PFOS-K通过MAPK通路抑制GH3细胞LH转录的机制研究

The mechanism study of PFOS-K inhibiting LH transcription of GH3 cells through MAPK pathway

目的探索全氟化合物干扰垂体分泌促黄体生成素(luteinizing hormone,LH)的机制。方法本研究以大鼠腺垂体瘤GH3细胞系为模型,以全氟辛烷磺酸钾(perfluorooctane sulfonate sulfonate,PFOS-K)为受试物连续处理14 d,采用细胞活力检测试剂盒(CCK-8)分析细胞活性;采用蛋白质印迹法检测丝裂原活化蛋白激酶(MAPK)信号通路中c-Jun氨基末端激酶(JNK,包括非磷酸化JNK及磷酸化JNK,p-JNK)的蛋白表达情况;采用彗星实验的方法检测DNA损伤情况;采用实时荧光定量PCR方法检测促黄体生成素基因(LH)的转录水平。结果PFOS-K浓度低于10^(-6) mol/L不影响细胞活性;根据细胞活性试验确定机制研究受试物PFOS-K剂量分别为10^(-8)、10^(-7)和10^(-6) mol/L;与对照组相比,10^(-8) mol/L剂量组p-JNK 54KD亚基表达升高(P<0.05),各剂量组p-JNK 46 KD亚基表达均明显升高(P<0.05),各剂量组非磷酸化JNK46 KD亚基表达均明显降低(P<0.01),10^(-7)和10^(-6) mol/L剂量组处非磷酸化JNK54 KD亚基表达降低(P<0.01);受试物组Olive尾距、尾DNA含量、尾/头(长)均明显增加(P<0.05);受试物组LH基因的转录水平均明显降低(P<0.05);除p-JNK之外的所有指标,均呈剂量—反应关系。结论PFOS-K暴露可激活MAPK通路,造成DNA氧化损伤,抑制LH基因的转录。

Objective To investigate the mechanism of perfluorinated compounds interfering with pituitary secretion of luteinizing hormone(LH).Methods In this study,GH3 cell line of rat’s adenohypophysioma was used as model,and was treated with perfluorooctane sulfonate potassium salt(PFOS-K)as test material.CCK-8 was used to measure the cell viability after continuous exposure to PFOS-K for 14 days;The protein expressions of non-phosphorylated c-Jun amino-terminal kinase(JNK)and phosphorylated JNK(P-JNK)in the mitogen-activated protein kinase(MAPK)signaling pathway were detected by Western blot.DNA damage was detected by comet assay;The transcriptional level of luteinizing hormone(LH)gene was detected by real-time fluorescence quantitative PCR.Results The result showed that the cell activity was not affected when the concentration of PFOS-K was lower than 10^(-6) mol/L;In the mechanism study the dosages of PFOS-K were determined with 10^(-8),10^(-7) and 10^(-6) mol/L,respectively;Compared with the control group,the expression of p-JNK 54KD subunit increased in the 10^(-8) mol/L dose group(P<0.05),the expression of p-JNK 46 KD subunit in each dose group was significantly increased(P<0.05),the expression of non-phosphorylated JNK 46 KD subunit in each dose group was significantly reduced(P<0.01),the expression of non-phosphorylated JNK 54 KD subunit decreased in the 10^(-7) and 10^(-6) mol/L dose groups(P<0.01);Olive tail distance,tail DNA content,tail/head(length)were increased significantly in the dose group(P<0.05);The transcription level of LH gene was significantly decreased in dose group(P<0.05);all parameters above except p-JNK showed a dose-response relationship.Conclusion PFOS-K exposure can activate MAPK pathway,cause DNA oxidative damage,and inhibit the transcription level of LH gene.