抗炎合剂通过调控Beclin-1介导自噬减轻大鼠脓毒症急性肺损伤  

Anti-inflammatory mixture alleviates acute lung injury induced by sepsis in rats by modulating Beclin-1-mediated autophagy

作　　者：陆玮兰 闫国良[1] 沈一凡 李海涛[1] 吴赛 翁同锐 张蕊 火艳雯 Lu Weilan;Yan Guoliang;Shen Yifan;Li Haitao;Wu Sai;Weng Tongrui;Zhang Rui;Huo Yanwen(Department of Critical Care Medicine,Shanghai University of Traditional Chinese Medicine Affiliated Shanghai Hospital of Traditional Chinese Medicine,Shanghai 200071,China)

机构地区：[1]上海中医药大学附属市中医医院重症医学科,上海200071

出　　处：《中华危重病急救医学》2024年第7期717-722,共6页Chinese Critical Care Medicine

基　　金：上海市进一步加快中医药传承创新发展三年行动计划项目[ZY(2021-2023)-0209-11]。

摘　　要：目的探讨抗炎合剂对脓毒症急性肺损伤(ALI)大鼠的保护作用及其可能机制。方法将40只SD大鼠按随机数字表法分为假手术组、脓毒症ALI模型组(模型组)、3-甲基腺嘌呤(3-MA)对照组、抗炎合剂预处理组,每组10只。采用盲肠结扎穿孔术(CLP)建立脓毒症ALI大鼠模型;假手术组仅开腹、关腹,不给予穿孔结扎;两组均于术前连续3 d给予生理盐水灌胃和腹腔注射。3-MA对照组于制模前连续3 d给予生理盐水和自噬抑制剂3-MA 15 mg/kg腹腔注射。抗炎合剂预处理组于制模前连续3 d给予抗炎合剂8.8 mL/kg灌胃〔抗炎合剂组成:大黄15 g(后下)、黄连15 g、黄芩12 g、厚朴12 g、败酱草30 g〕,并腹腔注射生理盐水。各组于术后24 h取大鼠腹主动脉血处死,采用酶联免疫吸附试验(ELISA)检测血清炎症细胞因子白细胞介素(IL-1β、IL-6)水平。取肺组织,收集支气管肺泡灌洗液(BALF),采用ELISA法检测IL-1β、IL-6水平;检测肺湿/干质量(W/D)比值;苏木素-伊红(HE)染色后,光镜下观察肺组织病理学改变;采用蛋白质免疫印迹试验(Western blotting)检测肺组织自噬标志物微管相关蛋白1轻链3-Ⅱ/Ⅰ(LC3-Ⅱ/Ⅰ)和Beclin-1蛋白表达;透射电镜下观察肺组织自噬小体。结果与假手术组比较,模型组大鼠肺组织结构破坏严重,有大量炎症细胞浸润,肺W/D比值、血清和BALF中IL-1β、IL-6水平均明显升高,LC3-Ⅱ/Ⅰ比值和Beclin-1蛋白表达均明显下调,自噬小体明显增加。3-MA对照组大鼠肺组织损伤程度较模型组更严重,肺W/D比值和血清、BALF中炎症细胞因子水平进一步升高,肺组织LC3-Ⅱ/Ⅰ比值和Beclin-1蛋白表达仍较假手术组呈下调趋势,自噬小体较模型组减少。与模型组相比,抗炎合剂预处理组大鼠肺组织损伤明显减轻,少量炎症细胞浸润,肺W/D比值明显降低(7.07±1.02比11.33±1.85,P<0.05),血清和BALF中IL-1β、IL-6水平均明显下降〔IL-1β(ng/L):血清为26.04±3Objective To investigate the protective effects of an anti-inflammatory mixture on acute lung injury(ALI)induced by sepsis in rats,as well as its possible mechanisms.Methods A total of 40 Sprague-Dawley(SD)rats were randomly divided into the sham group,septic ALI model group(model group),3-methyladenine(3-MA)control group,and anti-inflammatory mixture pretreatment group,with 10 rats in each group.Cecal ligation and perforation(CLP)was performed to reproduce a septic ALI model.The rats in the sham group only underwent opening and closing the abdomen without perforation and ligation.Both groups were given saline gavage and intraperitoneal injection for 3 consecutive days before surgery.The 3-MA control group was given intraperitoneal injection of saline and autophagy inhibitor 3-MA 15 mg/kg for 3 consecutive days before modeling.The anti-inflammatory mixture pretreatment group was given 8.8 mL/kg of anti-inflammatory mixture by gavage[the composition of anti-inflammatory mixture:rhubarb 15 g(after the next),coptis chinensis 15 g,baical skullcap root 12 g,magnoliae cortex 12 g,dahurian patrinia herb 30 g]and saline intraperitoneal injection for 3 consecutive days before modeling.The rats in each group were anesthetized 24 hours after surgery and died due to abdominal aortic blood collection.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum inflammatory cytokines interleukins(IL-1βand IL-6).Lung tissue was taken and then the bronchoalveolar lavage fluid(BALF)was collected,and the levels of IL-1βand IL-6 were detected by ELISA.Lung wet/dry weight(W/D)ratio was measured.After hematoxylin-eosin(HE)staining,the histopathological changes of the lungs were observed under light microscopy.Western blotting was used to detect the expression of autophagy markers microtubule-associated protein 1 light chain 3-Ⅱ/Ⅰ(LC3-Ⅱ/Ⅰ)and Beclin-1 protein in lung tissue.Autophagosomes in lung tissue were observed with transmission electron microscopy.Results Compared with the sham group,the rats in the

关 键 词：脓毒症 急性肺损伤 抗炎合剂 炎症因子 自噬 

分 类 号：R459.7[医药卫生—急诊医学]