Effects Of Saikosaponin-d On The Proliferation And Activation Of HSC-T6Cells And Associated Estrogen Receptor Mechanisms

Background&AimsHepatic fibrosis, the pathological process of excessive extracellular matrix(ECM)deposition, is a reversible stage of many chronic liver diseases such as cirrhosis,hepatocellular carcinoma.Activated hepatic stellate cells(HSCs) observed morphologicalchanges, significant proliferation and cytokine synthesis are considered as the major sourceof ECM. The persistent activation of HSCs, triggered by exposure to ROS and subsequentOS, is the key to the complicated progression from liver fibrosis to cirrhosis which resultedfrom the regulation by numerous cellular, molecular and signaling pathways, especiallyTGFβ-Smad and MAPK. Clinically and experimentally, estradiol has been proved itsanti-fibrotic effect. But the side-effects of estradiol limit its clinical application, researchersbegin to find out alternative drugs.recent reports suggest that phytoestrogens such asisoflavones, genitein, quercetin, resveratrol and genistein can not only substitute for theeffect of estrogen and regulate the expression of ER and its subtypes, but also avoid theadverse reaction and inhibit the process of liver fibrosis. Saikosaponin-d(SSd),a triterpenesaponin extracted from one of the oldest and widely used herb Bupleurum falcatum L intraditional Chinese medicine, is the major active pharmacological components. SSd hasbeen demonstrated its estrogen-like effect in vitro and vivo. However, the effect of SSd onHSCs is still unestablished. Thus, we use Rat hepatic stellate cell lineHSC-T6toinvestigate the estrogen-like effects of SSd on HSC-T6as well as the associatedmechanisms.MethodsRat HSC-T6cell line was used, and cultured in phenol red-free DMEM with10%FBS.Cells were cultured into cell culture clusters in5%sFBS phenol red-free DMEM, andtreated with indicated concentrations SSd and E2for24h in order to select the bestconcentration for treatment. Then, cells were treated with SSd (5μM) and E2(1μM) forindicated time points in order to select the best time for treatment. Cells were also treatedwith DMSO, SSd (5μM) and E2(1μM) in the presence or absence of ICI-182780(1μM), anpure estrogen receptor antagonist, for24h. Oxidative stress was induce by H2O2(0.01mM)for4h. We detected the proliferation and cell cycle distribution of HSC-T6cells by MTTassay and Flow Cytometry respectively. Alpha-SMA was detectedbyimmunocytochemistry. Estrogen response element (ERE)-luciferase report gene was uesd to detect the induction of SSd on ER. ER expression was detected by RT-PCR andwestern blot analysis. TGFβ1,Hyp,TIMP-1and MMP-1were examined to observe theeffects of SSd on synthesis and degradation of the extracellular matrixc (ECM) by ELISAtest. MDA and SOD were examined by ELISA test, which indirectly reflected theendogenous ROS generation. The ROS generation was detected by Flow Cytometry. Wealso detected the phosphorylation of MAPK signal pathway by western blot analysis tofind out the molecular mechanism of these effects.Results1. SSd and E2could inhibit the proliferation, reduce S phase cells, stop cell cycle atG0/G1phase,induce ERE luciferase report gene expression and upregulate the expressionof ERα/β in HSC-T6cells in a dose and time dependent manner.2. SSd and E2could suppress oxidative stress induced proliferation and activation ofHSC-T6cells, reduce the TGFbeta1,Hyp and TIMP-1content in cell culturalsupernatant,increase MMP-1content, inhibit the generation of MDA and ROS, enhance theactivity of SOD,and attenuate the phosphorylation of MAPK signal pathway.3. All the effects demonstrated above could be block by an pure estrogen receptorantagonist ICI-182780.ConclusionSSd can act on ER and suppress the proliferation and activation of HSC-T6cells,reduce ECM deposition, the associated mechanism may be dependent on its anti-oxidativeeffects by inhibition TGFbeta1/MAPK signal pathway.