镉诱导HEK293细胞自噬和凋亡

Cd Cl2induces autophagy and apoptosis in HEK293 cells

目的探究镉(二氯化镉,Cd Cl2)能否诱导HEK293细胞自噬与凋亡以及细胞外调节蛋白激酶(ERK)1/2和AKT蛋白在自噬中的作用。方法将绿色荧光蛋白(GFP)-微管相关蛋白Ⅰ轻链3B(LC3B)重组质粒转染至HEK293细胞24 h后,以Cd Cl22,4,8和10μmol·L^(-1)诱导细胞12 h,荧光显微镜观察自噬情况;以Cd Cl22,4,8和10μmol·L^(-1)诱导未转染GFP-LC3B重组质粒的HEK293细胞12 h,透射电子显微镜下观察自噬泡;Western蛋白印迹检测LC3B-Ⅱ/Ⅰ的表达变化,以及ERK1/2和AKT蛋白的磷酸化表达;流式细胞仪检测Cd Cl2(≤10μmol·L^(-1))对HEK293细胞凋亡的影响。用3-MA(自噬抑制剂)预处理Cd Cl210μmol·L^(-1)诱导的HEK293细胞12 h,Western蛋白印迹检测细胞内激活型胱天蛋白酶3的表达。结果 Cd Cl2(≤10μmol·L^(-1))诱导HEK293细胞12 h,荧光显微镜下转染细胞出现绿色荧光点状聚集,电镜下观察到自噬泡,Western蛋白印迹检测到LC3B-Ⅱ/Ⅰ表达量增加(P<0.05,P<0.01),ERK1/2和AKT蛋白磷酸化水平均增加(P<0.05,P<0.01)。流式细胞仪检测出Cd Cl2(≤10μmol·L^(-1))诱导的HEK293细胞发生了细胞凋亡;加入3-MA20μmol·L^(-1)+Cd Cl210μmol·L^(-1)后,细胞自噬被抑制,激活型胱天蛋白酶3表达增加(P<0.01)。结论低浓度Cd Cl2(≤10μmol·L^(-1))能引起细胞自噬,可能通过ERK1/2和AKT蛋白介导细胞自噬;自噬与凋亡相伴发生,自噬可能抑制凋亡。

OBJECTIVE To evaluate the possibility that Cd Cl2 induces autophagy and apoptosis in HEK293 cells,and the role of extracellular regulated protein kinases（ERK1/2）and AKT proteins in autophagy. METHODS Green fluorescence protein（GFP）-light chain 3B（LC3B）expression plasmid was transfected into HEK293 cells. After 24 h,HEK293 cells were induced with Cd Cl22,4,8 and 10 μmol·L^-1for 12 h. The expression of GFP- LC3 B was detected by fluorescent microscopy. HEK293 cells were induced with Cd Cl22,4,8 and 10 μmol·L- 1without transfection of GFP-LC3 B for 12 h while autophagic vacuoles were observed by transmission electron microscopy. The expression of LC3B-Ⅱ/Ⅰproteins and the phosphorylation levels of ERK1/2 and AKT were analyzed by Western blotting. Apoptosis was detected by flow cytometry microscopy. HEK293 cells were treated with 3-MA 20 μmol·L^- 1＋Cd Cl210 μmol·L^- 1for 12 h before cleaved caspase 3 protein was detected by Western blotting. RESULTS When HEK293 cel s were exposed to Cd Cl2（≤10 μmol·L^-1）for 12 h,cytoplasmic GFP-LC3 B punctuates were observed under the fluorescence microscope,and autophagic vacuoles were observed under an electron microscope. The expression of LC3B-Ⅱ/Ⅰ,p-ERK1/2 and p-AKT proteins was significantly increased in Cd Cl2-induced cells（P〈0.05,P〈0.01）. Moreover,apoptosis was observed. The addition of3-MA 20 μmol·L^- 1＋Cd Cl210 μmol·L- 1enhanced apoptosis. Cleaved capase 3 protein expression was significantly increased（P〈0.01）. CONCLUSION Cd Cl2（≤10 μmol·L^-1）can induce autophagy in HEK293 cel s. ERK1/2 and AKT proteins might be associated with the activation of autophagy that is accompanied by apoptosis,suggesting that autophagy can inhibit apoptosis at certain concentrations of Cd Cl2.