The Study On The Effects Of NLRP3 Inflammasome Mediated Inflammation In Trimethyltin-induced Neurotoxicity

Objectives: Based on the c DNA microarray data of central nervous system injuries induced by trimethyltin,acrylamide and somanm,we analyzed the common mechanisms and potential common targets for several kinds of chemical toxic substances to cause central nervous system injuries.Changes to m RNA and protein of NLRP3 inflammasome complexes(NLRP3,ASC,Caspase-1)in models exposed to trimethyltin were observed in vivo and in vitro respectively to clarify the roles of the inflammasomes in the mechanism by which trimethyltin caused the injuries.The autophagic phenomena induced by trimethyltin were observed to discuss mediating roles of autophagy in activating NLRP3 inflammasomes when trimethyltin caused central nervous system injuries.Methods: 1.Pathological analysis and behavioral scoring were performed as modelling standards to model central nervous system injuries induced by trimethyltin,acrylamide and soman.After exposure to the toxic substances,hippocampus tissues from rats were collected at different time points to make chips for gene expression profiles.Specific and common signaling pathways were screened for the injuries caused by above three toxic agents with bioinformatic technologies like R(a programming language)for analyzing differential genes and KEGG pathways.2.Activation of microglia in rat hippocampus exposed to trimethyltin was detected by immunofluorescence,and the impacts of trimethyltin upon apoptosis were tested via flow cytometry.The impacts of trimethyltin upon cell viability were observed through MTT.RT-PCR,immunofluorescence,ELISA and western blot were used for measuring the expressions of m RNA and protein in NLRP3,ASC,caspase-1 and IL-1β of rats’ hippocampal tissues exposed to trimethyltin at different time points(0,1th,2nd,4th,7th day)and varying concentration(0,1,10,100 μM)of BV2 cells.3.Si RNA targeted intervention with NLRP3,and silencing,the impacts of NLRP3 upon cytotoxicity of trimethyltin were detected through MTT.4.The impacts of trimethyltin upon the formation of autophagosomes were observed under a transmission electron microscope.The impacts of trimethyltin upon expressions of P62 and LC3(two autophagy-related genes)were observed using RT-PCR and western blot.The interactions between P62 and NLRP3 inflammasome complex were explored through immunofluoresence,co-expression and co-immunoprecipitation.By intervening with autophagy,its regulatory roles in NLRP3 inflammasome were observed.Results: 1.Central nervous system injuries induced by three kinds of toxic substances were modelled.On the second day after exposure to trimethyltin,a significant increase was detected in aggression scores of rats.On the third and fourth day after exposure,the rats were rather Irritability.On the seventh day after exposure,evident pathological lesions were detected.Rats exposed to acrylamide were exposed to this toxic substance for 11 consecutive days.On the seventh day,there was a significant increase in gait scores,and lower limbs were moderately weak.On the eleventh day after exposure,the lower limbs were faint and the rats even couldn’t move.The pathological lesions were the most severe on the 11 th day after exposure to the toxic agent.After they were exposed to Soman for approximately 10 min,the rats were convulsive all over,and evident pathological lesions were detected 30 min after poisoning.All above 3 models were generally built in line with classical literature home and abroad,which indicated that the modeling was successful.2.After analyzing data about gene chips of expression profiles,it was found that trimethyltin,acrylamide and Soman led to activiation of several types of inflammatory signaling pathways,particularly NOD-like signaling pathways.In combination with early research findings of the research group,the most representative NLRP3 inflammasomes of the NOD-like family were treated as potential “common molecular targets” of brain damages arising from toxic chemical substances.Then,an emphasis was placed upon exploring the roles of NLRP3 in the mechanism for trimethyltin to induce nerve injuries.3.On the seventh day after exposure to trimethyltin,microglia was significantly activated in rats’ hippocampal tissues.Meanwhile,the expressions of NLRP3-ASC-Caspase-1(an inflammasome complex),IL-1β and IL-18 m RNA(downstream inflammatory factors)reached the maximum.The expression of NLRP3 increased significantly on the 7th day after the exposure just like the expression of m RNA.However,no significant change happened to the expression of IL-1β(inflammatory factor).In BV2 microglia exposed to trimethyltin,trimethyltin could induce apoptosis and inhibit the survival rate of cell dependent upon concentration.In contrast,the expressions significantly increased in NLRP3-Caspase-1 and IL-1β m RNA with the concentration of trimethyltin.Besides,a significant increase was also observed in the expression of NLRP3-ASC-Caspase-1,which was dependent upon concentration and consistent with the expression of m RNA.Nevertheless,no significant change happened to the expression of IL-1β.4.Targeted intervention with NLRP3 by si RNA significantly reduced cytotoxicity of trimethyltin.5.In BV2 microglia exposed to trimethyltin,the expressions of autophagy-related proteins increased with the concentration of the toxic agent.After exposing to the toxic agent,the autophagy-related adapter protein P62 interacted with the inflammasome complex NLRP3/ASC.The further administration of autophagy inducers contributed to a significant decline in the expression of NLRP3 and cytotoxicity of trimethyltin.Conclusions: 1.The chips of gene expression profiles and early experimental research preliminarily suggested that inflammatory responses and the representative NOD-like signaling pathways were one of “common injury mechanisms” and “common signaling pathways” for above three types of nerve toxicants.2.Representative NLRP3 inflammasome complex of NOD-like signaling pathways played important roles in the mechanism by which trimethyltin caused central nervous system injuries.The inflammatory responses and apoptosis induced after its activation might be the key mechanisms for the injuries induced by trimethyltin.3.Autophagy couldsignificantly downregulate the activity of NLRP3 for trimethyltin-induced central nervous system injuries,thus reducing cytotoxity of trimethyltin.4.NLRP3 might become one of “common targets” for multiple kinds of toxic chemical substances to cause central nervous system injuries.