对比剂通过内质网应激诱导人肾小管上皮细胞凋亡

The role of endoplasmic reticulum stress in apoptosis of human renal tubular epithelial cells induced by contrast media

目的探讨内质网应激在对比剂诱导人肾小管上皮细胞(HKC)凋亡中的作用。方法在缺血、缺氧(0.2%胎牛血清,5%CO2-95%N2饱合混合气体)环境下,以HKC为研究对象,将HKC细胞株随机分为3组:空白组、对比剂组(加入120g I/L碘普罗胺预处理2h)和N-乙酰半胱氨酸(NAC)组(加入10mmol/L NAC预处理2h,再加120g I/L碘普罗胺处理2h)。细胞增殖试剂盒(CCK-8)法检测细胞增殖情况,流式细胞术检测细胞凋亡,实时荧光定量聚合酶链反应(real-time PCR)检测细胞中半胱氨酸蛋白酶-12(caspase-12)、葡萄糖调节蛋白-78(GRP78)和CAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA表达水平,蛋白质印迹法(Western blot)检测细胞中caspase-12、GRP78和CHOP的蛋白表达水平。结果对比剂可呈时间依赖性抑制HKC活性,与0h相比,1hHKC活性显著降低(0.351±0.066比0.447±0.087,P<0.05);流式细胞术结果示对比剂可促进缺血缺氧HKC凋亡(P<0.05);real-time PCR结果示,对比剂组和NAC组GRP78和CHOP的mRNA水平明显高于空白组(P<0.05),对比剂组caspase-12的mRNA水平明显高于空白组和NAC组(P<0.05);Western blot结果示,对比剂明显增加了caspase-12、GRP78和CHOP的蛋白表达,而NAC明显降低了caspase-12的表达,但不能明显降低对比剂诱导的GRP78和CHOP高表达(P<0.05)。结论在缺血缺氧条件下,对比剂诱导HKC活性下降、凋亡,此作用可能与细胞内氧化应激及内质网应激相关。

Objective To investigate the effect of endoplasmic reticulum stress （ERS） on apoptosis of human renal tubular epithelial cells induced by contrast media（CM）. Methods After the pretreatment with ischemia and hypoxia [0. 2% fetal calf serum （FCS）, 5% CO2-95% N2 saturated mixed gas], human kidney tubular epithelial cell （HKC） lines were randomly divided into three groups： control group, contrast media group （pretreated with 120 g I/L iopromide for 2 hours） and N-acetylcysteine （NAC） group （pretreated with 10 mmol/L NAC for 2 hours and then treated with 120 g I/L iopromide for 2 hours）. The cell proliferation was detected by cell counting kit-8 （CCK-8）. The apoptosis of human renal tubular epithelial cells was assayed by flow cytometry. The mRNA expression levels of caspase-12, glucose-regulated protein-78 （GRP78） and CAAT/ enhancer binding protein homologous protein （CHOP） were detected by real-time fluorescence quantitative polymerase chain reaction （PCR}. The protein expression levels of caspase-12, GRP78 and CHOP were detected by Western blotting. Results The viability of HKCs was significantly inhibited in a time-dependent manner by contrast media. Compared with 0 hour, the viability of HKCs was significantly reduced at 1 hour（0. 351±0. 066 vs 0. 447±0. 087, P〈0.05）. The flow cytometry results showed that CM accelerated the apoptosis of HKCs under the hypoxia-ischemic condition （P〈0.05）. The real-time PCR results showed that the mRNA expression levels of GRP78 and CHOP in the contrast media group and NAC group were significantly higher than those in the control group （P〈0. 05 ）; the mRNA expression level of caspase-12 in the contrast media group was significantly higher than that in the control group and NAC group （P〈0.05）. Western blotting results showed that CM significantly increased the protein expressions of GRP78, CHOP and caspase 12, and NAC significantly decreased the protein expression of caspase-12, but could not decreased the high expressions of GRP78 and CHOP induced by contrast media （P〈0.05）. Conclusion Under the hypoxia-ischemic condition, contrast media can reduce the viability of HKCs and increase cell apoptosis, which probably related to intracellular oxidative stress and ERS.