Mitophagy Mediates NLRP3 Inflammasome Activation In Arsenicinduced Liver Insulin Resistance

Objective:Insulin resistance?IR?is the key event for inorganic arsenic-caused type2 diabetes.The inflammatory response mediated by activation of NOD-like receptor protein 3?NLRP3?inflammasome plays an important role in the development of IR.It has been reported that mitochondrial damage is one of upstream mechanisms of NLRP3inflammasome activation.However,it is not clear how mitochondrial damage can induce the activation of NLRP3 inflammasome in the arsenic-induced IR model.Therefore,this study explored the relationship between mitochondrial reactive oxygen species?mt ROS?,mitophagy,NLRP3 inflammasome in sodium arsenite?NaAsO₂?-treated Sprague-Dawley?SD?rats and Hep G2 cells.Methods:In vivo,SD rats were exposed to NaAsO₂ in the concentration of 0,2.5,5mg/kg BW by oral gavage for three months.Oral glucose tolerance test?OGTT?,insulin tolerance test?ITT?and insulin secretion experiments to determine whether IR occurs in rats.Periodic acid-schiff?PAS?staining,oil red O staining and triglyceride?TG?were used to observe the glycogen content and lipid accumulation in rat liver tissues;Hematoxylin-eosin?H&E?staining was used to observe the morphological structure of liver;The inflammatory,NLRP3-inflammasome,mitophagy and insulin signaling pathway-related proteins were detected with Western blot.In vitro,the human hepatoma cell line Hep G2 cells were used as research objects.NLRP3 inhibitor?MCC950?,mitophagy inhibitor?Cs A?and mt ROS scavenger?Mito-TEMPO?were used in Hep G2cells,and then treated with 4mM NaAsO₂ for 24 h.The levels of mt ROS were measured Mito SOX?Red.Mitochondrial membrane potential were measured JC-1.In Hep G2 cells,the inflammatory,NLRP3 inflammasome,mitophagy and insulin signaling pathway-related proteins were detected with Western blot.The insulin sensitivity analysis in Hep G2 cells was measured by glucose oxidase-peroxidase method.Results:In vivo,serum insulin level and HOMA-IR were increased in NaAsO₂-treated compared with the control group.Rats treated with NaAsO₂ showed significantly impaired glucose tolerance and insulin sensitivity compared with the control group in OGTT and ITT.Taken together,NaAsO₂ induced hepatic IR.PAS staining results showed that glycogen content decreased in rat liver after exposure of NaAsO₂.Oil Red O staining and TG results showed that lipid accumulation increased in rat liver after exposure of NaAsO₂.As shown in the H&E staining of liver tissue,NaAsO₂ caused inflammatory edema compared with the control group.Western blot results showed that the expression of inflammation related-related proteins?pro-IL-1b,IL-1b?11?IL-18?,inflammasome-related proteins?NLRP3,ASC,pro-caspase-1,caspase-1?and mitophagy-related proteins?PINK1,Parkin and LC3?were increased in NaAsO₂-treated compared with the control group.Compared to the control group,the expression of insulin signaling pathway-related protein p-IRS1?Ser 307?increased,and the expression of p-AKT1?Ser 473?protein decreased in rat liver after exposure of NaAsO₂.In vitro,compared to the control group,the levels of mt ROS were significantly increased in the NaAsO₂-treated group.In addition,the reduction of mitochondrial membrane potential under NaAsO₂-exposed cells.We found that the expression of NLRP3 inflammasome related proteins and mitophagy-related proteins in Hep G2 cells increased after treated with NaAsO₂.Compared to the control group,the expression of p-IRS1?Ser 307?was significantly increased and the expression of p-AKT1?Ser 473?was significantly decreased in the NaAsO₂-treated group.Glucose uptake was significantly decreased in NaAsO₂-treated group.Compared with the NaAsO₂ group,after intervention with NLRP3 inhibitor?MCC950?,mitophagy inhibitor?Cs A?,and mt ROS scavenger?Mito-TEMPO?,the above phenomenon was suppressed.Conclusion:NaAsO₂induced the level of mt ROS and the expression of mitophagy increased,which activated the NLRP3 inflammasome and tiggered inflammatory response,leading to occur IR.