Activation Of NLRP3 Inflammasome Exacerbates Diesel Exhaust Particulate-induced Neural Impairment

In recent years,epidemiological studies had established a link between diesel exhaust particles（DEPs）and neurological diseases.The high level of oxidative stress promotes the NLR family,pyrin domain containing 3（NLRP3）mediated inflammatory pathways,which is a key role in inflammation.Microglia-activated neuroinflammation has been shown to be involved in the initial process of neuronal injury.In this study,it is focused on the mechanism of neurological injury induced by DEPs exposure in vivo and in vitro.It was found that DEPs exposure promoted the process of inflammatory response by activating NLRP3inflammasome in microglia cells and injured the capacities of spatial learning and memory.However,inhibition of NLRP3 can block the DEP-induced neurological damage.The data suggested that blocking of NLRP3 inflammasome protect microglia from DEPs-mediated impairments in hippocampal learning and memory ability,providing new clues for further research on the molecular mechanism of DEP-induced neurological injury.DEPs exposure induced murine neurobehavioral impairment1.To investigate the effects of DEPs exposure on the nervous system,suspension of DEPs with concentrations of 0（phosphate buffer（PBS））,0.1 or 1μg/μL were prepared and exposed to mice for 14 consecutive days by intranasal inhalation.Morris water maze（MWM）test was used to determine the learning and memory ability of each group.All mice received training experiment（3 times a day for 4 consecutive days）before DEPs exposure.On the 1^stand 14^thday after DEPs exposure,all mice were trained using the MWM navigation task（3times daily for 4 consecutive days）.After this,the platform was removed and the probe trial was performed.The camera system was used to record the time and frequency of mice entering the platform area during the experiment,and the Noldus Ethovision XT software was used for analysis.The escapse latency was significantly increased in DEP-exposed groups until post-exposure days 16（p<0.05）compared to the control.In the probe trial,the frequency and duration in platform zone were significantly reduced on the 5^thand 19^thday after DEPs exposure（p<0.05）compared to the control.During MWM sessions,the swim speed of DEP-exposed mice was undistinguishable from that of control mice（p>0.05）.2.Histopathology showed that the morphology of neurons in brain tissue was unchanged;however,the lung,liver and intestinal tissues of mice exposed to 1μg/μL DEPs showed slight inflammatory infiltration.Molecular mechanism triggered by activated hippocampal microglia in DEP-induced neurotoxicityIn the above studies,the impairment in learning and memory ability caused by DEPs exposure was demonstrated.But there was no obvious damage to the brain neurons.To further clarify the key regulatory molecules involved in DEP-induced neurological injury,pathological and molecular experiments in vivo and in vitro were performed to explore the potential mechanism triggered by hippocampal microglia.1.After collection of murine brain tissues,immunohistochemistry（IHC）staining was performed to examine the abundance of microglia using antibody against Iba-1.Results showed that the number of Iba-1⁺microglia cells in the hippocampus was increased significantly on the 5^thand 19^thdays after exposure（p<0.01）.2.Reactive oxygen species（ROS）levels were detected by fluorescence spectrophotometer and flow cytometry in hippocampus of DEP-exposed miceand in mice microglia cells（BV2）,respectively.Both results showed that DEP exposure significantly increased ROS level（p<0.01）compared to the control.DEPs exposure also increased the nitrogen oxide（NO）levels in cell culture medium and malondialdehyde（MDA）levels in cell lysate,compared to the control（p<0.05）,while the activity of antioxidant enzymes,superoxide dismutase（SOD）,and catalase（CAT）,following DEPs exposure were indistinguishable from control（p>0.05）.3.m RNA levels of p65 and Nlrp3 in murine hippocampus were measured using q RT-PCR and found that DEP exposure led to sustained increase in the expression levels of p65 and Nlrp3 on the 5^thand 19^thday after exposure（p<0.05）.The number of NLRP3⁺cells was consistent with the q RT-PCR results（p<0.001）.The expression levels of NLRP3 in m RNA and protein in BV2 cells were also consistent with results in vivo（p<0.05）.4.Further,ELISA assay was used to assess the expression levels of inflammatory cytokines in the inflammasome pathway in vivo and in vitro.Corroborating the NLRP3expression results,levels of interleukin（IL）-1βand IL-18 also increased significantly in the hippocampus of DEP-exposed mice on the 5^thand 19^thday post exposure compared with those in control mice（p<0.05）.The results in vitro were consistent with those in vivo.5.To further explore mechanisms involved in DEP-induced neurotoxic effects,cellular lysates of BV2 were collected for metabolomics analysis.The results showed that DEPs（20μg/m L and 50μg/m L）exposure changed the expression levels of metabolites.Nine differentially expressed metabolites from DEP-exposed groups（20 and 50μg/m L）was enriched in the taurine and hypotaurine metabolism pathways.Nlrp3 knockout ameliorated the neurotoxic effect in mice induced by DEPs exposureTo fully evaluate the role of NLRP3 in DEP-induced neurological injury,DEP-exposed Nlrp3 knockout(Nlrp3^-/-)mice model was established to verify the key regulatory role of NLRP3.1.The expression level of NLRP3 in the established mice model was verified by IHC,and the results showed that the NLRP3 protein was not detected in Nlrp3^-/-mice.2.MWM showed that DEPs exposure did not affect the escape latency of Nlrp3^-/-mice compared to the wildtype（WT）control.In the probe trial,time spent in the target zone and preference for the platform area in Nlrp3^-/-mice with high dose of DEPs exposure were indistinguishable from those in the WT control group（p>0.05）.3.The number of activated microglia in WT and Nlrp3^-/-mice after DEPs exposure were assessed by IHC.Following DEPs inhalation,the microglia in the hippocampal CA1 region of Nlrp3^-/-mice maintained a predominant resting state and the number of Iba-1⁺cells slightly increased（2.11±0.60 cells/HPF）,but it was not significantly different from that in control group（p>0.05）.4.ELISA results showed that Nlrp3 knockout inhibited DEP-induced high expression levels of IL-1βand IL-18.In conclusion,this study established a mouse model to find that exposure to DEPs induced impairment in spatial learning and memory capability.Then,the molecular mechanisms that play a key role in this process were explored,which were verified in vivo and in vitro.These results suggest that acute exposure to DEPs can induce neuroinflammation in hippocampus,thus affecting the ability of learning and memory.Molecular mechanism studies found that NLRP3 was a major inflammation-related regulatory molecule in the neurotoxicity induced by DEPs.It was found that inhibiting the expression of NLRP3 blocked the activation of microglial inflammasome induced by DEPs exposure,and effectively ameliorates the impariment of learning and memory ability.Therefore,this study suggested that NLRP3 can be used as a potential target for the prevention and mitigation in DEP-induced neurologic injury,assisting to formulate interventions air pollution related neurologic injury.