自噬在脓毒症小鼠肺损伤中的作用

Role of autophagy in lung injury in septic mice

目的 探讨自噬在脓毒症小鼠急性肺损伤中的作用.方法 雄性C57BL/6小鼠36只,6周龄,体重20～ 25 g,采用随机数字表法,将其分为3组（n=12）：假手术组（S组）、脓毒症组（CLP组）和脓毒症＋自噬抑制剂3-甲基腺嘌呤组（CLP＋3-MA组）.采用盲肠结扎穿孔法制备脓毒症模型.CLP＋3-MA组术后1h时腹腔注射3-甲基腺嘌呤10 mg/kg.术后24 h时取颈动脉血样,行血气分析,计算氧合指数;取肺组织,行肺组织病理学评分,测定肺组织湿重/干重比值（W/D比值）,采用比色法测髓过氧化酶（MPO）活性,采用Western blot法测定自噬蛋白微管相关蛋白1轻链3Ⅱ型（LC3Ⅱ）、Beclin-1和溶酶体相关蛋白Rab7和LAMP2的表达,进行支气管肺泡灌洗,计数支气管肺泡灌洗液（BALF）总细胞和多形核中性粒细胞（PMN）.结果 与S组比较,CLP组和CLP ＋3-MA组肺组织病理学评分、W/D比值、MPO活性、BALF总细胞计数和PMN计数升高,氧合指数降低,CLP组肺组织LC3Ⅱ、Beclin-1、LAMP2和Rab7的表达上调（P＜0.05）.与CLP组比较,CLP＋3-MA组肺组织病理学评分、W/D比值、MPO活性、BALF总细胞计数和PMN计数升高,氧合指数降低,肺组织LC3Ⅱ、Beclin-1、LAMP2和Rab7的表达下调（P＜0.05）.结论 自噬参与了脓毒症小鼠急性肺损伤时的内源性保护机制.

Objective To investigate the role of autophagy in the lung injury in the septic mice.Methods Thirty-six male C57BL/6 mice, aged 6 weeks, weighing 20-25 g, were randomly divided into 3 groups （n=12 each） using a random number table： sham operation group （group S）;cecal ligation and puncture （CLP） group;CLP ＋ autophagy inhibitor 3-methyladenine （3-MA） group （group CLP＋3-MA）.Sepsis was produced by CLP.In group CLP＋3-MA, 3-MA 10 mg/kg was injected intraperitoneal at 1 h after operation.Arterial blood samples were taken at 24 h after operation for blood gas analysis, and the oxygenation index was calculated.The lungs were removed for microscopic examination of pathologic changes which were scored, and for determination of wet/dry lung weight ratio （W/D ratio） , myeloperoxidase （MPO） activity （using colorimetric method） and the expression of autophagy protein microtubule-associated protein 1 light chain 3 Ⅱ] （LC3 Ⅱ）, Beclin-1 and lysosomes-associated protein Rab7 and lysosome-associated membrane protein-2 （LAMP2） （by Western blot）.The lung was lavaged, and broncho-alveolar lavage fluid （BALF） was collected for determination of the total cell count and polymorphonuclear leukocyte （PMN） count.Results Compared with group S, the pathological score, W/D ratio, MPO activity, and the total cell and PMN counts in BALF were significantly increased, and oxygenation index was decreased in CLP and CLP ＋3-MA groups, and the expression of LC3 Ⅱ , Beclin-1, LAMP2 and Rab7 was up-regulated in group CLP （P〈0.05）.Compared with group CLP, the pathological score, W/D ratio, MPO activity, and the total cell and PMN counts in BALF were significantly increased, the oxygenation index was decreased, and the expression of LC3 Ⅱ , Beclin-1, LAMP2 and Rab7 was down-regulated in group CLP＋ 3-MA （P〈0.05）.Conclusion Autophagy is involved in the endogenous protective mechanism of acute lung injury in the septic mice.