抑制p38丝裂素活化蛋白激酶途径调控Th17和Treg失衡减轻脂多糖诱导的小鼠急性肺损伤  

p38MAPK inhibitor ameliorates lipopolysaccharide induced acute lung injury through regulating the balance of Treg cells and Th17 cells

作　　者：冯净净[1,2] 邵川 李俊卿[1,2] 施天昀 都勇 揭志军[1,2] 施劲东 FENG Jingjing;SHAO Chuan;LI Junqing;SHI Tianyun;DU Yong;JIE Zhijun;SHI Jindong(Department of Pulmonary and Critical Care Medicine,Shanghai Fifth People's Hospital,Fudan University,Shanghai 200240,P.R.China;Center of Community-Based Health Research,Fudan University,Shanghai 200240,P.R.China;Department of Pulmonary and Critical Care Medicine,Ningbo Medical Center Lihuili Hospital,Ningbo,Zhejiang 315040,P.R.China)

机构地区：[1]复旦大学附属上海市第五人民医院呼吸与危重症医学科,上海200240 [2]复旦大学社区健康研究中心,上海200240 [3]宁波市医疗中心李惠利医院呼吸与危重症医学科,浙江宁波315040

出　　处：《中国呼吸与危重监护杂志》2023年第1期44-51,共8页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：闵行区高层次专科骨干医师培养计划(2020MZYS12);复旦闵行康联体项目(2020FM09)。

摘　　要：目的 探讨p38丝裂素活化蛋白激酶(p38 mitogen activated protein kinase,p38MAPK)抑制剂是否通过调节Th17/Treg平衡减轻脂多糖(lipopolysaccharide,LPS)所致急性肺损伤(acute lung injury,ALI)。方法 将Balb/c小鼠随机分组为对照组、ALI组和干预组。对照组腹腔注射磷酸盐缓冲液,ALI组腹腔注射40 mg/kg体重的LPS,干预组腹腔注射不同药物浓度(0.5、1、2、5 mg/kg)的p38MAPK抑制剂SB203580药物。1 h后通过腹腔注射LPS造模,12 h后处死小鼠并取材。肺组织送苏木精–伊红(hematoxylin-eosin,HE)染色观察肺组织病理变化,检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中细胞分类计数和总蛋白水平。采用实时聚合酶链反应检测叉头框蛋白P3(forkhead box p3,Foxp3)和视黄酸受体相关孤儿受体γt(retinoic acid receptor-related orphan receptor-γt,RORγt)mRNA的表达量。采用酶联免疫吸附法检测肺组织匀浆中白细胞介素(interleukin,IL)-6、IL-10、IL-17、IL-23和转化生长因子β(transforming growth factor-β,TGF-β)及血清中IL-6、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的蛋白含量。流式细胞计数法检测脾脏组织匀浆中Th17细胞和调节性T细胞(regulatory cells,Treg)亚群。结果 在LPS诱导建立的ALI小鼠模型中,LPS可引起肺组织内炎性细胞浸润,BALF中细胞总数及蛋白水平升高(P<0.05),中性粒细胞和单核细胞比例升高。SB203580干预后肺组织病理损伤程度减轻,相应的BALF中炎性细胞浸润减少。ALI组血清中IL-6和TNF-α蛋白浓度较对照组明显升高,SB203580干预后炎性细胞因子下降。与ALI组比较,干预组Th17细胞及Th17分化相关细胞因子IL-17和IL-23表达下降,转录因子RORγt分泌受抑制,而Treg及IL-10和Foxp3相关分化因子表达有所升高,且与SB203580呈浓度依赖性。SB203580干预后Th17/Treg比值较LPS组明显降低(P<0.05)。结论 p38MAPK抑制剂可通过调节Treg细胞和Th17细胞失衡状态减轻LPS导致的ALObjective To investigate whether p38 mitogen activated protein kinase(p38MAPK) inhibitor can reduce acute lung injury(ALI) caused by lipopolysaccharide(LPS) by regulating Th17/Treg balance.Methods Balb/c mice were randomly divided into a control group,an ALI group and an intervention group.The mice in the control group were injected with phosphate-buffered saline,the mice in the ALI group were intraperitoneally injected with 40 mg/kg LPS,and the mice in the intervention group were injected with SB203580(0.5 mg/kg,1 mg/kg,2 mg/kg,5 mg/kg)intraperitoneally 1 h prior to the intraperitoneal injection of LPS.All mice were killed on 12 h later respectively.Hematoxylin-eosinstin staining was used to observe the pathological changes of lung tissue,and cell classification,counting,and total protein levels in bronchoalveolar lavage fluid(BALF) were detected.Transcript expression of forkhead box p3(Foxp3) and retinoic acid receptor-related orphan receptor-γt(RORγt) was detected by real-time polymerase chain reaction.Interleukin(IL)-6,IL-10,IL-17,IL-23 and transforming growth factor-β(TGF-β) in lung tissue and IL-6,tumor necrosis factor-α(TNF-α) in serum were measured by enzyme-linked immunosorbent assay.The Th17 and Treg subset distribution in spleen was determined by flow cytometry.Results Histopathological examination showed that LPS induced inflammatory cell infiltration in lung tissue,increased cell count and protein levels in BALF(P<0.05),and increased proportion of neutrophils and monocytes in the ALI mice.SB203580 significantly attenuated tissue injury of the lungs in LPS-induced ALI mice.Serum levels of IL-6 and TNF-α in the ALI group were significantly higher than those in the control group,and inflammatory cytokines were decreased after SB203580 intervention.Compared with the ALI group,the production of inflammatory cytokines associate with Th17,including IL-17,IL-23,RORγt was inhibited,and the production of cytokines associate with Treg,such as IL-10 and Foxp3 in lung tissue was increased in the intervent

关 键 词：P38MAPK信号通路 脂多糖 急性肺损伤 TREG细胞 Th17细胞 

分 类 号：R563.8[医药卫生—呼吸系统]