The Activation Of NLRP3 Inflammasome Pathway Is Involved In Acrylamide Induced Neurotoxicity

Objective:Multiple chemical threats such as chemical terrorist attacks,secondary chemical disasters,and sudden chemical leaks affect people's health and social security and stability,while existing drugs cannot be satisfied with the needs of medical treatment against multiple chemical threats.Therefore,it is particularly important to seek one or more effective combinations of drugs to fight against the damage caused by various chemical threats.Seeking a variety of common mechanisms of chemical poisoning,to screen one or more effective of drugs combinations is a new treatment strategy for protect against multiple chemical threats.Our previous work has shown that the expression of NLRP3 inflammasome pathway was significantly increased in central injury caused by various chemical agents.The purpose of this study was to investigate the mechanism of activation of NLRP3 pathway in brain injury induced by acrylamide.Based on the mechanism results,the effective therapeutic drugs targeting to NLRP3against acrylamide poisoning were screened.Thus,this study provide basis experiments for confirmation that NLRP3 will be a common target of central nervous system injury caused by numerous chemical agents,and these studies are important for treatment of chemical poisoning.Methods:1. In vitro experiments,BV2 cells were exposed to acrylamide.The cytotoxicity of BV2cells induced by acrylamide was observed by both MTT assay and LDH assay.Western blot was used to detect the protein expression of NLRP3 inflammasome pathway in BV2 cells exposed to acrylamide.Both of NLRP3 specific antagonist MCC950 and NLRP3 si RNA were used to block/silence the NLRP3 gene,respectively and then observed the changes of cytotoxicity and proteins expression of NLRP3inflammasome pathway induced by acrylamide.2. The changes in body weight,behavioral changes,pathological damage,Purkinje cells and apoptosis were observed in mice exposed to acrylamide.Western blot was used to detect the protein expression of NLRP3 inflammasome complex?NLRP3/ASC/pro-caspase-1?and downstream inflammatory factors?pro-IL-1?/IL-1?/IL-18?in cerebellum tissue of mice exposed to acrylamide.3.The changes in body weight,behavioral,pathological damage,Purkinje cells and apoptosis were observed in NLRP3^-/-mice or MCC950-pretreated mice exposed to acrylamide.Western blot was used to detect the protein expression of NLRP3inflammasome complex?NLRP3/ASC/pro-caspase-1?and downstream inflammatory factors?pro-IL-1?/IL-1?/IL-18?in cerebellum tissues mice,and as well as the effect on the expression of Nrf2 signaling pathway?Nrf2/Keap1/HO1/NQO1?and oxidative stress?lipid peroxide LPO and superoxide dismutase SOD?.Based on these results,the neuroprotective effects of glibenclamide on mice with acrylamide poisoning were investigated.Results:1.The cytotoxicity was significantly increased in a dose-dependent manner after treatment with different concentrations of acrylamide?0,0.5,1.25,2.5,5,10 m M?in BV2 cells,and the protein expression of NLRP3 inflammasome complex?NLRP3/ASC/pro-caspase-1?and its mediated downstream inflammatory factors?pro-IL-1?/IL-1?/IL-18?were significantly increased in BV2 cells after acrylamide?2.5 m M?exposure.The NLRP3-specific antagonist MCC950 and si RNA NLRP3 intervention can significantly protect the cytotoxicity caused by acrylamide by inhibiting the expression of NLRP3 inflammasome complex and its downstream inflammatory factors after acrylamide exposure.2. Mice showed significant weight loss on the 3th day exposed to acrylamide?50mg/kg/d?,and showed obvious significant gait instability,muscle weakness,ataxia symptomson the 7th day after acrylamide-exposed.Purkinje cells were lost and apoptosis on the cerebellum of mice on 10th day after acrylamide-exposed.In addition,protein expression of NLRP3 inflammasome complex?NLRP3/ASC/pro-caspase-1?and its mediated downstream inflammatory factors?pro-IL-1?/IL-1?/IL-18?were significantly increased in mice cerebellum on day 10 after acrylamide exposure.3. Both the NLRP3-specific antagonist MCC950 and NLRP3 knockout mice can significantly protect the neurotoxicity caused by acrylamide by inhibiting the expression of NLRP3 inflammasome complex?NLRP3/ASC/pro-caspase-1?and its downstream inflammatory factors?pro-IL-1?/IL-1?/IL-18?after acrylamide-exposed.Further study found that knockout of the NLRP3 gene could up-regulation of the Nrf2antioxidant signaling pathway?Nrf2/Keap1/HO1/NQO1?,reduce LPO production,increase SOD activity,and show neuroprotective effects.In addition,NLRP3 targeted therapeutic drug glibenclamide could significantly improve the degeneration of mice cerebellar Purkinje neurons,and reduce ataxia.Conclusion:Both in vivo and in vitro studies have shown that the activation of NLRP3 inflammasome plays a dual role that in the mechanism of central inflammatory injury and oxidative stress caused by acrylamide.Activation of NLRP3 inflammasome and its mediate downstream inflammatory factors involved in the degeneration and reduce of Purkinje cells,ataxia and neuroinflammation damage by acrylamide-exposed;Blocking/inhibition of NLRP3 gene can significantly improve the symptoms of body weight,pathological damage,and ataxia,and can activate the Nrf2 antioxidant pathway to reduce oxidative stress damage,suggesting that NLRP3 antagonist can effectively prevent acrylamide-induced inflammatory damage and oxidative stress damage,which provides a scientific basis for a new treatment strategy for acrylamide poisoning,and also lays a theoretical foundation for develop broad spectrum chemical poisoning anti-toxic drugs that NLRP3 as a potential common target.