镉诱导小鼠十二指肠上皮细胞损伤及机制

Cadmium-induced injury of duodenal epithelial cells in mice and its mechanism

目的探讨在体和离体镉(Cd)暴露对小鼠十二指肠上皮细胞的损伤作用及其机制。方法①在体实验:C57BL/6小鼠连续30 d每天1次ig给予二氯化镉(CdCl2)10 mg·kg-1构建慢性Cd中毒模型;单次ig给予CdCl280 mg·kg-1构建急性Cd中毒模型,观察小鼠生存状况和生存率。分离培养急性和慢性Cd中毒小鼠的十二指肠上皮细胞,经E-钙黏蛋白免疫荧光鉴定为上皮细胞后,利用激光共聚焦显微镜检测细胞内Cd离子含量。②离体实验:分离培养正常C57BL/6小鼠的十二指肠上皮细胞,与CdCl22.5~100μmol·L-1共孵育24 h,CellTiter-Blue法检测细胞活力;与CdCl215μmol·L-1共孵育24 h,流式细胞术检测细胞周期;与CdCl230μmol·L-1共孵育3~12 h,Western印迹法检测细胞内活化的胱天蛋白酶3表达水平。结果①在体实验:与正常对照组相比,慢性Cd中毒小鼠活动状况正常,急性Cd中毒小鼠出现萎靡不振,进食减少和死亡现象,在急性Cd暴露第5天时,小鼠生存率降低至40%;急、慢性Cd中毒小鼠十二指肠上皮细胞Cd离子含量均显著增加(P<0.01)。②离体实验:CdCl2对体外培养的十二指肠上皮细胞活力具有抑制作用,其半数抑制浓度(IC50)为(24.55±0.84)μmol·L-1。与细胞对照组相比,CdCl215μmol·L-1使十二指肠上皮细胞细胞周期阻滞于G0/G1期(P<0.05),且CdCl230μmol·L-1处理6,9和12 h后,十二指肠上皮细胞中活化的胱天蛋白酶3表达水平显著上升(P<0.05,P<0.01)。结论经消化道进入体内的Cd会被十二指肠上皮细胞吸收并对其造成损伤,其机制可能与细胞周期阻滞和胱天蛋白酶3介导的细胞凋亡有关。

OBJECTIVE To investigate the effect of cadmium(Cd)exposure in vivo and in vitro on duodenal epithelial cells in mice and the mechanism.METHODS In vivo,C57 BL/6 mice were ig administered with CdCl210 mg·kg-1 once per day for 30 d to establish a chronic cadmium poisoning model,or were ig administered with a single dose of Cd Cl280 mg·kg-1 to establish an acute cadmium poisoning model before the survival status and survival rate of mice were observed.Duodenal epithelial cells of acute and chronic cadmium poisoning mice were isolated and cultured.The cel s were identified as epithelial cells by E-cadherin immunofluorescence.Then,the content of cadmium ion in duodenal epithelial cells was detected by confocal laser microscopy.In vitro,duodenal epithelial cells of normal C57 BL/6 mice were isolated and cultured,and incubated with CdCl22.5-100μmol·L-1 for 24 h.CellTiter-Blue was used to detect cell viability.Subsequently,the duodenal epithelial cells of normal mice were incubated with Cd Cl215μmol·L-1 for 24 h,and cel cycle was detected by flow cytometry.Then,the duodenal epithelial cells of normal C57 BL/6 mice were incubated with CdCl230μmol·L-1 for 3-12 h and the expression of cleaved-caspase 3 was detected by Western blotting.RESULTS Compared with the control group,the activities of mice with chronic cadmium poisoning were all normal.The mice with acute cadmium poisoning showed depression,less food intake and death.At the 5 thday of acute cadmium exposure,the survivalrate of mice decreased to 40%.The content of cadmium ion in duodenal epithelial cells of acute and chronic cadmium poisoning mice increased significantly(P<0.01).Furthermore,CdC l2 inhibited the viability of duodenal epithelial cells cultured in vitro and the half inhibitory concentration(IC50)was(24.55±0.84)μmol·L-1.Compared with the control group,CdCl2 blocked the cell cycle of duodenal epithelial cells at G0/G1 phase(P<0.05),and the expression of cleaved-caspase 3 in duodenal epithelial cells was significantly increased after CdCl2 treatment for 6,9 and 12 h(P<0.05,P<0.01).CONCLUSION Cadmium that enters the body through the digestive tract can be absorbed by duodenal epithelial cells and cause damage to the cells.The mechanism of cadmium-induced damage may be related to cell cycle arrest and caspase 3-mediated apoptosis.