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  

PS-MPs and DEHP Combined Induce Autophagy in Mouse Colonic Epithelial Cells via NO/iNOS/NF-κB Pathway

作　　者：艾婧竹 徐世文[1] AI Jingzhu;XU Shiwen(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150000,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150000

出　　处：《中国畜牧兽医》2024年第11期4678-4689,共12页China Animal Husbandry & Veterinary Medicine

基　　金：黑龙江省自然科学基金重点项目(ZD2023C002)。

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(IC 50),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200μg/L PS-MPs、100μmol/L DEHP、200μg/L PS-MPs+100μmol/L DEHP、200μg/L PS-MPs+100μmol/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IC 50分别为2315μg/L和1477μmol/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。【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(IC 50)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 200μg/L PS-MPs,100μmol/L DEHP,200μg/L PS-MPs+100μmol/L DEHP,and 200μg/L PS-MPs+100μmol/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 IC 50 of 2315μg/L and 1477μmol/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.

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 

分 类 号：S852.21[农业科学—基础兽医学]