Mechanism Of The Anti-fiber Soft Liver Particles Affect Proliferation Of Hepatic Stellate Cells

Objective: Activation and proliferation of hepatic stellate cells (HSC)is the key step in liver fibrogenesis. Therefore the strategy for terminating the proliferation of activated HSC might be an exciting therapy for patients with liver fibrosis. In previous research, we have found that KangXian Ruangan Granule (KXR) had good antifibrotic effects on clinical liver cirrhosis and experimental hepatic fibrosis, and could inhibit the proliferation collagen synthesis and TGF P i expression of HSC. The aim of this study was to investigate the role of KXR in inducing apoptosis, mediating the proliferation effects of PDGF and the signaling transduction of PDGF to further explore its possible antifibrotic mechanism at cellular and molecular lever.Methods: 1. The influence of KXR on HSC proliferation and apoptosis: we used various concentrations of KXR (The final concentration was 40rng/mK 20mg/mK lOmg/mK 5mg/ml. 2.5mg/mK 1.25mg/ml, 0.625mg/ml respectively) and its medicated sera (5%, 10%, 20%) to incubate with the rat hepatic stellate cells line-HSC-Tg for 48 hours. The proliferation was measured with MTT assay, the cell cycles and apoptosis was analyzed by flow cytometry, and we also examined the apoptosis with TUNEL method. The expression of bax^ Bcl-2 was detected with cell immunocytochemistry stain.2. The influence of KXR on the proliferation and signaling transduction induced by PDGF: using serum free cultured method, HSC were treated with KXR for 24 hours, and stimulated with PDGF-BB for 24 hours, and added the same concentration of KXR, 3 hours later, stimulated with PDGF-BB for 5 minutes, then collected the cells. The proliferation of HSC was examined with MTT assay. The cell cycle was analyzed with flow cytometry. Intracellular Ca2+ concentration was measured with calcium probe. Tyrosine phosphorylation protein MEK-1 and PI3-K was detected with western blotting enhanced chemiluminescent(ECL) method.Results: 1. In proliferation: Whether drug or medicated sera of KXR could markedly inhibit the proliferation of HSC, and in the safe concentration, the effects of the group of 5mg/ml KXR and the group of 10% medicated sera was especially obvious, which further proved the proliferation inhibition effect of KXR (P<0.01); serum free culture indicated KXR could attenuate HSC growth, with dose dependant(PO.Ol). Flow cytometry analysis indicated KXR could inhibit DNA synthesis of S and G2/M phases induced by PDGF, blocked HSC cell cycle beyond the GI phase, and existed dose dependent relation (P<0.01).2. In apoptosis both drug and medicated sera of KXR could induce apoptosis (PO.05), down regulate the expression of Bcl-2, a molecular of cell survival, and up-regulate the expression of Bax, a death-promoting molecule (PO.01).3. KXR could inhibit the increasing of intracellular calcium concentration induced by PDGF-BB: the [Ca2+l of 5mg/ml, 2.5mg/ml,1.25mg/ml groups was 208.40 ?11.99nmol/L. 285.97 ?5.83nmol/L, 330.77 ?12.44nmol/L respectively, which wereobviously lower than PDGF group (380.33 ?11.05nmol/L,P<0.01).4. Treatment of HSC with KXR resulted in an inhibition of the PDGF-BB-inducedexpression of Tyrosine phosphorylated preotein, MEK-1, and PI3-K.Conclusion:The mechanism of KXR inhibiting HSC activation and proliferation was including the following aspects:1. Blocked HSC through Gl/S restriction print, interrupted the cell cyclin;2. Down regulated the ratio of Bcl/Bax to increase the sensitivity of apoptosis and induced HSC apoptosis.3. Inhibited intracellular signal transudation of PDGF in HSC to block the biological effect of PDGF.