Bupivacaine Regulates DRG Autophagic Injury Through ROS/TUG1/IRS1 Pathway In High Glucose Environment

BackgroundWith the rapid increase of the diabetes,the number of patients who need to receive regional nerve block and LA is also increased.Diabetes patients are often accompanied by peripheral neuropathy.The current study suggests that patients with diabetes have increased neurological complications after applicated of LA,which involved reactive oxygen species(ROS),neuronal necrosis,apoptosis,and excessive autophagy.LincRNA taurine upregulated gene 1(TUG1)is closely related to the development of diabetic nephropathy and nervous system development.Insulin receptor substrate(IRS1)is a key mediator of insulin growth factor regulating glucose metabolism,the AKT/mTOR pathway plays an important role in autophagy.However,studies on whether ROS burst,TUG1,and IRS 1 serine phosphorylation in neurons have a mutual regulation relationship between LA and peripheral neurotoxicity in diabetes are still lack.Studies have shown that the classic drug of LA,bupivacaine(BP),can lead to increased phosphorylation of IRS1 in DRG neurons in high glucose environments,but whether ROS mediates TUG1 expression and IRS1 pathway,how to mediate needs to further study.ObjectiveTo explore whether BP regulates autophagic damage of DRG neurons through ROS/TUG1/IRS1 pathway in high glucose environment,which provides a theoretical basis and target in clinical prevention and treatment of neurotoxicity of LA in diabetic.MethodsChapter 1 ROS-related autophagic damage induced by BP in DRG neurons in high glucose environmentIn the high glucose environment,before treated by BP,the ROS inhibitor N-acetyl-L-cysteine(NAC)was pretreated,then the ROS level,cell viability,autophagosome of DRG neurons were detected.Analyzed for the relationship and regularity of ROS-mediated autophagic flow in BP-induced neuronal damage.Chapter 2 BP induces autophagic damage of DRG neurons through TUG1/IRS1 signaling pathway in high glucose environmentBefore BP treated in high glucose environment,pretreatment with autophagy lysosomal inhibitor chloroquine and(or)siRNA-TUG1,thenTUG1 expression,expression of key molecules of autophagy and IRS1/AKT/mTOR insulin signaling pathway were detected.Analyzed for the relationship between TUG1 and insulin signaling pathway in autophagy.Chapter 3 Bupivacaine regulates DRG autophagic injury through ROS/TUG1/IRS1 pathway in high glucose environmentBefore BP treated in high glucose environment,pretreatment with NAC,mTOR inhibitor Rapamycin,TUG1 expression,ROS-related autophagic flow,IRS/AKT/mTOR signaling pathway and autophagy protein expression,ROS levels were detected.Further analyzed for the regulation relationship and regularity of ROS-related autophagic flow,TUG 1,and IRS 1 signaling pathways.ResultsBP increase ROS level and phosphorylation of IRS 1 serine,inhibit downstream AKT/mTOR pathway,increased autophagosome and autophagy substrate P62 expresion,decreased cell viability in DRG neurons in high glucose environment.Compared with HG+BP group,ROS inhibitor NAC or TUG1 siRNA pretreatment reduce TUG 1 expression and IRS1 serine phosphorylation,improved downstream AKT/mTOR pathway inhibition,decreased autophagy level,and increased cell viability;compared with HG+BP group,mTOR inhibitor Rapamycin positive feedback increased ROS levels and TUG1 expression,inhibite the IRS1 signaling pathway,increase autophagy levels,and autophagic damage in cells.Conclusion1.BP block the autophagic flow and induce the autophagosome aggregate in DRG neurons in high glucose environment;2.BP induced autophagic damage of DRG neurons through ROS/TUG1/IRS1 signaling pathway in high glucose environment;3.After BP treat in high glucose environment,mTOR protein activity can feedback inhibite ROS/TUG1 pathway activation;4.After BP treat in high glucose environment,the aggregate of autophagosome can feedback the activate of TUG1/IRS1 pathway,further aggravate DRG neuron injury.