The Role Of PINK1/Parkin-mediated Mitophagy In Inorganic Arsenic Induced Hepatic Immunotoxicity

Objectives In this study,dendritic cells（DCs）,which are at the core of adaptive immune response,were used to explore the effects of inorganic arsenic on the function of hepatic dendritic cells in vivo and in vitro,and focused on how mitophagy mediated by PTEN-induced kinase 1（PINK1）and E3 ubiquitin protein ligase（Parkin）played a role in inorganic arsenic-induced DCs-mediated liver immunotoxicity.Furthermore,it provides a new idea for the effective control and treatment of liver injury caused by arsenism.Methods 1):Healthy C57BL/6 female mice were fed adaptively for a week.After random grouping,the mice were given 10 mg/kg sodium arsenite（Na As O₂）.The liver tissues were collected at 6,24,48 and 72 h,respectively.The related indexes of mitophagy protein were determined by western blot（WB）.The transcriptional activities of mitohagy related genes,tumor necrosis factor-α（Tnf-α）,interleukin（Il）-4,Il-10,Il-6,Il-1β,Il-12,Il-23 and transforming growth factor-β（Tgf-β）were detected by real-time q PCR.The contents of hydrogen peroxide（H₂O₂）,glutathione（GSH）and malondialdehyde（MDA）in mouse liver were detected by biochemical kit.2):Mouse bone marrow dendritic cells（BMDCs）induced in vitro were divided into control group,lipopolysaccharide（LPS）positive stimulation group and inorganic arsenic+LPS treatment group according to the purpose of the experiment.The protein expression of PINK1/Parkin-mediated mitophagy pathway was detected by WB.Real-time q PCR was used to detect the transcriptional activity of PINK1/Parkin-mediated mitophagy-related proteins and cytokines,and enzyme-linked immunosorbent assay（ELISA）was used to detect the secretion of cytokines.3):Mice and BMDCs exposed to inorganic arsenic were treated with mitophagy inhibitor cyclosporine A（Cs A）.The related indexes in the above 1)and 2)were detected respectively to explore how PINK1/Parkin-mediated mitophagy played a role in DCs-mediated liver immunotoxicity induced by inorganic arsenic.Results 1 After the mice were given one-time oral gavage of Na As O₂ for 6,24,48 and72h,the mice livers developed redox imbalance,which manifested as H₂O₂ content exhibited an initial increase followed by a decreasing trend and peaked at 6h,with a statistically significant difference compared with the control group（P<0.05）.GSH content showed a first decreasing and then increasing trend and peaked at 24 h,which was statistically significant（P<0.05）compared with the control group.Using real-time q PCR to detect the transcriptional activity of inflammatory cytokine genes in liver tissue,we found that the transcript levels of the genes for Tnf-α,Il-1β,Il-6,Il-4,Il-10,Il-12,and Il-23 showed an initial increase followed by a decrease,and peaked at 6 or 24 h,showing statistically significant differences（P<0.05）from those of the control group.2 At 6 h,0～3μM As had no obvious cytotoxicity to BMDCs,while 3～10μM inorganic arsenic had a significant effect on BMDCs at 24 h.Compared with LPS group,inorganic arsenic significantly inhibited the m RNA transcription of BMDCs phenotypic molecules and chemokines（P<0.05）.Compared with LPS group,inorganic arsenic significantly inhibited the m RNA transcription of BMDCs phenotypic molecules CD80 and CD86 and chemokines CCR7（P<0.05）.3Compared with the control group,inorganic arsenic significantly induced the expression of PINK1 and parkin protein in liver,and reached the peak at 24 h.Compared with LPS group,the expression of PINK1 protein in BMDCs exposed to inorganic arsenic increased significantly after being stimulated by LPS for 6 h and 24 h.Compared with As+LPS group,Cs A inhibited the expression of PINK1 protein induced by inorganic arsenic in BMDCs.4Compared with As+LPS group,Cs A intervention enhanced the inhibitory effect of As on MHC-Ⅱand CCR7,and alleviated the inhibitory effect of As on CD86.Compared with inorganic arsenic-exposed BMDCs,Cs A inhibited pro-inflammatory cytokines TNF-α,IL-6 and IL-1β,Th17-inducible cytokines IL-23 and Treg-inducible cytokines IL10,and up-regulated Th1-inducible cytokines IL-12.5 Compared with the Na As O₂ exposure group,intraperitoneal injection of 10mg/kg Cs A significantly inhibited the expression of PINK1protein in the liver of mice induced by inorganic arsenic,and the damage of mitophagy induced by PINK1/Parkin further enhanced the gene transcriptional activities of Il-1β,Il-6and Il-10 induced by arsenic.However,the transcriptional level of Tnf-αgene was not significantly affected.Conclusions 1 Inorganic arsenic induces liver immunotoxicity and PINK1/Parkin-mediated mitophagy.2 Inorganic arsenic induces BMDCs dysfunction and PINK1/Parkin-mediated mitophagy activation.3 PINK1/Parkin-mediated mitophagy aggravates liver immunotoxicity and BMDCs dysfunction induced by inorganic arsenic.Figure 14;Table 9;Reference 124...