PFOA通过ERK MAPK途径损伤Sertoli细胞紧密连接

PFOA disrupts tight junctions between Sertoli cells via activation of ERK MAPK signal pathway

目的 探讨PFOA对Sertoli细胞紧密连接的影响及分子机制。方法 体外分离纯化Sertoli细胞,通过细胞毒性实验,观察PFOA对原代Sertoli细胞生长影响;利用流式细胞术anexin-Ⅴ/PI双染法检测PFOA对Sertoli细胞凋亡的影响;利用双室培养模型,监测跨上皮电阻抗（TER）,观察PFOA对紧密连接功能的影响;采用Western blot法检测紧密连接相关蛋白及ERK的表达。结果 0~100μmol/L剂量的PFOA对细胞生长无明显影响（P〉0.05）;与对照组比,50μmol/L的PFOA未见有明显的促凋亡作用[凋亡率为（9.7%±3.2）%],P〉0.05;但可明显降低Sertoli细胞的TER值[（37.35±5.2）Ωcm2],联合ERK特异性抑制剂PD98059处理后TER值升高[（49.85±6.4）Ωcm2],（P〈0.05）;50μmol/L的PFOA可下调紧密连接蛋白claudin-11、ZO-1和occludin的表达,并上调pERK的表达（P〈0.05）,且可被PD98059明显抑制（P〈0.05）。结论 PFOA可能通过ERK MAPK途径损伤Sertoli细胞间的紧密连接。

Objective To explore the role of perfluorooctanoic acid （PFOA） in the tight junction function between Sertoli cells and its mechanism. Methods Primary Sertoli cells were isolated from 10 days old male ICR mice. Cytotoxicity assay,transepithelial electrical resistance（TER） analysis, flow cytometry, and immunoblotting analysis were used to explore the effects of PFOA on the Sertoli cells. Results There were no significant toxic effects observed after PFOA treatment at the dose range from 0 to 100 μl/L for 12 to 24 hours（P 〉0. 05 for all）. In addition,50 μmol/L of PFOA did not change the apoptosis rate of Sertoli cells （9. 7% ± 3.2% ）. However, TER values were significantly decreased （ 37. 35 ±5.2 Ωcm^2 for PFOA group ,49. 85 ±6.4Ωcm^2 for PFOA＋PD90859 group; P 〈 0. 05 ）. Meanwhile, claudin-11, zonula occludens-1, and occludin significantly decreased, while phosphorylated extracellular signal-regulated kinase （p-ERK） increased after the treatment of 50 μmol/L PFOA（ P 〈 0. 05 ）, which could be inhibited by PD90859. Conclusion PFOA disrupts tight junctions between Sertoli cells via activation of extracellular regulated protein kinase（ERK）/ mitogen-activated protein kinase（MAPK） signal pathway.