The Role And Mechanism Of Mitochondrial Transporter SFXN1 In Hepatic Stellate Cell Activation And Liver Fibrosis

Aims:Hepatic fibrosis is a pathological process in which the liver is exposed to various factors both in vivo and in vitro。Abnormal deposition of extracellular matrix forms during the chronic repair process,which leads to the destruction of the original structure and function of the liver and the formation of fibrous scars.It is the common adverse outcome of various chronic liver diseases.Hepatic stellate cell activation is considered to be the core event of hepatic fibrosis and plays an important role in extracellular matrix synthesis and cytokine secretion.Sideroflexin 1(SFXN1),a highly conserved mitochondrial inner membrane protein in eukaryotes,plays an important role in biological processes such as mitochondrial serine and iron transport.In recent years,studies have reported that SFXN1 plays an important role in sideroblastic anemia,lung adenocarcinoma,and myocardial hypertrophy.However,the role of SFXN1 in liver fibrosis has not yet been reported.This study aimed to investigate the role and regulatory mechanism of SFXN1 in hepatic stellate cell activation and liver fibrosis,which is expected to provide new targets and strategies for the treatment of liver fibrosis.Methods:1.Liver tissue specimens from cirrhotic and hepatic hemangioma patients were collected,and immunohistochemical staining technique and RT-qPCR were used to detect the expression and localization of SFXN1 in liver tissues,while immunohistochemistry(IHC)and Western Blot techniques were used to detect the expression of SFXN1 in mouse CCl4 model and rat TAA model.A rat primary hepatic stellate cell model was selected to evaluate the changes in SFXN1 expression during hepatic stellate cell activation in vitro.2.After in vitro lentiviral infection to establish interfering/overexpressing SFXN1 human hepatic stellate cell line LX-2 cells,Western Blot,RT-qPCR were used to reveal the correlation between SFXN1 and hepatic stellate cell activation.Mitochondrial Fe2+ dye was used to verify the transport function of SFXN1 on mitochondrial Fe2+,and the effect of SFXN1 on mitochondrial dysfunction in hepatic stellate cells was examined by Mitosox dye,JC-1 dye,and transmission electron microscopy.Cells overexpressing SFXN1 were then treated with the mitochondrial iron chelator,mitochondrial ROS scavenger,and NLRP3 inhibitor,respectively,to investigate the potential mechanism of SFXN1-induced activation of hepatic stellate cells.3.In vitro,we stimulated LX-2 cells with various cytokines known to promote hepatic stellate cell activation in order to find out the cytokine that capable to significantly stimulated SFXN1 elevation.We made literature retrieval and predict the transcription factor through the JASPAR database.Then,we used siRNA to interfere with this transcription factor to explore the possible upstream mechanisms of SFXN1.4.We established a mouse CCl4 model and injected AAV virus targeting to interfere with SFXN1 gene expression in hepatic stellate cells to verify the efficiency of viral interference and the improvement of fibrosis level.5.We also established a mouse CCl4 model with a novel NLRP3 inhibitor,OLT1177,and a positive control drug-MCC950 to study their effect on liver fibrosis.Results:1.SFXN1 was upregulated in patients with liver fibrosis and cirrhosis and was positively correlated with hepatic stellate cell activation index(a-SMA).Similar findings were revealed in the mouse CCl4 model and the rat TAA model.Upregulation of the SFXN1 gene was also observed in isolated rat primary hepatic stellate cells during their selfactivation in vitro.2.Intervention of the SFXN1 gene in the human hepatic stellate cell line LX-2 resulted in decreased activity of hepatic stellate cells,and overexpression of SFXN1 resulted in increased activity.SFXN1 transports intracytoplasmic Fe2+ into mitochondria,inducing mitochondrial dysfunction,causing activation of NLRP3 inflammatory vesicles,and ultimately leading to hepatic stellate cell activation.3.IL-1β significantly stimulated hepatic stellate cell activation and SFXN1 expression in a dose-dependent manner.IL-1β promotes SFXN1 expression through up-regulation of transcription factor c-myc.Then,SFXN1 facilitated downstream mtROS/NLRP3/IL-1βsignaling pathway.4.AAV6-GFAP-shSFXN1 virus effectively inhibited inflammatory vesicle activation,ameliorated liver fibrosis,and decreased deposition of collagen in a mouse CCl4 model.5.The novel NLRP3 inhibitor OLT1177 significantly ameliorated liver fibrosis in the mouse CCl4 model,and the efficacy of the high-dose group seemed to be better than that of MCC950.Conclusions:This study revealed that SFXN1 may play a role in promoting hepatic stellate cell activation by participating in mitochondrial iron transport that affects mitochondrial dysfunction,which in turn activates NLRP3 inflammasome.In addition,IL-1β can stimulate upregulation of the SFXN1 and form positive feedback.Interfering with hepatic stellate cell SFXN1 expression using adeno-associated virus or the novel NLRP3 inhibitor OLT1177 significantly alleviated liver fibrosis in a mouse CCl4 model,providing a novel target and intervention for the future treatment of liver fibrosis.