硫化氢对脂多糖致ALI大鼠肺组织线粒体的影响

Effects of hydrogen sulfide on mitochondria of lung in rats with ALI induced by lipopolysaccharide

目的观察硫化氢（H2S）对脂多糖（LPS）致急性肺损伤（ALI）大鼠肺组织线粒体超微结构及功能的影响。方法40只健康雄性SD大鼠按随机数字表法分为对照组、LPS损伤组及低、中、高剂量NaHS组，每组8只。LPS损伤组经舌下静脉注射LPS5mg／kg，低、中、高剂量NaHS组注射LPS3h后腹腔注射0．78、1．56和3．12mg／kgNaHS2mL／kg，对照组经舌下静脉注射2mL／kg生理盐水。制模后6h处死大鼠取肺组织，采用低温差速离心法提取肺组织线粒体，透射电镜下观察线粒体超微结构改变；采用硫代巴比妥酸法测定线粒体丙二醛（MDA）含量；采用黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH—Px）和三磷酸腺苷酶（ATP酶）活性；应用全波长酶标仪测定线粒体活性和肿胀度。结果透射电镜下显示，对照组线粒体结构基本正常；LPS损伤组线粒体明显肿胀，嵴数量减少或消失，板层小体结构融合或消失，粗面内质网脱颗粒现象明显；低剂量NaHS组线粒体超微结构损伤程度减轻，中、高剂量NaHS组则明显减轻。与对照组比较，LPS损伤组线粒体MDA含量明显升高（nmol／mg：26．30±1．45比11．16±1．20），SOD、GSH—Px和ATP酶活性明显降低[SOD（U／mg）：18．78±1．13比27．44±1．97，GSH—Px（U／nag）：63．91±1．99比128．15±3．47，ATP酶（U／mg）：4．83±0．25比9．92±0．65]；线粒体活性明显降低（A值：0．164±0．025比0．319±0．045），线粒体肿胀度明显升高（A值：0．182±0．012比0．273±0．023），差异有统计学意义（均P〈0．01）。与LPS损伤组比较，低、中、高剂量NaHS组线粒体MDA含量明显降低（nmol／mg：21．89±1．23、17．63±1．56、12．19±1．30比26．30±1．45），SOD、GSH—Px和ArrP酶活性明显升高[SOD（U／rag）：20．13±0．85、21．38±1．22、24．05±1．56比18．78±1．13，GSH—Px（U／rag）：82．06±1．65、101．45±2．14、117．80±2．12比63．91±1．99，ATP酶（U／mg）：5．34±0．23、7．06±0．37、8．78±0．44比4．83±0．25]；线粒体活性明显升高（A值：0．194±0．018、0．230±0．032、0．297±0．038比0．164±0．025），而线粒体肿胀度明显降低（A值：0．195±0．008、0．219±0．017、0．249±0．018比0．182±0．012），差异均有统计学意义（均P〈0．05）；且NaHS的保护作用呈剂量依赖性。结论LPS致ALI大鼠肺组织线粒体结构损伤、功能减弱；H2S可能通过降低LPS诱导的肺组织线粒体氧化损伤，从而保护线粒体结构和功能，并存在一定的量效关系。

Objective To observe the effects of hydrogen sulfide （H2S） on structure and function of mitochondria of lung in rats with acute lung injury （ALl） induced by lipopolysaccharide （LPS）. Methods Forty healthy male Sprague-Dawley （SD） rats were randomly divided into control group, LPS injury group, and low-, middle-, and high-dose NariS groups, with 8 rats in each group. The rats in LPS injury group were given LPS 5 mg/kg via sublingual vein, and those in low-, middle-, and high-dose NariS groups were challenged by LPS for 3 hours followed by intraperitoneally injection of 0.78, 1.56 and 3.12 mg/kg NariS respectively in a volume of 2 mL/kg. The rats in control group were given 2 mL/kg normal saline via sublingual vein. The rats were sacrificed at 6 hours after model reproduction, and the lung tissues were harvested on time. The mitochondria in lung tissues were isolated with differential centrifugation. The lung mitochondria ultra structures were observed with electron microscope. The content of malondialdehyde （MDA） in mitochondria was determined with thiobarbituric acid method, and the activities of superoxide dismutase （SOD）, glutathione peroxidase （GSH-Px）, and adenosine triphosphatase （ATPase） were determined with xanthine oxidase method. The mitochondrial activity and swelling were determined by muhiskan spectrum. Results It was shown by transmission electron microscope that the mitochondrial structure in the control group was normal. The mitochondria in rat lung cells was swollen with disrupted or disintegrated cristae, the osmiophilic lamellar bodies had fused or disappeared, and rough endoplasmie reticulum degranulation phenomenon was obvious in LPS injury group. The mitochondrial damage was slightly mitigated in the low-dose NariS group, and it was significantly mitigated in the middle-dose and high-dose NariS groups. Compared with control group, the MDA content in lung mitochondria in LPS injury group was significantly increased （nmol/mg： 26.30±1.45 vs. 11.16±1.20）, and SOD, GSH-Px, and ATPase activities were significantly decreased [SOD （U/mg）： 18.78± 1.13 vs. 27.44± 1.97, GSH-Px （U/mg）： 63.91 ± 1.99 vs. 128.15 ± 3.47, ATPase （U/mg）： 4.83 ± 0.25 vs. 9.92 ± 0.65]; as well as the activity of the mitochondria was significantly decreased （A value： 0.164 ± 0.025 vs. 0.319 ± 0.045）, and the swelling of the mitochondria was significantly increased （A value： 0.182 ±0.012 vs. 0.273 ±0.023）, all with significantly statistical differences （all P 〈 0.01）. Compared with LPS injury group, the MDA contents in low-, middle-, and high-dose NariS groups were significantly decreased （nmol/mg： 21.89± 1.23, 17.63± 1.56, 12.19± 1.30 vs. 26.30± 1.45）, and the SOD, GSH-PX, and ATPase activities were significantly increased [SOD （U/mg）： 20.13 ±0.85, 21.38 ± 1.22, 24.05±1.56 vs. 18.78 ± 1.13; GSH-Px （U/rag）： 82.06 ± 1.65, 101.45 ± 2.14, 117.80 ± 2.12 vs. 63.91 ± 1.99; ATPase （U/mg）： 5.34 ± 0.23, 7.06 ± 0.37, 8.78 ± 0.44 vs. 4.83 ± 0.25]; as well as the activity of the mitochondria was markedly increased （A value： 0.194 ± 0.018, 0.230±0.032, 0.297 ± 0.038 vs. 0.164± 0.025）, and the swelling of mitoehondria was markedly decreased （A value： 0.195 ± 0.008, 0.219 ± 0.017, 0.249 ± 0.018 vs. 0.182 ± 0.012）, all with significantly statistical differences （all P 〈 0.05）. Moreover, the protective effect of NariS showed a dose-dependent manner. Conclusion It could be concluded that LPS induce mitochondrial structural damage and functional impairment in rats with ALI induced by LPS, and H2S have a beneficial effect against ALl induced by LPS with decreasing the mitochondrial lipid peroxidation level and protecting the cell structure and function, and the effect is correlated with the dosage.