Lin Rui, Pian Yajing, Zhang Chaoqin, Zhou Li, Ren Xiangmei
Department of Nutrition, School of Public Health, Xuzhou Medical University, Xuzhou 221004, China.
Wei Sheng Yan Jiu. 2022 Jul;51(4):632-637. doi: 10.19813/j.cnki.weishengyanjiu.2022.04.022.
To investigate the regulation mechanism of N-acetylcysteine(NAC) on cadmium-induced apoptosis of mouse testicular interstitial cells based on protein kinase B pathway(AKT pathway).
Mouse testicular mesenchymal cells(TM3) were divided into fourgroups according to different treatment, control group, cadmium group(Cd, 5, 10, 20, 30, 40 and 50 μmol/L), NAC group(NAC, 500 μmol/L) and NAC+Cd group(500 μmol/L NAC+20 μmol/L Cd). Cells of NAC+Cd group were pretreated with NAC for 30 min, and then combined with cadmium for 24 h. Cell viability was determined by CCK8. Hoechst staining was used to determine cell morphology. Cell apoptosis rate was analyzed by flow cytometry. Malondialdehyde(MDA) and glutathione(GSH) were measured simultaneously. Western blot was used to detect the expression levels of AKT protein, B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(Bax).
Cadmium inhibited the proliferation of TM3 cells in a dose-effect relationship. Cell morphology observation showed that with the increase of cadmium concentration, the cells shrank, became round and even fell off, and appeared dense nuclear staining. The MDA level in Cd group was(1.56±0.11) μmol/mg prot, which was significantly higher than that in control group(P<0.01). Compared to the control group, the level of GSH was significantly decreased to(1.28±0.25) μmol/mg prot(P<0.01). NAC pretreatment could reduce the MDA content and increase the GSH level, and the difference was statistically significant compared with the Cd group(P<0.01). Western blot result showed that NAC pretreatment significantly increased levels of phosphorylated AKT and Bcl-2, the levels were 0.65±0.05 and 0.45±0.03, respectively(P<0.01). The Bax/Bcl-2 ratio was 1.54±0.15, which was significantly lower than that of the Cd group(P<0.01).
NAC can inhibit cadmium-mediated TM3 cell damage and apoptosis, which may be related to the improvement of oxidative stress state, activation of TM3 AKT pathway and reduction of Bax/Bcl-2 ratio.
基于蛋白激酶B通路（AKT通路）探讨N-乙酰半胱氨酸（NAC）对镉诱导的小鼠睾丸间质细胞凋亡的调控机制。
将小鼠睾丸间质细胞（TM3）根据不同处理分为四组，对照组、镉组（Cd，5、10、20、30、40和50 μmol/L）、NAC组（NAC，500 μmol/L）和NAC+Cd组（500 μmol/L NAC+20 μmol/L Cd）。NAC+Cd组细胞先用NAC预处理30分钟，然后与镉共同作用24小时。采用CCK8法测定细胞活力。用Hoechst染色法观察细胞形态。通过流式细胞术分析细胞凋亡率。同时测定丙二醛（MDA）和谷胱甘肽（GSH）含量。采用蛋白质免疫印迹法检测AKT蛋白、B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的表达水平。
镉对TM3细胞增殖的抑制作用呈剂量效应关系。细胞形态观察显示，随着镉浓度的增加，细胞皱缩、变圆甚至脱落，细胞核染色加深。镉组MDA水平为（1.56±0.11）μmol/mg prot，显著高于对照组（P<0.01）。与对照组相比，GSH水平显著降低至（1.28±0.25）μmol/mg prot（P<0.01）。NAC预处理可降低MDA含量，提高GSH水平，与镉组相比差异有统计学意义（P<0.01）。蛋白质免疫印迹结果显示，NAC预处理显著提高了磷酸化AKT和Bcl-2的水平，分别为0.65±0.05和0.45±0.03（P<0.01）。Bax/Bcl-2比值为1.54±0.15，显著低于镉组（P<0.01）。
NAC可抑制镉介导的TM3细胞损伤和凋亡，这可能与改善氧化应激状态、激活TM3细胞的AKT通路及降低Bax/Bcl-2比值有关。
