PS-MPs and DEHP Combined Induce Autophagy in Mouse Colonic Epithelial Cells via NO/iNOS/NF-κB Pathway
[Objective]This study was aimed to investigate the mechanism of combined exposure of polystyrene microplastics(PS-MPs)and di(2-ethylhexyl)phthalate(DEHP)in inducing autophagy in mouse colonic epithelial cells(MCEC)via NO/iNOS/NF-κB pathway.[Method]According to the half inhibitory concentrations(IC5o)of PS-MPs and DEHP on MCEC measured by CCK-8 method,MCEC were divided into control,PS-MPs,DEHP,combination,and inhibition groups.They were treated with culture medium containing 200 μg/L PS-MPs,100 μmol/L DEHP,200 μg/L PS-MPs+100 μmol/L DEHP,and 200 μg/L PS-MPs+100 μmol/L DEHP+5 nmol/L bortezomib(PS-341)for 24 h,respectively.Then,the NO content and iNOS activity were detected by biochemical kit,autophagosomes were detected by MDC method,and the expression of mRNA and protein of autophagy and NO/iNOS/NF-κB pathway-related genes were detected by immunofluorescence staining,Real-time quantitative PCR and Western blotting,respectively.[Result]Both PS-MPs and DEHP could inhibite the viability of MCEC with IC50 of 2 315 μg/L and 1 477 μmol/L,respectively.Compared with control group,the NO content,iNOS activity and the expression of mRNA and protein of genes related to NO/iNOS/NF-κB pathway in PS-MPs,DEHP and combination groups were significantly increased(P＜0.05),the fluorescence intensity of MDC method and immunofluorescence staining were significantly enhanced(P＜0.05),and the expression of mRNA and protein of the autophagy-related genes(Beclin1,ATG5 and LC3-B)were significantly upregulated(P＜0.05),PS-341 could significantly alleviate the changes of the above detection indexes caused by PS-MPs and DEHP.[Conclusion]The combined exposure to PS-MPs and DEHP could activate iNOS,lead to a sharp increase in NO content,and upregulate of NO/iNOS/NF-κB pathway,which induced autophagy in MCEC.
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