Objective To observe the effect of metformin on mitochondrial oxidative stress in lung tissue of rats with acute lung injury induced by sepsis.Methods The rat model of sepsis was established by cecal ligation and perforation (CLP).Forty male Wistar rats were randomly divided into four groups:normal control group (Sham group),control group (Sham/M group),sepsis group (CLP group),sepsis treatment group (CLP/M group).Each group was divided into 6 h,12 h,24 h subgroups according to observation time points.Sham/M group was injected with metformin (100 mg/kg) and the same group as Sham group 1.5 h before operation.The sham rats underwent surgery,but without ligation and puncture.CLP/M group was injected with metformin (100 mg/kg) by intragastric administration 1.5 h before operation.The levels of TNF-α and IL-1 β in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA).The activity of mitochondrial superoxide dismutase (SOD) was measured by xanthine oxidase method.The expression of AMPK protein was detected by protein immunoblotting test (Western blotting).The pathological changes of lung tissue were observed by HE staining.Results ①There were no significant differences in TNF-α and IL-1β levels between the Sham group and the Sham/M group at 12 h and 24 h (P ＞0.05).At 12 h and 24 h,the CLP group and the CLP/M group were significantly (P ＜ 0.01).The levels of TNF-α and IL-1β in lung tissue of CIP/M group were significantly lower than those in CLP group (P ＜ 0.01),and the level of TNF-α and IL-1β in lung tissue of CLP (P ＜ 0.05).②The levels of SOD in the mitochondria of Sham group and Sham/M group were significantly lower than those in Sham group (P ＜ 0.01),CLP/M (P ＜ 0.05).③The expression of AMPK protein in Sham group and Sham/M group was not significantly different (P ＞ 0.05),CLP/M and CLP group were higher than Sham group (P ＜ 0.05);CLP/M group was significantly higher than CLP group (P ＜ 0.05).④The pathological morphology and ultrastructural injury of CLP group were heavier under light microscope.After treatment with metformin,the pathological morphology and ultrasonography microstructure damage has been reduced.Conclusion Metformin may protect the acute lung injury induced by sepsis,which may be related to the decrease of TNF-α and IL-1β inflammatory mediators,the increase of SOD level and the activation of AMPK protein.%目的 观察二甲双胍对脓毒症急性肺损伤大鼠肺组织线粒体氧化应激的影响.方法 采用盲肠结扎穿孔法(CLP)建立脓毒症大鼠模型.取60只Wistar雄性大鼠,随机分成四组:正常对照组(Sham组)、对照处理组(Sham/M组)、脓毒症组(CLP组)和脓毒症处理组(CLP/M组).每组再分为6h、12 h和24h三个观察时间点.Sham组仅开腹取出盲肠翻动后还纳腹腔,不进行结扎和穿孔;Sham/M组动物在术前1.5h通过灌胃灌入二甲双胍(100 mg/kg),其他同Sham组.CLP/M组动物在术前1.5h通过灌胃灌入二甲双胍(100 mg/kg),其他同CLP组.采用酶联免疫吸附双抗体夹心(ELISA)测定法检测肺组织中TNF-α、IL-1β的表达水平,黄嘌啉氧化酶法测定线粒体超氧化物歧化酶(SOD)活性,蛋白质免疫印记试验(Westren Blot)检测AMPK蛋白的表达,HE染色法观察肺组织病理学变化.结果 ①Sham组大鼠与Sham/M组大鼠肺组织TNF-α、IL-1β水平在12 h、24 h时差异无统计学意义(P＞0.05);在12 h、24h时,CLP组和CLP/M组大鼠肺组织TNF-α、IL-1β水平较Sham组明显升高(P＜0.01);同时CLP/M组大鼠肺组织TNF-α、IL-1β水平较CLP组明显降低(P＜0.01);②Sham组和Sham/M组大鼠线粒体中SOD水平差异无统计学意义(P ＞0.05);CLP组大鼠线粒体中SOD水平较Sham组明显下降(P＜0.01),CLP/M组较CLP组SOD水平明显上升(P＜0.05);③AMPK蛋白表达水平在Sham组和Sham/M组中差异无统计学意义(P＞0.05),CLP/M和CLP组较Sham组明显升高(P＜0.05);且CLP/M组较CLP组明显升高(P＜0.05);④光镜下见CLP组肺的病理形态及超微结构损伤均较重,二甲双胍干预后,肺的病理形态及超微结构损伤有所减轻.结论 二甲双胍对脓毒症诱导的急性肺损伤大鼠有一定的保护作用,这可能与减少TNF-α、IL-1β等炎症介质释放,提高SOD水平,以及激活AMPK蛋白表达等有关.
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