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目的探讨N-乙酰半胱氨酸对氟化钠(NaF)介导睾丸支持细胞内质网应激的抑制作用。方法将原代培养大鼠睾丸支持细胞进行分组:0μg/ml NaF(对照组)、6μg/ml NaF组、12μg/ml NaF组、24μg/ml NaF组、N-乙酰半胱氨酸(2 mmol/L NAC)组、6μg/ml NaF+2 mmol/L NAC组、12μg/ml NaF+2 mmol/L NAC组和24μg/ml NaF+2 mmol/L NAC组,各组以相应药物孵育24 h。四甲基偶氮唑蓝(MTT)法测定各组细胞生长活性;荧光探针法分析各组细胞内活性氧(ROS)水平;免疫印迹法(Western blot)检测内质网应激相关蛋白GRP78、PERK和CHOP的表达。结果 2 mmol/L NAC有效提高了细胞存活率(P<0.01);NAC明显抑制了NaF介导的细胞内ROS水平的升高,与NaF组相比,各NAC组的ROS水平均显著降低(P<0.01);NaF诱导内质网应激相关蛋白GRP78、PERK和CHOP表达明显上调,经2mmol/L NAC处理后,其有效拮抗了GRP78、PERK和CHOP蛋白表达的上调(P<0.01)。结论 ROS激活了内质网应激相关信号通路,而NAC通过减少ROS的产生,拮抗了NaF介导的睾丸支持细胞内质网应激。
Objective To investigate the inhibitory effect of N-acetylcysteine on the endoplasmic reticulum stress mediated by sodium fluoride (NaF) in testis-supporting cells. Methods Primary cultured rat sertoli cells were divided into groups: 0μg / ml NaF (control group), 6μg / ml NaF group, 12μg / ml NaF group, 24μg / ml NaF group, N-acetylcysteine / L NAC group, 6μg / ml NaF + 2mmol / L NAC group, 12μg / ml NaF + 2mmol / L NAC group and 24μg / ml NaF + 2mmol / . Cell viability was measured by MTT assay. ROS levels in each group were analyzed by fluorescent probe method. Endoplasmic reticulum stress-related protein GRP78 was detected by Western blot. , PERK and CHOP expression. Results NAC at 2 mmol / L increased cell viability significantly (P <0.01). NAC significantly inhibited NaF-mediated increase of intracellular ROS levels. Compared with NaF group, ROS levels in NAC group were significantly decreased P <0.01). The expression of GRP78, PERK and CHOP in endoplasmic reticulum was upregulated by NaF, and the up-regulation of GRP78, PERK and CHOP protein was antagonized by 2mmol / L NAC treatment (P <0.01). Conclusion ROS activates the endoplasmic reticulum-stress-related signaling pathway, whereas NAC antagonizes NaF-mediated endoplasmic reticulum stress in testicular support cells by reducing ROS production.