MTOR Regulates Sorafenib-induced Activated HSC Ferroptosis

Objective:Liver fibrosis plays a central role in the development of various chronic liver diseases,which main pathological manifestations are excessive deposition of the Extracellular matrix(ECM)by activated hepatic stellate cell(HSC).Therefore,inhibiting activation of HSCs or inducing its death is an effective treatment for hepatic fibrosis.Ferroptosis is a new type of programmed cell death,and inducing ferroptosis of HSCs can delay the development of liver fibrosis.However,its specific mechanismneeds to be further explored.Mammalian target of rapamycin(mTOR)is a serine/threonine protein kinase that is involved in the process of iron death in liver cancer cells,kidney cancer cells and other cells.The purpose of this study was to explore the effect and possible mechanismof mTOR on sorafenib-induced activated HSC ferroptosis to improve liver fibrosis and to provide potential therapeutic strategies for alleviating liver fibrosis.Methods:Initially,PDGF-BB induced HSC-T6 activation.The protein expression levels of α-smooth muscle actin(α-SMA),Collagen type I alpha-1(COL1α1)and GPX4 in HSC-T6 cells were detected by western blot.The effect of sorafenib on the viability of activated HSC-T6 cells was detected by CCK8.The levels of Iron,MDA and GSH were detected by the kit,and the level of ROS were detected by DCFH-DA fluorescent probe.In the following experiments,the protein expressions of mTOR and p-mTOR in HSC-T6 cells were detected by western blot after the addition of mTOR inhibitor rapamycin or mTOR activator MHY1485.The viability of HSC-T6 cells was detected by CCK8.Indicators of ferroptosis were detected by the kit or the fluorescent probe.Finally,Western blot analysis was performed to determine the effect of sorafenib on the protein expressions of α-SMA and COL1α1 in HSC-T6 cells.Results:1.Sorafenib triggers activated HSC ferroptosis.PDGF-BB induced HSC-T6 activation,while sorafenib inhibited the viability HSC-T6 cell in a concentration-dependent manner.The protein expression levels of α-smooth muscle actin(α-SMA)and Collagen type I alpha-1(COL1α1)in HSC-T6 cells were reduced by sorafenib.These results suggest that sorafenib can inhibit the activation of HSC-T6 cells.Further studies found that sorafenib significantly reduced the protein expression of GPX4,a marker of ferroptosis.In addition,compared with PDGF-BB group,the levels of Iron,MDA and ROS in sorafenib group were increased,while the levels of GSH were significantly decreased.These results indicate that sorafenib inhibits HSC activation and is associated with HSC ferroptosis during this process.2.Sorafenib inhibits the phosphorylation of mTOR.Sorafenib decreased the protein expression of p-mTOR in activated HSC-T6 cells.3.mTOR inhibitor strengthens sorafenib-induced HSC ferroptosis and thus enhances its anti-hepatic fibrosis effect.After treatment of HSC-T6 cells with an mTOR inhibitor,the viability of HSC-T6 cells were decreased.Compared with sorafenib group,the levels of GPX4 and GSH were further reduced,but the levels of Iron,ROS,MDA were increased.Compared with sorafenib group,the protein expressions of liver fibrosis markers,α-SMA and COL1α1,were reduced after the mTOR inhibitor treatment.4.mTOR inhibitor strengthens sorafenib-induced HSC ferroptosis and thus restrains its anti-hepatic fibrosis effect.Compared with sorafenib group,the decline in cell viability of HSC-T6 was significantly reversed after treatment with mTOR agonists,and the levels of GPX4 protein and GSH were significantly increased.Compared with sorafenib group,α-SMA and COL1α1 levels were increased in the mTOR agonist group.Conclusion: Sorafenib induced HSC ferroptosis and delayed the progression of liver fibrosis.Inhibition of mTOR signaling pathway further enhanced HSC ferroptosis,while activation of mTOR inhibited the effect of sorafenib.