Study On Chemical Composition And Anti-liver Fibrosis Effect Of Anemones Raddeanae Rhizoma Before And After Processing

Objective:Hepatic fibrosis（HF）is the common basic pathological feature of various chronic liver diseases.Continuous hepatic fibrosis will gradually develop into cirrhosis and even liver cancer.Therefore,it is urgent to find effective anti-hepatic fibrosis drugs.Group early identified Anemones Raddeanae Rhizoma（RAR）have the function of the anti-inflammatory,liver cancer,vinegar,main effect is better,hint at both ends can be used for liver disease,so in this paper,to determine the best process of Anemones raddeanae rhizoma（RAR）stir-frying with vinegar,compare the changes of chemical composition and toxicity before and after RAR stir-frying with vinegar,explore the basis of the pharmacological substance,potential target and pathway of RAR anti-HF,and study the effect and mechanism of raw and vinegar processed RAR anti-HF.Methods:1.Single factor investigation,cross experimental design and other methods were used to determine the steaming time,rice vinegar dosage,temperature,time and other parameters.2.UHPLC-Q-Orbitrap/MS combined to compare the chemical composition changes of raw and vinegar processed RAR.Survival,weight gain and food intake,histopathological changes of viscera,liver and kidney function indexes of ICR mice were measured,and acute toxicity of RAR and vinegar RAR in different doses was investigated.3.SD rats after gavage administration,serum samples were collected.Swiss Target Prediction and TCMSP database were used to obtain blood component targets,Genecards and OMIM database were used to obtain HF targets,Cytoscape 3.8.1 software was used to construct the blood component-target-HF action network at both ends,and PPI network was constructed based on STRING database.GO enrichment analysis and KEGG enrichment analysis were performed to screen the potential targets and pathways of anti-HF at both ends.4.CCl₄-induced HF model in SD rats was constructed and colchicine was used as positive control.Different concentrations of RAR and vinegar RAR were administered.Serum cytokines such as TNF-αand IL-1βand HF indexes such as HA and LN were determined by ELISA.Antioxidant indexes such as MDA and SOD were determined by kit method.Liver function indexes such as AST and ALT were determined by biochemical analysis.HE staining and Masson staining were used to detect the degree of HF in rats.Immunohistochemistry was used to detect the expression of TGF-β1,α-SMA and other proteins in rat liver tissues by raw and vinegar processed RAR.RT-q PCR was used to detect the expression of m RNA related to HF in rat liver tissues by raw and vinegar processed RAR.Western-blot was used to detect the expression of TGF-β1,α-SMA,et al in rat liver tissues.5.HSC-T6 cells induced by TGF-β1 were used as in vitro model of HF.The proliferation of HSC-T6 cells was determined by CCK-8 method,the effect of flow cytometry on h SC-T6 cell cycle was detected by Hoechst staining and JC-1 staining to detect h SC-T6 cell apoptosis and mitochondrial membrane potential changes.The expression of hepatic fibrosis related m RNA in HSC-T6 cells was detected by RT-q PCR,and the expression of hepatic fibrosis proteins including TGF-β1 andα-SMA in HSC-T6cells was detected by Western-blot.Results:1.The vinegar processing technology of the tips of both ends is:clean the tips of both ends every 100g,add 20g rice vinegar,mix them well,dry them for 2h,Stir-frying at120℃for 15min,take them out until down to room temperature.2.A total of 32 compounds were identified from the RAR,including 29 negative ion model,3 positive ion model,and 22 differential compounds,including 20 negative ion model and 2 positive ion model,The content of raddeanoside R₃and other compounds decreased,while the content of oleanolic acid and other compounds increased.With the decrease of drug concentration,the toxic reaction of RAR and its concoction became smaller,,the weight of the blank group increased rapidly,and the intake of food and water was normal.In the high-concentration administration group,the toxic reactions such as death,drowsiness and diarrhea occurred,and no substantial organ damage occurred in all mice.The maximum non-toxic reaction dose of the RAR was 31.30g/kg,about 69 times that of the adult dose.The vinegar RAR was 47.99g/kg,about 106 times of the adult dose.3.26 compounds,mostly triterpenoid saponins,were identified from the decoction of The two tip,among which 19 were prototypical blood components.The target sites of 18blood components were obtained based on Swiss Target Prediction and TCMSP database.6897 HF targets were obtained by Genecards and OMIM database,and 369 non-repeating targets were found to act on HF by integrating with blood transfusion targets,constructing blood transfusion component—target—HF network.Targets closely related to HF include Akt1,STAT,MAPK3,etc.Target proteins mainly act on protein tyrosine kinase activity,amide binding,etc.The main pathways may be PI3K-Akt signaling pathway,Calcium signaling pathway,Proteoglycans in cancer,,etc.4.raw and vinegar processed RAR can reduce the pathological damage of liver tissue induced by CCl₄,reverse the increase of spleen and liver index caused by CCl₄,reduce the levels of AST,ALT,ALP,TBIL,DBIL,TBA and GGT in serum,reduce the content of MDA and improve the activity of SOD.Decreased the levels of serum cytokines TNF-αand IL-1β,increased the level of IL-10,decreased the HF indexes of PC-III,LN,Col-IV and HA,and decreased the m RNA and protein expressions of Col-1a1,α-SMA,TGF-β,SMad2 and Smad3.The expression of Smad7 m RNA and protein was enhanced,inhibited the phosphorylation of PI3K-Akt-mTOR signaling pathway proteins.5.The raw and vinegar processed RAR,Raddeanin A（RDA）,Oleanolic acid（OA）inhibited the proliferation of HSC-T6 cells,blocked the cell cycle in G0/G1 phase,induced apoptosis of HSC-T6 cells,and decreased m RNA and protein expressions of TGF-β1,α-SMA,Col-1a1,Smad2 and Smad3.The expression of Smad7 m RNA and protein was enhanced,inhibited the phosphorylation of PI3K-Akt-mTOR signaling pathway proteins.Conclusion:1.The chemical constituents of the RAR were changed and the toxicity was reduced after stir-frying with vinegar.2.The raw and vinegar processed RAR have significant therapeutic effects on CCl₄-induced HF,which may be the regulation of TGF-β1/Smad signaling pathway and PI3K/Akt/mTOR signaling pathway.