激活大麻素2型受体对脓毒症小鼠急性肺损伤的影响:自噬在其中的作用

Effect of activating cannabinoid receptor 2 on sepsis-induced acute lung injury in mice: the role of autophagy

目的评价激活大麻素2型受体(CB2R)对脓毒症小鼠急性肺损的影响及自噬在其中的作用。方法SPF级雄性C57BL/6小鼠24只,8~10周龄,体重20~25g,采用随机数字表法分为4组(n=6):假手术组(Sham组)、脓毒症组(Sep组)、脓毒症+CB2R激动剂HU308组(Sep+HU308组)、脓毒症+HU308+自噬抑制剂3-甲基腺苷组(Sep+HU308+3-MA组)。采用盲肠结扎穿孔法构建脓毒症模型。Sep+HU308组和Sep+HU308+3-MA组于术后15min时腹腔注射HU3082.5mg/kg,15min后Sep+HU308+3-MA组腹腔注射3-甲基腺苷10mg/kg。术后12h时取肺组织,HE染色观察病理学结果,并进行肺损伤评分;采用RT-PCR法检测TNF-α、IL-18和IL-1β的mRNA表达水平;采用免疫组织化学法测定自噬相关蛋白Atg5的表达;采用Westernblot法检测微管相关蛋白1轻链3(LC3)、Beclin-1和p62的表达水平,计算LC3Ⅱ/LC3Ⅰ比值。结果与Sham组比较,其余3组肺组织TNF-α、IL-18和IL-1β的mRNA、Atg5表达上调,LC3Ⅱ/LC3Ⅰ比值升高,肺损伤评分升高,Sep组和Sep+HU308组Beclin-1表达上调,p62表达下调,Sep+HU308+3-MA组p62表达上调(P<0.05);与Sep组比较,Sep+HU308组和Sep+HU308+3-MA组肺组织TNF-α、IL-18和IL-1β的mRNA表达下调,Atg5表达上调,肺损伤评分降低,Sep+HU308组LC3Ⅱ/LC3Ⅰ比值升高,Beclin-1表达上调,p62表达下调,Sep+HU308+3-MA组Beclin-1表达下调,p62表达上调(P<0.05);与Sep+HU308组比较,Sep+HU308+3-MA组肺组织TNF-α、IL-18和IL-1β的mRNA表达上调,Atg5和Beclin-1表达下调,LC3Ⅱ/LC3Ⅰ比值降低,p62表达上调,肺损伤评分升高(P<0.05)。结论激活CB2R可减轻脓毒症小鼠急性肺损伤,部分机制可能与增强自噬,减轻炎症反应有关。

Objective To evaluate the effect of activating cannabinoid receptor 2(CB2R) on sepsis-induced acute lung injury and the role of autophagy in mice. Methods Twenty-four SPF male C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups(n=6 each) using a random number table method: sham operation group(group Sham), sepsis group(group Sep), sepsis plus CB2R agonist HU308 group(group Sep+ HU308) and sepsis plus HU308 plus autophagy inhibitor 3-methyladenine group(group Sep+ HU308+ 3-MA). Sepsis was induced by cecal ligation and puncture in anesthetized mice. HU308 2.5 mg/kg was intraperitoneally injected at 15 min after surgery in Sep+ HU308 and Sep+ HU308+ 3-MA groups, and 15 min later 3-MA 10 mg/kg was intraperitoneally injected in group Sep+ HU308+ 3-MA. Lung tissues were obtained at 12 h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of tumor-necrosis factor-alpha(TNF-α), interleukin-18(IL-18) and IL-1β mRNA(by real-time polymerase chain reaction), expression of autophagy-related protein 5(Atg5)(by immuno-histochemistry), and expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1 and p62(by Western blot). The ratio of LC3Ⅱ to LC3Ⅰ(LC3Ⅱ/LC3Ⅰ ratio) was calculated. ResultsCompared with group Sham, the expression of TNF-α, IL-18 and IL-1β mRNA was significantly up-regulated, and LC3Ⅱ/LC3Ⅰratio and lung injury score were increased in the other three groups, the expression of Beclin-1 was up-regulated, and the expression of p62 was down-regulated in group Sep and group Sep+ HU308, and the expression of p62 was significantly up-regulated in group Sep+ HU308+ 3-MA(P<0.05). Compared with group Sep, the expression of TNF-α, IL-18 and IL-1β mRNA was significantly down-regulated, the expression of Atg5 was up-regulated, and lung injury score was decreased in group Sep+ HU308 and group Sep+ HU308+ 3-MA, LC3Ⅱ/LC3Ⅰratio was increased, the expression of Beclin-1 was up-regulated, and the expression of p62 was down-regulated in group Sep+ HU308, and the expression of Beclin-1 was down-regulated, and the expression of p62 was up-regulated in group Sep+ HU308+ 3-MA(P<0.05). Compared with group Sep+ HU308, the expression of TNF-α, IL-18 and IL-1β mRNA was significantly up-regulated, the expression of Atg5 and Beclin-1 was down-regulated, LC3Ⅱ/LC3Ⅰratio was decreased, the expression of p62 was up-regulated, and lung injury scores were increased in group Sep+ HU308+ 3-MA(P<0.05). Conclusion Activating CB2R can alleviate acute lung injury in septic mice, and the mechanism may be partially related to enhancing autophagy and reducing inflammatory responses.