Protective Effect And Mechanism Of Mongolian Medicine Honghua-3 On Alcohol-induced Liver Injury In Mice

Objective Network pharmacology was used to predict the material basis and molecular mechanism of Honghua-3 in the treatment of ALD disease.To explore the protective effect and potential pharmacological mechanism of Honghua-3 on alcoholic liver injury mice.Methods 1.The active ingredients of Honghua-3 were collected by TCMSP database and screened by oral bioavailability(OB)and pharmacodynamics(DL).The potential targets of compounds were predicted by reverse pharmacophore matching method and drug similarity method,and then the compound-target network was constructed.ALD disease related targets were collected by OMIM database and Dis Ge NET database,and then disease target interaction network diagram was constructed.Finally,the biological function and related signal pathway were analyzed by DAVID online tool.2.ICR mice were randomly divided into the normal group,the model group,the positive group(177mg/kg)and the low,medium and high dose groups(0.65g/kg,1.3g/kg,1.95g/kg)of Honghua-3.In addition to the normal group,the other groups were given 50 °Erguotou for 4 weeks to establish the model of alcoholic liver injury in mice.Except the normal group and the model group,each group was given corresponding drug intervention.The levels of AST,alt,ALP,BILD,TBIL,TC,TG,LDL-C,HDL-C in serum and the contents of SOD,MDA,GSH,IL-6,IL-1 ? IL-1,TNF – ? in liver tissue of mice in each group were detected after the last administration;the levels of caspase-3,Bax,BCL and CYP2E1 protein were detected by immunohistochemistry;the pathological changes of liver cells were observed by HE staining.3.Proteomic analysis: TMT(tandem mass tags)technology was used to analyze the protein expression profile of liver tissue of mice with alcoholic liver injury,and the protein was quantitatively analyzed.Finally,a series of functional analysis of the screened differentially expressed proteins were conducted,including GO,KEGG functional enrichment analysis and interaction network analysis.4.Metabolomics analysis : The urine samples of ALD mice treated with Honghua-3 were analyzed by UHPC-MS/MS to obtain the metabolite profile.Principal component analysis(PCA)and orthogonal correction partial least squares discriminant analysis(OPLS-DA)were used to identify the specific markers of Honghua-3 powder in the treatment of ALD,and the pathway enrichment of different metabolites was analyzed.5.Intestinal flora analysis: the v3-v4 hypervariable region of bacteria 16 S r RNA was sequenced based on Illumina Nova sequencing method.Based on the species annotation,the diversity of hetero and hetero was further calculated and the differences between groups were compared,revealing the regulatory effect of Honghua-3 on the structure and function of intestinal flora in ALD mice.Results 1.Network analysis results showed that 47 active compounds screened from Honghua-3 directly or indirectly acted on 64 key targets of ALD disease.A total of 17 biological processes and 32 KEGG pathways were involved(P<0.05,FDR<0.05),suggesting that Honghua-3 was mainly used to treat ALD through the intervention of chemical carcinogenesis,metabolism of xenobiotics by cytochrome P450,Drug metabolism-cytochrome P450,TNF signaling pathway,cytokines and Inflammatory Response and PPAR signaling pathway.2.Compared with the model group,Honghua-3 can reduce the content of AST,alt,ALP,TC,TG and LDL-C,increase the content of HDL-C,increase the content of SOD and GHS,and decrease the content of MDA,TNF-? and IL-6;It can reduce the expression of CYP2E1,caspase-3 and bax protein,increase the expression of Bcl-2 protein,and improve the pathological symptoms such as punctate necrosis and inflammatory cell infiltration.3.The results of proteomics showed that compared with the model group,84 differential proteins were screened in Honghua-3 group,including 44 down regulated proteins and 40 up regulated proteins;Go enrichment analysis showed that these differentially expressed proteins were mainly involved in various metabolic processes,redox processes and cellular protein modification,mainly distributed in cytoplasm,nucleus,extracellular region,endoplasmic reticulum and mitochondria.KEGG enrichment analysis showed that Honghua-3 mainly regulated signal pathways such as metabolic pathways,chemical carcinogenesis,steroid hormone biosynthetic,arachidonic acid metabolism,drug metabolism-cyclochrome P450,glutathione metabolism and metabolism of xenobiology by cyclochrome P450.4.The results of metabolomics experiments showed that in the positive ion mode and negative ion mode,the metabolic profiles of normal group,model group and Honghua-3 group were completely separated,in the negative ion mode,the metabolites of Honghua-3 group and normal group were overlapped,which indicated that alcohol could make metabolism disorder in mice,and Honghua-3 had therapeutic effect on ALD.According to VIP > 1.0,FC > 1.5 or FC < 0.667 and P value < 0.05,239 potential biomarkers for the treatment of ALD were screened out.The results of metabolic pathway annotation and enrichment analysis of different metabolites showed that Honghua-3 group mainly regulated metabolic pathways such as steroid hormone biosynthesis,drug metabolism-cytochrome P450,metabolic pathways,Bill secret and inflammatory mediator regulation of TRP channels to achieve the treatment of ALD.5.The sequencing results of 16 Sr RNA showed that the analysis of Beta Diversity showed that Honghua-3 could improve the bacterial Diversity caused by alcohol.Further studies showed that,at the level of phylum,Honghua-3 improved the bacterial abundance of Bacteroides and Firmicutes,down-regulated the content of Proteobacteria,and promoted the growth of intestinal symbiotic bacteria.On the genus level,Bacteroides,streptococcus,helicobacter,Odoribacter and secret-enterobacteriaceae increased significantly.Uule-lachnospiraceae,Alloprevotella,and Lactobaculum were significantly reduced.Conclusion Honghua-3 has a protective effect on alcoholic liver injury in mice.The mechanism may play a therapeutic role by reducing the activity of ALT,AST and other enzymes in serum,reducing the release of inflammatory factors,regulating the apoptosis of liver cells,inhibiting the abnormal activation of CYP2E1,and improving the lipid metabolism and the structure of intestinal flora.The material basis and core target of Honghua-3 were predicted by network pharmacology,which provided a scientific basis for elaborating the mechanism of action of Honghua-3 on ALD.