N-乙酰半胱氨酸对大鼠肺缺血/再灌注损伤时肺细胞凋亡的保护作用
[Protective effect of N-acetylcysteine against pneumocyte apoptosis during ischemia/reperfusion injury of lung in rats].
作者信息
Zhang Zheng, Shen Huau, Qin Hai-dong, Xu Ying, Ma Ming-zhou, Bao Lei, Wang Hong
机构信息
Intensive Care Unit, Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing 210006, Jiangsu, China.
出版信息
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2012 Feb;24(2):111-5.
OBJECTIVE
To investigate the effect of N-acetylcysteine (NAC) on apoptosis of pneumocytes and expression of caspase-3 during lung ischemia/reperfusion 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 reperfusion), 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 Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. Malondialdehyde (MDA, thiobarbituric acid) and superoxide dismutase (SOD, xanthine oxidase) of lung tissue were measured. Expression of caspase-3 in lung was determined by reverse transcription-polymerase chain reaction (RT-PCR), and the changes in ultrastructure 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 (nmol/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)%], 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 LIRI 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.
目的
探讨N - 乙酰半胱氨酸（NAC）对大鼠肺缺血/再灌注损伤（LIRI）过程中肺细胞凋亡及半胱天冬酶 - 3（caspase - 3）表达的影响，以探究NAC在肺细胞凋亡中的可能作用。
方法
将42只雄性Sprague - Dawley大鼠随机分为三组：假手术组、LIRI组（通过夹闭肺门45分钟，随后再灌注3小时或6小时制备LIRI）和NAC组（在LIRI前腹腔注射NAC 150 mg/kg）。在LIRI后3小时或6小时采集肺组织标本。采用膜联蛋白V - 异硫氰酸荧光素（FITC）和碘化丙啶（PI）染色后，用流式细胞仪测定肺组织凋亡率。检测肺组织丙二醛（MDA，硫代巴比妥酸法）和超氧化物歧化酶（SOD，黄嘌呤氧化酶法）水平。通过逆转录 - 聚合酶链反应（RT - PCR）测定肺组织中caspase - 3的表达，并用电镜观察肺组织超微结构变化。
结果
与假手术组相比，LIRI组在3小时和6小时时肺细胞凋亡率显著升高[（25.60 ± 3.22）% 对 （2.19 ± 0.48）%，（26.01 ± 4.50）% 对 （2.55 ± 0.36）%]，MDA含量（nmol/mg）显著增加（3.26 ± 0.32对0.73 ± 0.23，3.53 ± 0.46对1.08 ± 0.42），SOD活性（U/mg）显著降低（32.80 ± 3.82对60.51 ± 6.81，33.44 ± 3.24对64.19 ± 6.60），肺组织中caspase - 3 mRNA表达显著上调（0.717 ± 0.037对0.216 ± 0.046，0.744 ± 0.046对0.227 ± 0.037，均P < 0.01）。与LIRI组相比，NAC组肺细胞凋亡率显著降低[（14.42 ± 1.61）% 对 （25.60 ± 3.22）%，（10.02 ± 1.64）% 对 （26.01 ± 4.50）%]，MDA含量（nmol/mg）显著降低（1.75 ± 0.33对3.26 ± 0.32，2.15 ± 0.25对3.53 ± 0.46），SOD活性（U/mg）显著升高（42.76 ± 2.06对32.80 ± 3.82，44.94 ± 3.11对33.44 ± 3.24，均P < 0.01）。与LIRI组相比，NAC组肺组织中caspase - 3表达显著下调（0.441 ± 0.038对0.717 ± 0.037，0.410 ± 0.037对0.744 ± 0.046，均P < 0.01）。NAC组肺组织超微结构变化比LIRI组更轻。LIRI 3小时和6小时后，caspase - 3表达与凋亡率及MDA含量呈正相关（3小时：r = 0.9036，0.9216；6小时：r = 0.9655，0.9650，均P < 0.01），但凋亡率与SOD活性呈负相关（3小时：r = -0.9511，6小时：r = - 0.9574，均P < 0.01）。
结论
在LIRI早期，NAC可显著调节caspase - 3，从而抑制细胞凋亡，保护肺组织免受LIRI损伤。
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