NLRP3 Activation Regulated By MiR-143/PUMA:Implications For Methamphetamine-induced Neuroinflammatory Response

Objective: Methamphetamine is an addictive drug widely abused worldwide,which can significantly activate microglia and induce the release of inflammatory cytokines and inflammatory mediators,leading to a neuroinflammatory response.NLRP3 inflammasome is a cytoplasmic protein complex composed of NLRP3,apoptosis-related mottled proteins,and cysteine aspartate protease 1(caspase-1).The activation of NLRP3 inflammasome can also induce neuroinflammatory responses,but the regulatory mechanism and regulatory network between methamphetamine,microglia and NLRP3 inflammasome remain unknown.PUMA(P53 up-regulated modulator of apoptosis),the downstream target gene of P53,contains only BH3 domain protein,which is the most common induction of apoptosis at present.Whether PUMA participates in methamphetamine-mediated neuroinflammatory response is still a little known problem.Micro RNA(miRNA)is a small non-coding RNA molecule.Mi R-143 is related to cardiac morphology,tumor development and glucose metabolism,etc.However,its role in mediating the neuroinflammatory response induced by methamphetamine has not been explored.This study started with exploring the regulatory mechanism of neuroinflammatory response,and clarified the role and related molecular mechanisms of NLRP3 activation induced by miR-143/PUMA in methamphetamine-mediated neuroinflammatory response.This study provided a new strategy for clinical treatment of diseases,and provided a new perspective for the selection of drug targets.Methods: In vitro level,the expression of microglia activation related inflammatory factors induced by methamphetamine,such as NLRP3,iNOS,IL-1 beta and cleaved caspase 1 were examined,through the cultivation of the mice BV2 cells,using Western blot,ELISA and RT-PCR experiments,and si RNA or over-expression of miR-143/PUMA method to seek miR-143 / PUMA regulating methamphetamine to microglia activation mechanism;In vivo level,18 male C57BL/6J mice,18 male PUMA KO C57BL/6J mice and 12 male mice with gene mutation miR-143+/-male mice were randomly divided into normal saline group(WT n=6,PUMA KO n=6,miR-143+/-n=6),methamphetamine group(WT n=6,PUMA KO n=6,miR-143+/-n=6)and LPS treatment group(WT n=6,PUMA KO n=6).miR-143 was microinjected into the mouse brain.Using gene knockout techniques,PUMA KO mice and miR-143 +/-mice were used to determinate the expression of inflammatory factors above.Combining the methods of molecular biology,Western blot,Ch IP assay and immunohistochemistry,the activation of microglia cells in the hippocampal brain region of mice was observed,and the expressions of inflammatory factors NLRP3,i NOS,il-1 expression and cleaved caspase 1 were determined.Then the effect of miR-143/PUMA in the activation of NLRP3 induced by methamphetamine was elaborated.Results: 1)In cell level,PUMA is involved in regulating the activation of microglia by regulating the activation of NLRP3 inflammasome.2)Methamphetamine can increase the protein expression level of PUMA by inhibiting miR-143.3)PUMA si RNA induced reduction of methamphetamine-induced microglial cell activation and significantly down-regulated expression of NLRP3 inflammasome.4)PUMA overexpression induced microglial cell activation enhancement induced by methamphetamine and significantly up-regulated expression of NLRP3 inflammasome.5)In body level,microglial cell activation induced by methamphetamine was significantly reduced in PUMA KO mice,and the expression of NLRP3 inflammasome was significantly down-regulated.6)In miR-143+/-mice,the activation of microglial cell induced by methamphetamine was significantly increased,and the expression of NLRP3 inflammasome was significantly up-regulated.7)Using anti-miR-143 lentivirus,NLRP3 inflammasome was significantly up-regulated.While using PUMA si RNA simultaneously,NLRP3 inflammasome was significantly down-regulated.Conclusions: This study focused on the neuroinflammatory response characterized by microglia activation and NLRP3 inflammasome activation by methamphetamine,and revealed the role of miR-143/PUMA axis in this neuroinflammatory response.It may provide a new molecular mechanism for the occurrence and development of neuroinflammatory response induced by methamphetamine abuse.And target PUMA or NLRP3 inflammasome,it may provide a new strategy for relieving the neuroinflammatory injury caused by methamphetamine in the brain and for the selection of clinical therapeutic drugs.