Zhang Ling, Wang Yixiang, Yi Lingna, Huang Dingyu, Zhang Yuqian, Mirqami Kunuduz Ayi, Cheng Yi, Ren Quanlong
Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Public Health, Wuhan University of Science and Technology, Wuhan 430065, China.
Wei Sheng Yan Jiu. 2023 May;52(3):489-496. doi: 10.19813/j.cnki.weishengyanjiu.2023.03.025.
To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms.
The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 μmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 μmol/L) were added to cell culture in the presence/absence of CdCl_2(10 μmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot.
Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 μmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P<0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P<0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P<0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P>0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P<0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P<0.05).
Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.
研究自噬在氯化镉（CdCl₂）诱导的小鼠精母细胞（GC-2 spd）细胞凋亡中的作用，并探讨其潜在的分子机制。
用不同浓度的CdCl₂（0、5和10 μmol/L）处理细胞24 h。采用Hoechst33342染色和单丹磺酰尸胺（MDC）检测自噬体和凋亡小体的形成。通过TUNEL染色检测镉处理细胞的凋亡情况。在存在/不存在CdCl₂（10 μmol/L）的情况下，向细胞培养物中加入自噬抑制剂3-甲基腺嘌呤（3-MA）（60 μmol/L）、凋亡抑制剂半胱天冬酶抑制剂Z-VAD-FMK（zVAD-FMK）（50 nmol/L）、自噬诱导剂雷帕霉素（RAPA）（50 nmol/L）和溶酶体抑制剂氯喹（CQ）（10 μmol/L），处理GC-2 spd细胞24 h。通过蛋白质免疫印迹法检测自噬相关蛋白LC3、P62以及促凋亡蛋白裂解的半胱天冬酶-3和裂解的半胱天冬酶-9的表达水平。
CdCl₂处理24 h后，自噬体聚集，凋亡细胞数量增加。蛋白质免疫印迹结果显示，在5和10 μmol/L CdCl₂暴露组中，与对照组（5.50±0.56）相比，LC3II/LC3I的蛋白表达水平分别增加到9.23±0.81和12.15±0.80（P<0.05），LC3II蛋白表达水平分别增加到3.35±0.14和3.47±0.32，与对照组（2.35±0.34）相比（P<0.05），P62蛋白表达水平分别增加到1.48±0.12和1.80±0.22，与对照组（0.83±0.09）相比（P<0.05）。与CdCl₂处理组相比，3-MA处理后LC3II/LC3I、LC3II、P62、裂解的半胱天冬酶-9和裂解的半胱天冬酶-3的蛋白表达水平分别降至0.90±0.07（CdCl₂组：1.47±0.06）、1.57±0.14（CdCl₂组：2.45±0.29）、0.82±0.05（CdCl₂组：1.44±0.18）、0.18±0.01（CdCl₂组：0.28±0.01）和0.61±0.84（CdCl₂组：1.15±0.04）（P<0.05）。与CdCl₂处理组相比，zVAD-FMK处理后裂解的半胱天冬酶-9和裂解的半胱天冬酶-3的蛋白表达水平分别降至0.12±0.01（CdCl₂组：0.28±0.01）和0.34±0.01（CdCl₂组：1.15±0.04）（P<0.05），而LC3II/LC3I、LC3II和P62的蛋白表达水平无显著变化（P>0.05）。与CdCl₂处理组相比，RAPA增强了镉诱导的LC3II/LC3I、LC3II和P62蛋白表达，分别为2.22±0.21（CdCl₂组：1.56±0.06）、3.72±0.21（CdCl₂组：2.97±0.15）和2.41±0.19（CdCl₂组：1.52±0.35）（P<0.05）。蛋白质免疫印迹结果显示，与CdCl₂组相比，CdCl₂和CQ处理组中LC3II/LC3I、LC3II、P62和裂解的半胱天冬酶-3的蛋白表达水平分别增加到3.21±0.31（CdCl₂组：2.09±0.25）、4.49±0.43（CdCl₂组：2.72±0.26）、2.59±0.19（CdCl₂组：1.84±0.19）和2.43±0.23（CdCl₂组：1.50±0.27）（P<0.05）。
氯化镉通过抑制自噬体-溶酶体融合并促使自噬体异常聚集，诱导小鼠精母细胞凋亡。
