Zhang Zheng, Shen Huau, Qin Hai-dong, Xu Ying, Ma Ming-zhou, Bao Lei, Wang Hong
Intensive Care Unit, Affiliated Nanjing First Hospital of Nanjing Medical University, Nanjing 210006, Jiangsu, China.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2012 Feb;24(2):111-5.
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.
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.
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.
During early period of LIRI, caspase-3 was significantly deregulated by NAC, therefore the cellular apoptosis was inhibited, thus protecting lung tissue from LIRI.
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。
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。
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）。
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。
