| ObjectivesTo study the effect of total saponins of Schizocapsa plantaginea Hance(TSSPH)on hepatic fibrosis in rats induced by CCl4 and its inducing activation on hepatic stellate cells,and investigate the molecular mechanisms of these effects in vitro and in vivo.Methods1?Hepatic stellate cell line was used in the experiments of studying the effect of TSSPH on the activation of hepatic stellate cells:MTT assay method was used to observe the inhibitory effect of TSSPH on proliferation of HSC-T6 cells.Hoechst 33258 staining was used to observe the effect of TSSPH on the apoptotic morphology of HSC-T6 cells.The apoptosis in HSC-T6 cells treated with TSSPH was detected by flow cytometry(FCM).2?Used rat liver fibrosis model established by intragastric carbon tetrachloride method to study the anti-fibrosis effect of TSSPH:Serum AST?ALT and hydroxyproline(Hyp)were measured by biochemical detection method.The four indexes of liver fibre(C ?,PC?,LN,HA)in serum were measured by enzyme linked immunosorbent assay.The location of liver fibrous tissues were observed by Masson staining.Liver sections were stained with HE?Masson and observed for pathologic changes.Liver ultrastructural observed by a transmission electron microscope.3?The effects of TSSPH on TGF-?1/Smad signaling pathway were studied by using liver fibrosis rat liver tissue and HSC-T6 cells:The expressions of mRNA of a-smooth muscle actin(a-SMA),transforming growth factor ?1(TGF-?1),Smad4 and Smad7 in liver were detected by RT-PCR.The expression of ?-SMA?TGF-?1?Smad2/3 and Smad7 protein was detected by immunohistochemistry.The expression of apoptosis-related proteins and TGF-(31/Smad signal pathway of HSC-T6 cells treated with TSSPH was detected by Western blot(WB)method.Results1?TSSPH effectively inhibited the proliferation of HSC-T6 cells,and also induced apoptosis and caspase cascade in HSC-T6TSSPH significantly inhibited the proliferation of HSC-T6 cells,and the IC50 of 24 h were 3.841?g/mL.Hoechst 33258 staining showed that the nucleus of the control group was clear and complete,showing light blue fluorescence.In the administration group,significant morphological changes were observed,such as apoptotic bodies.The results of FCM showed that TSSPH induced apoptosis of HSC-T6 cells in a dose-dependent manner.The results of WB showed that the expression in HSC-T6 cells of Bax protein was up-regulated by TSSPH,and the expression of Bcl-2 and caspase-3,-9 protein was down-regulated.2?TSSPH significantly reduced liver tissue fibrosis and reduced collagen content and improve liver fibrosis contentsTSSPH could reduce the levels of AST and ALT in the blood of rats with hepatic fibrosis.The TSSPH-H group was the most obvious(P<0.05),the effect was better than that of colchicine group;The content of the four indexes of liver fibrosis and the level of hydroxyproline on the serum of liver fibrosis model group were significantly higher than those of the normal control group(P<0.01,P<0.05).In the experimental animals of the TSSPH intervention group,we also found that fibrosis in the serum samples of rats after 4 weeks of TSSPH intervention.The index content was significantly lower than that of the fibrotic model group,and the collagen content was reduced(P<0.01,P<0.05).Combined with pathological results,it was confirmed that TSSPH could significantly reduce liver damage caused by liver fibrosis.3?TSSPH regulated ?-SMA gene and protein expression in the liver to reduced collagen depositionThe results of RT-PCR showed that the expression of a-SMA mRNA in the model group was significantly higher than that in the normal control group.Compared with the model group,the expression of a-SMA mRNA in the liver tissue of the fibrotic rats was significantly higher than that in the model group(P<0.01).The expression of a-SMA protein in liver tissue of normal rats was observed by immunohistochemical staining.Immunohistochemistry showed the weak expression.Hepatic fibrosis induced by CCl4 showed abnormal proliferation and activation of liver cells,and the content of a-SMA in collagen effector increased,and its expression was significantly enhanced.It was mainly strongly expressed near the fiber bundle,and it was signifi cantly inhibited by TSSPH intervention.Hepatic stellate cell proliferation activates and reduces collagen deposition.4?TSSPH could effectively reduce the expression of TGF-?1,Smad 2/3 and other genes and increase the expression of Smad 7 protein in liver fibrosis rats.The results of immunohistochemistry showed that TGF-?1 and Smad2/3 were negatively expressed in the liver of normal rats,and Smad7 was positive.When the liver developed fibrotic lesions,the model group not only had TGF-?1 promoting liver fibrosis signal transduction.The expression of Smad2/3 was increased(P<0.01),the inhibitory Smad7 was weakly and positively expressed(P<0.01),and the positive staining of TGF-?1 and Smad2/3 in TSSPH-L group was compared with the model group.Compared with the model group,the Smad7 positive staining rate was significantly increased(P<0.01),which inhibited the TGF-?1/Smad signaling pathway.RT-PCR showed that the model was compared with the normal control group.The expression of Smad4 and TGF-?1 mRNA was significantly increased,and the expression of Smad7 mRNA was significantly decreased.Compared with the model group,the expression of TGF-?1 and Smad4 mRNA in the liver tissue of fibrotic rats was significantly decreased after TSSPH administration(P<0.01).The expression level of Smad7 mRNA was significantly increased(P<0.01).The expression of this signaling pathway-related protein was consistent with the trend of mRNA expression,indicating that TGF-?1/Smad signaling pathway may be one of the regulatory mechanisms of TSSPH against liver fibrosis.5?TSSPH was significant effect in the expression of TGF-?/Smad signaling pathway related proteinTSSPH significantly increased the expression of of Smad7,and the expression of TGF-?1 and Smad2/3,Smad7 protein was down-regulated.ConclusionTSSPH can inhibit the activation of hepatic stellate cells and anti-hepatic fibrosis through inhibiting TGF-(31/Smad-related signaling pathway. |
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