血红素加氧酶-1对大鼠Ⅱ型肺泡上皮细胞凋亡及水通道蛋白-1表达的影响

摘要

目的 探讨血红素加氧酶-1(HO-1)对过氧化氢(H2O2)诱导的氧化损伤大鼠Ⅱ型肺泡上皮细胞(AECⅡ)凋亡及水通道蛋白-1(AQP-1)表达的影响.方法 取雄性SD大鼠肺组织,将分离、纯化的AECⅡ细胞原代培养24 h后分为4组.正常组加入生理盐水；H2O2损伤组加入0.5 mmol/L H2O2处理；HO-1对照组加入1 μmol/L HO-1处理；HO-1保护组同时加入1μmol/L HO-1及0.5 mmol/L H2O2处理,将处理后的细胞继续培养2h.分别于处理前及处理后1、3、6、12h取细胞悬液,采用蛋白质免疫印迹试验(Western blotting)检测AQP-1表达,用流式细胞仪检测细胞凋亡率.结果 H2O2损伤组AQP-1表达水平随时间延长逐渐下降,各时间点明显低于正常组；HO-1对照组和HO-1保护组AQP-1表达水平随时间延长呈上升趋势,各时间点高于其他各组,尤以HO-1保护组明显高于H2O2损伤组(1 h:60.81±5.78比46.21 ±4.81,3 h:63.05 ±9.61比39.32±4.96,6 h:92.59±8.21比36.82±4.32,12 h:86.16±14.84比34.88±2.66,均P＜0.05).正常组和HO-1对照组细胞凋亡率无明显变化；H2O2损伤组细胞凋亡率随时间延长逐渐升高,各时间点均显著高于正常组；HO-1保护组细胞凋亡率于3h内逐渐升高,6h后下降并保持稳定,各时间点均显著低于H2O2损伤组[1 h:(9.04±2.17)％比(15.14±2.47)％,3 h:(12.90±2.04)％比(22.37±4.84)％,6 h:(10.42±1.68)％比(27.83±3.93)％,12 h:(11.97± 1.91)％比(33.63±6.61)％,均P＜0.05].H2O2损伤组AQP-1与细胞凋亡率呈显著负相关(r=-0.723,P＜0.001),并存在回归关系[y=672.548(0.914)x,R2=0.597]；HO-1保护组AQP-1与细胞凋亡率无显著相关性(r=0.210,P=0.193),但存在回归关系[y=e(3.130-59.654/x),R2=0.225].结论 HO-1可上调H2O2氧化损伤AECⅡ细胞的AQP-1表达水平并降低细胞凋亡率；上调AQP-1表达可能是HO-1抗氧化损伤机制之一.%Objective To explore the effect of heme oxygenase-1 (HO-1) on apoptosis and expression of aquaporin-1 (AQP-1) in primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) in rats with hydrogen peroxide (H2O2) induced oxidative damage.Methods Lung tissue of male Sprague-Dawley (SD) rats was collected.Primary AEC Ⅱ were isolated,purified,and cultured for 24 hours,then they were divided into four groups:① normal group (treated with normal saline); ② H2O2 injury group (treated with H2O2 0.5 mmol/L); ③ HO-1 control group (treated with HO-1 1 μmol/L); ④ HO-1 protection group (treated with HO-1 1 μmol/L and H2O2 0.5 mmol/L).Cells of each group were cultured for 12 hours after various treatment.The cell suspension was collected before and 1,3,6,12 hours after treatment,the expression of AQP-1 was determined by Western blotting and the apoptosis rate was assessed with flow cytometer.Results The expression of AQP-1 in H2O2 injury group was significantly declined with time,and was lower than that in normal group at each time point after treatment.The expression of AQP-1 in HO-1 control group and HO-1 protection group was significantly increased with time,and was higher than that of other groups at each time point after treatment.The expression of AQP-1 in HO-1 protection group was significantly up-regulated compared with that in H2O2 injury group (1 hour:60.81 ± 5.78 vs.46.21 ± 4.81,3 hours:63.05 ± 9.61 vs.39.32 ± 4.96,6 hours:92.59 ± 8.21 vs.36.82 ±4.32,12 hours:86.16 ± 14.84 vs.34.88 ± 2.66,all P＜0.05).No significant difference in apoptosis rate was found between normal group and HO-1 control group.The apoptosis rate in H2O2 injury group was increased with time,and was significantly higher than that of normal group at each time point.The apoptosis rate in HO-1 protection group was gradually increased within 3 hours after treatment,then decreased and remained stable after 6 hours,while it was significantly lower than that of H2O2 injury group at each time point [1 hour:(9.04 ± 2.17)％ vs.(15.14 ± 2.47)％,3 hours:(12.90 ± 2.04)％ vs.(22.37 ± 4.84)％,6 hours:(10.42 ± 1.68)％ vs.(27.83 ± 3.93)％,12 hours:(11.97 ± 1.91)％ vs.(33.63 ± 6.61)％,all P＜0.05].A negative correlation was found between AQP-1 and apoptosis rate in H2O2 injury group (r=-0.723,P＜0.001),and a regression correlation was found [y=672.548(0.914)x,R2=0.597].AQP-1 was not correlated with apoptosis rate in HO-1 protection group (r=0.210,P=0.193),but a regression correlation was found [y=e (3.130-59.654/x),R2=0.225].Conclusions HO-1 could increase the expression of AQP-1 in H2O2 injured AEC Ⅱ of rat,and lower its apoptosis rate.Increase in the expression of AQP-1 may be the underlying mechanism of anti-oxygenation property of HO-1.