DHX58 Inhibits Hepatic Ischemia/Reperfusion Injury By Suppressing Hepatocyte Ferroptosis

With the development of society and economy,more and more patients died of liver diseases worldwide.Common liver diseases include hepatocarcinoma,cirrhosis and so on.And liver surgery is the only effective treatment for end-stage liver diseases,including liver resection and liver transplantation.Ischemia/reperfusion（I/R）injury is a significant cause of liver injury during liver resection or transplantation.Under the ischemia condition,hepatocytes died directly due to the interruption of oxygen supply and the depletion of intracellular glycogen and ATP.During reperfusion stage,reactive oxygen species（ROS）stimulation and local inflammation caused by the restoration of blood supply aggravate hepatocyte death.However,the damage-associated molecular pattern（DAMP）released by hepatocyte death,in turn,promotes the outbreak of inflammation mainly manifested as the infiltration of macrophages and neutrophils,and the release of inflammatory cytokines,which further aggravates hepatocyte death.The pathological manifestations of liver are hepatocyte swelling,steatosis,vacuolar degeneration and spotty necrosis.Serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）are increased.Therefore,hepatic I/R injury is currently considered to be an important risk factor for liver injury after liver surgery,but little is known about its occurrence and development mechanism.Currently,it is believed that a variety of cell death modes in the process of hepatic I/R are important causes of liver injury,including necrosis,apoptosis,necroptosis,ferroptosis,etc.Therefore,the intervention of hepatocyte death and its potential mechanism research are very important for the clinical treatment of hepatic I/R injury.Ferroptosis is a regulated cell death,which is newly discovered by Stockwell et al in2012.Ferroptosis can be induced by small molecule drugs,such as Erastin and RSL3,and inhibited by Liproxstatin-1 and Ferrostatin-1.Ferroptosis is characterized by the accumulation of iron-dependent lipid peroxides,and it occurs when the accumulation of lipid peroxides exceeds the maximum range the cell can tolerate.The morphological characteristics manifested by electron microscope were mitochondrial shrinkage,increased mitochondrial membrane density,decreased mitochondrial cristae and broken mitochondrial outer membrane.Ferroptosis has been shown to exist in a variety of pathophysiological processes,including tumor occurrence and development,degenerative diseases,stroke,and ischemia-reperfusion injury.The underlying biological mechanism of ferroptosis is the imbalance of ROS homeostasis system.On the one hand,lipids undergo Fenton reaction under the involvement of ferric bivalent,thus producing excess ROS.On the other hand,the ROS scavenging system mediated by solute carrier family 7 member 11（SLC7A11）/glutathione peroxidase 4（GPX4）can not remove the excess ROS in time,which eventually leads to cell death.However,the current understanding of the regulatory mechanism of ferroptosis in hepatocytes is not thorough.Therefore,to perfect the precise regulation of ferroptosis in hepatocytes can provide an important basis for the treatment of hepatic I/R injury.DDX-/DHX-family molecules are critical for RNA metabolism in the body,including RNA recognition,modification,splicing,transport,degradation and translation.DDX-/DHX-family molecules in hepatocytes also play a very important role in liver pathophysiological processes.For example,DEx D/H-box helicase 58（DDX58）,previously discovered by our group,can regulate the hepatocarcinogenesis at two different levels.On the one hand,DDX58 can inhibit hepatocarcinogenesis in diethylnitrosamine（DEN）-induced hepatocarcinogenesis mouse model.On the other hand,DDX58 can promote hepatocarcinogenesis in Stelic Animal Model（STAM）induced by streptozocin（STZ）and high-fatty diet（HFD）.Its specific molecular mechanism is that DDX58 can inhibit janus kinase 2（JAK2）-signal transducers and activators of transcription 3（STAT3）association and the interleukin 6（IL-6）effect signaling pathway by binding with STAT3 in DEN-induced hepatocarcinogenesis mouse model.While in NASH-induced hepatocarcinogenesis mouse model,DDX58 inhibits the association of AMP-activated protein kinaseα（AMPKα）and HMG-Co A reductase（HMGCR）by binding with AMPKα,thus suppressing the phosphorylation of HMGCR.Ultimately,it promotes cholesterol synthesis and hepatocarcinogenesis.DEx H-box helicase 58（DHX58）,also known as laboratory of genetics and physiology（LGP2）,is one of RIG-I-like helixases（RLH）,which is similar to DDX58.The RLH family is a typical family of pattern recognition receptors.Interferon（IFN）stimulation or virus infection can significantly promote the expression of RLH,which play a key role in antiviral innate immune response.After the RNA virus infects cells,viral components such as viral RNA is recognized by the RLH,which activates the downstream antiviral innate immune signaling pathway and prompts immune cells to express pro-inflammatory cytokines and type I interferon,finally clearing the viral infection.The molecular structure of DHX58 is mainly composed of two domains:RNA helicase domain and C-terminal repressed domain.The RNA helicase domain is the conserved domain of the DEx D/H family,which is mainly responsible for hydrolyzing ATP,binding and releasing RNA.The repressed domain at the C-terminal is generally thought to play an inhibitory role.The RNA helicase domain and the C-terminal repressed domain recognize double-stranded RNA and 5’triphosphorylated-RNA in a synergistic manner.Compared with other RLH,DHX58 lacks the caspase activation and recruitment domain（CARD）at the N-terminal,which transmits antiviral innate immune signals downwards.Hence,DHX58 is thought to be a regulator of DDX58 and melanoma differentiation-associated gene 5（MDA5）-mediated innate immune signaling pathway.Although DHX58 lacks the CARD domain,it has the strongest RNA binding ability among the RLH family,so it is also considered as a non-classical RNA-binding protein（RBP）.However,the role of DDX-/DHX-family molecules in hepatic I/R injury remains unknown.By comprehensively analyzing the RNA expression level and RNA expression difference of RNA-seq results,we found that,in mouse hepatic I/R-induced livers,DHX58 in DDX-/DHX-family molecules was highly expressed and significantly decreased compared with wild mouse liver samples.The increase of ROS production is the most obvious feature in the occurrence and development of hepatic I/R.Therefore,we further studied whether the decreased expression of DHX58 in hepatic I/R models was dependent on the excessive ROS production in the microenvironment.In vitro,we stimulated mouse primary hepatocytes and human hepatocyte line HHL5 with hydrogen peroxide to simulate ROS,and found that the expression of DHX58 was significantly reduced.Furthermore,after ROS was cleared in vivo and in vitro with butylated hydroxyanisole（BHA）and N-acetylcystein（NAC）,the expression of DHX58 recovered significantly.Hence,ROS that produced during hepatic I/R could inhibit DHX58expression.Then we constructed Dhx58 hepatocyte-specific dificiency mice.We found that Dhx58 hepatocyte-specific dificiency mice,compared with littered-control mice,showed significantly increased liver damage in hepatic I/R models.These results indicated that DHX58 can inhibit hepatic I/R injury.We further used single-cell sequencing to analyze liver samples from Dhx58 hepatocyte-specific dificiency mice and littered-control mice,alone or in combination with hepatic I/R treatment.It was found that Dhx58 hepatocyte-specific dificiency significantly promoted the activation of ferroptosis signaling pathway in hepatic I/R mouse models.Further,we analyzed the related indicators of ferroptosis and found that Dhx58 hepatocyte-specific dificiency significantly promoted ferroptosis of hepatocyte in hepatic I/R mouse models.These results strongly suggested that Dhx58 hepatocyte-specific dificiency is likely to promote hepatic I/R-induced liver injury by promoting ferroptosis of hepatocyte.Then,by injecting necroptosis inhibitor,apoptosis inhibitor and ferroptosis inhibitor into hepatic I/R mouse models,we found that ferroptosis inhibitor completely reversed the aggravation of hepatic I/R injury caused by Dhx58 hepatocyte-specific dificiency.It was further confirmed that Dhx58 hepatocyte-specific dificiency aggravated hepatic I/R injury by promoting ferroptosis of hepatocyte.To investigate the mechanism by which DHX58 inhibited ferroptosis of hepatocyte,and considering the strong RNA-binding capacity of DHX58,RIP-seq analysis was performed on the livers of wild-type mice,and proteomic analysis was performed on the livers of Dhx58^hep-/-and control(Dhx58^fl/fl)mice.RIP-seq results showed that 8432 genes were bound to DHX58 protein,and proteomic analysis indicated that 214 proteins were differentially expressed after Dhx58 hepatocyte-specific dificiency.Through taking intersection of DHX58-binding genes and differential proteins in the livers of Dhx58^hep-/-and control(Dhx58^fl/fl)mice,and then taking intersection with the ferroptosis gene set,Gpx4,Hspb1 and Aox1 were identified as the downstream targets of DHX58 to regulate ferroptosis.Further,Gpx4 was identified as the downstream target of DHX58 by comprehensive analysis of glutathione metabolism.We also confirmed that DHX58overexpression significantly promoted GPX4 protein expression in hepatocytes by western blot experiment,and further confirmed that DHX58 promoted GPX4 protein expression by enhancing Gpx4 m RNA translation efficiency through ribosome profiling assay.The mRNA metabolism in eukaryotic cells is important for controlling gene expression,and this process is strictly regulated by post-transcriptional modification of m RNA,such as N6-methyladenosine（m6A）modification,which is the most common in RNA modification.The m6A modification of RNA is edited by a series of methyltransferases,including methyltransferase like 3（METTL3）,methyltransferase like14（METTL14）and Wilms’-tumor-1-associating protein（WTAP）.They are also known as"m6A writers".The m6A modification can also be cleared by demethylases called"m6A erasers",including fat mass and obesity associated gene（FTO）and Alk B homolog5（ALKBH5）.RNA modified with m6A can be recognized by"m6A readers",including YTH domain family molecules（YTHDF1,YTHDF2,YTHDF3,YTHDC1 and YTHDC2）.After recognizing the m6A site of RNA,they regulate RNA metabolism and gene expression by regulating RNA splicing,transport,translation and stability.It has been reported that YTHDC2 can regulate the stability and the translation process of RNA in an m6A-dependent manner,but the role of m6A recognition process mediated by YTHDC2 in ferroptosis is still limited.We identified the molecules associated with DHX58 in the resting state by IP-MS,and found that DHX58 was bound to YTHDC2,which had the highest protein score.Co-IP also confirmed that DHX58 could constitutively bind to YTHDC2,and the binding sites are C-terminal domain（CTD）of DHX58 and R3H domain of YTHDC2.Using Me RIP-seq,we confirmed that Gpx4m RNA is modified with m6A,and YTHDC2 can promote GPX4 protein expression in an m6A-dependent manner.We then demonstrated by ribosome profiling that YTHDC2promoted GPX4 protein expression by promoting Gpx4 m RNA translation efficiency.Hence,DHX58 could recruit YTHDC2 to recognize Gpx4 m RNA modified with m6A in the resting state,thus promoting GPX4 protein expression by promoting Gpx4 m RNA translation efficiency,and ultimately inhibiting ferroptosis of hepatocyte and hepatic I/R injury caused by it.IFN has a variety of effects.In addition to playing a key role in the antiviral innate immune response,it can also inhibit the development of tumors.In response to IFN stimulation,the expressions of a series of IFN-stimulated genes（ISG）increase in cells.DHX58 is a classic ISG,which increases significantly after IFN stimulation.Considering the effect of DHX58 on inhibiting ferroptosis in hepatocytes and alleviating hepatic I/R injury,we studied the effect of IFN on hepatic I/R injury.Through a series of ferroptosis and liver injury indicators,we found that IFN could alleviate hepatic I/R injury by inhibiting ferroptosis of hepatocyte,and this process was dependent on the expression of DHX58.Therefore,hepatic DHX58 may be a potential intervention target for hepatic I/R injury.IFN can induce the expression of DHX58 and treat hepatic I/R injury.In summary,in this study,we found that hepatic DHX58 could bind to Gpx4 m RNA and recruit YTHDC2 to recognize Gpx4 m RNA in an m6A-dependent manner,thus promoting GPX4 protein expression by promoting Gpx4 m RNA translation efficiency.ROS inhibited GPX4 expression by suppressing DHX58 expression during hepatic I/R,thus promoting ferroptosis of hepatocyte and ferroptosis-related hepatic I/R injury.Our study revealed the mechanism by which DHX58 inhibited ferroptosis of hepatocyte and hepatic I/R injury,providing a new potential basis for hepatic I/R intervention.