25 g of turtle shell, 25 g of Angelica sinensis, 30 g of safflower, 30 g of peach kernel, 10 g of liquorice.

[0064] The specific preparation method includes: S1: Weigh Astragalus membranaceus, turtle shell, Angelica sinensis, safflower, peach kernel and liquorice according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add water with a weight 10 times that of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0065] Example 9 Preparation of the composition of the present invention

[0066] 30 g of Astragalus membranaceus, 15 g of turtle shell, 9 g of Angelica sinensis, 18 g of safflower, 18 g of peach kernel, 5 g of liquorice.

[0067] The specific preparation method includes: S1: Weigh Astragalus membranaceus, turtle shell, Angelica sinensis, safflower, peach kernel and liquorice according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add water with a weight 8 times that of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0068] Example 10 Preparation of the composition of the present invention

[0069] 20 g of Astragalus membranaceus, 5 g of turtle shell, 5 g of Angelica sinensis, 10 g of safflower, 10 g of peach kernel, 3 g of Salvia miltiorrhiza, 3 g of Hirudo, 5 g of Eupolyphaga seu Steleophaga, 1 g of liquorice.

[0070] The specific preparation method includes: S1: Weigh Astragalus membranaceus, turtle shell, Angelica sinensis, safflower, peach kernel, Salvia miltiorrhiza, Hirudo, Eupolyphaga seu Steleophaga and liquorice according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add water with a weight 9 times that of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0071] Example 11 Preparation of the composition of the present invention

[0072] 50 g of Astragalus membranaceus, 25 g of turtle shell, 25 g of Angelica sinensis, 30 g of safflower, 30 g of peach kernel, 20 g of Salvia miltiorrhiza, 20 g of leech, 25 g of Eupolyphaga seu Steleophaga, 10 g of liquorice.

[0073] The specific preparation method includes: S1: Weigh Astragalus membranaceus, turtle shell, Angelica sinensis, safflower, peach kernel, Salvia miltiorrhiza, leech, Eupolyphaga seu Steleophaga and liquorice according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add water 8 times the weight of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0074] Example 12 Preparation of the composition of the present invention

[0075] 30 g of Astragalus membranaceus, 15 g of turtle shell, 9 g of Angelica sinensis, 18 g of safflower, 18 g of peach kernel, 10 g of Salvia miltiorrhiza, 5 g of leech, 10 g of Eupolyphaga seu Steleophaga, 5 g of liquorice.

[0076] The specific preparation method includes: S1: Weigh Astragalus membranaceus, turtle shell, Angelica sinensis, safflower, peach kernel, Salvia miltiorrhiza, leech, Eupolyphaga seu Steleophaga and liquorice according to the weight ratio for standby; S2: Mix the Chinese medicinal materials in step S1, add water 9 times the weight of the medicinal materials and soak for 0.5 hour, decoct twice, each time for 1 hour, filter the two decoctions, discard the medicinal residues, and reserve the medicinal liquid; S3: Heat and concentrate the medicinal liquid obtained in step S2 into an extract, then vacuum-dry the extract to obtain a dry extract, and pulverize the dry extract to obtain dry extract powder; S4: Add pharmaceutically acceptable excipients to the dry extract powder obtained in step S3 to prepare a pharmaceutically common pharmaceutical preparation.

[0077] The beneficial effects of the present invention are illustrated by the following test examples

[0078] The drug of the present invention involved in Test Example 1 is a decoction prepared according to the dosage ratio of Example 5 (each milliliter of decoction contains 2 g of crude drug), temporary generic name: Qijia Fuzheng Tongluo Formula.

[0079] The drug of the present invention involved in Test Example 2 is a ready-to-drink granule prepared by mixing the formula granules of each raw material drug of Example 5 according to their dosage ratio, temporary generic name: Qijia Rougan Formula.

[0080] Test Example 1

[0081] I. Experimental study on the anti-rat liver fibrosis of the drug composition of the present invention

[0082] 1 Experimental animals and materials

[0083] 1.1 Experimental animals

[0084] In this invention, 108 healthy specific pathogen-free (SPF) male SD rats of the same batch, weighing 180 - 200 g, were selected and provided by Chengdu Dashuo Laboratory Animal Co., Ltd. The quality certificate number of the experimental animals is: 51203500010152, and the license number is: SCXK(Sichuan)2015 - 030. Before the experiment started, all the rats were adaptively fed with regular diet for 1 week.

[0085] 2 Experimental methods

[0086] 2.1 Modeling method for liver fibrosis rat model

[0087] In this invention, 10 rats were set as the blank group and were fed with regular diet and water. The remaining 98 rats were all established with liver fibrosis rat models by the composite modeling method of "subcutaneous injection of CCL4 peanut oil + 10% alcohol feeding". During the modeling process, it was necessary to judge whether the model was successful. At the end of the 4th week, 6th week, and 8th week respectively, 2 rats were randomly sacrificed, and liver tissues were taken for HE and Masson staining to judge the effect of modeling.

[0088] 2.2 Treatment plan

[0089] 2.2.1 Grouping and medication plan

[0090] After judging that the modeling was successful, the liver fibrosis model rats were divided into the model group (Model group, Model, N = 15), the Biejia Jianwan group (BiejiaJian wan group, BW, N = 14), the high-dose group of Qijia Fuzheng Tongluo formula (RCF high-dose group, RCF-HD, N = 15), the middle-dose group of Qijia Fuzheng Tongluo formula (RCF middle-dose group, RCF-MD, N = 14), and the low-dose group of Qijia Fuzheng Tongluo formula (RCF low-dose group, RCF-LD, N = 15); the rats in the blank group (Control group, Control, N = 10). They were respectively gavaged with the corresponding drugs once a day. The gavage time was fixed at 9:30 - 10:30 in the morning, and the course of treatment was 6 weeks. The administration doses were calculated according to the body surface area of the rats with reference to "Pharmacological Experiment Methodology" (Third Edition). For adults with a body weight of 70 kg, the treatment plan is as follows:

[0091] Blank group (Control): Gavage with normal saline, 1 ml / 100 g;

[0092] Model group (Model): Gavage with normal saline, 1 ml / 100 g;

[0093] Group of Biejiajian Pills (BW): The rats were intragastrically administered with the suspension of Biejiajian Pills, 0.67 ml / 100 g (converted according to the adult dosage in the drug instruction manual).

[0094] Group of High-dose Qijia Fuzheng Tongluo Prescription (RCF-HD): The rats were intragastrically administered with the decoction of Qijia Fuzheng Tongluo Prescription, 1.2 ml / 100 g.

[0095] Group of Medium-dose Qijia Fuzheng Tongluo Prescription (RCF-MD): The rats were intragastrically administered with the decoction of Qijia Fuzheng Tongluo Prescription, 0.6 ml / 100 g.

[0096] Group of Low-dose Qijia Fuzheng Tongluo Prescription (RCF-LD): The rats were intragastrically administered with the decoction of Qijia Fuzheng Tongluo Prescription, 0.3 ml / 100 g.

[0097] 2.3 Sampling method

[0098] After the intragastric administration treatment was completed, all the rats were fasted and water-deprived for 1 day before sampling. After anesthesia by intraperitoneal injection of 3% sodium pentobarbital, blood was collected from the heart and serum and plasma were separated for detecting molecular biological indexes, liver function, serum fibrosis markers and other indexes. The liver was taken, weighed and recorded, and the color and texture of the liver were observed. Several pieces of liver tissue were taken for molecular biological detection, HE and Masson staining.

[0099] 2.4 Observation indexes and detection methods

[0100] 2.4.1 General condition of rats

[0101] During the experiment, the diet and water intake of the rats were recorded every day, and the activity level, hair color glossiness and other conditions were observed. The body weight of the rats was weighed and recorded every week. In addition, after the rats were sacrificed, the liver was taken and weighed to calculate the liver index, and the liver index = (wet liver weight ÷ body weight) × 100%.

[0102] 2.4.2 Liver function

[0103] Alanine substrate method, aspartate substrate method and bromocresol green method were used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (ALB) in the serum of rats respectively.

[0104] 2.4.3 Liver fibrosis indexes

[0105] Radioimmunoassay was used to detect the amino-terminal propeptide of type III procollagen (PⅢNP), type IV procollagen (PCⅣ), hyaluronic acid (HA) and laminin (LN) in the serum of rats.

[0106] 2.4.4 Pathological morphology of liver tissue

[0107] Pathological morphology of liver tissue was observed after HE staining and Masson staining.

[0108] 2.5 Statistical methods

[0109] In this invention, SPSS 20.0 software is used for statistical analysis of data. For data conforming to normal distribution and having homogeneity of variance, one-way analysis of variance is adopted. For data conforming to normal distribution but not having homogeneity of variance, independent samples T-test is used. For data not obeying normal distribution, the Mann-Whitney U method in non-parametric test is selected for testing, and P<0.05 is taken as the judgment standard for significant statistical differences between groups.

[0110] 3 Research results

[0111] 3.1 Research results of Qijia Fuzheng Tongluo Prescription in anti-liver fibrosis of rats

[0112] 3.1.1 Comparison of general conditions of rats in each group

[0113] The rats in the blank group had good mental state, sensitive response, shiny hair, good weight gain, normal diet and drinking water, formed stools during the gavage process, and no death occurred, with 10 rats remaining.

[0114] The rats in the model group were thinner and more irritable than those in the blank group, with sparse and sallow-colored hair. The weight gain was significantly slower than that in the blank group (P<0.05), the stools were soft, and 6 rats died during the gavage period, with 9 rats remaining.

[0115] The rats in the high-dose, medium-dose, low-dose groups of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group had relatively sensitive responses, yellowish hair, normal diet, drinking water and stools, but their weight gains were all relatively slow, significantly lower than that in the blank group (P<0.05), and there was no significant statistical difference compared with the model group (P>0.05). During the gavage period, 3 rats died in the high-dose group of Qijia Fuzheng Tongluo Prescription, with 12 rats remaining; 1 rat died in the medium-dose group of Qijia Fuzheng Tongluo Prescription, with 13 rats remaining; 4 rats died in the low-dose group of Qijia Fuzheng Tongluo Prescription, with 11 rats remaining; 3 rats died in the Biejiajian Pills group, with 11 rats remaining.

[0116] 3.1.2 Liver index

[0117] This study found that compared with the blank group, the liver index of the rats in the model group was significantly increased (P<0.05). The liver indexes of the high-dose, medium-dose, low-dose groups of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group were all significantly lower than that in the model group (P<0.05). There was no significant statistical difference between the high-dose group of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group (P>0.05), as shown in Figure 1 (a).

[0118] 3.1.3 Liver function conditions of rats in each group

[0119] The present invention detected the liver function-related indicators in the sera of rats in each group: ALT, AST, and ALB. The results are shown in Figure 1 (b), (c), (d). Compared with the blank group, the ALT and AST in the model group were significantly increased (P<0.05). The ALT and AST in the high-dose, medium-dose, and low-dose groups of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group were significantly lower than those in the model group (P<0.05), and there was no statistical difference compared with the blank group (P>0.05). There was no statistical difference in ALT and AST among the high-dose, medium-dose, and low-dose groups of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group (P>0.05). There was no statistical difference in ALB among the groups (P>0.05).

[0120] 3.1.4 Conditions of liver fibrosis markers in the sera of rats in each group

[0121] This study detected the fibrosis markers in the sera of rats in each group by radioimmunoassay. The results are shown in Figure 1 (e), (f), (g), (h). Compared with the blank group, the serum PⅢP, PCⅣ, HA, and LN in the model group of rats were significantly increased (P<0.05). Compared with the model group, the PⅢP, PCⅣ, HA, and LN in the high-dose group of Qijia Fuzheng Tongluo Decoction were significantly lower than those in the model group (P<0.05), and the HA content in the high-dose group of Qijia Fuzheng Tongluo Decoction was significantly lower than that in the Biejiajian Pills group (P = 0.03); the HA in the low-dose group of Qijia Fuzheng Tongluo Decoction was significantly lower than that in the model group (P = 0.009), the HA in the medium-dose group of Qijia Fuzheng Tongluo Decoction was significantly lower than that in the model group (P = 0.001), and there was no statistical difference from the blank group (P = 0.06); compared with the model group, the PCⅣ, HA, and LN in the Biejiajian Pills group were significantly reduced (P<0.05).

[0122] 3.1.5 Comparison of pathological morphology of liver tissues in rats in each group

[0123] 3.1.5.1 Results of gross observation of liver tissues in rats in each group

[0124] The gross pictures of liver tissues in rats in each group are shown in Figure 2 (b). Among them, the liver tissue surface of rats in the blank group was smooth, purple-red in color, soft and elastic in texture, and without nodules. The liver tissue volume of rats in the model group was slightly increased, the color became lighter, showing yellowish-brown, the texture was harder, and the surface was uneven, with nodules of different sizes distributed. The liver tissue volume of rats in the high-dose group of Qijia Fuzheng Tongluo Decoction, the low-dose group of Qijia Fuzheng Tongluo Decoction, and the Biejiajian Pills group was slightly increased, showing dark red, with a relatively hard texture, and scattered small nodules on the surface. The liver tissue volume of rats in the medium-dose group of Qijia Fuzheng Tongluo Decoction was increased, the color became lighter, showing dark yellowish-brown, and scattered small nodules were distributed on the surface.

[0125] 3.1.5.2 HE staining results in each group

[0126] The HE staining results of the liver tissues of rats in each group were as follows Figure 2 (a) on the left.

[0127] Blank group: The hepatic lobule structure was clear, the hepatic cords were arranged neatly, the cytoplasm of hepatocytes was abundant, the morphological structure was normal, the hepatic sinusoids were not significantly dilated or compressed, and a small amount of lymphocyte infiltration was seen around the bile ducts in the portal area.

[0128] Model group: A large number of hepatocyte necrosis, cell swelling, nuclear pyknosis and deep staining, cytoplasmic rarefaction and pale staining or vacuolization, or dissolution and replacement by a large amount of proliferated connective tissue were seen in the tissue, accompanied by more lymphocyte and neutrophil infiltration, and a large number of pseudo-lobule structures were visible; hepatocyte fatty degeneration was common in the tissue, and round vacuoles of different sizes and bile duct hyperplasia in the portal area were visible in the cytoplasm.

[0129] High-dose group of Qijia Fuzheng Tongluo Decoction: Hepatocyte fatty degeneration was common in the tissue, and a small amount of lipofuscin deposition was seen; bile duct hyperplasia was visible in the local portal area, accompanied by more lymphocyte infiltration.

[0130] Medium-dose group of Qijia Fuzheng Tongluo Decoction: Hepatocyte fatty degeneration was common in the tissue, round vacuoles of different sizes were visible in the cytoplasm, and lipofuscin deposition was rarely seen; hepatocyte necrosis and dissolution were visible in the local hepatic lobule and around the portal area, replaced by proliferated connective tissue, and accompanied by more lymphocyte infiltration; bile duct hyperplasia was common in the portal area.

[0131] Low-dose group of Qijia Fuzheng Tongluo Decoction: A large number of hepatocyte fatty degeneration was visible in the tissue, round vacuoles of different sizes were visible in the cytoplasm, and a small amount of lipofuscin deposition was seen; hepatocyte necrosis and dissolution were visible around the local central vein, replaced by proliferated connective tissue; dot-like lymphocyte infiltration was common around the bile ducts in the portal area.

[0132] Biejiajian Pills group: A large number of hepatocyte fatty degeneration was visible in the tissue, and a small amount of lipofuscin deposition was seen; focal hepatocyte necrosis and nuclear fragmentation were visible locally, accompanied by a small amount of lymphocyte infiltration; bile duct hyperplasia was common around the portal area, accompanied by lymphocyte infiltration and a small number of acidophilic bodies.

[0133] 3.1.5.3 Inflammatory activity and fibrosis scores of the liver tissues of rats in each group

[0134] As can be seen from Table 1, there were only a few punctate inflammations in the rats of the blank group, without fibrotic inflammation activity and the fibrosis degree score was mainly G1S0. In the model group, the liver tissue inflammation activity score of the rats was mainly G4, with severe portal area inflammation / lobular necrosis in most cases, and the fibrosis degree score was mainly S4. In the high-dose group of Qijia Fuzheng Tongluo Decoction, the liver tissue inflammation was mostly in G1 stage, and the fibrosis was mainly S2 and S3. In the medium-dose group of Qijia Fuzheng Tongluo Decoction, the inflammation was mainly G2 and G3, and the fibrosis was mainly S2 and S3. In the low-dose group of Qijia Fuzheng Tongluo Decoction, the liver inflammation activity was mostly G2, the fibrosis was mainly S3, and there was 1 case with a fibrosis score of S4. In the Biejiajian Pills group, the liver tissue inflammation was mainly G2, and the fibrosis was mainly in S2 and S3 stages.

[0135] Table 1 Inflammation activity and fibrosis degree scores of liver tissues of rats in each group

[0136]

[0137] 3.1.6 Masson staining results of liver tissues of rats in each group

[0138] The Masson collagen staining results of rats in each group are shown in Figure 2 (a) Right. No obvious collagen fiber hyperplasia was seen in the blank group. Model group: A large amount of collagen fiber hyperplasia was visible around the portal vein and central vein in the portal area of the tissue, and a large number of fibrous septa were formed, and a large number of false lobules were visible. High-dose group of Qijia Fuzheng Tongluo Decoction: A relatively large amount of collagen fiber hyperplasia was visible around the portal vein and central vein in the portal area of the tissue, and fibrous septa were formed. Medium-dose group of Qijia Fuzheng Tongluo Decoction: A large amount of collagen fiber hyperplasia was visible around the portal vein and central vein in the portal area of the tissue, and a large number of fibrous septa were formed, and relatively many false lobules were visible. Low-dose group of Qijia Fuzheng Tongluo Decoction: A large amount of collagen fiber hyperplasia was visible, and a large number of fibrous septa were formed, and local false lobules were visible. Biejiajian Pills group: A large amount of collagen fiber hyperplasia was visible around the portal vein and central vein in the portal area of the tissue, and a large number of fibrous septa were formed, and local false lobules were visible.

[0139] The collagen fiber area of liver tissues in each group was statistically analyzed using Image J software, and the results are as in Figure 2 (c). Compared with the blank group, the collagen fiber area of the model group and each treatment group increased significantly (P<0.05). Compared with the model group, the collagen fiber area of the high-dose group of Qijia Fuzheng Tongluo Decoction, the low-dose group of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group decreased significantly (P<0.05).

[0140] II. Experimental study on the regulation of miR-23a-3p in liver tissues of liver fibrosis rats by the pharmaceutical composition of the present invention

[0141] 1 Experimental animals and materials

[0142] 1.1 Experimental animals and grouping

[0143] After 8 weeks of subcutaneous injection of CCl4 to establish a model and 6 weeks of intragastric administration for treatment, the treatment protocol was the same as that in "2.2.1 Grouping and Medication Protocol" under item 1. After the end of intragastric administration, 6 rats were taken from each of the blank group, the model group, the high-dose group of Qijia Fuzheng Tongluo Decoction, the medium-dose group of Qijia Fuzheng Tongluo Decoction, the low-dose group of Qijia Fuzheng Tongluo Decoction, and the Biejiajian Pills group, and qRT-PCR was used to detect the expression of miR-23a-3p in liver tissues.

[0144] 2 Experimental methods

[0145] 2.1 qRT-PCR

[0146] 2.1.1 Tissue homogenization

[0147] Take 10 mg of liver tissue and add it to 300 μl of lysis buffer RL pre-added with β-mercaptoethanol, and use an MP Fastprep-24 homogenizer to prepare tissue homogenate.

[0148] 2.1.2 Total RNA extraction

[0149] In this invention, the TIANGEN RNAprep Pure Tissue Kit (DP431) was used to extract total RNA from rat liver tissues.

[0150] 2.1.3 RNA purity and concentration detection

[0151] In this study, a spectrophotometer was used to detect the concentration of total RNA. The results showed that the amount of 28S rRNA in all samples was about twice that of 18S rRNA, and the RNA integrity was good, so the next experiment could be carried out.

[0152] 2.1.4 Primer design

[0153] Search for the target gene in NCBI (https: / / www.ncbi.nlm.nih.gov / pubmed). After finding the mRNA of the gene, copy the coding region sequence in the CDS, then design primers for the target gene in Primer Primer 6.0, and recheck the designed primers in Primer-BLAST. The primer design of miRNA adopts the stem-loop method.

[0154] Table 2 Gene names and primer sequences

[0155]

[0156] 2.1.5 Reverse transcription reaction

[0157] 2.1.5.1 Genomic DNA removal reaction

[0158] Prepare the reaction mixture on ice according to the following components. To ensure the accuracy of reaction mixture preparation, when conducting each reaction, first prepare the Master Mix in an amount equal to the number of reactions + 2, then aliquot it into each reaction tube, and finally add the RNA sample.

[0159] Table 3 Preparation of the reaction system for removing genomic DNA

[0160]

[0161] Incubate at room temperature for 5 minutes.

[0162] 2.1.5.2 RNA reverse transcription to synthesize DNA

[0163] To ensure the accuracy of reaction mixture preparation, when conducting each reaction, first prepare the MasterMix in an amount equal to the number of reactions + 2, then aliquot 10 μl into each reaction tube. The reaction mixture preparation is carried out on ice. After gentle mixing, centrifuge and immediately perform the reverse transcription reaction using a gradient PCR instrument. The reverse transcription conditions are set as 37°C for 15 min, 85°C for 5 s, and 4°C indefinitely. The synthesized cDNA is stored at -80°C for the next experiment.

[0164] Table 4 Preparation of the reverse transcription reaction system

[0165]

[0166] * When detecting miR-23a-3p, add the miR-23a-3p Specific Primer when preparing the reverse transcription reaction system.

[0167] 2.1.5.3 Real-time fluorescence quantification

[0168] Prepare the PCR reaction solution on ice. Considering the pipetting error, prepare the premix in an amount 10% more than the total reaction volume when preparing the PCR reaction solution. The formula for the PCR reaction solution is shown in Table 5.

[0169] Table 5 Preparation of the quantitative reaction system

[0170]

[0171] After preparing the reaction solution, add the samples to the PCR 96-well plate in sequence. Each sample is measured in 3 replicates, and the difference in Ct values between replicates is < 0.5.

[0172] In this study, the SYBR Green method was used to detect the expression level of the target gene, and the PCR amplification reaction was carried out according to the reaction conditions in the following table.

[0173] Table 6 Amplification conditions

[0174]

[0175] 2.2 miRNA Target Gene Prediction and Research on Binding Sites

[0176] The target genes of miR-23a-3p were predicted based on TarBase v.8 (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8%2Findex) and TargetScan (targetscan.org / ), and the GO functions and KEGG pathways of the target genes were analyzed through http: / / enrich.shbio.com / index / ga.asp. RNA22 (https: / / cm.jefferson.edu / rna22 / Interactive) and relevant literature were combined to identify the target genes and study the binding sites between the target genes and miRNAs.

[0177] 2.3 Statistical Methods

[0178] This experiment used 2 -ΔΔCt for miRNA relative quantitative analysis. The Ct value is the number of cycles when the gene reaches logarithmic amplification. △Ct = the difference between the Ct values of the target gene and the internal reference gene, △△Ct = the difference between the △Ct of each experimental group and the △Ct of the blank group, and the fold change = 2 -△△Ct . The inter-group differences were statistically analyzed using SPSS 20.0 statistical software and independent sample T-tests, and P < 0.05 was used as the criterion for judging statistical differences between groups.

[0179] 3 Experimental Results

[0180] 3.1 Expression of miR-23a-3p in the Liver Tissues of Rats in Each Group

[0181] In this invention, qRT-PCR was used to detect the expression of miR-23a-3p in the liver tissues of rats in each group, and the results are shown in Figure 3 (a). Compared with the blank group, the expression of miR-23a-3p in the liver tissues of rats in the model group was significantly decreased (P = 0.002). Compared with the model group, the expression of miR-23a-3p in the high-dose group of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group was significantly higher than that in the model group (P = 0.006, P = 0.005), and there was no statistical difference compared with the blank group (P > 0.05). There was no statistical difference in the expression of miR-23a-3p in the medium-dose group and low-dose group of Qijia Fuzheng Tongluo Decoction compared with the model group (P > 0.05).

[0182] 3.2 Bioinformatics Analysis of miR-23a-3p

[0183] The target genes of miR-23a-3p were predicted by TarBase v.8, TargetScan, etc., involving a total of 1,744 target genes. For the results of GO function analysis, see Figure 3 (b), and for the KEGG pathway analysis of the target genes, see Figure 3 (c). Through the combination of bioinformatics analysis and literature research, this study found that CXCL12 is one of the important target genes regulated by miR-23a-3p.

[0184] III. Experimental study on the anti-hepatic fibrosis of the pharmaceutical composition of the present invention based on the miR-23a-3p / CXCL12 / CXCR4 / TXNIP / NLRP3 inflammasome pathway

[0185] 1 Experimental animals and materials

[0186] 1.1 Experimental animals

[0187] For the blank group, model group, high-dose group of Qijia Fuzheng Tongluo Prescription, medium-dose group of Qijia Fuzheng Tongluo Prescription, low-dose group of Qijia Fuzheng Tongluo Prescription, and Biejiajian Pills group, there were 6 rats in each group. qRT-PCR, Western blot, immunohistochemistry, and ELISA experiments were carried out, and the methods of drug gavage and sample collection were the same as those in Test Example 1.

[0188] 2 Experimental methods

[0189] 2.1 RT-qPCR

[0190] 2.1.1 Tissue homogenization, total RNA extraction, RNA purity and concentration detection, reverse transcription reaction, and PCR reaction were as described in 2.1 of Test Example 2.

[0191] 2.1.2 Primer design

[0192] The method was referred to as "2.1.4 Primer design" in "II. Experimental study on the regulation of miR-23a-3p in the liver tissue of rats with hepatic fibrosis by the pharmaceutical composition of the present invention", and the gene names and primer sequences are shown in Table 7.

[0193] Table 7 Gene names and primer sequences

[0194]

[0195] 2.2 Western blot

[0196] Experimental method:

[0197] Take 50 mg of tissue, add 500 μl of protein lysate (containing PMSF), ultrasonically disrupt it, and place it on ice for lysis for 30 min.

[0198] Centrifuge the lysate at 4°C and 12,000 g for 10 min. Take the supernatant and transfer it to another new EP tube for later use.

[0199] Prepare the standard and working solution: dilute the BSA standard into several concentration gradients according to Table 11, using the same diluent as the sample to be tested. Ensure that the volume of the standard at each dilution concentration is sufficient for 3 repeated tests.

[0200] Table 8 Preparation of BSA standards

[0201]

[0202]

[0203] Preparation of BCA working solution: Prepare the working solution at a ratio of 50:1 between reagent A and reagent B. When reagent B is added to reagent A, turbidity can be observed. After stirring, the turbidity will quickly disappear, and a clear working solution will be obtained, which can be stored stably at room temperature for several days.

[0204] Take 25 μl of each protein standard and protein sample to be tested at each dilution concentration and add them to a microplate (detection range = 20-2000 μg / ml).

[0205] Add 200 μl of working solution to each well and shake on a shaker for 30 seconds to mix thoroughly.

[0206] The 96-well plate was sealed and incubated at 37°C for 30 min.

[0207] After the incubation, take out the plate and cool it to room temperature, and use a microplate reader to detect the absorbance of the sample at 562nm or near this wavelength. If the absorbance at 562nm is low, the incubation time can be increased to 2h.

[0208] The actual absorbance of the sample is obtained by subtracting the average absorbance of the blank standard at 562 nm from the absorbance of each standard and the protein sample to be tested at 562 nm.

[0209] Use EXCEL to make a standard curve, with the average absorbance of each standard at 562nm after blank correction as the X-axis and the known standard concentration (μg / ml) as the Y-axis. Calculate the protein concentration of each sample to be tested according to the standard curve, and dilute each sample to the same concentration with 5×SDS-PAGE loading buffer, boil on a 100℃ metal bath for 10 minutes, and pack after protein denaturation, and store at -20℃ for later use. The preparation of 5×SDS-PAGE loading buffer sample buffer (10ml) is shown in Table 9.

[0210] Table 9 Preparation of 5×SDS-PAGE loading buffer sample buffer

[0211]

[0212] Preparation of gel. For protein samples, first run the internal reference protein β-actin of each sample, adjust the total protein concentration in each sample to a certain level, and then determine the separating gel with the optimal concentration according to Table 10.

[0213] Table 10 Relationship between protein molecular weight and separating gel concentration

[0214]

[0215] Fix the two glass plates in the electrophoresis device on the gel-casting support. Prepare 5 ml of separating gel according to Table 11, mix well and add it, then add 1 ml of isopropanol to flatten the surface of the separating gel. After the separating gel gels, pour off the isopropanol and wipe the residual isopropanol with a tissue. Prepare 2 ml of stacking gel according to Table 12, mix well and add it, carefully insert the comb (do not introduce air bubbles). After the gel forms, remove the comb in the electrophoresis buffer.

[0216] Table 11 Preparation of 5 ml separating gel

[0217]

[0218]

[0219] Table 12 Preparation of stacking gel

[0220]

[0221] Loading and electrophoresis. Add 5 μL of maker, 5 - 10 μL of sample, and 5 μL of loading buffer in sequence. Add 1×SDS-PAGE electrophoresis buffer, insert the positive and negative electrodes. After running the stacking gel at 90 V, adjust the voltage to 130 V to run the separating gel. When the sample is close to the bottom of the gel, turn off the voltage.

[0222] Table 13 Preparation of 5×SDS-PAGE electrophoresis buffer

[0223]

[0224] Transfer membrane. Soak the PVCF membrane in methanol for 2 min, and then wash it once with the transfer solution. From the negative electrode to the positive electrode in sequence are: filter paper, gel, PVCF membrane, filter paper (no air bubbles should be introduced between each layer). At 4 °C, run at 90 V for 1 h - 1.5 h.

[0225] Preparation of 1000 ml transfer working solution: 100 ml of 10× transfer buffer + 200 ml of ethanol + 700 ml of secondary water. After preparation, store at 4°C.

[0226] Table 14 Preparation of 10× Transfer Buffer (without ethanol)

[0227]

[0228] Blocking. Incubate with 5% BSA solution at 37°C for 0.5 - 1 h. Do not wash and proceed directly to primary antibody incubation.

[0229] Primary antibody incubation. Dilute the primary antibody with PBS-T (PBS solution containing 0.1% Tween 20) according to the antibody instruction manual. Incubate overnight at 4°C or for 1.5 h at room temperature. Then wash with PBS-T 3 times, 5 min each time.

[0230] Secondary antibody incubation. Dilute the secondary antibody with PBS-T (containing 1‰ Tween-20) according to the antibody instruction manual. Incubate at room temperature or 37°C for 1 h. Then wash with PBS-T 3 times, 5 min each time.

[0231] ECL color development. Mix equal volumes of color development solutions A and B and drop them on the PVDF membrane placed on the plastic wrap, completely covering the PVDF membrane. Incubate at room temperature for 3 min.

[0232] Gel imaging. Place the PVDF membrane face up in the Bio-rad VersaDoc (VersaDoc 3000 model) gel imaging system. Adjust the exposure time from 5 to 300 s, continuous exposure, stepping 5 s. Use Quantity one analysis software to perform quantitative analysis on the digital photos.

[0233] 2.3 Enzyme-linked immunosorbent assay (ELISA)

[0234] Collect 2 ml of whole blood using an EDTA anticoagulant blood collection tube, invert and mix well, centrifuge at 3000 rpm for 15 min, and take the supernatant for detection. In this study, the ELISA method was used to detect the expression level of TGF-β1 in rat plasma.

[0235] 2.4 Immunohistochemistry

[0236] 2.4.1 Paraffin-embedded sections

[0237] Take fresh liver tissue and fix it in 4% paraformaldehyde for 24 h, then dehydrate it with gradient concentrations of alcohol, paraffin-embed it, and section it.

[0238] 2.4.2 Deparaffinization of paraffin sections

[0239] Place the sections into xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and then wash with distilled water.

[0240] 2.4.3 Antigen retrieval

[0241] Place the tissue sections in a retrieval box filled with citric acid antigen retrieval buffer (pH 6.0) and perform antigen retrieval in a microwave oven. Heat at medium power for 5 min until boiling, turn off the heat and keep warm for 5 min, then turn to medium-low power for 10 min. During this process, prevent the buffer from evaporating excessively and do not let the sections dry. After natural cooling, place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each time.

[0242] 2.4.4 Block endogenous peroxidase

[0243] Put the sections into 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each time.

[0244] 2.4.5 Serum blocking

[0245] Drop 3% BSA in the immunohistochemical circle to evenly cover the tissue and block at room temperature for 30 min.

[0246] 2.4.6 Add primary antibody

[0247] Gently shake off the blocking solution, and drop the prepared Collagen I and α-SMA primary antibodies at ratios of 1:400 and 1:2000 on the sections. Place the sections flat in a wet box and incubate overnight at 4°C. Add a small amount of water in the wet box to prevent antibody evaporation.

[0248] 2.4.7 Add secondary antibody

[0249] Place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each time. After slightly drying the sections, drop HRP-labeled goat anti-rabbit secondary antibody in the circle to cover the tissue and incubate at room temperature for 50 min.

[0250] 2.4.8 DAB color development

[0251] Place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each time. After slightly drying the sections, drop freshly prepared DAB color development solution in the circle. Control the color development time under a microscope. The positive reaction shows brownish-yellow color. Rinse the sections with tap water to terminate the color development.

[0252] 2.4.9 Counterstain the cell nuclei

[0253] Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiating solution for several seconds, rinse with tap water, blue with hematoxylin blueing solution, and rinse with running water.

[0254] 2.4.10 Dehydration and mounting

[0255] Put the sections into 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and xylene I for 5 minutes in sequence for dehydration and clearing. Take the sections out of xylene, let them dry slightly, and mount with neutral balsam.

[0256] 2.4.11 Microscopic examination and image acquisition and analysis. Randomly select 200-fold fields of view for each section within each group for photographing. Try to make the tissue fill the entire field of view as much as possible when photographing, ensure that the background light of each photo is consistent, use Image-pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) for image analysis, select the same brownish-yellow as the unified standard for judging the positivity of all photos, analyze each photo to obtain the integrated optical density value (IOD) of the positivity of each photo and the pixel area (AREA) of the tissue in the area to be measured, and calculate the surface density value (IOD / AREA). The larger the value, the higher the positive expression level. Picture interpretation: Hematoxylin stains the cell nucleus blue, and the positive expression shown by DAB is brownish-yellow.

[0257] 2.5 Statistical methods

[0258] Use SPSS 20.0 statistical software for data analysis. For data that conform to both normal distribution and homogeneity of variance, use analysis of variance. For data that conform to normal distribution, use T-test. For data whose distribution does not conform to normal distribution, use non-parametric test. When P < 0.05, there is a statistically significant difference in the comparison between groups. Use 2-ΔΔCt for relative mRNA quantification analysis. The Ct value is the number of cycles when the gene reaches logarithmic amplification. △Ct = the difference between the Ct values of the target gene and the internal reference gene. △△Ct = the difference between the △Ct of each experimental group and the △Ct of the blank group. Fold Change = 2-△△Ct. The inter-group differences are analyzed using SPSS 20.0 statistical software.

[0259] 3 Results

[0260] 3.1 Expression of CXCL12 / CXCR4 signaling axis in liver tissues of fibrotic rats

[0261] The relative expression level of CXCL12 mRNA in the liver fibrosis model group was significantly higher than that in the blank group (P = 0.00). After drug treatment, the relative expression levels of CXCL12 mRNA in the high, medium, and low dose groups of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group decreased. Among them, the CXCL12 mRNA in the liver tissues of rats in the high dose group of Qijia Fuzheng Tongluo Prescription, the medium dose group of Qijia Fuzheng Tongluo Prescription, and the Biejiajian Pills group was significantly lower expressed than that in the model group (P < 0.05), and there was no statistical difference compared with the blank group (P = 0.07), as shown in Figure 4 (a). The expression of CXCR4 mRNA in the liver tissues of rats in each group is shown in Figure 4 (b). Compared with the blank group, the expression of CXCR4 mRNA in the liver tissues of rats in the model group was significantly increased, and the difference was statistically significant (P = 0.000). Compared with the model group, the expression of CXCR4 mRNA in the high dose group of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group was significantly decreased, and the differences were statistically significant (P = 0.001, P = 0.007). There was no statistical difference in the expression of CXCR4 mRNA between the high dose group of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group (P = 0.348).

[0262] The results of Western blot showed that compared with the blank group, the expression of CXCL12 in the model group was significantly increased (P = 0.00). The expression of CXCL12 in the liver tissues of rats in each group after drug treatment decreased effectively. Compared with the model group, the protein expressions of CXCL12 in the high dose group, medium dose group of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group were significantly decreased, and the differences were statistically significant (P < 0.05). There was no statistical difference in the expression of CXCL12 in the liver tissues of rats between the high dose group of Qijia Fuzheng Tongluo Prescription and the Biejiajian Pills group and the blank group, and there was also no statistical difference between the two, as shown in Figure 4 (c) and 4(e).

[0263] Compared with the blank group, the protein expression of CXCR4 in the liver tissues of rats in the model group was significantly increased, and the difference was statistically significant (P = 0.039). Compared with the model group, the expressions of CXCR4 in the high dose group and the Biejiajian Pills group of Qijia Fuzheng Tongluo Prescription were significantly decreased (P < 0.05), and there was no statistical difference compared with the blank group (P > 0.05). The expressions of CXCR4 in the medium dose group and low dose group of Qijia Fuzheng Tongluo Prescription were decreased compared with the model group, but the differences were not statistically significant (P > 0.05), as shown in Figure 4 (d) and 4(e).

[0264] 3.2 Expression of α-SMA, CollagenⅠ and TGF-β1 in rats in each group

[0265] α-SMA and CollagenⅠare one of the markers of hepatic stellate cell (HSC) activation. In this study, the protein and mRNA expression levels of α-SMA and CollagenⅠin the liver tissues of rats in each group were detected. The results of qRT-PCR are shown in Figure 5 (a) and 5(b). Compared with the blank group, the expression of CollagenⅠmRNA in the model group of rats was significantly increased, and the difference was statistically significant (P = 0.018). Compared with the model group, the expression of CollagenⅠmRNA in the high-dose, medium-dose groups of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group was significantly decreased after treatment (P < 0.05). There was no statistical difference in the expression of CollagenⅠmRNA between the low-dose group of Qijia Fuzheng Tongluo Decoction and the model group (P > 0.05). The results of Western blot are shown in Figure 5 (c), (d), and (e) showed that compared with the blank group, the protein of CollagenⅠin the liver tissues of the model group of rats was significantly highly expressed (P = 0.000). Compared with the model group, the expression of CollagenⅠin the high-dose, medium-dose, low-dose groups of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group was significantly decreased, and the difference was statistically significant (P < 0.05).

[0266] Compared with the blank group, the expression of α-SMA mRNA in the model group was significantly increased (P = 0.016). Compared with the model group, the expression of α-SMA mRNA was significantly decreased after treatment with the high-dose, medium-dose of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills (P < 0.05). Although the expression level of α-SMA mRNA in the low-dose group of Qijia Fuzheng Tongluo Decoction decreased to some extent, there was no statistical difference compared with the model group (P = 0.082), and there was no statistical difference in the expression of α-SMA mRNA among the treatment groups. The results of Western blot showed that compared with the blank group, α-SMA in the model group was significantly highly expressed (P = 0.000). Compared with the model group, the expression of α-SMA in the high-dose, medium-dose, low-dose groups of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group in the liver tissues of rats was significantly decreased (P < 0.05), and there was no statistical difference compared with the blank group (P > 0.05).

[0267] In addition, immunohistochemistry was used in this study to detect the expression and localization of α-SMA and CollagenⅠin the liver tissues of rats in each group. The results are as shown in Figure 5 (f), (g), and (h). Compared with the blank group, α-SMA and CollagenⅠin the model group were significantly highly expressed (P < 0.05). Compared with the model group, the expression of α-SMA and CollagenⅠwas significantly decreased after treatment with the high-dose group of Qijia Fuzheng Tongluo Decoction and the Biejiajian Pills group, and the difference was statistically significant (P < 0.05).

[0268] In this invention, ELISA was used to detect the expression of TGF-β1 in rats in each group. The results are shown inFigure 5 (i), compared with the blank group, the content of TGF-β1 in the plasma of rats in the model group was significantly increased (P = 0.002). Compared with the model group, the content of TGF-β1 in the high-dose group and medium-dose group of Qijia Fuzheng Tongluo Decoction was significantly decreased, and the difference was statistically significant (P < 0.05).

[0269] 3.3 Expression of key proteins and mRNAs in the TXNIP / NLRP3 inflammasome pathway in the liver tissues of rats in each group The expression of mRNAs in the TXNIP / NLRP3 inflammasome pathway in the liver tissues of rats in each group is shown in Figure 6 (a), (b), (c), (d), and the Western blot results are as Figure 6 (e), (f), (g), (h), (i). Compared with the blank group, the mRNA expression of TXNIP in the liver tissues of rats in the model group was significantly up-regulated, and the difference was statistically significant (P = 0.049). Compared with the model group, the expression of TXNIP mRNA in the high-dose, medium-dose, and low-dose groups of Qijia Fuzheng Tongluo Decoction did not show a significant decrease; the Western blot results showed that compared with the blank group, the expression of TXNIP in the model group was significantly increased (P < 0.05). Compared with the model group, the expression of TXNIP in the high-dose group, medium-dose group, and Biejiajian Pills group of Qijia Fuzheng Tongluo Decoction was significantly decreased, and the difference was statistically significant (P < 0.05).

[0270] Compared with the blank group, the mRNA expression of NLRP3, ASC, and Caspase 1 in the liver tissues of rats in the model group was significantly up-regulated (P < 0.05). Compared with the model group, the expression of NLRP3, ASC, and Caspase 1 mRNA in the high-dose group of Qijia Fuzheng Tongluo Decoction and Biejiajian Pills group was significantly decreased, and the difference was statistically significant (P < 0.05). The Western blot results showed that compared with the blank group, the expression of NLRP3 and ASC in the model group was significantly up-regulated (P < 0.05), and there was no statistical difference in the expression of pro-caspase 1 between the model group and the blank group (P > 0.05); compared with the model group, the expression of NLRP3, ASC, and pro-caspase 1 in the high-dose group, medium-dose group, and Biejiajian Pills group of Qijia Fuzheng Tongluo Decoction was significantly down-regulated (P < 0.05), and the expression of NLRP3 in the low-dose group of Qijia Fuzheng Tongluo Decoction was significantly increased compared with the model group (P < 0.05).

[0271] 3.4 Expression of IL-1β in rats in each group

[0272] After the activation of the NLRP3 inflammasome, IL-1β will be released to activate HSCs. In this study, the Western blot method was used to detect the expression of IL-1β in the liver tissues of rats in each group. The results are shown in Figure 6(j), compared with the blank group, the expression of IL-1β in the liver tissue of rats in the model group was significantly up-regulated (P < 0.05). Compared with the model group, the expression of IL-1β in the high-dose, medium-dose groups of Qijia Fuzheng Tongluo Prescription and Biejiajian Pills group was significantly decreased (P < 0.05). There was no significant difference in the expression of IL-1β between the high-dose and medium-dose groups of Qijia Fuzheng Tongluo Prescription and Biejiajian Pills group (P > 0.05).

[0273] IV. Study on the regulation of PI3K / AKT / mTOR pathway by the pharmaceutical composition of the present invention and its influence on HSC autophagy and activation

[0274] 1 Experimental animals and materials

[0275] 1.1 Sources of animals and cell lines

[0276] The rat HSC-T6 cell line was provided by Beina Biotechnology. SPF-grade SD rats, male, weighing 150 g - 180 g, a total of 32 rats were used for the preparation of drug-containing serum. They were provided by Chengdu Dashuo Experimental Animal Co., Ltd., with the certificate number 51203500014953 and the license number SCXK (Sichuan) 2020-030. The rats were housed in the Animal Experiment Center of the Basic Medical College of Chengdu University of Traditional Chinese Medicine. After 7 days of adaptive feeding, the corresponding experiments were carried out. All operations involving animal experiments were carried out in accordance with the ethical norms of experimental animals.

[0277] 1.4 Preparation of drug-containing serum of Qijia Fuzheng Tongluo Prescription

[0278] Composition and preparation of traditional Chinese medicine compound: Prepared according to the method of Example 5.

[0279] Animal grouping: After 7 days of adaptive feeding of rats, they were randomly divided into a blank control group and low-, medium-, and high-dose groups of Qijia Fuzheng Tongluo Prescription. The low-, medium-, and high-dose groups of Qijia Fuzheng Tongluo Prescription were referred to the equivalent dose conversion between humans and rats in "Methodology of Traditional Chinese Medicine Pharmacological Experiments".

[0280] ① Blank control group: 8 rats, intragastrically administered with equal-dose normal saline, 2 times / day (8 am and 5 pm), continuously intragastrically administered for 7 days.

[0281] ② Low-dose group of Qijia Fuzheng Tongluo Prescription: A total of 8 rats, 0.57 g / 100 g, administration volume: 1 ml / 100 g (rat body weight), 2 times / day (8 am and 5 pm), continuously intragastrically administered for 7 days.

[0282] ③ Medium-dose group of Qijia Fuzheng Tongluo Prescription: A total of 8 rats, 1.13 g / 100 g, administration volume: 1 ml / 100 g (rat body weight), 2 times / day (8 am and 5 pm), continuously intragastrically administered for 7 days.

[0283] ④ High-dose group of Qijia Fuzheng Tongluo prescription: 8 rats in total, 2.26g / 100g, administration volume: 1ml / 100g (rat body weight), 2 times / day (8 am and 5 pm), continuous gavage for 7 days.

[0284] One hour after the last oral gavage on the 7th day (fasting but not water for 12 hours before the last administration), rats were intraperitoneally injected with 3% sodium pentobarbital, anesthetized, and the abdominal aorta was cut open for blood collection. The process was aseptic. The collected blood was kept still at 37°C for 2 hours, centrifuged (3500rpm, 30min), and the upper serum was sucked with a sterile pipette tip, mixed and transferred to a centrifuge tube, inactivated in a 56°C water bath for 30 minutes, and the serum after water bath was further sterilized by filtering with a 0.22μm microporous filter membrane in a clean bench, divided into vials, and frozen in a -80°C refrigerator for use.

[0285] 2. Experimental Methods

[0286] 2.1 Recovery, culture, passage and cryopreservation of primary rat HSCs

[0287] 2.1.1 Cell recovery

[0288] Place the primary rat HSC cell line in the cryopreservation tube in a 37℃ water bath for about 1 minute. When the ice mass in the cryopreservation tube is dissolved by 90%, quickly move it into the clean bench, move the cells to a 15ml centrifuge tube with 5ml culture medium, slowly drip it first and then speed up the dripping, centrifuge at 800rpm for 3min. Discard the culture medium and cryopreservation solution, leave the cell sediment at the bottom, add 3ml of pre-prepared high-glucose culture medium (containing 10% fetal bovine serum and 1% double antibody), and gently blow to resuspend the cells. Take 1ml and place it in a 10cm culture dish pre-added with 10ml culture medium, draw a "cross" horizontally to evenly distribute the cells in the culture dish, observe the cells under a microscope, and culture them in a cell incubator (5% CO2, 37℃). Change fresh culture medium the next day.

[0289] 2.1.2 Cell culture

[0290] Rat HSC-T6 cell line was cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody in a 37°C, 5% CO2 incubator. Fresh medium was replaced every 1 to 2 days, and cells were passaged when the cells grew to about 80% confluence.

[0291] 2.1.3 Cell passaging

[0292] Remove the medium, add 2 ml of PBS, wash twice, add 1 ml of trypsin and digest for 1 minute, add 3 ml of medium to terminate digestion, pipette the cells down, centrifuge at 800 rpm for 3 minutes, resuspend the cells with 3 ml of medium, take 1 ml and add it to a 10-cm culture dish pre-filled with 10 ml of medium, and place it in an incubator for culture.

[0293] 2.1.4 Cell cryopreservation

[0294] Prepare the cryopreservation solution in advance: 90% fetal bovine serum + 10% DMSO. After digesting the cells, centrifuge at 800 rpm for 3 minutes, discard the supernatant, add 1 ml of the cryopreservation solution, gently pipette and mix well, transfer to a cryotube, make a label, seal the tube mouth with sealing tape, place it in a gradient cooling box, cryopreserve at -80°C overnight, and transfer to a liquid nitrogen tank for cryopreservation.

[0295] 2.2 Detection of cell viability by CCK8 method to screen for the appropriate serum concentration

[0296] Inoculate rat HSC-T6 cells in a 96-well plate. When the cell growth reaches about 70%, add 2.5%, 5%, 10%, 15%, 20% blank rat serum (as experimental wells) DMEM medium to the 96-well plate inoculated with HSC-T6 cell line respectively (set 3 wells for each volume of blank rat serum), and set a control well (cell medium without blank rat serum) and a blank well (no cells and blank rat serum) as experimental controls. After treatment for 24 hours, add 10 μl of CCK8 (Cell Counting Kit-8) solution to each well, incubate in an incubator for 2 hours, and then detect the OD value at 450 nm to determine the appropriate rat serum concentration. Cell survival rate = [(OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of blank well)] * 100%.

[0297] 2.3 Experimental grouping and drug administration

[0298] According to the results of the CCK8 experiment, select 10% serum as the serum concentration for the following experimental interventions.

[0299] (1) Control group: Administer 10% blank rat serum.

[0300] (2) Model group: Incubate with 0.1 mg / L LPS for 24 hours.

[0301] (3) LPS + low-dose drug-containing serum group: Incubate with 0.1 mg / L LPS for 24 hours and treat with 10% low-dose drug-containing serum for 24 hours.

[0302] (4) LPS + medium-dose drug-containing serum group: Incubate with 0.1 mg / L LPS for 24 hours and treat with 10% medium-dose drug-containing serum for 24 hours.

[0303] (5) LPS + High-dose Drug-containing Serum Group: Incubate with 0.1 mg / L LPS for 24 h, and then treat with 10% high-dose drug-containing serum for 24 h.

[0304] (6) LY294002 + LPS + Drug-containing Serum Group: Add 20 μM LY294002 1 h in advance, then add 0.1 mg / L LPS, incubate for 24 h, and then treat with 10% high-dose drug-containing serum for 24 h.

[0305] (7) mTOR Inhibitor Group: Add Rapamycin at 200 nM simultaneously with 0.1 mg / L LPS, culture and incubate for 24 h, and then treat with 10% high-dose drug-containing serum for 24 h.

[0306] 2.4 Detection Methods and Detection Indicators

[0307] 2.4.1 Transmission Electron Microscopy

[0308] After rinsing the cells with PBS, make slices of 1 mm3 in size, place them in 2.5% glutaraldehyde and incubate at 4°C for 12 h. After rinsing with PBS, add 1% osmium tetroxide and fix at 4°C for 3 h. Dehydrate with ethanol and acetone, then embed in epoxy resin for 4 h, make slices of 50 nm in size, add uranium acetate and lead citrate for staining, and observe autophagosomes under a transmission electron microscope.

[0309] 2.4.2 Detection of Autophagic Flux

[0310] Select cells with good growth status and inoculate them on the cell slides in a 12-well plate, culture overnight in an incubator at 37°C with 5% CO2. After washing twice with PBS buffer, add DMEM (500 μl / well), and at the same time add 1 μl of HBAD-mcherry-EGFP-LC3 autophagy dual-label adenovirus. Incubate in an incubator at 37°C with 5% CO2 for 2 h, change the medium, add 1 ml of fresh medium, and culture for another 12 h. After the drug treatment is completed, take out the cell slides, wash them 3 times with PBS, 3 min each time, add 4% paraformaldehyde to fix for 15 min, wash them 3 times with PBS, 3 min each time, blot the PBS dry with absorbent paper, add hoechst 33258 staining solution, incubate at room temperature for 5 min, wash 3 times with PBS, 3 min each time, blot the liquid dry with absorbent paper and then add an anti-fluorescence quenching agent to seal the slides, and observe and take pictures under a laser confocal microscope. Yellow fluorescence and red fluorescence can be seen. If the process of autophagosome maturation into autolysosome is blocked, only the yellow fluorescence will increase.

[0311] 2.4.3 Western Blot Detection

[0312] (1) Protein Extraction and Sample Preparation of HSC Cells

[0313] HSC cells in the logarithmic growth phase with good growth status were cultured until they covered the bottom of the flask. Then they were digested with trypsin, centrifuged at 800 rpm for 3 min, resuspended, and counted. The cell density was adjusted to 2×105 / ml, and 100 μl of cells were seeded into each well of a 6-well plate for culture. After the drug treatment was completed, the supernatant was aspirated, 1 ml of pre-cooled PBS was added, and the cells were gently washed 3 times. Then the PBS solution was completely aspirated. The operation was carried out on ice. A pre-cooled cell lysis mixture containing RIPA, PMSF, and phosphatase inhibitor (RIPA:PMSF = 100:1) was added, and the cells were gently ground and scraped off with a cell scraper. The scraped cells were added to a prepared centrifuge tube (1.5 ml) and left standing on ice for 15 min. A low-temperature high-speed centrifuge was pre-cooled at 4°C, and the cells were centrifuged at 12,000 rpm for 30 min. The flocculent precipitate was completely removed, and the clear supernatant was collected and stored in a -20°C refrigerator for later use.

[0314] (2) Western Blot detection

[0315] Gel casting: After the glass plates were thoroughly washed with tap water and then rinsed with ddH2O and dried for later use. The prepared separating gel was poured along the glass plates, and isopropanol was added to press it flat. After it solidified, the isopropanol was removed. The stacking gel was prepared, TEMED was added and shaken well, and then the gel was poured to fill the remaining space, and a comb was inserted horizontally. After the gel above solidified, the comb was pulled out while holding the gel, and it was placed in the electrophoresis tank.

[0316] Loading: The pre-prepared protein sample stored in a -20°C refrigerator was taken out, dissolved and centrifuged to obtain the upper layer liquid, and the upper layer liquid was aspirated for loading.

[0317] Electrophoresis: The power supply was connected to start electrophoresis for 1.5 h at a voltage of 100 V until the bromophenol blue ran out of the bottom of the gel, and electrophoresis was terminated and membrane transfer was started.

[0318] Membrane transfer: Wear gloves to cut the PVDF membrane and mark it with a pen to distinguish the front and back sides. The cut membrane was placed in water and soaked for 2 h. The clip was opened, a sponge was placed, and the air bubbles were rolled away with a glass rod. The glass plate was gently pried off, the stacking gel was cut off, the separating gel was peeled off and covered with filter paper, the membrane was placed on the gel, then the filter paper was covered on the membrane, the air bubbles were removed, and then the sponge pad was covered, and the clip was closed. The whole process was completed in the transfer buffer. The clip was transferred to the transfer tank for membrane transfer.

[0319] Antibody Incubation: The transferred membrane was placed in a box containing blocking solution and blocked on a shaker at room temperature for 1 h. The diluted primary antibody was added for incubation overnight at 4°C. It was washed 3 times with TBST for 5 min each time to remove the unbound primary antibody. The diluted secondary antibody was added and incubated on a shaker at room temperature for 2 h. It was washed 5 times with TBST for 5 min each time to remove the unbound antibody, and then soaked in ddH2O and stored at 4°C for later use. Dilution concentration of primary antibody: β-actin: 1:1000; α-SMA: 1:1000; LC3: 1:1000; pAKT: 1:1000; pmTOR: 1:1000.

[0320] Chemiluminescence, Development and Fixation: After the membrane was taken out, the excess moisture on the membrane was blotted with absorbent paper and placed in a petri dish. The exposure substrate was added onto the membrane protein surface for exposure, development and fixation. The film was scanned and saved for record. The gray value of the tape was analyzed by ImageJ.

[0321] 2.5 Statistical Analysis

[0322] GraphPad Prism 5.0 software was used for statistical analysis of the data, with mean ± standard deviation representing measurement data. Appropriate statistical analysis methods were selected according to the different situations of the collected data. Analysis of variance was used for between-group comparison when the variances were homogeneous, and the rank sum test was used for analysis when the variances were inhomogeneous. P < 0.05 was considered statistically significant, P < 0.01 was considered significantly statistically different, and P < 0.001 was considered very significantly statistically different.

[0323] 3. Experimental Results

[0324] 3.1 Serum Containing Qijia Fuzheng Tongluo Formula Reduces Autophagy and Activation of HSC Cells

[0325] 3.1.1 Effects of Serum at Different Concentrations on the Viability of HSC Cells

[0326] The CCK8 detection results showed that compared with the control group, when the concentration of blank rat serum was above 15%, the cell viability was significantly reduced (P < 0.05). When the concentration of blank rat serum was between 0 and 10%, there was no significant difference in cell viability compared with the control group (P > 0.05), indicating that the concentration of blank rat serum between 0 and 10% had no significant effect on cell viability. Therefore, a serum concentration of 10% was selected. As Figure 7 (a).

[0327] 3.1.2 Serum Containing Qijia Fuzheng Tongluo Formula Reduces the Number of Autophagosomes

[0328] The HSC cells in the blank group had normal morphological structures. The cell nuclei were irregular polygons, with evenly distributed chromatin, clear and intact nuclear membranes. Organelles such as mitochondria and rough endoplasmic reticulum with complete structures were seen in the cytoplasm, and there were a small number of autophagosomes. Lipid droplets were visible in the cytoplasm of HSC cells, and the cell membranes were intact with rich microvilli structures. Compared with the blank group, there were more autophagosomes in the cytoplasm of cells in the model group. Compared with the model group, the number of autophagosomes decreased in the low, medium, and high-dose serum-containing drugs of Qijia Fuzheng Tongluo Formula. As Figure 7 (b).

[0329] 3.1.3 The serum-containing drug of Qijia Fuzheng Tongluo Formula hinders the fusion process of autophagosomes with lysosomes

[0330] By transfecting the autophagy dual-label adenovirus mcherry-EGFP-LC3 into HSC cells, the effect of the serum-containing drug of Qijia Fuzheng Tongluo Formula on the autophagy activation process was observed. EGFP shows green, mcherry shows red. In the picture after the overlay of the two, yellow spots indicate autophagosomes, and red spots indicate autophagolysosomes. If only the yellow spots increase, it indicates that the process of autophagosome maturation into autophagolysosome is blocked. Compared with the blank group, there were a large number of green and red spots in the model group, and the yellow fluorescence (P < 0.01) and red fluorescence spots (P < 0.001) after overlay increased significantly, with the increase in red spots being obvious, indicating an increase in autophagolysosomes. Compared with the model group, the number of red spots decreased in the low, medium, and high-dose serum-containing drug groups of Qijia Fuzheng Tongluo Formula (P < 0.001), indicating that the fusion process of autophagosomes with lysosomes is blocked. As Figure 7 (c), (d).

[0331] 3.1.4 The serum-containing drug of Qijia Fuzheng Tongluo Formula inhibits HSC activation

[0332] Compared with the blank group, the expression of α-SMA protein increased significantly in the model group (P < 0.001), and the LC3II / LC3I ratio decreased (P < 0.05). Compared with the model group, the expression of α-SMA protein decreased significantly in the low, medium, and high-dose serum-containing drug groups of Qijia Fuzheng Tongluo Formula (P < 0.001), and the LC3II / LC3I ratio increased (P < 0.001); there were no significant differences in the expression of α-SMA protein and the LC3II / LC3I ratio among the low, medium, and low-dose serum-containing drugs of Qijia Fuzheng Tongluo Formula (P > 0.05). As Figure 9 (a).

[0333] 3.2 Qijia Fuzheng Tongluo Formula affects HSC cell autophagy and activation by regulating the PI3K / AKT / mTOR pathway

[0334] 3.2.1 Transmission electron microscopy detection results

[0335] The results of transmission electron microscopy showed that the morphological structure of HSC cells in the blank group was normal. The cell nucleus was irregular polygon-shaped, chromatin was evenly distributed, the nuclear membrane was clear and intact. Organelles such as mitochondria and rough endoplasmic reticulum with intact structures were seen in the cytoplasm, with a small number of autophagosomes. Lipid droplets were present in the cytoplasm of HSC cells, and the cell membrane was intact with abundant microvillus structures. Compared with the blank group, there were more autophagosomes in the cytoplasm of cells in the model group. Compared with the model group, the number of autophagosomes decreased after the treatment with serum containing Qijia Fuzheng Tongluo Prescription. Compared with the group treated with serum containing Qijia Fuzheng Tongluo Prescription alone, the number of autophagosomes increased in the group of serum containing Qijia Fuzheng Tongluo Prescription + LY294002 and the group of Qijia Fuzheng Tongluo Prescription + Rapamycin. As Figure 8 (a).

[0336] 3.2.2 Detection results of autophagy activation process

[0337] The detection results of autophagy activation process showed that compared with the blank group, there were a large number of green and red spots in the model group, and the yellow and red spots increased after overlay, with the obvious increase in red spots (P < 0.05), aggregating around the nucleus. Compared with the model group, the red spots significantly decreased after overlay in the group of serum containing Qijia Fuzheng Tongluo Prescription (P < 0.05). As Figure 8 (b), (c).

[0338] Compared with the group treated with serum containing Qijia Fuzheng Tongluo Prescription alone, the red spots increased after overlay in the group of serum containing Qijia Fuzheng Tongluo Prescription + LY294002 (P < 0.001), aggregating around the nucleus; the red spots increased after overlay in the group of serum containing Qijia Fuzheng Tongluo Prescription + Rapamycin (P < 0.001). As Figure 8 (b), (c).

[0339] 3.2.3 WB detection results

[0340] Compared with the blank group, the protein expressions of pAKT and pmTOR decreased significantly in the model group (P < 0.001), while the protein expression of α-SMA increased (P < 0.01). Compared with the model group, the protein expressions of pAKT and pmTOR increased significantly in the group of serum containing Qijia Fuzheng Tongluo Prescription (P < 0.001), while the protein expression of α-SMA decreased (P < 0.001). Compared with the group treated with serum containing drug alone, the protein expression of pmTOR decreased in the group of serum containing Qijia Fuzheng Tongluo Prescription + Rapamycin (P < 0.001), while the protein expression of α-SMA increased (P < 0.001); the protein expressions of pAKT (P < 0.001) and pmTOR (P < 0.01) decreased, while the protein expression of α-SMA increased (P < 0.001) in the group of Qijia Fuzheng Tongluo Prescription + LY294002. As Figure 9 (b).

[0341] It can be seen from the above experimental results that the pharmaceutical composition of the present invention has a good therapeutic effect on liver fibrosis, provides a new method for the treatment of liver fibrosis, and the drug combination for the disease is simple, the medicine is simple and powerful, and is worthy of promotion and application.

[0342] Experimental Example 2 Study on the effect of Qijia Rougan prescription on improving non-alcoholic fatty hepatitis in rats and its anti-oxidative stress injury

[0343] 1 Experimental animals

[0344] 40 SD rats, weighing 260-280 g, with animal certificate number: SCXK (Xiang) 2019-0004, were kept in the animal room of the Experimental Center of the School of Basic Medical Sciences of Chengdu University of Traditional Chinese Medicine, ensuring a 12-h day / night cycle and kept in an environment of 21±2°C and 40% relative humidity.

[0345] 2 Experimental drugs and reagents configuration

[0346] The Qijia Rougan Formula is composed of astragalus, angelica, turtle shell, earthworm, safflower, peach kernel, curcuma, Tripterygium wilfordii, and raw licorice. All drugs are commissioned by Sichuan Boyitang Pharmacy Retail Chain to be prepared as decoction-free granules, which are sealed and stored. Before each gavage, they are dissolved in pure hot water to a 157.5 mg / ml solution, and fully dissolved and mixed with a magnetic stirrer for immediate use.

[0347] The positive control drug is bicyclol tablets, also known as Baiseno, produced by Beijing Union Pharmaceutical Factory (approval number: National Medicine Standard H20040467), the main ingredient of which is bicyclol synthesized on the basis of schisandrin. Preparation method of bicyclol tablet suspension: put bicyclol tablets into a mortar, grind repeatedly until fine powder, then collect and store in a cool and dry place. Before each intragastric administration, weigh the required amount of bicyclol fine powder, add appropriate amount of hot water and mix well, the final concentration is 787.5mg / ml, prepare and use immediately, and shake well.

[0348] Preparation of CCl4 solution: weigh the animals before each modeling, estimate the total amount needed for the day, add olive oil and carbon tetrachloride solution in a light-proof glass bottle in proportion to the required amount, use a sterile syringe to draw, and finally mix the two solutions thoroughly and prepare them for immediate use.

[0349] 3 Experimental methods

[0350] 3.1 Establishment of experimental animal model

[0351] 65 SPF - level SD rats, weighing 260 - 280 g, were fed with ordinary feed for 14 days for adaptation. Then, according to their behavioral status, diet, and body weight, heterogeneous rats were excluded and re - divided into 2 groups. Among them, there were 10 rats in the control group and 30 rats in the model group. The control group continued to be fed with ordinary feed, while the model group was fed with a 45% high - fat, high - cholesterol, choline - deficient diet (CDHFD). At 15:00 every Friday, all rats in the model group were subcutaneously injected with 20% CCl4 at a dose of 2 ml / kg according to their body weight. Before and after the injection, the puncture site was strictly disinfected with a sterilized cotton ball. The modeling time was 6 weeks. After successful modeling, the CDHFD feed was removed from the model group, and subcutaneous injection of 10% CCl4 was given once a week according to the previous method to prevent the self - healing of the model rats.

[0352] 3.2 Experimental grouping and drug administration

[0353] Grouping situation: After successful modeling of non - alcoholic steatohepatitis (NASH) rats, they were arranged in descending order of body weight and divided into 3 groups by combining the random number table method, namely the model group, the Qijia group, and the positive drug control group of bicyclol, with 10 rats in each group.

[0354] Drug administration situation: After successful modeling of all animals, gavage was started in the 7th week. Rats in the control group and the model group were gavaged with an appropriate amount of normal saline according to their body weight. The adult daily dose of Qijia Rougan Decoction free - decoction granules was weighed and calculated as 15 g. According to the conversion of the drug administration dose for experimental animals, calculated based on the body weight of a 60 - kg adult, the dose for rats was 6.3 times the adult dose, that is, 1.575 g / kg; the dose of bicyclol was converted to 7.875 mg / kg for rats according to the adult daily dose of 75 mg. Before each gavage, an appropriate amount of Qijia free - decoction granules was weighed, added with hot pure water, and fully mixed and dissolved with a magnetic stirrer. It was prepared and used immediately. The gavage time was 4 weeks. All rats were gavaged with a unified gavage volume of 100 g / ml (that is, calculated according to the body weight of the rats, 1 ml was given for every 100 g) or normal saline.

[0355] 4. Experimental results

[0356] 4.1 Effects of Qijia Fuzheng Tongluo Prescription on serum liver function of NASH rats

[0357] As Figure 10, Animal experiments have shown that Qijia Rougan Recipe has a significant protective effect on the liver tissue of NASH rats and can reduce liver function damage in NASH rats. Compared with the control group, the levels of ALT, AST, and ALP in the model group, Qijia group, and bicyclol group were all increased (P < 0.05); compared with the model group, the levels of ALT, AST, and ALP in the Qijia group and bicyclol group were all decreased. Among them, except that the decrease in serum ALP level in the bicyclol group was not statistically significant compared with the model group, the decrease levels in the other groups and all indicators were significant (P < 0.05). The levels of ALT, AST, and ALP in the Qijia group were slightly lower than those in the bicyclol group, but the difference was not statistically significant.

[0358] 4.2 Effect of Qijia Fuzheng Tongluo Recipe on the pathological morphology of liver tissue in NASH rats

[0359] As Figure 11 (a), The liver surface of the control group rats was smooth, with a moderate volume, bright color, reddish-brown color, elastic touch, and tough texture; the liver volume of the model group rats was significantly increased, the surface was rough, with obvious granularity, the color was yellowish-brown, and the texture was greasy; the liver size of the Qijia group and bicyclol group rats had no obvious change compared with the model group, but the surface was smoother, the capsule toughness was improved, the color was bright and shiny, and the greasy feeling was reduced.

[0360] As Figure 11 (b), HE staining of liver tissue showed that the hepatocytes of the control group rats were arranged neatly, and the hepatic lobules formed hepatic cell cords radially from the central vein to the periphery, with a complete structure, and no inflammatory cell infiltration or ballooning degeneration was seen; the hepatic lobule structure of the model group rats was severely damaged, the hepatic cords disappeared, a large number of inflammatory cells infiltrated, local eosinophilic bodies were generated, and a large number of fat vacuoles and cell ballooning degeneration were visible between the hepatic lobules, indicating successful modeling; the hepatocyte structure of the Qijia group and bicyclol group was relatively complete, and the radial strip-shaped hepatic cords were still visible, the fat vacuoles were relatively reduced, and the inflammatory cells mainly surrounded the hepatic lobules.

[0361] As Figure 11 (c), The results of Oil Red O staining of liver tissue in each group of rats showed that no obvious red-stained lipid droplets were seen in the control group rats; red-stained lipid droplets were diffusely distributed in the intercellular spaces of the hepatocytes in the model group rats, especially large red round granules were piled up in the portal area; compared with the model group, the number and size of lipid droplets in the Qijia group and bicyclol group rats were significantly reduced, the distribution was sparse, and the granules were smaller.

[0362] As Figure 11(d). The Masson staining results of the liver tissues of rats in each group showed that there was no abnormal collagen fiber deposition in the control group, and the hepatic lobule structure was normal; in the model group, a large amount of collagen fiber deposition was visible, the hepatic lobules were surrounded by coarser fiber septa, pseudo-lobules were formed, and there were more fat vacuoles; compared with the model group, the fibrosis in the Qijia group and the bicyclol group was significantly improved, the fiber septa and fat vacuoles were both reduced, and the arrangement of hepatocytes was more regular.

[0363] As can be seen from the above results, Qijia Rougan Recipe can reduce serum transaminases, significantly reduce lipid deposition, liver tissue inflammation and collagen fiber deposition in the treatment of NASH.

[0364] In summary, when the pharmaceutical composition of the present invention is used for treating liver fibrosis, it can significantly reduce liver tissue inflammation, reduce collagen fiber deposition in liver tissue, significantly reduce the liver index of rats, significantly reduce the numerical values of related indicators such as serum liver function ALT and AST in rats, significantly down-regulate the numerical values of related indicators such as serum PⅢP, PCⅣ, HA and LN, significantly inhibit the NLRP3 inflammasome pathway in liver tissue, inhibit the activation of hepatic stellate cells, the core link of liver fibrosis, and reduce the autophagy and activation of hepatic stellate cells; when used for treating non-alcoholic steatohepatitis, it can reduce serum transaminases and significantly reduce lipid deposition, liver tissue inflammation and collagen fiber deposition. These results indicate that the pharmaceutical composition of the present invention has good therapeutic effects on non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

Claims

1. A pharmaceutical composition for treating non-alcoholic fatty liver disease and liver fibrosis, characterized in that: It is prepared from the following raw medicinal materials in the following weight ratios: Astragalus membranaceus 15 - 50 parts, turtle shell 5 - 25 parts, angelica sinensis 5 - 25 parts, safflower 10 - 30 parts, peach kernel 10 - 30 parts, rhizoma sparganii 5 - 25 parts, rhizoma zedoariae 5 - 25 parts, ground beetle 5 - 25 parts, liquorice 1 - 10 parts.

2. The pharmaceutical composition according to claim 1, wherein: It is prepared from the following raw medicinal materials in the following weight ratios: Astragalus membranaceus 25 - 35 parts, turtle shell 10 - 20 parts, angelica sinensis 7 - 13 parts, safflower 15 - 25 parts, peach kernel 15 - 25 parts, rhizoma sparganii 10 - 20 parts, rhizoma zedoariae 10 - 20 parts, ground beetle 8 - 12 parts, liquorice 2 - 7 parts.

3. The pharmaceutical composition according to claim 2, characterized in that: It is prepared from the following raw medicinal materials in the following weight ratios: Astragalus membranaceus 30 parts, turtle shell 15 parts, angelica sinensis 9 parts, safflower 18 parts, peach kernel 18 parts, rhizoma sparganii 15 parts, rhizoma zedoariae 15 parts, ground beetle 10 parts, liquorice 5 parts.

4. A method for preparing the pharmaceutical composition according to any one of claims 1-3, characterized in that: It includes the following steps: S1: Weigh the raw medicinal materials according to the ratio, mix them, add 8 - 10 times the amount (g / g) of water, soak for 0.5 - 1 hour, decoct twice, each time for 1 - 1.5 hours, filter, combine the filtrates, concentrate into an extract, dry under vacuum, and pulverize to obtain a dry extract powder; S2: Add the dry extract powder obtained in step S2 to pharmaceutically acceptable excipients to prepare a preparation.

5. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a medicament for treating non - alcoholic fatty liver disease.

6. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a medicament for treating liver fibrosis.

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