| Objective:To clarify the effect of the Zhuang medicine Yi-Shan-Hong（YSH）on hepatic fibrosis rats,and to elucidate its mechanism of action through experimental and analytical techniques such as proteomics,molecular biology,serum pharmacochemistry,and network pharmacology,providing scientific basis for the clinical application of the Zhuang medicine YSH in treating liver fibrosis.Methods:（1）Sixty male SD rats were randomly divided into a normal control group,a model group,a silybin group,a high-dose group of YSH,a medium dose group of YSH,and a low-dose group of YSH.A hepatic fibrosis model was prepared by intraperitoneal injection of 40%carbon tetrachloride（CCl4）2 m L/Kg,twice a week for 8 weeks.After 24 hours of initial modeling,the positive control group was given 42 mg/kg silybin by gavage,while the high,medium,and low dose groups of YSH were given 12,6,and 3 g/kg by gavage,each once a day,for a total of 8 weeks of treatment.At the end of treatment,Sirius red staining was used to evaluate the degree of liver tissue fibrosis in rats.ELISA was used to detect serum hepatic fibrosis indicators such as collagen Ⅲ pre protein（PC-Ⅲ）,type Ⅳ collagen（Col Ⅳ）,hyaluronic acid（HA）,and laminin（LN）to evaluate the degree of liver fibrosis;HE staining method was used to evaluate the pathological changes of rat liver cells,serum biochemical method was used to detect the liver function of each group of rats:alanine aminotransferase（AST）,aspartate aminotransferase（ALT）,alkaline phosphatase（AKP）.Biochemical method was used to detect the changes in oxidative stress indicators in liver homogenate:superoxide dismutase（SOD）,malondialdehyde（MDA）,and glutathione peroxidase（GSH-PX）.（2）Using the 4D label-free quantitative proteomics analysis method,the protein changes in the liver tissue of rats in the control group,model group,and high-dose group of YSH were analyzed globally.Using a P-value of less than 0.05 as the standard,proteins with a protein ratio greater than 1.5 are considered upregulated,and proteins with a protein ratio less than 1/1.5 are considered downregulated.Gene ontology（GO）classification analysis and Kyoto Encyclopedia of Genomes and Genomes（KEGG）pathway enrichment analysis were performed on the differentially expressed proteins,and subcellular localization of the differentially expressed proteins was demonstrated.Real-time quantitative PCR（q RT-PCR）and Western Blotting（WB）immunoblotting experiments were used to verify the mRNA and protein expression levels on two pathways enriched with differentially expressed proteins in the liver tissues of six groups of rats.（3）Using ultra-high performance liquid chromatography tandem quadrupole time-of-flight high-resolution mass spectrometry（UPLC-Q-TOF-MS）technology,serum pharmacochemical analysis was performed on the serum of rats treated with Zhuang medicine YSH.Qualitative analysis was conducted on the main blood components of Zhuang medicine YSH,and combined with network pharmacology,the possible mechanism of improving liver fibrosis was elucidated.Results:（1）Both YSH and the positive control drug silybin can significantly improve the level of hepatic fibrosis caused by CCl4 modeling in rats,reduce collagen accumulation in liver tissue,improve the disorder of liver fiber septum and lobular structure caused by modeling,and significantly reduce the levels of hepatic fibrosis markers HA,LN,PC-Ⅲ,and Col Ⅳ.Alleviate liver cell swelling,degeneration,and inflammatory infiltration,and improve liver function and reduce levels of ALT,AST,and AKT in rats;Reduce liver oxidative stress response,significantly reduce oxidative stress indicator MDA,significantly increase SOD and GSH-PX levels to regulate liver oxidative stress response.Among them,high-dose YSH has the best effect,and the effect decreases with decreasing dosage.（2）Proteomics identified a total of 50581 peptide segments and 5797proteins,with 4801 comparable proteins.After modeling,compared with the control group,a total of 543 proteins were significantly upregulated and 636proteins were significantly downregulated in rat liver tissue.Compared with the liver tissue of rats after modeling,there were a total of 191 proteins significantly upregulated and 204 proteins significantly downregulated in the YSH treatment group.The differential proteins between the model group and the normal group,as well as between the YSH group and the model group,are more localized in the cytoplasm.The KEGG pathway enrichment analysis results showed that there were a total of 37 pathways enriched in differential proteins between the model group and the normal group,including pathways related to liver fibrosis,such as alanine,asparagine and glutamate metabolism,linolenic acid metabolism,glycine,serine and threonine metabolism,valine,leucine and isoleucine degradation,lysine degradation,etc;There are a total of 18 pathways enriched in differentially expressed proteins between the YSH treatment group and the model group,including DNA replication,vitamin B6metabolism,linolenic acid metabolism,cell cycle,ABC transporters,tryptophan metabolism,bile secretion,and other pathways related to liver fibrosis.Among them,differential proteins in the DNA replication（map03030）signaling pathway were highly upregulated in the model group,while significantly downregulated in the YSH group;The protein levels in the tryptophan metabolism（map00380）signaling pathway were significantly reduced in the model group and significantly upregulated in the YSH group.（3）The mRNA expression of proteins MCM7,MCM3,FEN1,PCNA,MCM4,RFC2,AND MCM5 on the DNA replication pathway in the model group rats was significantly increased,while the mRNA levels of these proteins were significantly downregulated at high,medium,and low doses of YSH;High doses of YSH can significantly reduce the protein expression levels of MCM7,PCNA,FEN1,and RFC2 that were elevated after modeling.However,the medium dose of YSH only reversed the protein expression of PCNA and RFC2,while the low dose only reversed the protein expression of FEN1.The mRNA expression of proteins AOX3,GCDH,IDO2,AFMID,TDO2,KMO,and AADAT on the tryptophan metabolism signaling pathway in the model group rats was significantly reduced in the modeling group.However,high and medium doses of YSH reversed these abnormal expressions.Low doses of YSH failed to reverse the mRNA expression of AOX3,GCDH,AND IDO2.The expression levels of GCDH and TDO2proteins were consistent with mRNA expression.The regulation of these protein expressions by Zhuang medicine YSH is dose-dependent.（4）Preliminary identification has yielded 25 blood components of the Zhuang medicine YSH,including 16 in positive ion mode and 13 in negative ion mode.Mainly including flavonoids,organic acids,terpenes,alkaloids,etc.The hepatic fibrosis related targets that these blood entering components are most likely to intervene in include estrogen receptorsα、Heat shock protein90α、Protein kinase B,nuclear receptor co activating factor 1,epidermal growth factor receptor,etc.may improve hepatic fibrosis by regulating signaling pathways such as alcoholic liver disease（hsa04936）,hepatitis B（hsa05161）,NF kappa B（hsa04064）,HIF-1（hsa04066）,as well as regulating biological functions such as nuclear receptor activity,ligand activated transcription factor activity,and DNA binding transcription factor binding.Conclusions:（1）YSH and the positive control drug silybin have a significant alleviating effect on CCl4 induced hepatic fibrosis in rats.High dose YSH has the best therapeutic effect,as it can reduce the level of liver tissue fibrosis,reduce liver function damage and oxidative stress response,and reduce pathological changes such as inflammation and infiltration in liver tissue.（2）Proteomic analysis revealed significant changes in protein expression in rat liver tissue after CCl4 modeling.After administration of YSH,the expression of some proteins was adjusted.These proteins are mainly localized in the cytoplasm.KEGG pathway enrichment analysis showed that these differently expressed proteins involved multiple pathways related to liver fibrosis;The specific mechanism of action of YSH may involve signaling pathways such as DNA replication and tryptophan metabolism.（3）For the first time,UPLC-Q-TOF-MS method was used to preliminarily identify serum compounds in rats after oral administration of the Zhuang medicine YSH.25 blood entering components were preliminarily identified,mainly including flavonoids,organic acids,terpenes,coumarins,alkaloids,and other compounds.These blood components may work together through multiple targets,pathways,and biological processes to improve liver fibrosis. |
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