| Nonylphenol(NP)is a typical environmental neurotoxicant,which has the characteristics of wide application,serious pollution and high toxicity.At present,the mechanism of neurotoxicity caused by NP is unclear.A study have reported that NP exposure activated Toll-like Receptor 4(TLR4)on the membrane of microglia,triggering neuroinflammation.Pyroptosis mediated by the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome is a newlydiscovered programmed cell death with inflammatory response,which is a hot spot in the research on the mechanism of various neurological/psychiatric diseases.TLR4 is the first initiating signal of pyroptosis mediated by NLRP3 inflammasome.Whether the inflammatory response caused by NP induces the occurrence of neural pyroptosis has not been reported.Micro RNAs(miRNAs)are abundant in the brain and are tightly controlled in neurodevelopment,precisely located in susceptible populations or early stages of life,and are often used as biomarkers in the early stages of disease development.However,the regulatory role of miRNAs in NP’s neurotoxicity has not been reported.Whole transcriptome sequencing showed that NP exposure downregulated miR-542-3p in the brain;Bioinformatics analysis and literature reports: miR-542-3p can target and regulate TLR4.The substantia nigra which is one of microglial specific localizing structures has a large number of microglia,which is particularly susceptible to the impact of environmental toxicants.In vivo experiments,SD rats exposed to NP(80 mg/kg/d)for 90 days found that the microglia in the substantia nigra were activated,the expression of miR-542-3p was down-regulated,and the genes/proteins of TLR4 and pyroptosis-related indicators increased.So it was judged that NP exposure induced pyroptosis in this brain region.Microglia are innate immune cells in the brain,the core cells of the neuroinflammatory response.Therefore,we wondered whether miR-542-3p could be involved in NP-induced microglial pyroptosis by regulating TLR4? First,in vitro experiments verified that the changes of miR-542-3p,TLR4 and pyroptosis-related indexes after NP exposure to BV2 microglia were consistent with those in vivo.Then,the regulation of miR-542-3p on TLR4 was clarified by inhibiting and overexpressing miR-542-3p in BV2 cells;finally,the changes of TLR4 and pyroptosis-related indexes were detected after overexpressing miR-542-3p.In conclusion,the above studies were used to determine whether NP leads to microglial pyroptosis through the miR-542-3p/TLR4/NLRP3 regulatory network,and to explore the regulatory targets for NP neurotoxicity.Part Ⅰ Nonylphenol Exposure Induced Microglial Pyroptosis and Affected MiR-542-3p/TLR4 in the Substantia Nigra of RatsObjective: To explore the effects of nonylphenol(Nonylphenol,NP)on the exercise capacity of rats,microglial pyroptosis and miR-542-3p/TLR4 in the midbrain substantia nigra after oral administration of nonylphenol(NP).Methods: Thirty-six 36-day-old SD rats were randomly divided into 3 groups,including: control group(corn oil),NP group(80 mg/kg·bw/day,NP)and LPS group(n = 12 per group).(1)The content of NP in the midbrain was detected by high performance liquid chromatography.(2)Y-maze test,Tield test,and Rota-rod test were used to detect the spatial learning/memory and motor ability of rats.(3)After whole transcriptome sequencing,miRNAs with difference in expression between control group and exposure group were selected through Difference Fold(|log2(Fold change)|>1)and Significant Level(P < 0.05);Targetscan and miRDB databases were used to identify the target genes.(4)The pathological changes and ultrastructure of the substania nigra after NP exposure were observed by HE staining,immunofluorescence and transmission electron microscopy.The miRNA/m RNAs expression levels of miR-542-3p,TLR4 and pyroptosis indicators(NLRP3,GSDMD,caspase-1 and IL-1β)were detected by RT-PCR.(5)The protein expressions of TLR4,NLRP3,GSDMD-N,ASC oligomer/monomer,pro-caspase-1 and caspase-1 were detected by Western Blot.(6)The content of IL-1β was detected by ELISA.Results:(1)NP accumulation in the midbrain was significantly higher in the NP group than that of the control and LPS groups(P < 0.05).(2)Compared with the control group,the frequency of the rats in the NP group entering the novelarm of the Y maze and the center of the field test decreased(P < 0.05);In the rota-rod test,the duration of rats in the control group,NP group and LPS group decreased in turn(P < 0.05).(3)Wholetranscriptome sequencing of miRNAs in the brain tissue of NP-exposed rats showed that compared with the control group,the expression of 14 miRNAs was different(P <0.05),among which miR-542-3p was down-regulated;Bioinformatics analysis showed that miR-542-3p A binding site exists with the TLR4 m RNA sequence;Combined with the literature,TLR4 is the target gene of miR-542-3p.(4)Compared with the control group,the neural cells in the NP and LPS groups were arranged irregularly with morphological abnormalities;the plasma membrane was ruptured,there were nucleus pyknosis and necrosis.(5)Compared with the control group,the microglia were activated in the NP and LPS groups;the cell bodies were enlarged;the neurites became thicker and smaller,and even retracted in an "amoeba-like" appearance.(6)Compared with the control group,the microglial membrane was damaged,the cytoplasm was swollen in NP and LPS groups;the nuclei were irregular in shape;the heterochromatins were reduced,which gathered in the nuclear membrane;the boundary of the nuclear membrane fractured,which was accompanied by vacuolizations;the number of damaged mitochondria was increased.(7)Compared with the control group,the expression of miR-542-3p in the substania nigra of the NP and LPS groups decreased sequentially,while the m RNA expressions of TLR4,NLRP3,caspase-1,GSDMD and IL-1β increased(P < 0.05).(8)Compared with the control group,the protein levels of TLR4,NLRP3,pro-caspase-1/caspase-1,GSDMD-N and ASC oligomer/monomer of the NP and LPS groups increased(P < 0.05).(9)The concentration of IL-1β increased in the control,NP and LPS groups sequentially(P < 0.05).Conclusion: NP exposure could led to the decline of spatial learning and memory and whole-body mobility ability in rats.NP activated microglia with the occurrence of pyroptosis,and down-regulated the expression level of miR-542-3p in the substania nigra.Part Ⅱ The Mechanism of MiR-542-3p Regulating TLR4 Involved in BV2 Microglial Pyroptosis induced by NonylphenolObjective: To explore the function of miR-542-3p regulating TLR4 in BV2 microglial pyroptosis induced by Nonylphenol.Methods:(1)In vitro part one: Contorl group,LPS group,and NP group;In vitro part two: mimics NC group,miR-542-3p mimics group,mimics NC +NP group and miR-542-3p mimics+NP group.(2)NP concentration in BV2 microglia was determined by CCK8.(2)The protein expression of ASC in BV2 cells was detected by immunofluorescence.(3)The genes expressions of miR-542-3p,TLR4,NLRP3,GSDMD,caspase-1 and IL-1β were detected by RT-PCR.(4)The protein levels of TLR4,NLRP3,GSDMD-N,caspase-1 and IL-1β were detected by Western Blot.Results: Part Ⅰ:(1)The NP exposure concentration of BV2 microglia was 40 μM.(2)Compared with the control group,the fluorescence intensity of ASC increased,and ASC speck spots existed in the NP and LPS groups(P < 0.05).(3)Compared with the control group,miR-542-3p was down-regulated in the NP and LPS groups,while TLR4,caspase-1 and IL-1β gene levels were increased in the NP and LPS groups(P < 0.05).(4)Consistent with the LPS group,NP exposure increased the protein expressions of TLR4,NLRP3,GSDMD-N,caspase-1 and IL-1β in BV2 cells.(P < 0.05).These results confirmed that NP exposure induced pyroptosis in BV2 cells.Part Ⅱ:(1)Transfecting miR-542-3p mimics can significantly increase the expression of miR-542-3p in BV2 cells,while transfecting miR-542-3p inhibitors can downregulate the expression of miR-542-3p(P < 0.05).(2)Transfecting of the miR-542-3p mimics inhibited TLR4 m RNA expression,whereas transfecting of the miR-542-3p inhibitors increased TLR4 m RNA expression(P < 0.05).(3)Compared with the mimics NC group,the ASC fluorescence intensity of BV2 cells in the mimics NC+NP group was increased;compared with the mimics NC+NP group,the ASC fluorescence intensity of the miR-542-3p mimics+NP group was significantly decreased(P < 0.05).(4)NP exposure can reduce the expression of miR-542-3p,and overexpression of miR-542-3p can inhibit the down-regulation of miR-542-3p caused by NP exposure(P < 0.05).Compared with mimics NC,overexpression of miR-542-3p can reduce the m RNA expression of TLR4,NLRP3 and caspase-1(P < 0.05);Compared with mimics NC+NP,overexpression of miR-542-3p can inhibit the exposure of NP The resulting increased TLR4,NLRP3 and IL-1β m RNA expression(P < 0.05).Conclusion: Up-regulation of miR-542-3p targeting TLR4 could alleviate NPinduced microglial pyroptosis by inhibiting thet activation of NLRP3 inflammasome. |
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