Multi-Target Based Mechansim Investigation Of Fuzheng Huayu Formula In Treating Liver Fibrosis

Liver fibrosis is a pathological change caused by chronic liver injury.It is a necessary stage for the development of chronic liver disease to cirrhosis,which can seriously affect liver function,produce various complications,and even endanger life.Hepatic fibrosis is characterized by excessive accumulation of extracellular matrix protein(ECM),while hepatic stellate cells with fibrotic and proliferative activation status are the main source of ECM,thus inhibiting hepatic stellate cell activation is one of the important strategies for the treatment of liver fibrosis.Fuzheng Huayu Recipe was developed by Shanghai University of Traditional Chinese Medicine for liver fibrosis and blood stasis.The compound consists of Salvia miltiorrhiza Bge.,Cordyceps sinensis(Berk.)Sae.,Prunus persica(L.)Batsch,Pinus massoniana Lamb.,pentaphyllum(Thunb.)Makino and Schisandra chinensis(Turcz.)Baill.The Salvia miltiorrhiza Bge.in the blood is used as a monarch;the Cordyceps sinensis(Berk.)Sae.tonic deficiency,qi and blood,Prunus persica(L.)Batsch kernel to help Salvia miltiorrhiza Bge.blood stasis,a total of ministerial medicine;Pinus massoniana Lamb.Yiqi Runzao,pentaphyllum(Thunb.)Makino heat detoxification,a total of adjuvant;Schisandra chinensis(Turcz.)Baill.sour,for menstruation.The combination of various medicines,promoting blood circulation and removing blood stasis,and replenishing the liver to achieve liver fibrosis effect.At present,the research on the chemical substance basis and anti-liver fibrosis mechanism of Fuzheng Huayu Recipe is not comprehensive.The network pharmacology,characterized by holistic and systematic nature,focuses on exploring the relationship between drugs and diseases from the whole,which coincides with the principles of TCM's holistic view,syndrome differentiation and prescription.Therefore,based on the research of Fuzheng Huafang Chemical Substance Group,this study used the combination of network pharmacology and multi-omics to explore the mechanism of Fuzheng Huayu formula against liver fibrosis.The research content of this paper is mainly divided into the following three parts:1.Rapid identification of chemical components in the body and outside of Fuzheng Huayu Recipe by UHPLC-Q-TOF/MS method.The UHPLC-Q-TOF/MS method was used to rapidly identify the chemical components in vitro and in vivo of Fuzheng Huayu Recipe.The mass spectrometryinformation collected in the positive and negative ion modes was established,and the chemical analysis of Fuzheng Chemical Recipe was established for data analysis.A total of 153 in vitro chemical components and 49 in vivo chemical components were identified in Fuzheng Huayu Recipe,and the next step was to apply network pharmacology methods to explore the effective components and mechanism of anti-liver fibrosis in Fuzheng Huayu Recipe.2.Combined network pharmacology and transcriptomics methods to explore the active constituents and mechanism of action of Fuzheng Huayu Recipe on inhibiting hepatic stellate cell activationBased on the chemical substance group of Fuzheng Huafang,the potential active ingredients of the compound were screened by text mining.Then,combined with similarity matching and molecular docking methods,40 active components and 79 potential targets against hepatic fibrosis were screened from Fuzheng Huayu Recipe.To improve the accuracy of target identification,we combined the transcriptomics data to construct a "component-target-difference gene protein" network,reducing 79 potential targets to 31 species.The results of enrichment analysis of target proteins indicate that Fuzheng Huayu Recipe can inhibit hepatic stellate cell activity by regulating HIF-1,PI3K-Akt,FoxO and chemokine signaling pathways,and thus has the effect of treating liver fibrosis.Finally,we used the CCK8 method to detect the activation of hepatic stellate cells by 15 potential active ingredients in Fuzheng Huayu Recipe.The results showed that dihydrotanshinone I,Schisandrin A,Schisandrin B,Salvianolic acid A,salvianolic acid B,tanshinone IIA,cordycepin and kaempferol can significantly reduce the activity of hepatic stellate cells(rat hepatic stellate cell line-T6 cells and human hepatic stellate cell line-LX-2 cells).Further,anti-fibrotic effect was exerted,wherein dihydrotanshinone I had the strongest inhibitory activity(IC50=5.6 ?M for T6 cells;IC50 = 14.4 ?M for LX-2 cells).Among the 31 potential target proteins screened,we selected peroxisome proliferative activated receptor ?(PPARG)for experimental and cellular level verification.The results showed that schisandrin B,salvianolic acid A and kaempferol could directly bind to PPARG,reduce the viability of hepatic stellate cells(T6 cells and LX-2 cells)and exert anti-fibrotic effects.3.Based on the "protein-metabolite interaction" strategy to study the multi-target mechanism of dihydrotanshinone I inhibiting the activation of hepatic stellate cellsBased on the above studies,we used network pharmacology methods such as text mining,similarity matching and molecular docking to identify potential targets of 182dihydrotanshinone I against liver fibrosis.In order to more accurately locate the target protein,we further combined the metabolome.Based on the data,a network of "dihydrotanshinone I-hepatic fibrosis-related protein-metabolite-related proteinmetabolites" was constructed.After network analysis,the most relevant targets of dihydrotanshinone I,STAT3 and P53,were screened.Finally,through in vitro and in vivo experiments,specifically flow cytometry,surface plasmon resonance(SPR)analysis,Western blotting,metabolomics experiments and thioacetamide-induced liver fibrosis rats The interaction between dihydrotanshinone I and STAT3 and P53 was verified by experiments.The results showed that dihydrotanshinone I inhibited the activation of hepatic stellate cells by targeting STAT3 and P53,thereby alleviating the development of liver fibrosis.