The Effects Of Transient Receptor Potential Melastaln7Channel Protein On Apoptosis Of Rat Hepatic Stellate Cell Line HSC-t6

Liver fibrosis is a liver repair response to chronic injury，and is one of the commonestpathological of liver. Activation and proliferation of HSC are the key steps for liverfibrosis. The activated HSC secretes large amounts of extra cellular matrix （ECM） afterchanging into myofibroblast, which results in unbalance of composition anddegradation of ECM. A lot of ECM deposited in the liver and eventually evolved into alarge number of liver cirrhosis and even liver failure. Recent studies suggest that liverfibrosis is a reversible disease, remove damage factors or application anti fibrosis drug,with the recovery time is extended, liver fibrosis can partially or completely reverserecovery, showing deposition of collagen degradation, the liver structure and liverfunction recovery. The hepatic stellate cells （hepatic stellate cell, HSC） apoptosis playa key role in hepatic fibrosis. Research shows that during recover of HF, the activationof HSC expressed tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）and other apoptosis stimulating factors to promote HSC apoptosis, enhancing ECMdegradation, thereby inhibiting the formation of hepatic fibrosis. Therefore, induceapoptosis of hepatic stellate cells promote the liver fibrosis as an important way torecovery of HF.At present, it has been demonstrated that PDGF could enhance NHE activity of HSC by increasing PH value to promote proliferation. It may mediate by Ca2+-CaM andPKC pathway. Aldosterone antagonist canrenone inhibited the proliferation of HSC byblocking NHE activity; NHE selective inhibitor cariporide could markedly suppressHSC activity induced by PDGF in vitro and vivo, which has noting to do withPI3K/Akt and ERK pathway. The above research has demonstrated that theproliferation of HSC induced by PDGF could be suppressed by inhibitor of NHE. Ionchannels of HSC will become a novel target for proliferation and apoptisis of HSC.Transient receptor potential melastain7（TRPM7） channels, members of transientreceptor potential （TRP） channels superfamily, are bifunctional proteins with dualstructure of both ion channels and protein kinases on cell surface, participating in manyimportant physical processes such as proliferation, migration and apoptosis. We havedemonstrated that downexpression of TRPM7could inhibited the activation andproliferation of HSC markedly. However, whether there is an influence of TRPM7onHSC apoptosis and the exact mechanism is unclear. This study chooses rat hepaticstellate cell lines HSC-T6as object to explore the influence of TRPM7on HSCapoptosis. The study includes the following three parts:1. Expression of TRPM7in rat hepatic stellate cell line HSC-T6.Through cultured rat hepatic stellate cell line HSC-T6in vitro, detect the expressionlevels of TRPM7in HSC-T6using RT-PCR and Western blot. Results suggest thatactivated HSC expressed more TRPM7than the normal. Gd³⁺or2-APB not onlyinhibit the expression of TRPM7mRNA and protein （P<0.01）. SpecificTRPM7-siRNA could markedly downregulate the expression of TRPM7.2. Inhibition of TRPM7on the effects of rat hepatic stellate cell HSC-T6.Assay Gd³⁺or2-APB on HSC proliferation rate by MTT colorimetric, evaluate the levels of Caspase-3、α-SMA、col-1mRNA by RT-PCR，detect the expression levels ofCaspase-3by Western blot，Caspase-3assay kits measured the change of activity，cellapoptosis was measured by Flow Cytometry. Results suggest that Gd³⁺or2-APB notonly inhibit the activation of HSC in certain of range but also inhibit. Moreover, Gd³⁺or2-APB could inhibit the expression of α-SMA and col-1mRNA，which arebiomarkers of HF（P<0.01）.Specific TRPM7-siRNA was transfected by LipofectamineTM2000to downexpress oftransient receptor potential melastatin7（TRPM7） in rat hepatic stellate cell HSC-T6.Cell apoptosis was measured by Flow Cytometry. Caspase-3protein expression wereanalyzed by Western blotï¼›Using RT-PCR to evaluate Caspase-3and Bid mRNAexpression. Results suggest that suppression TRPM7-siRNA significantly increased theapoptosis of HSC、the expression of Caspase-3protein and Bid mRNA compared withthe normal or control.3The Effects of TRPM7on the apoptosis of rat hepatic stellate cell HSC-T6induced by TRAIL.Addition TRAIL to induce apoptosis of activated HSC，we found that apoptosis rate ofHSC，Caspase-3mRNA、protein and activity increased. While combining TRAIL withGd³⁺or2-APB, all the results above increased markedly（P<0.01）.In summary, this study has demonstrated that Gd³⁺or2-APB increased the apoptoticrate in activated HSC-T6induced by TRAIL compared with TRAIL free, anddependently decreased the expression of biomarkers of HF those used to promote HFprogression. Silencing expression of TRPM7could significantly increase the apoptosisof HSC, which may mediate by Bid. It is suggested that TRPM7is one of importanttargets of HSC apoptosis. Our study opens a new avenue to treatment of HF.