N-乙酰半胱氨酸对大鼠肺缺血／再灌注损伤诱导细胞凋亡的保护机制

Protective effect of N-acetvlcvsteine against pneumocyte apoptosis during ischemia/reperfusion injury of lung in rats

目的研究N-乙酰半胱氨酸（NAC）对大鼠肺缺血／再灌注损伤（LIRI）中细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）表达的影响，并探讨其可能机制。方法雄性sD大鼠42只，按随机数字表法均分为假手术组、LIRI组（阻断肺门45min后再开放）、NAC组（于LIRI前腹腔注射NAC150mg／kg）3组，各组分别于5h、6h取肺组织，用膜联蛋白V-异硫氰酸荧光素（AnnexinV—FITC）和碘化丙啶（e1）双染法通过流式细胞仪检测凋亡细胞并计算凋亡率，测定丙二醛（MDA）浓度（硫代巴比妥法）和超氧化物歧化酶（SOD）活性（黄嘌呤氧化酶法），用逆转录-聚合酶链反应（RT—PCR）技术检测caspase-3mRNA表达，电镜下观察肺脏超微结构的改变。结果与假手术组相比，LIRI组3h、6h肺组织细胞凋亡率明显增加[（25．60±3．22）％比（2．19±0．48）％，（26．01±4．50）％比（2．55±0．36）％]，MDA浓度（nmol／mg）升高（3．26±0．32比0．73±0．23，3．53±0．46比1．08±0．42），SOD活性（U／mg）下降（32．80±3．82比60．51±6．81，33．44±3．24比64．19±6．60），肺组织caspase-3raRNA表达显著增加（0．717±0．037比0．216±0．046，0．744±0．046比0．227±0．037），差异均有统计学意义（均P〈0．01）；与URI组相比，NAC组3h、6h肺组织细胞凋亡率明显减少[（14．42±1．61）％比（25．60±3．22）％，（10．02±1．64）％比（26．01±4．50）％]，MDA浓度（nmol／mg）明显下降（1．75±0．33比3．26±0．32，2．154-0，25比3．53±0．46）．SOD活性（U／mg）明显升高（42．764-2．06比32．80±3．82，44．94±3．11比33．444-3．24），肺组织caspase-3mRNA表达显著减少（0．441土0．038比0．717±0．037，0．410±0．037比0．744±0．046），差异均有统计学意义（均P〈0．01）。NAC组肺组织超微结构损害较LIRI组明显减轻。在LIRI3h、6h时，caspase-3与肺组织细胞凋亡率、MDA呈显著正相关，与S01）呈显著负相关（3h：r值分别为0．9036、0．9216、-0．9511，6h：r值分别为0．9655、0．9650、-0．9574．均P〈0．01）。结论在LIRI早期，NAC可下调caspase-3表达从而抑制细胞凋亡的发生，起到保护肺组织的作用。

Objective To investigate the effect of N-acetvlevsteine （NAC） on apoptosis of pneumoeytes and expression of caspase-3 during lung ischemia/repeffusion injury （LIRI） in rats, and to explore the possible role of NAC in pneumocyte apoptosis. Methods Forty-two male Sprague-Dawley rats were randomly divided into three groups： sham operation group, LIRI group （ LIRI was produced by 45 minutes of clamping of the pulmonary hilum followed by 3 hours or 6 hours of repeffusion）, and NAC group （NAC 150 mg/kg was injected intraperitoneally before LIRI）. Lung specimens were harvested 3 hours or 6 hours after LIRI. Apoptosis rate in lung tissue was determined with flow cytometer after AnnexinV-fluoreseein isothiocyanate （FITC） and propidium iodide （PI） staining. Malondialdehyde （MDA, thiobarbituric acid） and superoxide dismutase （SOD, xanthineoxidase） of lung tissue were measured. Expression of caspase-3 in lung was determined by reverse transeription-polymerase chain reaction （RT-PCR）, and the changes in uhrastructure of lung tissue were observed by electron microscope. Results Compared with that of the sham operation group, apoptosis rate of pulmonary cells was significantly increased at 3 hours and 6 hours in LIRI group [ （25.60±3.22）% vs. （2.19±0.48）% , （26.01±4.50）% vs. （2.55±0.36）%, the content of MDA （nmal/mg） was significantly increased （3.26±0.32 vs. 0.73±0.23, 3.53±0.46 vs. 1.08±0.42）, and the activity of SOD （U/mg） was significantly lowered （32.80±3.82 vs. 60.51±6.81, 33.44±3.24 vs. 64.19±6.60）, and the expression of caspase-3 mRNA in lung tissue was significantly up-regulated （0.717±0.037 vs. 0.216±0.046, 0.744±0.046 vs. 0.227 ±0.037, all P〈 0.01 ）. Compared with that of the LIRI group, apoptosis rate of pulmonary cell was significantly decreased [ （ 14.42± 1.61）% vs. （25.60±3.22）%, （10.02 ±1.64）% vs. （26.01± 4.50）% 1, content of MDA （nmol/mg） was lowered significantly （ 1.75 ±0.33 vs. 3.26±0.32, 2.15±0.25 vs. 3.53± 0.46）, and activity of SOD （U/mg） was significantly elevated （42.76± 2.06 vs. 32.80± 3.82, 44.94± 3.11 vs. 33.44 ± 3.24, all P〈0.01 ） in NAC group. The expression of caspase-3 in lung tissue was remarkably down-regulated compared with that of L1R1 group （0.441± 0.038 vs. 0.717 ±0.037, 0.410 ± 0.037 vs. 0.744±0.046, both P〈0.01 ）. The ultrastructure changes in lung tissue were milder in NAC group than in LIRI group. Positive correlation was found between the expression of caspase-3 and apoptosis rate and the content of MDA （3 hours： r=0.9036, 0.9216; 6 hours： r=0.9655, 0.9650, all P〈0.01 ）, but negative correlation was found between apoptosis rate and activity of SOD （3 hours： r=-0.9511, 6 hours： r=-0.9574, both P〈0.01 ） after LIRI 3 hours and 6 hours. Conclusion During early period of LIRI, caspase-3 was significantly deregulated by NAC, therefore the cellular apoptosis was inhibited, thus protecting lung tissue from LIRI.