活性氧在热打击诱导肠上皮细胞凋亡中的作用

摘要

Objective To observe the oxidative stress, integrity of lysosome and apoptosis of intestinal epithelial cells 6 (IEC-6) after heat stress, and explore the pathogenesis of intestinal damage caused by heat stress.Methods In the heat stress groups,the cells were incubated at 43℃ for 1 hour, then, further incubated at 37℃ and 5% CO2 for 0, 1, 3, 6 and 12 hours respectively; in the medicine intervention group, the cells were pretreated with the medicine 1h before heat stress; while in control group, the cells were incubated at 37℃ and 5% CO2. The amount of reactive oxygen species (ROS) was assayed with 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) staining. The stability of lysosome membrane was checked by AO staining. Apoptosis was analyzed by flow cytometry using annexinⅤ-FITC/PI staining, CCK-8 assay was used to assess cellular viability.Results Compared with control group, cell viability decreased and apoptosis increased at 1 h after heat stress, which was the most obvious at 12h after rewarming (P<0.05). While ROS and pale cells increased immediately after heat stress and the increase become the most obvious (P<0.05). The cell viability in E-64 pretreatment group was significantly improved such as apoptosis reduction, compared with heat stress group (P<0.05).Conclusion Heat stress could induce robust increase of ROS, which mediates lysosome damage and results in cell apoptosis, thus suggesting that ROS-lysosome pathway may play an important role in intestinal injury in heat stress.%目的 观察热打击对大鼠肠上皮细胞(IEC-6)氧化应激反应、溶酶体损伤及细胞凋亡的影响,探讨中暑热损伤过程中肠道损伤的机制.方法 将IEC-6细胞分为对照组(细胞仅置于37℃、5%CO2细胞培养箱中孵育)、热打击组(细胞置于43℃、5%CO2细胞培养箱中1h,之后分别置于正常细胞培养箱中继续孵育0、1、3、6、12h)、药物干预组(于热打击前分别采用溶酶体抑制剂E-64和活性氧清除剂NAC预处理细胞1h).采用DCFH-DA法检测细胞内活性氧(ROS)的表达,吖啶橙(AO)染色法检测溶酶体膜稳定性,AnnexinⅤFITC/PI双染色法检测细胞凋亡率,CCK-8法检测细胞活力.结果 与对照组比较,热打击后复温1h即导致IEC-6细胞活力下降,凋亡增加,且在复温后12h时最为明显(P<0.05);热打击后即刻即引起细胞内ROS表达及"苍白细胞"数量增加,且在复温后12h时最为明显(P<0.05).与热打击组比校,采用NAC对细胞进行预处理可明显提高热打击后细胞活力,减轻溶酶体损伤,并降低细胞凋亡率(P<0.05);采用E-64对细胞进行预处理可明显提高热打击后细胞活力,并降低细胞凋亡率(P<0.05).结论 ROS介导了热打击诱导肠上皮细胞凋亡的调控,这可能是中暑导致肠道黏膜屏障功能损伤的重要机制之一.