PS-MPs和DEHP联合暴露通过NO/iNOS/NF-κB通路诱导小鼠结肠上皮细胞自噬的作用机制研究

doi:10.16431/j.cnki.1671-7236.2024.11.003

摘要/Abstract

摘要： 【目的】探究聚苯乙烯微塑料(PS-MPs)和邻苯二甲酸二(2-乙基己基)酯(DEHP)联合暴露通过NO/iNOS/NF-κB通路诱导小鼠结肠上皮细胞(MCEC)自噬的作用机制。【方法】依据CCK-8法测定的PS-MPs和DEHP对MCEC的半数抑制浓度(IC₅₀)，将MCEC分为5组：对照组、PS-MPs组、DEHP组、联合组和抑制组，各组MCEC分别用培养基及含有200 μg/L PS-MPs、100 μmol/L DEHP、200 μg/L PS-MPs+100 μmol/L DEHP、200 μg/L PS-MPs+100 μmol/L DEHP+5 nmol/L硼替佐米(PS-341)的培养基处理24 h后，采用生化试剂盒检测NO含量及iNOS活性，通过MDC法检测自噬小体，利用免疫荧光染色、实时荧光定量PCR和Western blotting检测自噬，以及NO/iNOS/NF-κB通路相关基因的mRNA和蛋白表达水平。【结果】 PS-MPs和DEHP均可抑制MCEC的活力，且IC₅₀分别为2 315 μg/L和1 477 μmol/L。与对照组相比，PS-MPs、DEHP、联合组细胞中NO含量、iNOS活性及NO/iNOS/NF-κB通路相关基因的mRNA和蛋白表达水平均显著升高(P＜0.05)，MDC法与免疫荧光染色的荧光强度均显著增强(P＜0.05)，MCEC的自噬相关基因(Beclin1、ATG5、LC3-B)的mRNA和蛋白表达水平显著上调(P＜0.05)；PS-341可显著缓解PS-MPs和DEHP对MCEC暴露引起的上述检测指标的变化。【结论】联合暴露于PS-MPs和DEHP可激活iNOS，使NO含量激增，上调NO/iNOS/NF-κB通路，从而诱导小鼠结肠上皮细胞自噬。

关键词: 聚苯乙烯微塑料(PS-MPs); 邻苯二甲酸二(2-乙基己基)酯(DEHP); NO/iNOS/NF-κB通路; 小鼠结肠上皮细胞(MCEC); 自噬

Abstract: 【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 (IC₅₀) 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.

Key words: polystyrene microplastics (PS-MPs); di(2-ethylhexyl) phthalate (DEHP); NO/iNOS/NF-κB pathway; mouse colonic epithelial cells (MCEC); autophagy

中图分类号:

S852.21

艾婧竹, 徐世文. PS-MPs和DEHP联合暴露通过NO/iNOS/NF-κB通路诱导小鼠结肠上皮细胞自噬的作用机制研究[J]. 中国畜牧兽医, 2024, 51(11): 4678-4689.

AI Jingzhu, XU Shiwen. PS-MPs and DEHP Combined Induce Autophagy in Mouse Colonic Epithelial Cells via NO/iNOS/NF-κB Pathway[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(11): 4678-4689.

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