Application of icaritin in hepatic fibrosis

Abstract

The invention discloses application of icaritin in hepatic fibrosis, and belongs to the technical field of hepatic fibrosis drugs. According to the invention, a CCL4-induced SD rat hepatic fibrosis model is adopted to evaluate the treatment effect of icaritin on hepatic fibrosis, and experiments find and confirm the inhibition effect of icaritin on hepatic fibrosis; in-vivo and in-vitro experiments prove that the icaritin has an inhibiting effect on activity of hepatic stellate cells and liver inflammation and can effectively intervene in the process of hepatic fibrosis. According to the application of the icaritin in the hepatic fibrosis, the provided compound icaritin has great potential for inhibiting the hepatic fibrosis, an effective traditional Chinese medicine monomer component medicine is provided for treating the hepatic fibrosis, the clinical new adaptation application of the icaritin is expanded, and the icaritin has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of liver fibrosis drugs, and in particular to the application of icariin in liver fibrosis. Background Art

[0002] According to the pathological changes and clinical manifestations of liver fibrosis (including liver cirrhosis), it is summarized by TCM disease names, and is mostly classified as "accumulation" and "flank pain". The basic TCM syndrome pathogenesis of liver fibrosis is that deficiency leads to accumulation, and deficiency of vital energy leads to blood stasis. "Blood stasis is the body (symptom) of accumulation, and deficiency is the root (root) of accumulation". Deficiency of vital energy is mainly manifested as deficiency of both qi and yin; blood stasis is mainly manifested as blood stasis blocking the collaterals. Its basic syndrome type is deficiency of qi and yin, and blood stasis blocking the collaterals. Western medicine believes that liver fibrosis is a common pathological process of chronic liver diseases caused by multiple causes such as viral hepatitis, alcoholism, and non-alcoholic fatty liver disease. Its essence is that various causes induce hepatocytes and Kupffer cells to over-secrete cytokines, reactive oxygen species, chemokines and growth factors, promote the proliferation, synthesis and secretion of extracellular matrix (ECM) by hepatic stellate cells (HSC), and accumulate them to form fibrous scars, leading to abnormal changes in liver tissue structure, and then forming liver fibrosis.

[0003] Liver fibrosis is a reversible process. Early effective anti-fibrosis treatment can prevent or slow down its progression to cirrhosis or even liver cancer. From the perspective of the pathogenesis of liver fibrosis, the reversal of liver fibrosis mainly involves: treating the primary disease or removing the pathogenic factors, eliminating liver inflammation, inhibiting collagen fiber formation, promoting the apoptosis of activated HSC or converting it back to the quiescent state, or directly promoting the degradation of fibrous tissue.

[0004] At present, the treatment of liver fibrosis mainly includes the treatment of the cause of liver fibrosis and anti-fibrosis treatment. Western medicine only treats the cause of liver fibrosis, and there is no drug that directly treats liver fibrosis on the market. At present, traditional Chinese medicine for the treatment of liver fibrosis, including Fuzheng Huayu Tablets and Anluo Huaxian Pills, has been used clinically. However, on the one hand, its clinical efficacy is limited, and on the other hand, due to the complex composition of traditional Chinese medicine, its specific molecular mechanism is not fully understood, which limits its clinical application prospects. Therefore, finding effective anti-fibrosis drugs with clear mechanisms is an urgent clinical need.

[0005] Icaritin (ICT) is the dried leaves of the herbaceous plant Epimedium of the Berberidaceae family. It is pungent, sweet and warm in nature and enters the liver and kidney meridians. It has the functions of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is often used for kidney yang deficiency, impotence and spermatorrhea, tendon and bone weakness, rheumatic pain, etc. ICT is an isopentenyl flavonoid compound extracted from Epimedium, which has multiple pharmacological effects such as anti-inflammatory, anti-tumor, and immunomodulatory. It has been proven to have anti-tumor effects in malignant tumors such as breast cancer, gastric cancer, and liver cancer.

[0006] The intestine and liver are closely connected in both anatomy and biological functions, forming the "gut-liver axis". In recent years, with the application of gene sequencing technology, the diversity of fecal intestinal flora in patients with cirrhosis has decreased, the content of beneficial bacteria such as Clostridium and Akkermansia in the intestinal flora has decreased, and the levels of harmful bacteria such as Bacillus and Proteobacteria have increased. The proliferation and activation of HSC are key factors in the formation of liver fibrosis. A healthy intestinal barrier can prevent harmful bacteria and their products (such as lipopolysaccharide) from entering the blood, but when the barrier is damaged, these bacteria and products enter the liver through the portal vein, activate Kupffer cells, and induce chronic inflammation, which in turn promotes the activation of HSC and liver fibrosis.

[0007] Some scholars also believe that after liver fibrosis occurs, the increased portal vein pressure may lead to impaired intestinal blood return and reduced bile acid supply, which in turn leads to intestinal flora imbalance and intestinal mucosal barrier damage. Some scholars have restored intestinal flora disorders by taking oral probiotics based on Acinetobacter muciniphila and found that it can restore intestinal barrier function and inhibit the development of liver fibrosis. After eliminating the pathogenic factors of chronic liver disease, if the intestinal microbiome disorder and intestinal barrier are not improved, bacterial translocation will continue to stimulate chronic inflammation and the progression of liver fibrosis.

[0008] Currently, icariin has been conditionally approved as a treatment for hepatocellular carcinoma for the treatment of patients with advanced liver cancer who are not suitable for standard treatment (Guidelines for the Diagnosis and Treatment of Primary Liver Cancer 2024 Edition). However, the effect of icariin on inhibiting liver fibrosis and its mechanism are still unclear. Summary of the invention

[0009] The purpose of the present invention is to provide an application of icariin in liver fibrosis, aiming to prove the inhibitory effect of icariin on the mechanism of liver fibrosis through animal experiments, and preliminarily explore its mechanism which may be related to intestinal flora and liver metabolism.

[0010] To achieve the above object, the present invention provides an application of icariin in liver fibrosis, wherein the application comprises at least one of the following 1) to 6):

[0011] 1) Icariin inhibits pathological changes associated with liver fibrosis by regulating the abundance and diversity of intestinal flora;

[0012] 2) Icariin inhibits liver fibrosis by improving bile acid metabolism;

[0013] 3) Icariin inhibits liver fibrosis by intervening in the linoleic acid metabolic pathway;

[0014] 4) Icariin improves liver function in rats with CCL4-induced liver fibrosis;

[0015] 5) Icariin improves liver fibrosis in rats with CCL4-induced liver fibrosis;

[0016] 6) Icariin improves fatty degeneration of hepatocytes in rats with CCL4-induced liver fibrosis.

[0017] Preferably, the intestinal flora includes s__Ligilactobacillus_murinus, s__GGB27906_SGB40342, s_Akkermansia_muciniphila, and s_Coriobacteriaceae_bact erium; the abundance of s__Ligilactobacillus_murinus and s__GGB27906_SGB40342 is negatively correlated with the degree of liver fibrosis, and the abundance of s_Akkermansia_muciniphila and s_Coriobacteri aceae_bacterium is positively correlated with the degree of liver fibrosis.

[0018] Preferably, the drug contains icariin as the single or main active ingredient.

[0019] Preferably, the liver function test includes ALT and AST.

[0020] Preferably, the liver fibrosis includes HA, LN, PCIII liver fibrosis indicators in the blood and HE staining, Masson staining of liver tissue and their standardized scores.

[0021] Therefore, the application of icariin in the present invention in inhibiting carbon tetrachloride-induced rat liver fibrosis has the following beneficial effects:

[0022] (1) The present invention adopts CCL 4 The induced SD rat liver fibrosis model was used to evaluate the therapeutic effect of icariin on liver fibrosis. The experiment found and confirmed the inhibitory effect of icariin on liver fibrosis. In vivo and in vitro experiments confirmed that icariin has an inhibitory effect on the activity of hepatic stellate cells and liver inflammation, and can effectively intervene in the process of liver fibrosis.

[0023] (2) The prior art mainly focuses on the direct anti-tumor effect of icariin, and has not yet explored in depth the effect of icariin on liver fibrosis. The present invention proposes for the first time the synergistic effect of icariin and intestinal flora in inhibiting liver fibrosis, providing new theoretical support for the research on the medicinal application of icariin.

[0024] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0026] Figure 1 A is HE staining of liver tissue of the normal group 4 weeks after modeling in Example 1, and B is HE staining of liver tissue of the model group 4 weeks after modeling;

[0027] Figure 2 This is a test flow chart of the model group after successful modeling in Example 1;

[0028] Figure 3 The liver, body weight, liver coefficient and biochemical function of SD rats in each group, among which A is the gross specimen of liver of SD rats in each group; B is the change of body weight of SD rats in each group; C is the liver coefficient of SD rats in each group; D is the ALT level at 4 weeks of the experiment (before drug administration); E is the ALT level at 6 weeks of the experiment (2 weeks after drug administration); F is the ALT level at 8 weeks of the experiment (4 weeks after drug administration); G is the AST level at 4 weeks of the experiment (before drug administration); H is the AST level at 6 weeks of the experiment (2 weeks after drug administration); I is the AST level at 8 weeks of the experiment (4 weeks after drug administration);

[0029] Figure 4 are the liver pathological results and fibrosis indexes, among which A is the result of HE staining of liver of SD rats in each group; B is the result of Masson staining of liver of SD rats in each group; C is the score of liver fibrosis; D is the score of liver fatty degeneration; E is the NAS score; F is the level of hyaluronidase; G is the level of laminin; H is the level of type III procollagen;

[0030] Figure 5 This is the diversity analysis of intestinal flora of SD rats in each group, where A is Chao1 index; B is Shannon index; C is Simpson index;

[0031] Figure 6This is the analysis of the differential bacterial flora of each group of SD rats, where A is the significantly differential bacteria among the three groups of SD rats at the phylum level; B is the statistical graph of A; C is the significantly differential bacteria among the three groups of SD rats at the species level, and D is the statistical graph of C;

[0032] Figure 7 The KEGG enrichment results of differential bacterial communities, where A is the average abundance enrichment analysis diagram; B is the LDA value enrichment analysis diagram;

[0033] Figure 8 The correlation between the TOP6 differential bacteria and the degree of liver fibrosis, among which A is s__Ligilact obacillus_murinus bacteria; B is s__Lactobacillus_intestinalis bacteria; C is s__Lactobac illus_johnsonii bacteria; D is s__Akkermansia_muciniphila bacteria; E is s__Coriobacter iaceae_bacterium bacteria; F is s__GGB27906_SGB40342 bacteria;

[0034] Fig. 9 This is the result analysis of liver non-targeted metabolomics, where A is the OPLS-DA analysis of the normal control group and the model control group; B is the OPLS-DA analysis of the model control group and the high-dose icariin; C is the 200 permutation test analysis corresponding to A; D is the 200 permutation test analysis corresponding to B; E is the differential metabolite screening volcano between the normal control group and the model control group; F is the differential metabolite screening volcano between the high-dose icariin group and the model control group; G is the differential metabolic pathway between the normal control group and the model control group; H is the differential metabolic pathway between the high-dose icariin group and the model control group;

[0035] Fig.10 The figure shows the effect of icariin on the inhibition rate of HSC-T6 (hepatic stellate cells), wherein A is the cell activity of each group 24 hours after icariin administration; B is the morphology of each group of cells under a microscope 24 hours after icariin administration. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0037] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0038] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0039] Embodiment 1

[0040] (1) Establish a liver fibrosis SD rat model. The process is as follows Figure 1 As shown in Part A:

[0041] 44 SD rats (200 g / rat) were selected and kept in an SPF clean environment at 24°C. They were randomly divided into 2 groups: a normal control group of 10 rats and a modeling group of 34 rats.

[0042] The rats in the modeling group were intraperitoneally injected with 50% CCL4 olive oil solution, with an injection volume of 2 mL / kg, twice a week for 4 consecutive weeks. The normal control group was given an equal volume of olive oil. After 4 weeks of modeling, the liver tissues of 4 rats (2 in the normal control group and 2 in the modeling group) were fixed and pathologically examined, showing pathological characteristics of liver fibrosis.

[0043] Elisa was used to analyze the serum liver function (ALT, AST) and liver fibrosis indexes of each group of rats before drug administration. The results were as follows Figure 1 As shown in parts A and B, liver function (ALT, AST) and liver fibrosis indexes were significantly higher than those in the normal control group, indicating that the model was successfully established.

[0044] (2) Grouping and dosing regimen, such as Figure 2 As shown: After the model was successfully established, the model group was randomly divided into 4 groups, 8 in each group. That is, the model control group (0.5% CMC-Na solution), the low-dose group (icariin 1mg / kg / d), the medium-dose group (icariin 4mg / kg / d), and the high-dose group (icariin 16mg / kg / d). Another 8 normal control groups were given 0.5% CMC-Na solution. A total of 40 mice. The drug was administered by gavage once a day, and the experimental endpoint was 4 weeks after the drug.

[0045] Drug dosage basis and preparation of test drug:

[0046] High dose of icariin for rats: rat gavage volume: 1mL / 100g=10mL / kg, i.e., preparation concentration: 1.6g / mL; if 96.9mg of icariin is weighed, dilute to 60.6mL with 0.5% CMC-Na solution.

[0047] Medium dose for rats: oral gavage volume for rats: 1 mL / 100 g = 10 mL / kg, i.e., prepared concentration: 0.4 g / mL; draw 20 mL from the high dose and make up to 80 mL with 0.5% CMC-Na solution.

[0048] Low dose for rats: oral gavage volume for rats: 1mL / 100g=10mL / kg, i.e., prepared concentration: 0.4g / mL; draw 20mL from the medium dose and make up to 80mL with 0.5% CMC-Na solution.

[0049] (3) Detection indicators: The rats' hair, mental state, body weight and food intake were observed and monitored. Blood was collected before administration and 4 and 8 weeks after administration and centrifuged at 4000 rpm for 10 min. The supernatant serum was used to detect serum liver function (ALT, AST) using a kit. The degree of liver fibrosis was evaluated by HE staining and Masson staining. Liver inflammatory factors, fibrosis markers and immune responses related to intestinal flora were detected by ELISA, Western blot and other techniques.

[0050] ① Metagenomics: Tianjin Novogene Technology Co., Ltd. was commissioned to conduct 16S rDNA amplicon sequencing and analysis of intestinal flora.

[0051] a. Experimental procedure: DNA sample detection: At 8 weeks of the experiment, feces of SD rats in the normal control group, model control group and high-dose icariin group were collected for fecal DNA concentration, determination and 16S rRNA sequencing. DNA was extracted from the feces using a DNA extraction kit and then the fecal DNA concentration was detected.

[0052] b. Library construction and sequencing: Take 1μg of genomic DNA from the sample, randomly break it into fragments of about 350bp in length using a Covaris ultrasonic crusher, and then construct the library. The entire library preparation is completed through end repair, A-tailing, sequencing adapter addition, purification, PCR amplification and other steps. After the library is constructed, AATI is used to detect the integrity of the library fragments and the size of the inserted fragments. If it meets expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (library effective concentration>3nM) to ensure the quality of the library. After the library inspection is qualified, different libraries are sequenced after pooling according to the effective concentration and the target amount of data off the machine.

[0053] c. Bioinformatics analysis: Fastp (https: / / github.com / OpenGene / fastp) was used to preprocess the raw data obtained by sequencing to obtain the effective data for subsequent analysis. Bowtie2 software (http: / / bowtie-bio.sourceforge.net / bowti e2 / index.shtml) was used by default, and Metaphlan 4 was called by HUMAnN 3 to compare with the Uniprot database (http: / / www.uniprot.org) to obtain the species classification results and UniRef90ID. According to the correspondence between UniRef90ID and other functional databases, the annotation results of each functional library were obtained. Then, the gene sequences were annotated with species, compared with species differences, analyzed for species composition and species richness. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate gene functions.

[0054] ② Liver metabolomics analysis

[0055] During the 8-week experiment, the rats were fasted for 12 h and then anesthetized according to their body weight. The rats were killed by cervical dislocation, and the livers were removed and stored in a -80°C ultra-low temperature refrigerator for later use.

[0056] A. Sample Preparation

[0057] The collected liver samples were placed on ice and 50% methanol buffer was added to extract metabolites. 20 μL of the sample was extracted with 120 μL of ice-cold 50% methanol, vortexed for 1 min, reacted at room temperature for 10 min, and the extraction mixture was stored at -20 ° C for 12 h. After centrifugation at 3500 r / min for 20 min, the upper clear liquid and body fluid were aspirated into a clean 96-well plate. 100 μL was taken in a 1.5 mL centrifuge tube, 400 μL of pre-cooled ice acetonitrile was added, and vortexed for 3 min; centrifuged at 14000 rpm at 4 ° C for 10 min, and the supernatant was taken; nitrogen was blown dry, 100 μL of 80% methanol was re-dissolved, and the centrifuged at 14000 rpm at 4 ° C for 10 min, and the supernatant was taken; LC-MS analysis was performed. Preparation of QC samples: Take 5 μL from each sample, gently shake and mix, add 4 times the volume of acetonitrile, treat the QC sample in the same way as the above samples, and take the supernatant for injection.

[0058] b. UHPLC-Orbitrap MS analysis conditions

[0059] Chromatographic conditions: U3000 ultra high performance liquid phase system equipped with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used, and the mobile phase was acetonitrile (A) and 0.1% formic acid aqueous solution (B) for gradient elution. The elution gradient was: 0-7 min, 5%-100% A; 7-10 min, 100%-100% A; 10-10.1 min, 100%-5% A; 10.1-13 min, 5%-5% A; the flow rate was 0.3 mL min -1 , injection volume 2 μL, column oven set to 35 °C.

[0060] c. Mass spectrometry conditions: Mass spectrometry analysis was performed on a Thermo Fisher-Orbitrap MS mass spectrometer, with a high-energy electrospray ionization source (HESI source) and Full-MS / DD-MS in positive and negative ion modes. 2 The ion source parameters were: spray voltage 3.5 kV in positive ion mode, 2.8 kV in negative ion mode, capillary temperature 320 °C, ion source auxiliary temperature 350 °C, sheath gas (N 2 ) flow rate is 35L / h, auxiliary gas (N 2 )The flow rate was 10 L / h, the collision energy was set to 20 V, 40 V, 60 V, the scanning range was m / z 100-1500, the resolution was 60000, and the RF Lens (%) was 70.

[0061] Test result analysis:

[0062] 1) Icariin can improve the body weight and biochemical function of rats with liver fibrosis model

[0063] (1) Figure 3 Part A in the middle shows the degree of liver fibrosis in SD rats in each group. It can be seen that the liver surface of the model group is rough and yellow, and as the concentration of icariin increases, the liver surface gradually becomes smooth, indicating that icariin can significantly improve liver fibrosis in rats with CCL4-induced liver fibrosis model.

[0064] (2) Figure 3 Part B and Table 1 show the weight changes of SD rats in each group. It can be seen that the weight of rats in the normal control group gradually increased, and the weight of rats in the model control group decreased significantly (P<0.001). Compared with the model control group, the icariin 1, 4, and 16 mg / kg groups showed different degrees of increase in rat weight after 1 week, 2 weeks, 3 weeks, and 4 weeks of administration (P>0.05).

[0065] Table 1 Effects on body weight (g) of rats with liver fibrosis induced by carbon tetrachloride (mean ± SD, n = 8)

[0066]

[0067] Note: Compared with the normal control group: ###, P<0.001.

[0068] (3) Figure 3 Part C shows the liver coefficient of SD rats in each group. It can be seen that the liver coefficient of rats in the model control group was significantly reduced (P<0.01). Compared with the model group, the icariin 16mg / kg group significantly reduced the liver coefficient of rats after 4 weeks (P<0.01), and the improvement rate reached 23.2%.

[0069] (D) Figure 3 Part D in the figure shows the ALT level at week 4 of the experiment (before administration). Figure 3 Part E in the middle shows the ALT level at 6 weeks of the experiment (2 weeks after drug administration); Figure 3 Part F in the middle shows the ALT level at 8 weeks of the experiment (4 weeks after drug administration); Figure 3 Part G in the figure shows the AST level at week 4 of the experiment (before administration). Figure 3 The H part in the middle shows the AST level at 6 weeks of the experiment (2 weeks after medication); Figure 3 Part I shows the AST level at 8 weeks of the experiment (4 weeks after medication).

[0070] It can be seen that 4 weeks after modeling, compared with the normal control group, the serum ALT and AST of the model control group rats were significantly increased (P<0.001), indicating that the liver had obvious hepatocyte necrosis after modeling. Compared with the model control group, the serum ALT and AST of icariin 4 and 16 mg / kg were significantly reduced (P<0.05) after 6 weeks (2 weeks after drug administration) and 8 weeks (4 weeks after drug administration), indicating that icariin can improve the liver function of rats with liver fibrosis model.

[0071] 2) Effect of icariin on food intake in rats with hepatic fibrosis model

[0072] The food intake of rats in the normal control group showed a trend of gradual increase with the increase of age, and the food intake of rats in the model control group decreased significantly before administration, 1 week, 2 weeks, 3 weeks, and 4 weeks after administration (P<0.05). Compared with the model control group, the food intake of the icariin 4 mg / kg group increased significantly after 4 weeks of administration (P<0.05), and the food intake of the icariin 16 mg / kg group increased significantly after 3 weeks and 4 weeks of administration (P<0.05). The results are shown in Table 2:

[0073] Table 2 Effects on food intake (g / rat) of rats with liver fibrosis induced by carbon tetrachloride (mean±SD, n=8)

[0074]

[0075] Note: #, compared with the normal control group, P<0.05; *, compared with the model control group, P<0.05.

[0076] 3) Effect of icariin on liver fibrosis in rats with liver fibrosis model

[0077] like Figure 4 As shown in A, the liver of SD rats in the normal control group had normal lobular structure with central veins and radial hepatic cords, while the liver of SD rats in the model control group had normal lobular structure with central veins and radial hepatic cords. 4 After 4 weeks of administration, necrosis occurred in the center of the liver lobule, lipid droplets were deposited in the liver cells, inflammatory cells infiltrated, and lipid degeneration occurred. Icariin inhibited these pathological changes, which were manifested as reduced liver cell degeneration, inflammatory cell aggregation, and lipid deposition, indicating that icariin may alleviate CCl 4 Induced hepatic steatosis in SD rats.

[0078] like Figure 4 As shown in B, Masson trichrome staining showed that compared with the normal control group, the model control group was treated with CCl 4 This led to connective tissue hyperplasia, obvious structural distortion, and deposition of fibrous collagen between the portal vein and the lobule in the liver of SD rats, indicating that liver fibrosis has been established. Icariin treatment significantly reduced the accumulation of collagen fibers, suggesting that icariin has a protective effect on liver fibrosis.

[0079] like Figure 4 As shown in C, it is the liver fibrosis score, that is, the "METAVIR score" standard liver tissue fibrosis staging diagnosis, that is, F0: no fibrosis; F1: portal area fibrosis, no fibrous septa; F2: a small amount of fibrous septa formation; F3: a large amount of fibrous septa formation leading to lobular structure deformation; F4: cirrhosis.

[0080] like Figure 4 D in the middle shows the liver steatosis score, that is, the hepatocyte steatosis score in the NAS scoring system, which is divided into three levels according to the degree of hepatocyte steatosis. The proportion of hepatocyte steatosis is 6%-33%, with a score of 1 point; the proportion of hepatocyte steatosis is 33%-66%, with a score of 2 points; the proportion of hepatocyte steatosis exceeds 66%, with a score of 3 points.

[0081] like Figure 4The NAS score is calculated by the weighted sum of hepatocyte fatty degeneration (0-3 points), intralobular inflammation (0-3 points) and hepatocyte ballooning degeneration (0-2 points).

[0082] like Figure 4 F, G, and H show the levels of three liver fibrosis indicators, namely hyaluronidase (HA), laminin (LN), and type III procollagen (PCⅢ), indicating that icariin may alleviate CCl 4 The liver fibrosis indicators of induced SD rats were significantly reduced.

[0083] 4) Effect of icariin on intestinal flora in rats with liver fibrosis model

[0084] In order to explore the effect of icariin on the intestinal flora of the liver fibrosis model, the fecal flora distribution of the normal control group, model control group, and high-dose icariin group of rats at the end of the experiment was detected by metagenomics. The results showed that:

[0085] ① Icariin can improve species richness in rats with liver fibrosis model

[0086] There were 402 OTUs in the three groups of samples, of which 33 OTUs were unique to the normal control group and 37 OTUs were unique to the model control group, indicating that Ccl 4 The induced liver fibrosis SD rat model caused a decrease in the diversity of intestinal flora in SD rats, and the administration of icariin helped to restore this diversity to a certain extent.

[0087] like Figure 5 A, B, and C show the results of the determination of the α diversity index Chao1 index, Shannon index, and Simpson index of the three groups of rats at the end of the experiment. It can be seen that compared with the model control group, icariin can increase the α diversity index.

[0088] The beta diversity assessed by Bray-Curtis distance indicated that the three groups were clearly separated by PCA, and the PCA results showed that the two principal coordinates extracted cumulatively explained 48.84% of the variance, with PC1 and PC2 explaining 26.21% and 22.23%, respectively.

[0089] To further identify the signature microbial taxa of each group, LEfSe (LDA>4, p<0.05) analysis was performed to explore the effects on the intestinal bacteria of rats from phylum to species, and a total of 70 differentially abundant taxa were identified, including 27 in the control group, 19 in the model group, and 24 in the icariin group. Surface icariin administration can increase intestinal flora taxa.

[0090] At the species level, the biomarker groups in the control group included Lactobacillus rhamnosus (Ligilactobacillus_murinus), GGB50207_SGB70279, GGB1503_SGB2083, GGB27906_SGB40342, and the model group included Olsenella_SGB42251, GGB29545_SGB42337, GGB29473_SGB42252. After icariin administration, the main dominant bacterial flora included the beneficial bacteria Lactobacillus_johnsonii (Lactobacillus_johnsonii), Phascolarctobacterium_faecium (Phascolarctobacterium_faecium), Ruminococcus_gnavus (Ruminococcus_gnavus), GGB31835_SGB45211.

[0091] The results of the study showed that icariin has a regulatory effect on the structure of intestinal microbial communities in rats with liver fibrosis.

[0092] ②Differential bacterial flora analysis

[0093] like Figure 6 As shown, A and B are the significantly different bacteria among the three groups of SD rats at the phylum level; C and D are the significantly different bacteria among the three groups of SD rats at the species level.

[0094] At the phylum level, compared with the normal control group, CCl 4 The abundance of Firmicutes in the intestinal microorganisms of rats with induced liver fibrosis decreased, while that of Bacteroidetes increased, with no statistical significance; compared with the model control group, icariin treatment increased the relative abundance of Firmicutes in the intestinal flora of rats.

[0095] At the species level, compared with the normal group, the levels of Ligilactobacillus murinus and Lactobacillus johnsonii in the model group decreased significantly, while those of Lactobacillus intestinalis, Akkermansia muciniphila, and Coriobacteriaceae bacterium decreased significantly and increased significantly.

[0096] ③Enrichment analysis based on multiple databases

[0097] Through the composition and difference analysis of KEGG metabolic pathways, the differences and changes in the metabolic pathways of the functional genes of the microbial communities between different grouping samples were observed. Figure 7 Shown are the KEGG enrichment results of differential bacterial communities.

[0098] It can be seen that the main metabolic pathways of differential bacterial enrichment in the icariin group are Teichoic acid biosynthesis, Central carbon metabolism in cancer, and Secondary bile acid biosynthesis. It is speculated that icariin may inhibit liver fibrosis by improving bile acid metabolism.

[0099] ④Correlation analysis between differential bacteria and liver fibrosis

[0100] In order to evaluate the relationship between the relative abundance of microbiota and the degree of liver fibrosis, the correlation between s__Ligilactobacillus_murinus, s__Lactobacillus_intestinalis, s__Lactobacillus_johnsonii, s__Akkermansia_muciniphila, s__Coriobacteriacea e_bacterium, s__GGB27906_SGB40342 (differential TOP6 bacteria) and the degree of liver fibrosis was compared by Spearman correlation analysis, as shown in Figure 2 . Figure 8 Species A to F are shown.

[0101] The results showed that the abundance of s__Ligilactobacillus_murinus and s__GGB27906_SGB40342 bacteria was negatively correlated with the degree of liver fibrosis, while s_Akkermansia_muciniphila and s_Coriobacteria ceae_bacterium were positively correlated with the degree of liver fibrosis ( Figure 7 Among them, the abundance of s_Coriobacterium riaceae_bacterium was most significantly positively correlated with the degree of liver fibrosis ( Figure 8 Middle E).

[0102] 5) Effect of icariin on liver metabolomics in a liver fibrosis model

[0103] ① Screening of differential metabolites

[0104] Partial least squares discriminant analysis (OPLS-DA) was used to identify the differences in metabolites and eliminate the effects of individual differences on body metabolism. The OPLS-DA model was established to evaluate the model's explanatory rate (R) and predictive ability (Q). Fig. 9 shown.

[0105] Fig. 9 A and B are OPLS-DA analysis of the pairwise comparison of each sample. It can be seen that the metabolic changes between the normal control group and the model control group are significantly different. Under the OPLS-DA model, the clustering effect of the sample points in each group is good. Then, the model validity is further tested by 200 permutation tests. The 200 permutation test analysis is as follows Fig. 9 As shown in C and D, it can be seen that R 2 Y is very close to 1, and the established model is consistent with the actual situation of the sample data.

[0106] Differential metabolites were screened based on the standard t-test P value < 0.05 and the variable projection importance (VIP) of the first principal component of the OPLS-DA model > 1.

[0107] Fig. 9 E in the middle is the differential metabolite screening volcano between the normal control group and the model control group; F is the differential metabolite screening volcano between the high-dose icariin group and the model control group. Each point in the volcano plot represents a metabolite, the abscissa represents the fold change of each substance in the group (taking the logarithm with base 2), and the ordinate represents the P value of the t-test (taking the negative number of the logarithm with base 10). The color of the scattered points represents the final screening results, with significantly upregulated metabolites represented in red, significantly downregulated metabolites represented in blue, and non-significantly different metabolites in gray.

[0108] It can be seen that compared with the normal control group, 197 differential metabolites in the model control group were significantly upregulated, and 169 differential metabolites were significantly decreased. Compared with the model control, 55 differential metabolites were significantly upregulated and 168 differential metabolites were significantly decreased after icariin treatment.

[0109] right Fig. 9 The data of E and F were analyzed by pathway enrichment analysis using the Metabo Analyst open source platform, and the related metabolic pathways were screened by differential metabolites, such as Fig. 9 As shown in G and H. It can be seen that compared with the normal control group, the metabolic pathways affected by the metabolites in the model control group are linoleic acid metabolism, glyoxylate and dicarboxylate metabolism, and compared with the model control group, the metabolic pathways affected by the metabolites after high-dose icariin treatment are related to linoleic acid metabolism. The common pathway between the two is linoleic acid metabolism, which suggests that icariin intervenes in the linoleic acid metabolic pathway.

[0110] 6) In vitro experiment: Effect of icariin on the inhibition rate of HSC-T6 (hepatic stellate cells)

[0111] The CCK8 experiment was used to observe the inhibitory effect of icariin at gradient concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128 μM) on hepatic stellate cells (HSC-T6).

[0112] like Fig.10 As shown in A, the cell activities of each group after icariin administration for 24 hours; Fig.10 B shows the morphology of cells in each group under a microscope 24 hours after icariin administration. It can be seen that as the concentration of icariin increases, the growth of HSC-T6 cells is significantly inhibited.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The use of icariin in liver fibrosis, characterized in that: The application includes at least one of the following 1) to 6): 1) Icariin inhibits pathological changes associated with liver fibrosis by regulating the abundance and diversity of intestinal flora; 2) Icariin inhibits liver fibrosis by improving bile acid metabolism; 3) Icariin inhibits liver fibrosis by intervening in the linoleic acid metabolic pathway; 4) Icariin improves liver function in rats with CCL4-induced liver fibrosis; 5) Icariin improves liver fibrosis in rats with CCL4-induced liver fibrosis; 6) Icariin improves fatty degeneration of hepatocytes in rats with CCL4-induced liver fibrosis.

2. The use of icariin in liver fibrosis according to claim 1, characterized in that: The intestinal flora includes s__Ligilactobacillus_murinus, s__GGB27906_SGB40342, s_Akkermansia_muciniphila, and s_Coriobacteriaceae_bacterium; the abundance of s__Ligilactobacillus_murinus and s__GGB27906_SGB40342 is negatively correlated with the degree of liver fibrosis, while the abundance of s_Akkermansia_muciniphila and s_Coriobacteriaceae_bacteriu m is positively correlated with the degree of liver fibrosis.

3. The use of icariin in liver fibrosis according to claim 1, characterized in that: The medicine uses icariin as the single or main active ingredient.

4. The use of icariin in liver fibrosis according to claim 1, characterized in that: The liver function tests include ALT and AST.

5. The use of icariin in liver fibrosis according to claim 1, characterized in that: The liver fibrosis includes HA, LN, PCIII liver fibrosis indicators in the blood and HE staining, Masson staining and standardized scores of liver tissue.

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