The Mechanism Of M~6A Modification In Regulating Ferroptosis Of Hepatic Stellate Cells To Alleviate Liver Fibrosis And The Interventions Of Dihydroartemisinin

Liver fibrosis,which is a pathological condition characterized by excessive deposition of extracellular matrix(ECM),results from liver injury of one or more damaging factors.It is reported that liver fibrosis is a key stage in the progression of various chronic liver diseases to cirrhosis or even liver cancer.The activated hepatic stellate cells(HSCs)are the main source of ECM and thus play a key role in the pathogenesis of liver fibrosis.Inhibition or clearance of activated HSCs may reverse liver fibrosis.However,there are no clinical drugs available for the treatment of liver fibrosis.Therefore,it is urgent to explore the mechanism of HSC activation and develop therapeutic drugs to scavenge HSCs for liver fibrosis therapy.Ferroptosis,a novel iron-dependent programmed cell death phenotype,is thought to be associated with disease progression.Distinct from apoptosis,necrosis and autophagy,glutathione(GSH)depletion,glutathione peroxidase 4(GPX4)inactivation,lipid reactive oxygen species(ROS)accumulation,and lipid peroxidation are the main events of ferroptosis.Links between ferroptosis and various human diseases have also been established.Interestingly,our previous study indicated that induction of ferroptosis in HSCs may be a potential treatment for liver fibrosis,but the mechanism was not elucidated.In this study,we found that N6-methyladenosine(m6A)modification plays an important role in HSC ferroptosis.Importantly,m6A modification is the most abundant post-transcriptional regulatory mechanism in eukaryotic mRNA.Base on this,we innovatively put forward a scientific hypothesis that m6A modification mediates HSC ferroptosis ameliorates liver fibrosis.Exploring the post-transcriptional regulatory mechanism of m6A modification-mediated ferroptosis in HSC may provide effective therapeutic targets for liver fibrosis.In this study,we confirmed that ferroptosis inducers can inhibit the viability of HSCs and trigger ferroptotic events.Importantly,ferroptosis inhibitor but not apoptosis inhibitor and necrosis inhibitor could inhibit HSC ferroptosis.Interestingly,dot blot and m6A modification quantification kits showed that ferroptosis-inducing compounds could significantly up-regulate the total levels of m6A modification in HSCs.Western blot and real-time PCR showed that the upregulation of methylase METTL4 and the downregulation of demethylase FTO were responsible for the increased m6A modification in HSC ferroptosis by ferroptosis inducer treatment.Furthermore,reduction of m6A modification by METTL4 knockdown or FTO overexpression could impair the classical ferroptotic events in HSCs.Overall,these data showed that up-regulation of m6A modification may contribute to HSC ferroptosis.To further determine the molecular mechanism by which m6A modification promotes HSC ferroptosis,we performed RNA sequencing(RNA-seq)to identify the target genes.HSCs were overexpressed with demethylase FTO and then treated with ferroptosis inducers.The results showed that ferroptosis-related genes such as SLC7A11,SLC3A2,and GPX4 were screened out.These positive outcomes validated our screen approach.Interestingly,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis fully indicated that autophagy signaling was potentially regulated by m6A modification during HSC ferroptosis.In addition,MeRIP qPCR showed that the level of m6A modification in BECN1 was significantly increased.The reduction of m6A modification by FTO over-expression or METTL4 down-regulation markedly reduced the protein expression of BECN1 without large differences in other autophagy-associated proteins.Furthermore,the conversion of LC3-II and the formation of autophagosome were suppressed during the reduction of m6A modification.Whereas overexpression of BECN1 could attenuate the inhibitory effect of METTL4 downregulation or FTO overexpression on HSC ferroptosis.Taken together,BECN1-mediated activation of the autophagy pathway is a potential regulatory mechanism for m6A modification-induced ferroptosis in HSCs.It has been reported that the m6A reader protein can bind to m6A-modified RNA,thereby affecting the biological function of the RNA.In this study,we indicated that YTHDF1 is a key m6A reader protein that regulates HSC ferroptosis.Moreover,down-regulation of YTHDF1 suppressed BECN1-induced ferroptosis in HSCs.Western blot showed that down-regulation of YTHDF1 reduced the protein level of BECN1 in HSCs under ferroptosis-inducing compound treatment.Noteworthy.YTHDF1 triggered autophagy activation by promoting the stabilization of BECN1 mRNA via recognizing the m6A binding site within BECN1 coding regions.The mutation of m6A binding sites could abrogate the YTHDF1-mediated BECN1 mRNA stability,and in turn prevented HSC ferroptosis.Next,CCl4-induced liver fibrosis of mouse was constructed and treated it with the ferroptosis inducer erastin.Histopathological analysis showed that erastin significantly alleviated the pathological damage of liver fibrosis in mice.In addition,HSC-specific inhibition of m6A modification blocked HSC ferroptosis,thereby destroying the ameliorative effect of erastin in mouse with liver fibrosis.Over all,we revealed that m6A reader protein YTHDF1 promoted BECN1 mRNA stability,thus triggering autophagy activation,and eventually causing HSC ferroptosis to alleviate liver fibrosis.Artemisinin is a sesquiterpene lactone compound with antimalarial activity extracted from the traditional Chinese medicine Artemisia annua,and dihydroartemisinin(DHA)is the most active derivative of this compound.We found that DHA inhibited the activation of HSCs by inducing HSC ferroptosis,whereas ferroptosis inhibitors significantly attenuated the inhibitory effect of DHA on HSC activation.Interestingly,the level of autophagy was increased in DHA-induced ferroptosis,and the autophagy inhibitor 3-MA significantly abolished HSC ferroptosis by DHA treatment.Mechanistically,DHA inhibited the expression of m6A modification-related demethylase FTO,resulting in the increased m6A modification levels in HSCs.Moreover,the m6A modification level of BECN1 mRNA was significantly upregulated in DHA-induced HSC ferroptosis.More importantly,YTHDF1 was identified as a key m6A reader protein for BECN1 mRNA stability.Noteworthy,we further confirmed that YTH domain is essential for YTHDF1 to prolong the half-life of BECN1 mRNA in HSCs by DHA treatment.In mice,DHA treatment alleviated liver fibrosis by triggering HSC ferroptosis.HSC-specific inhibition of m6A modification and autophagy could impair DHA-induced HSC ferroptosis in murine of liver fibrosis.In summary,this study elucidated the role and mechanism of m6A modification regulating HSC ferroptosis in improving liver fibrosis,and the intervention effect of dihydroartemisinin.Our results indicated that m6A modification may be the critical post-transcriptional regulatory mechanism of HSC ferroptosis.We also found that DHA could regulate m6A modification and inhibit HSC activation,by which to provide a theoretical basis for DHA in the treatment of liver fibrosis.