氢气对PC12细胞缺氧复氧时内质网应激的影响

Effect of hydrogen on endoplasmic reticulum stress during hypoxia-reoxygenation in PC12 cells

目的探讨氢气对PCI2细胞缺氧复氧时内质网应激的影响。方法PCI2细胞采用随机数字表法，将其随机分为4组，正常对照组：细胞常规培养25h；阳性对照组：细胞正常培养1h后，用氢气饱和的RPMI-1640培养基继续培养24h；缺氧复氧组：细胞缺氧1h后复氧24h；氢气组：细胞缺氧1h后，用氢气饱和的RPMI-1640培氧基复氧24h。PCI2细胞加入含Na2S2O4终浓度为5．0mmol／L的RPMI-1640培养液，5％CO2培养箱37℃孵育1h；更换正常RPMI．1640培养液，继续培养24h，制备PCI2细胞缺氧复氧模型。采用WST-1法测定细胞相对增殖率，采用硫代巴比妥酸法测定MDA浓度，采用免疫组化法检测caspase-3表达，采用RT-PCR法检测活化转录因子4（ATF4）mRNA和C／EBP同源蛋白（CHOP）mRNA的表达。结果与正常对照组和阳性对照组比较，缺氧复氧组细胞相对增殖率降低，MDA浓度升高，caspase-3、ATF4mRNA和CHOPmRNA的表达上调，氢气组ATF4mRNA和CHOPmRNA的表达上调（P〈0．05）；正常对照组和阳性对照组间细胞相对增殖率、MDA浓度、caspase-3、ATF4mRNA和CHOPmRNA的表达比较差异无统计学意义（P〉0．05）；与缺氧复氧组比较，氢气组细胞相对增殖率升高，MDA浓度降低，caspase-3、ATF4mRNA和CHOPmRNA的表达下调（P〈0．05）。结论氢气可能通过抑制内质网应激，降低细胞凋亡，减轻PCI2细胞缺氧复氧损伤。

Objective To investigate the effect of hydrogen on endoplasmic reticulum stress during hypoxia-reoxygenation （H/R） in PC12 cells. Methods PC12 cells were randomly divided into 4 groups： normal control group （group NC）, positive control group （group PC）, H/R group and hydrogen group （group H）. In group NC, the cells were cultured routinely for 25 h. In group PC, the cells were cultured routinely for 1 h and then in RPMI-1640 culture medium saturated with hydrogen for 24 h. In H/R group, the cells were exposed to 1 h of hypoxia followed by 24 h of reoxygenation. In group H, the ceils were exposed to 1 h of hypoxia and then reoxygenated in RPMI-1640 culture medium saturated with hydrogen for 24 h. Hypoxia-reperfusion was produced by 1 h exposure of cells to 5% CO2 in an incubator at 37℃ in RPMI-1640 culture medium containing Na2S2O4 with the final concentration of 5.0 mmol/L followed by 24 h reoxygenation in the normal RPMI-1640 culture medium. The relative rate of cell proliferation was detected by WST-1. The concentration of MDA was determined by thiobarbituric acid method. The expression of caspase-3 was determined by immuno-histoehemistry. The expression of activating transcription factor-4 （ATF4） mRNA and C/EBP homologous protein （CHOP） mRNA was determined by RTPCR.Results Compared with groups NC and PC, the relative rate of cell proliferation was significantly decreased, MDA concentration was significantly increased, and the expression of caspase-3, ATF4 mRNA and CHOP mRNA was up-regulated in group H/R, and the expression of ATF4 mRNA and CHOP mRNA was up-regulated in group H （ P 〈 0.05） . There was no significant difference in the relative rate of cell proliferation, MDA concentra- tion, and the expression of caspase-3, ATF4 mRNA and CHOP mRNA between groups NC and PC （ P 〉 0.05）. Compared with group H/R, the relative rate of cell proliferation was significantly increased, MDA concentration was significantly decreased, and the expression of caspase-3, ATF4 mRNA and CHOP mRNA was down-regulated in group H （P 〈 0.05）. Conclusion Hydrogen can decrease cell apoptosis and attenuate H/R injury to PC12 cells through inhibiting endoplasmic reticulum stress.