Role Of NLRP3 On Lead-induced Neuroinflammation And Possible Mechanism

BackgroundWith the development of modern civilization,heave metal lead(Pb)-induced environmental pollution is taken into consideration and becomes a worldwide public health problem.Pb toxicity causes function disorders or afunction in multiple organs and systems,and Pb-induced neurotoxicity,especially to Pb neurotoxicity in childhood,is the hoy spot of research.Our previous studies have shown that Pb exposure in the early time induced Pb neurotoxicity by multiple mechanisms,and Pb-induced microglial activation and neuroinflammation play an important role in Pb neurotoxicity.Since the conception of inflammasome was put forward at 2002,great attention is paid on the role of inflammasomes in neurological diseases.Studies have shown that inflammasomes-mediated neuroinflammation plays a great part in the progress and prognosis of neurological diseases,and NLRP3 inflammasome receives most attention.NLRP3 mainly expresses in microglia in the central nervous system,and when stimulated,NLRP3 can be activated,leading to the cleavage of Caspase-1 and maturation of interleukin 1 beta.Recent studies have shown the activation of NLRP3 in multiple neurological diseases or disorders,whether NLRP3 is involved in Pb-induced neuroinflammation is still unknown.NLRP3 can be regulated by multiple mechanisms,and present research spot aims at homeostasis and transcriptional regulation and signal transduction.Autophagy is one of the most important mechanisms to maintain cellular physiological function.Recent studies showed that autophagy can mediate the activation of NLRP3,but whether autophagy is involoved in Pb-induced neuroinflammation is unclear;Studies showed that NF-?B signaling could regulate the activation of NLRP3,and our study tries to find whether NF-?B signaling play a part in Pb-induced NLRP3 activation.AimsBy establishing Pb exposure in vivo and in vitro model,we firstly confirmed the phenomenon of Pb-induced microglial activation and neuroinflammation;by investigating the expression of NLRP3 in vivo and in vitro,we secondly tried to testify the effect of Pb on the activation of NLRP3 in microglia,and to further examine the underlying mechanism via autophagy and NF-?B signaling pathways.Our research tried to shed a new light on the mechanism of Pb neurotoxicity.Methods1.By establishing Pb exposure in vivo and in vitro model,using immunofluorescence staining,molecular biological experiments,etc.,we tried to detect the morphology of microglia,release of inflammatory cytokines and the the damage status of neurons after Pb treatment.2.By the abovementioned Pb exposure in vivo and in vitro model,we dectected the expression of NLRP3;by NLRP3 konck-out mice and RNA interference technology,we next tried to find the role of NLRP3 in Pb-induced neuroinflammation,3.By live cell imaging system and other experiments,we aimed at finding the alteration of autophagy after Pb treatment,and detecting the role of autophagy in Pb induced neuroinflammation and NLRP3 activation by inhibitor and RNA interference.4.By investigating the phosphorylation of NF-?B,we tried to examine the effect of NF-?B signaling in Pb-induced NLRP3 activation and autophagy disorder.Results1.The results of Pb exposure in vivo and in vitro model showed that Pb exposure could induce microglial activation and neuroinflammation.Compared with the control group,blood lead levels of lead exposure groups were significantly higher than those of control group(P<0.05).Water mirros maze tests showed that latency periods of the platform in the lead-exposed groups were significantly longer than those in control group(P <0.05).The percentage of TUNEL positive cells in lead group was significantly higher than that in the control group(P <0.05),and the percentage of activated microglia in the lead group was significantly increased(P<0.05).The results of primary microglia and BV2 cells showed that the proportion of CD68-positive cells increased significantly after lead treatment,as well as the cell body and the mRNA expression and release of inflammatory factors(P <0.05).2.NLRP3 inflammasome is involved in Pb-induced neurotoxicity.In vivo results showed that lead exposure could significantly increase the expression of NLRP3 in hippocampus and increase the expression of Caspase-1 and IL-1?(P <0.05).Lead or LPS treatment could significantly increase the expression of NLRP3 and related protein in BV2 cells.NLRP3-siRNA treatment could inhibit the expression of NLRP3 and downstream molecules in BV2 cells and decrease mRNA level and release of IL-1?(P <0.05).NLRP3 knockout mice were used to established a lead exposure model.The results showed that wild type and NLRP3 knockout mice had no significant difference;the administration of lead changed motor abilities in NLRP3 knockout mice,and knockout of NLRP3 did not increase the level of apoptosis in hippocampus,but could inhibit the number of lead-induced apoptosis.NLRP3 knockout did not affect the number of microglia,but affected the morphology of microglia after lead exposure;knockout of NLRP3 could reduce the activation of NLRP3 in vivo and reduce the expression of some inflammatory factors.3.Pb induced neuroinflammation is mediated by microglial autophagy.After exposure to lead,the accumulation of LC3-positive vesicles in BV2 cells was significantly increased and the expression of multiple autophagy-related proteins was increased(P <0.05).3-MA or Atg5-siRNA could significantly inhibit the expression of autophagy and inhibit the expression of inflammatory factors in BV2 cells,and reduce the protein expression of NLRP3 and downstream proteins.4.Pb induced NLRP3 activation by,at least in part,phosphorylation of NF-?B.The phosphorylation of NF-?B in hippocampus of mice was significantly increased after exposure to lead(P<0.05).The phosphorylation of NF-?B was inhibited by PDTC in BV2 cells,and the expression of NF-?B and NLRP3 and downstream proteins induced by lead were significantly decreased(P <0.05).The addition of 3-MA could partially inhibit NF-?B phosphorylation significantly and reduced the activation of NLRP3.Conclusion1.Data of Pb exposure in pregnant period suggested that Pb mainly decreased ability of spatial learning memory and increased the cell apoptosis of hippocampus neurons;Studies of in vitro and in vivo experiments confirmed that Pb could induce microglia activation by transforming morphology and releasing multiple inflammatory cytokines,which suggested that Pb-induced neuroinflammation,to a great concent,led to Pb neurotoxicity and neural death.2.Pb exposure increased the expression of NLRP3 and its downstream Caspase-1 and IL-1? in vivio and in vitro,and inhibition of NLRP3 by RNA interference and transgenic mice could markedly mitigate Pb-induced neuroinflammation,which suggested that NLRP3 played an important role in Pb-induced neuroinflammation.3.Pb exposure could induce the abnormal upregulation of autophagic related proteins,which promoted the activation of microglia;inhibition of autophagy could attenuate the activation of microglia induced by Pb,and the mechanism may be mediated by inhibition of NLRP3 activation.4.The underlying signaling pathway of Pb-induced NLRP3 activation was mediated by NF-?B signal.These data suggested that Pb-induced neuroinflammation and NLRP3 activation can be mediated by transcriptional levels and signal pathways.Taken together,our research confirmed Pb-induced neuroinflammation and underlying mechanism,and firstly found the role of NLRP3 and microglial autophagy in Pb-induced neuroinflammation,and preliminarily confirmed the role of autophagy/NF-?B in Pb-caused neuroinflammation and NLRP3 activation.Our study supplied theoretical foundation for further research of Pb neurotoxicity,and may shed a new light on the prevention of Pb neurotoxicity.