Toxic effects of permethrin on HMC3 microglia and its associated mechanism
[Background]Permethrin is a commonly used pyrethroid insecticide and has been found to be potentially neurotoxic.Microglia are innate immune cells in the central nervous system and are involved in the development of a range of neurodegenerative diseases.[Objective]To observe possible toxic effects of permethrin on human microglia clone 3(HMC3)in vitro and explore associated mechanism.[Methods]HMC3 were treated with 0,10,25,and 55 μmol·L-1 permethrin for 72 h.Cell cycle and apoptosis were measured using flow cytometry.Cyclin-dependent kinase 1(CDK1),cyclin-dependent kinase inhibitor 1A(CDKN1A),cyclin B2(CCNB2),cellular tumor antigen p53(p53),factor-related apoptosis(FAS),caspase 3(CASP3),and H2A histone family member X(H2AX)were detected by quantitative re-al-time PCR(qPCR).The differential genes and enrichment pathways of HMC3 after 0 and 25 μmol·L-1 permethrin treatment was analyzed by RNA sequencing.HMC3 was treated by 0,10,25,and 55 μmol·L-1 permethrin for 72 h.The content of nitric oxide(NO)in the supernatant was detected using Griess reagent.The secretion level of interleukin-6(IL-6)was detected by enzyme linked immunosorbent assay(ELISA).The mRNA expression levels of mitogen-activated protein kinase(MAPK)pathway(including MAPK1,MAPK8,and MAPK14),in-terleukin-1β(IL-1β),IL-6,and matrix metalloproteinase(MMP)families(including MMP1,MMP2,MMP3,and MMP9)were detected by qPCR.The protein expressions of phosphorylated p38 mitogen-activated protein kinase(p-p38),phosphorylated extracellular signal-reg-ulated kinase(p-ERK),IL-1β,IL-6,and MMP1 were detected by Western blot.[Results]HMC3 was arrested in G2/M phase after 0,10,25,and 55 μmol·L-1 permethrin treatment for 72 h,of which there was a statistically significant difference between the 55 μmol·L-1 permethrin treatment group and the control group(P<0.01),and the mRNA expression of CDKN1A was up-regulated according to the qPCR(P<0.05).There was no statistically significant difference in the proportions of apoptosis between the groups(P>0.05).The RNA sequencing showed that the differential genes were enriched in the MAPK pathway,and the mRNA expressions of MAPK1,MAPK8,and MAPK14 were up-regulated after the permethrin treatment at 55 μmol·L-1 compared to the control group by qPCR(P<0.05).The Western blot revealed that,compared to the control group,the levels of p-p38 and p-ERK were increased after the 10 μmol·L-1 permetrin treatment(P<0.05),the p-ERK level was increased after the 25 μmol·L-1 permetrin treatment(P<0.05),and the p-p38 level was up-regulated after the 55 μmol·L-1 permetrin treatment(P<0.05).The secretion of NO in the supernatant of HMC3 increased after permetrin treatment compared to the control group(P<0.05),the mRNA and protein expressions and the secretion of IL-6 showed an upward trend,the mRNA and protein expressions of IL-1β were up-regulated(P<0.05),and the mRNA and protein ex-pressions of MMP1 were up-regulated in the 25 and 55 μmol·L-1 permethrin groups(P<0.05).[Conclusion]Permethrin inhibits HMC3 cell proliferation in vitro,induces cell cycle arrest,activates MAPK pathway,and promotes the ex-pression of inflammatory factors IL-1β and MMP1,which may be one of the mechanism of neurotoxicity induced by permethrin.
permethrinmicrogliainterleukin-1βmatrix metalloproteinasemitogen-activated protein kinaseinflammatory response
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