Preliminary Research Of Purifying Damp,Heat,Blood Stasis And Toxin On TLR4and TGF-β/Smad Signal Pathway Of Hepatic Fibrosis

Objective:The purpose of our research was to investigate TLR4and TGF-β/Smad signal pathway mechanism of action of serum containing Qing Hua decoction in the activated Hepatic Stellate Cells-T6strains,and then to verify the theory of "purifying damp,heat,blood stasis and toxin" proposed by Professor Zhou Zhongying.The result provided some foundations for the future clinical applications.Method:As an experimental group,serum containing Qing Hua decoction was devided into three mass concentration.And serum containing Colchicine was used as positive control and serum containing Huangqi Yiguan decoction as comparision of strengthening vital Qi and eliminating pathogen.The anti-proliferative acpacity of serum containing Qing-Hua decoction,influenced on HSC-T6cultured by macrophage conditioned medium in vitro,was detected used by MTT assay.The expression of TLR4and fibrosis associated proteins in the TGF-β/Smad pathway were evaluated by RT-PCR and Western Blot after treatment with serum containing Qing Hua decoction on activated HSC-T6cultured by macrophage conditioned medium in vitro.Result:(1) Serum containing Qing Hua decoction inhibited the proliferation of activated HSC-T6in a time and dose-dependent manner by MTT assay.(2)The expression level of TGF-β1and TLR4were significantly decreased by RT-PCR assay in the activated HSC-T6treated by serum containing Qing Hua decoction after48hours,and inhibition effect showed dose-dependent manner.(3) The experimental result of Western Blot assay indicated that the expression level of Smad3and Smad4were down-regulated and Smad7were up-regulated in the activated HSC-T6treated by serum containing Qing Hua decoction after48hours, and inhibition effect showed concentration-dependent manner.Conclusion:(1) Serum containing Qing Hua decoction could inhibit the proliferation of activated HSC-T6.(2) Serum containing Qing Hua decoction may constitutively suppress the expression of TGF-β1and TLR4,whereafter activated Smads protein family in the activated HSC-T6,and then to inhibit the activation of TGF-β/Smad signal transduction pathway and the occurrence of endotoxemia mediated by Lipopolysaccharide,which suggested that TLR4and TGF-β/Smad pathway plays an important roles in supressing fibrosis caused of activated HSC-T6by serum containing Qing Hua decoction.