ABSTRACT
Objective To investigate the molecular mechanism of Loureirin A mediated anti-hepatic fibrosis by evaluting its effects on proliferation , secretion ofα-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1), and expression of rat hepatic stellate cells in vitro . Methods Primary hepatic stellate cells were isolated and cultured from Sprague-Dawley rats. After activating and inducing primary hepatic stellate cells from qHSC to aHSC, the activated hepatic stellate cells model in vitro was established. Then we observed the morphological changes of static hepatic stellate cells and activated hepatic stellate cells with inverted phase contrast microscope. Cultured hepatic stellate cells were treated with different concentrations of loureirin A and the inhibitory rate of HSCs proliferation was measured by MTT assay. The expression of Frizzled-4 was measured by western blot analysis. The content ofα-SMA and TGF-β1 in the cultured HSCs'supernatant were measured by enzyme-linked immunosorbent assay (ELISA) . Results Loureirin A the proliferation of inhibited activated hepatic stellate cells in a time-dose-dependent manner compared with the control group,IC50=0.30 μg/μL. After loureirinA treatment of the HSCs, western blot analysis showed that Frizzled-4 expression level was obviously lower than control group. Loureirin A also inhibitedα-SMA and TGFβ1 (P<0.05) secretion in the cultured HSCs'supernatant in different degree by the assay of ELISA. Conclusions The molecular mechanism of Loureirin A and Wnt signaling pathway mediated anti-hepatic fibrosis and anti-angiogenesis may involve down-regulation the expression of Frizzled-4, inhibiting the synthesis and secretion ofα-SMA,TGF-β1and the proliferation of HSCs.
ABSTRACT
Objective To investigate the expression of integrin avβ3 in both of hepatic stellate cell (HSC)and fibrotic liver tissue,and to demonstrate whether integrin avβ3 is a phenotypical receptor of activated HSC.Methods HSC were isolated from Sprague-Dawley rats and activated by prolonged cultural.The rats were injected with 175 mg of thioacetamide twice a week for 12 weeks to induce liver fibosis.The expressions of avβ3 and a-SMA were identified by immunocytochemical staining.The expression of avβ3 in HSC,normal and fibrotic tissues were examined by real-time PCR and Western blot.Results The expression of avβ3 in activated HSC were up regulated and levels of mRNA and protein were increased to 18 and 5.2 folds on day 14 compared with day 1 and were also higher than control(t=2.39,P＜0.05;t=2.74,P＜0.05).The immunochemistry staining showed that integrin avβ3 was expressed on membrane of activated HSCs.The avβ3 and a-SMA were expressed in portal vein in normal liver,but were also expressed in portal and fibrotic.Conclusions The integrin avβ3 is up-regulated in activated HSC both in vitro and in vivo.It is a phenotypical receptor of activated HSC which involved in liver fibrosis.