Sorafenib Attenuates Liver Fibrosis By Triggering Hepatic Stellate Cell Ferroptosis Via HIF-1α/SLC7A11 Pathway

Background and objective:Liver fibrosis is a reversible pathophysiological process,the essence of which is the disruption of the balance between synthesis and degradation of collagen-rich extracellular matrix（ECM）,leading to excessive production and deposition of ECM,causing the formation of liver scar tissue and disrupting the physiological structure of the liver.Activated hepatic stellate cells（HSCs）are the main source of ECM.Therefore,inhibition of HSC activation or induction of HSC death is an effective strategy for the treatment of liver fibrosis.Ferroptosis as a novel form of programmed cell death,which is evidenced by the accumulation of iron-driven lipid reactive oxygen species（ROS）.Notably,the induction of ferroptosis in HSC inhibits the deposition of ECM,which offers a new perspective for the liver fibrosis therapy.Nevertheless,the potential relationship between liver fibrosis and ferroptosis has been poorly studied.Thus,the present study is aimed at exploring and refining the role of ferroptosis in the treatment of liver fibrosis.Sorafenib is an ferroptotic inducer.At the same time,sorafenib attenuates liver fibrosis by inhibiting HSC proliferation or inducing HSC apoptosis to inhibit the accumulation of ECM.Then,in addition to the existing mechanisms,it remains to be investigated whether sorafenib as an ferroptotic inducer can attenuates liver fibrosis by inducing ferroptosis in HSC.In this study,we investigated the role of sorafenib in HSC ferroptosis and further explored the signaling pathway of HSC ferroptosis in the anti-fibrotic effects of sorafenib.Methods:（1）CCl₄-induced liver fibrosis model was established in mice,treated with sorafenib（2.5,5,10 mg/kg）administration.The effects of sorafenib on HSC ferroptosis（PTGS2,SLC7A11 and GPX4）and ECM expression in mouse liver were detected by immunohistochemistry and Western blot.（2）In vitro,exposure sorafenib（5,10,15μM）to HSC-T6 cells and cell viability was detected by CCK-8.Subsequently,the apoptosis inhibitor ZAVD-FMK,necrosis inhibitor NSA,ferroptotic inhibitor Fer-1 and DFO were exposed to sorafenib-induced HSC-T6 cells and cell viability was examined,which were to explore whether ferroptosis was involved in the inhibition of cell viability by sorafenib.Transmission electron microscopy（TEM）,kits and Western blot were used to assess the effects of sorafenib on HSC ferroptotic events（Iron,SLC7A11,GSH,GPX4,ROS and MDA）and HSC activation（α-SMA,COL1α1 and fibronectin）.（3）In HSC-T6 cells,Fer-1 and DFO were used to inhibit cell ferroptosis,and Western blot was used to detect changes in ferroptotic indicators（Iron,SLC7A11,GSH,GPX4,ROS and MDA）and liver fibrosis indicators（α-SMA,COL1α1 and fibronectin）to further investigate HSC ferroptosis in the anti-fibrosis of sorafenib.（4）The level of HIF-1αduring sorafenib-induced ferroptosis in HSCs was measured via Western blot and immunofluorescence.HIF-1αsi RNA,plasmid and stabilizer were treated with HSC-T6 cells,and SLC7A11,Iron,GSH,GPX4,ROS and MDA were detected by Western blot and immunofluorescence,to revel the HSC ferroptotic-related signaling pathways.Results:（1）Compared to normal mice,the CCl4-induced mice showed ALT and AST levels in serum increased,collagen deposition andα-SMA,COL1α1 and fibronectin protein levels significant increased,indicating that the liver fibrosis model was successfully established.After sorafenib administration,serum ALT and AST levels were significantly decreased,collagen deposition was reduced andα-SMA,COL1α1 and fibronectin levels were significantly decreased,indicating that sorafenib reduced CCl₄-induced liver injury and fibrosis in mice.In addition,PTGS2 expression was increased and SLC7A11 and GPX4 expression was decreased in the sorafenib-induced mice compared to the CCl₄ induction,indicating that sorafenib attenuated liver fibrosis and was accompanied by HSC ferroptosis.（2）Cell viability was declined dose-dependently in HSC-T6 cells treated with sorafenib.Subsequently,the apoptosis inhibitor ZVAD-FMK,ferroptotic inhibitor Fer-1 and DFO all reversed the inhibitory effect of sorafenib on HSC viability.These results suggest that sorafenib induces ferroptosis in HSCs.Further results showed that Sorafenib-treated HSCs exhibited morphological reduction in cell mitochondrial volume and mitochondrial cristae disruption,and SLC7A11,GPX4 and GSH significant reduced,and Iron,ROS and MDA significant increased,which further confirmed that Sorafenib induced ferroptosis in HSC-T6 cells in vitro.Finally,α-SMA,COL1α1 and fibronectin protein levels were significantly down-regulated in sorafenib-induced HSC compared to vehicle,suggesting that sorafenib reduced the accumulation of ECM.（3）Sorafenib induced ferroptosis in HSC-T6 cells,while the addition of two ferroptotic inhibitors,Fer-1 and DFO,significantly reversed sorafenib-induced ferroptosis in HSC.Subsequently,the results of liver fibrosis-related markers showed that sorafenib reduced the accumulation of ECM,while the ferroptotic inhibitors Fer-1 and DFO were significantly up-regulated.These data further suggest that sorafenib attenuates fibrosis by inducing ferroptosis in HSCs.（4）HIF-1αprotein expression was significantly elevated in the livers of CCl₄-treated mice as well as in HSC-T6 cells compared to the control group.HIF-1αprotein expression was significantly reduced after sorafenib treatment.Subsequently,HIF-1αstabilizers DMOG and DFO were exposed to sorafenib-treated HSC-T6 cells,and it was found that DMOG and DFO significantly increased HIF-1αexpression in the nucleus of HSCs.Moreover,DMOG and DFO significantly attenuated sorafenib-induced HSC ferroptosis,accompanied by elevated SLC7A11.These results suggest that HIF-1αmay regulate SLC7A11 in HSC ferroptosis.Subsequently,HIF-1αoverexpression and si RNA plasmid were exposed to HSC-T6 cells showed that elevated HIF-1αpromoted SLC7A11 expression and inhibited HSC ferroptosis,which in turn attenuated the anti-fibrotic effect of sorafenib.Conclusion:Sorafenib triggers HSC ferroptosis via HIF-1α/SLC7A11 signaling,which in turn attenuates liver injury and fibrosis.