| Objective Exosomal microRNAs are involved in the development and progression of Alzheimer's disease,while the long-term chronic lead exposure can also cause neurological damage similar to Alzheimer's disease,but the role of exosomal microRNAs in lead neurotoxicity has not been reported.In this study,the lead workers were first studied,and high-throughput sequencing methods were used to analyze changes in serum-derivied exosome microRNA expression profiles to screen for key differential microRNAs.A lead-exposed rat model and an in vitro microglia-infected model were established to preliminarily elucidate the effect of key exosomal microRNAs on the signaling pathway involved in target genes,then to explore the role of key exosomal microRNAs in lead-induced neuroinflammation injury.The current study will enrich the mechanism of lead-induced neuroinflammation and provide new targets for protection strategies and monitoring measures for lead workers.Methods 1 A total of 42 lead exposed workers and 32 non-lead exposed workers in a battery factory were selected as subjects.Serum samples from 8 lead exposed workers and 4 control groups were used for exosomes microRNA sequencing.2 High-throughput sequencing was used to detect theprofile of exosome microRNA,analyze and screen key differential microRNAs(microRNA-124,microRNA-506).Realtime-PCR was applied to verify the expression of microRNA.3 Simple correlation analysis was used to analyze the correlation between the expression of serum exosome microRNA-124,microRNA-506 and neuroinflammation and neurobehavioral changes.Hierarchical regression analysis was applied to analyze the mediating effect.4 A total of 60 4-6 week healthy male SPF SD rats were randomly divided into control group,low-lead exposure group and highlead exposure group.Rats in low-lead exposure group and high-lead exposure group were drank lead acetate solution containing water at dose of 250 mg/L and 500 mg/L respectively.Rats in control group rats were drank with 500 mg/L sodium acetate solution containing.The exposure periodswere 24 weeks.5 Morris water maze and open field test were used to detect spatial learning,memory and exploratory function of rats;ICP-MS was used to detect blood lead level;Realtime-PCR was used to detect the expression of microRNA-124 and microRNA-506 in serum and cerebrospinal fluid exosomes of rats;6 Realtime-PCR and Western blot were used to detect the mRNA and protein expression of key proteins of SOCS3,JAK/STAT pathway(JAK2,p JAK2,STAT3,p STAT3)in cortex and microglial cell line BV-2 of rats.The TNF-? and IL-1?contents were detected by ELISA.7 miR-124 mimimics and mimics negative control(NC)were transfected into BV-2 by Lipofectamine 2000 for knocking down the miR-124 mRNA expression.Results 1 The serum exosome characterization showed that the shape of serum exosome was oval with double-layer structure of lipid molecule,with an average of 159.5 nm and the CD63 expression,a marker protein of exosome.2 The results of high-through put sequencing analysis of microRNA in serum exosomes of lead-exposed workers showed that 79 microRNAs were differentially expressed in the exosomes of lead-exposed workers(P<0.05),of which 54 were up-regulated and 25 were down-regulated;GO functional enrichment analysis showed that the functions of differentially expressed genes were mainly concentrated in protein binding,metal ion binding,nuclear transcription,transcriptional regulation.KEGG functional enrichment analysis showed taht target genes were enriched in MAPK signaling pathway,insulin signaling pathway,AMPK signaling pathway,JAK/STAT signaling pathway and so on.AndmiR-124 and miR-506 also showed the top increses in terms of change folds.The results of mediating effect showed that miR-124 and mir-506 played a partial mediating role in the changes of neurobehavior,emotional state and serum IL-1? induced by lead exposure,while they played a complete mediating role in the increase of serum TNF-? induced by lead exposure.The bioinformatic analysis showed that miR-124 and miR-506 have common target genes AKT3,GRB2,PK3R3,SOCS1,SOCS2,NRAS,SOCS3,TNF,PDPK1,RELA and PLCG1 which associated with JAK/STAT,NF-kappa B,m TOR and TCR signaling pathway that regulated several immune inflammations signaling pathways.Simultaneously,among which,SOCS3 participated in JAK/STAT and NF-kappa B signaling pathways.The serum levels of miR-124 and miR-506 in lead-exposed workers were higher than that in control,which were consistent with the microRNA sequencing.3 The correlation analysis found that serum-derivied exosomes miR-124 and miR-506 expression was positively correlated with emotion scores.miR-124 and miR-506 were negatively correlated with scores of series addition and subtraction,visual retention scores,memory scanning,aim chasing scores.There was a correlation with the NBI scores and miR-124/506.Meanwhile,miR-124 and miR-506 were also related with TNF-? and IL-1? contents in serum.4 The blood lead levels of rats in low-lead exposure group and high-lead exposure group were 0.17±0.04 ug/m L and 0.32±0.09 ug/m L,respectively,which were significantly higher than that in control group(0.08±0.02ug/m L).Moreover,the escape latency in two lead exposure groups was both higher than that in control group.The total distance and central space residence time the number of horizontal pass and standing timesof lead exposure rats in open field experiment were lower than those in control group,and the high-lead exposure group was significantly lower than that in low lead exposure group.The number and time ratio of new object explorationin lead exposed rats were significantly lower than those in the control group.5The expression levels of miR-124 and miR-506 in serum exosomes of rats in low-lead exposure group and high-lead exposure group were higher than those in control group(P<0.05),but only the expression of microRNA-124 in cerebrospinal fluid exosomes was higher than that in control group.There was no significant difference in the expression of microRNA-506 between low-lead exposure group and high-lead exposure group(P>0.05).6 The mRNA expression level of SOCS3 in the cortex of the low lead exposure group and the high lead exposure group was lower than that of the control group(P<0.05).The expression level of cortical SOCS3 was low in the low lead exposure group and the high lead exposure group.The difference was statistically significant(P<0.05).There was a statistically significant difference in the expression of p JAK2 and p STAT3 between the control group and the lead exposure group(P<0.05).The contents of TNF-?and IL-1? protein in the lead exposed group and high lead exposed rats were increased,the difference was statistically significant(P<0.05).7The expression of SOCS mRNA in BV-2 cells exposed to 20 ?g/m L CSF exosome of lead-exposed ratwas significantly decreased(P<0.05).The expression of SOCS3 was significantly decreased after 12 and24 hours,and the phosphorylation levels of JAK2 and STAT3 were significantly increased(P<0.05).The level of TNF-? and IL-1? were significantly increased in the supernatant of the cells after 6h lead exposure,and the difference was statistically significant(P<0.05).8 The transfection of miR-124 mimics to BV-2 increased the expression of miR-124 in BV-2,respectively(P<0.05);miR-124 mimics transfected BV-2 cells for 24 h,The mRNA expression level of SOCS3 decreased significantly,the expression of SOCS3 protein decreased,the phosphorylation level of JAK2 and STAT3 increased significantly,the difference was statistically significant(P<0.05).The TNF-?and IL-1? in cell supernatant were significantly increased(P<0.05).Conclusion Lead exposure did results in the changes in microRNA expression profiles in serum exosomes,especially for exosomal miR-124 and miR-506.The differentially expressed exosome miR-124 and miR-506 are closely related to neurobehavioral damage and inflammation factor IL-1? and TNF-?.miR-124 and miR-506 played a partial mediating role in the changes of neurobehavior,emotional state and serum IL-1? induced by lead exposure,while they played a complete mediating role in the increase of serum TNF-? induced by lead exposure.miR-124 can activate JAK/STAT signaling pathway by inhibiting SOCS3 expression and enhancing IL-1? and TNF-? release.It involved in the development and progression of lead neutrophilic injury,and suggested that exosome miR-124 can be a new target for exploring the mechanism and therapeutic approach of lead neurotoxicity.Figure40;Table50;Reference 299... |
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