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目的 研究邻苯二甲酸二(2-乙基己基)酯[Di(2-ethylhexyl) phthalate,DEHP]暴露后诱导的神经毒性及其分子机制。方法 以12.5、25、50和100μmol/L的DEHP处理3D类脑器官，通过免疫荧光检测神经标志物(GFAP、PAX6、PSD95、Synapsin-1、MAP2和Nestin)的表达，免疫组化检测cleaved Caspase-8、p-RIPK1和RNF4的表达。将RNF4shRNA表达载体导入SKNSH细胞，检测DEHP处理组和对照组相关蛋白的表达水平，TUNEL染色和Hoechst 33258染色检测细胞凋亡。结果 3D类脑器官暴露在梯度浓度的DEHP的培养液24 h后，PAX6、PSD95、Synapsin-1、MAP2和Nestin的表达随着DEHP浓度上升呈剂量依赖性下降，cleaved Caspase-8、RNF4、p-RIPK1、GFAP和细胞凋亡水平呈剂量依赖性上升。在SKNSH细胞中敲低RNF4表达后，再将其暴露于DEHP，其上调的cleaved Caspase-8、RNF4和p-RIPK1恢复到了正常水平，细胞凋亡受到了抑制。结论 DEHP暴露上调RNF4的表达，促进磷酸化RIPK1上调，激活Caspase-8，引发细胞凋亡，造成神经元减少最终诱发神经毒性。敲低RNF4的表达后，挽救了DEHP造成的神经毒性，因此，RNF4可作为防治DEHP暴露导致的神经系统疾病的潜在靶点。
Abstract：Objective To investigate the neurotoxicity and its molecular mechanisms induced by di-(2-ethylhexyl) phthalate(DEHP). Methods 3D brain organoids were treated with DEHP. The expression of neural markers( GFAP,PAX6,PSD95,Synapsin-1,MAP2,Nestin) was detected by immunofluorescence. The level of apoptosis in the brain organoids was assessed by TUNEL staining. The levels of cleaved Caspase-8,p-RIPK1,and RNF4 were evaluated by immunohistochemistry. The RNF4 shRNA expression vector was transfected into SKNSH cells to detect the expression levels of related proteins in both DEHP-treated group and the control group. Apoptosis was assessed by TUNEL staining and Hoechst 33258 staining. Results After 24 hours exposure to 12. 5,25,50,and 100 μmol/L DEHP,the expression of PAX6,PSD95,Synapsin-1,MAP2,and Nestin in the 3D brain organoids decreased in a dose-dependent manner. The levels of cleaved Caspase-8,RNF4,p-RIPK1,GFAP,and apoptosis rate increased in a dose-dependent manner. In SKNSH cells,knockdown of RNF4 expression restored the upregulation of cleaved Caspase-8,RNF4,and p-RIPK1 to normal condition and inhibited apoptosis induced by DEHP exposure. Conclusion DEHP to could up-regulate the expression of RNF4,promote the phosphorylation of RIPK1,activate Caspase-8,and induce apoptosis as well. All above would lead to neuronal loss. Knockdown of RNF4 expression rescues DEHP-induced neurotoxicity.Therefore,RNF4 can be considered as a potential therapeutic target for preventing and treating DEHP-induced neurological disorders.
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基本信息:
DOI：10.16421/j.cnki.1002-3127.2025.05.008
中图分类号:R994.6
引用信息:
[1]李建军,戴思雨,周璐,等.RNF4通过上调磷酸化RIPK1介导增塑剂DEHP诱发的神经毒性[J].毒理学杂志,2025,39(05):328-336.DOI:10.16421/j.cnki.1002-3127.2025.05.008.
基金信息:
湖南省教育厅优秀青年项目(24B0087); 湖南省自然科学基金面上项目(2025JJ550565)