Study On The Molecular Mechanism Of Japanese Encephalitis Virus Induced Ferroptosis In Neurons

Japanese encephalitis virus(JEV)belongs to the Flaviviridae,and is a serious infectious disease that is mainly transmitted through mosquitos and can infect humans and animals.JEV mainly invades the central nervous system during its pathogenic process and can induce severe neuroinflammatory reactions,leading to nerve damage.However,the specific mechanism of neuronal damage caused by JEV is still unclear.Ferroptosis is a newly identified programmed cell death characterized by iron-dependent superoxide accumulation.A large number of studies have shown that ferroptosis plays a key role in the occurrence of many diseases and the pathogenic process of pathogenic microorganisms,but its role in the pathogenic process of JEV still poorly reported.Therefore,in this study,we focused on JEV to explore whether JEV can induce ferroptosis in neurons,and further reveal its molecular mechanism,clarify the role of ferroptosis in JEV pathogenesis,and provide new scientific clues for elucidating the pathogenesis mechanism of JEV and the development of therapeutic drugs.The main research contents are as follows:1.JEV infection induces ferroptosis in neuronsThis study found that after JEV infected human neuroblastoma cell line(SH-SY5 Y cells)and primary mouse neurons,the m RNA level of the signature gene of ferroptosis PTGS2 was significantly upregulated,and JEV infection could cause lipid peroxidation in these cells,and ferroptosis inhibitors could inhibit neuronal death and lipid peroxidation induced by JEV infection.In addition,the levels of PTGS2 m RNA and lipid peroxidation in the brain tissues of JEV infected mice were also significantly upregulated.These results show that JEV infection can induce ferroptosis in neurons.In order to investigate whether this phenomenon is specific for cell species,we examined the phenomenon of ferroptosis after JEV infection with other susceptible cell lines(He La,BHK-21 and BV2),and found that JEV infection with BHK-21 cells can cause ferroptosis,but infection with He La cells and BV2 cells does not,indicating that JEV induced ferroptosis has cell selectivity.2.JEV induces ferroptosis in SH-SY5 Y cells by inhibiting the GSH-GPX4 pathwayTo investigate the molecular mechanism of ferroptosis in neurons caused by JEV infection,we examined the signaling pathway related to glutathione peroxidase 4(GPX4),an important negative regulator of ferroptosis,and found that JEV infection inhibited the expression of GPX4 in SH-SY5 Y cells and significantly downregulated the level of GSH,demonstrating that JEV may inhibit the expression of GPX4 by reducing the level of GSH in neuronal cells GPX4 expression.To further determine the role of GPX4 in JEV-induced ferroptosis,we constructed SH-SY5 Y cell line with GPX4 overexpression,and found that after GPX4 overexpression,JEV-induced ferroptosis was significantly inhibited.The above results prove that JEV can disrupt the antioxidant system in neuronal cells by inhibiting the GSH-GPX4 signaling axis,thereby leading to ferroptosis.3.JEV induces ferroptosis in SH-SY5 Y cells through ACSL4-mediated lipid peroxidationIn addition to the GSH-GPX4 signaling pathway,Acyl-Co A synthetase long-chain family member(ACSL4)is also an important signaling molecule that regulates ferroptosis.This study found that JEV infection promoted the expression of ACSL4 in SH-SY5 Y cells,and the expression of its upstream Yes-related protein(YAP1)also increased significantly.To further investigate the role of the YAP1-ACSL4 signaling pathway in JEV induced ferroptosis,SH-SY5 Y of YAP1 and ACSL4 knockout were infected with JEV,and found that YAP1 and ACSL4 knockout inhibited lipid peroxidation caused by JEV infection.The above results suggested that JEV infection caused the disorder of polyunsaturated fatty acid synthesis in neuronal cells by promoting the expression of YAP1 as well as ACSL4,which promoted lipid peroxidation and led to the occurrence of ferroptosis.4.JEV pr M and E proteins are involved in neuronal ferroptosisIn order to explore the main components of JEV induced ferroptosis,JEV was inactivated by ultraviolet irradiation and then inoculated into SH-SY5 Y cells.It was found that the inactivated virus could still induce ferroptosis,indicating that its structural proteins may play a role.Further,lentivirus expressing various structural proteins was transduced into SH-SY5 Y cells,and it was found that the expression of pr M and E proteins could induce ferroptosis,while protein C did not,demonstrating that the pr M and E proteins of JEV are the ones that induce neuronal ferroptosis key protein.5.Ferroptosis participates in regulating JEV induced neuroinflammatory response and pathogenesisIn order to investigate whether f ferroptosis is related to JEV replication and induced neuroinflammatory response,this study used the ferroptosis inhibitor Liproxstatin-1 to treat SH-SY5 Y cells and primary mouse neurons after JEV infection,and found that ferroptosis inhibitors inhibited the replication of JEVs in SH-SY5 Y and inhibited the expression of inflammatory factors in primary neurons and glial cells infected with JEV.In order to further investigate the role of ferroptosis in JEV pathogenesis,in this study,ferroptosis inhibitor injection into JEV-infected mice,and found that Liproxstatin-1 can significantly improve the survival rate of JEV infected mice,and alleviate the clinical symptoms and brain tissue inflammatory response caused by JEV infection.These results suggest that ferroptosis promotes neuroinflammatory responses induced by JEV and plays a key role in its pathogenesis.In conclusion,this study proved that JEV infection can induce ferroptosis in neurons,preliminarily elucidates the molecular mechanism of ferroptosis induced by JEV,and revealed the role of ferroptosis in the pathogenesis of JEV,providing important scientific clues and molecular targets for the elucidating of the pathogenesis of JEV and the development of therapeutic drugs.