自噬在氯化镉诱导小鼠初级精母细胞凋亡中的作用

Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells

目的探究自噬在氯化镉(cadmium chloride,CdCl_(2))诱导小鼠初级精母细胞(GC-2 spd)凋亡中的作用及其潜在作用机制。方法以不同浓度CdCl_(2)(0、5和10μmol/L)染毒GC-2 spd细胞24 h。Hoechst33342染色法和单丹磺酰戊二胺法分别检测凋亡小体和自噬体;TUNEL荧光染色检测细胞凋亡情况;在不含/含CdCl_(2)(10μmol/L)的细胞培养液中分别加入自噬抑制剂3-甲基腺嘌呤(60μmol/L)、凋亡抑制剂胱天蛋白酶家族抑制剂(50 nmol/L)、自噬激动剂雷帕霉素(50 nmol/L)和溶酶体抑制剂氯喹(10μmol/L)处理GC-2 spd细胞24 h,Western blot检测细胞内自噬相关蛋白LC3、P62以及促凋亡蛋白cleaved Caspase-3和cleaved Caspase-9的表达水平。结果CdCl_(2)染毒细胞24 h后,自噬体聚集且凋亡细胞增多。Western blot结果显示,5和10μmol/L CdCl_(2)染毒组LC3II/LC3I蛋白表达水平与对照组(5.50±0.56)相比分别升高为9.23±0.81和12.15±0.80(P<0.05);LC3II蛋白表达水平与对照组(2.35±0.34)相比分别升高为3.35±0.14和3.47±0.32(P<0.05);P62蛋白表达水平与对照组(0.83±0.09)相比分别升高为1.48±0.12和1.80±0.22;与CdCl_(2)组相比,3-MA处理后LC3II/LC3I、LC3II、P62、cleaved Caspase-9和cleaved Caspase-3蛋白的表达水平分别下降为0.90±0.07(CdCl_(2)组为1.47±0.06)、1.57±0.14(CdCl_(2)组为2.45±0.29)、0.82±0.05(CdCl_(2)组为1.44±0.18)、0.18±0.01(CdCl_(2)组为0.28±0.01)和0.61±0.84(CdCl_(2)组为1.15±0.04)(P值均<0.05);与CdCl_(2)组相比,zVAD-FMK处理后cleaved Caspase-9和cleaved Caspase-3的表达水平分别下降为0.12±0.01(CdCl_(2)组为0.28±0.01)和0.34±0.01(CdCl_(2)组为1.15±0.04)(P值均<0.05),对LC3II/LC3I、LC3II和P62的表达水平无显著影响(P值均>0.05)。与CdCl_(2)组相比,RAPA能增强CdCl_(2)诱导的LC3II/LC3I、LC3II和P62蛋白表达水平,分别为2.22±0.21(CdCl_(2)组为1.56±0.06)、3.72±0.21(CdCl_(2)组为2.97±0.15)和2.41±0.19(CdCl_(2)组为1.52±0.35)(P值均<0.05)。Western blot结果显示,与CdCl_(2)组比较,CdCl_(2)与CQ处理组LC3II/LC3I、LC3II、P62和cleaved Caspase-3的表达水平明显增加为3.21±0.31(CdCl_(2)组为2.09±0.25)、4.49±0.43(CdCl_(2)组为2.72±0.26)、2.59±0.19(CdCl_(2)组为1.84±0.19)和2.43±0.23(CdCl_(2)组为1.50±0.27)(P值均<0.05)。结论氯化镉可能通过抑制自噬体-溶酶体融合阻滞自噬通量,促使自噬体异常聚集,诱导小鼠初级精母细胞发生凋亡。

OBJECTIVE 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.METHODS The cells were treated with different concentrations of CdCl_(2)(0,5 and 10μmol/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)(60μmol/L),apoptotic inhibitorCaspase inhibitor Z-VAD-FMK(zVAD-FMK)(50 nmol/L),autophagy inducer rapamycin(RAPA)(50 nmol/L)and lysosomal inhibitor chloroquine(CQ)(10μmol/L)were added to cell culture in the presence/absence of CdCl_(2)(10μmol/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.RESULTS 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 10μmol/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).CONCLUSION Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosomelysosomal fusion and prompting abnormal aggregation of autophagosomes.