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24h后分为4组。正常组加入生理盐水；H2O2损伤组加入0．5mmol／LH2O2 处理；HO-1对照组加入1μmol／LHO-1处理；HO-1保护组同时加入1μmol／LHO-1及0．5mmol／LH2O2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（Westernblotting）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（1h：60．81±5．78比46．21±4．81，3h：63．05±9．61比39．32±4．96，6h：92．59±8．21比36．82±4．32，12h：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[1h：（9．04±2．17）％比（15．14±2．47）％，3h：（12．90±2．04）％比（22．37±4．84）％，6h：（10．42±1．68）％比（27．83±3．93）％，12h：（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，R^2=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），R^2=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）; ②H2O2 injury group （treated with H2O2 0.5 mmol/L）; ③ HO-1 control group （treated with HO-1 1μmol/L）; ④ HO-1 protection group （treated with HO-1 1 μmol/L and H2O2 0.5 retool/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-regnlated 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, R^2 = 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）, R^2=0.225 ]. Conclusions HO-1 could increase the expression of AQP-1 in H2O2 injured AEC 1/ 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.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2013年第6期351-355,共5页
Chinese Critical Care Medicine
基金
贵州省科委科技计划项目（SY2008-3052）
贵州省教育厅自然科学研究项目（黔教科2007042）
