短链脂肪酸抑制M1型肺泡巨噬细胞极化的作用机制研究  

Study on the mechanism of action of short-chain fatty acid in inhibiting M1 type alveolar macrophage polarization

作　　者：陈健[1,2] 周卫东 马金兰 马立兵 杨晓军 Chen Jian;Zhou Weidong;Ma Jinlan;Ma Libing;Yang Xiaojun(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Stem Cell and Regenerative Medicine,Ningxia Hui Autonomous Region,Yinchuan 750004,China;Department of Critical Care Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学第一临床医学院,银川750004 [2]宁夏回族自治区干细胞与再生医学重点实验室,银川750004 [3]宁夏医科大学总医院重症医学科,银川750004

出　　处：《中华急诊医学杂志》2024年第4期522-528,共7页Chinese Journal of Emergency Medicine

基　　金：宁夏回族自治区自然科学基金项目(2023AAC03559)。

摘　　要：目的探讨短链脂肪酸(Short-chain fatty acid,SCFA)丁酸钠(Sodium butyrate,NaB)对脂多糖(Lipopolysaccharide,LPS)诱导M1型肺泡巨噬细胞极化的影响及其作用机制。方法小鼠肺泡巨噬细胞(MH-S)随机(随机数字法)分为对照组(Control组)、丁酸钠组(NaB组)、LPS组、LPS+NaB组(LB组)、LPS+NaB+腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)组(LC组)。通过qRT-PCR检测MH-S细胞中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、白细胞分化抗原86(CD86)、诱导型一氧化氮合酶(iNOS)和小鼠肺上皮细胞(MLE-12)中闭锁小带蛋白1(ZO-1)、紧密连接蛋白4(Claudin-4)、闭合蛋白(Occludin)的mRNA表达水平;ELISA检测MH-S细胞培养基上清液IL-6、IL-1β、TNF-α蛋白含量;Western blot测定MH-S细胞AMPK、P-AMPK、核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)的蛋白表达;流式细胞术测定M1型巨噬细胞相关标记物CD86和iNOS的表达。结果(1)qRTPCR和ELISA结果一致,与LPS组相比,LB组加入NaB后M1型巨噬细胞相关促炎细胞因子IL-6、IL-1β、TNF-α显著降低(均P<0.05);(2)qRT-PCR和流式细胞术的结果一致,与LPS组相比,LB组加入NaB后M1型巨噬细胞相关极化指标CD86、iNOS显著降低(均P<0.05);(3)Western blot检测AMPK/Nrf2/HO-1信号通路的表达,与LPS相比,LB组加入NaB后增强了P-AMPK/AMPK、Nrf2(nucleus)、HO-1的表达(均P<0.05);与LB组相比,LC组降低了P-AMPK/AMPK、Nrf2(nucleus)、HO-1的表达(均P<0.05);流式细胞术结果显示,与LPS组相比,LB组加入NaB后iNOS+表达水平显著降低(P<0.05);与LB组相比,LC组加入Compound C逆转了NaB对iNOS+的抑制作用(P<0.05);(4)MLE-12细胞qRT-PCR结果显示,与LPS组相比,LB组加入NaB后Z0-1、Claudin-4、Occludin明显增高(均P<0.05)。结论SCFA通过激活AMPK/Nrf2/HO-1信号通路,抑制LPS诱导的M1型肺泡巨噬细胞极化,改善了炎症反应。Objective To investigate the effect of short-chain fatty acid(SCFA)sodium butyrate(NaB)on the polarization of lipopolysaccharide(LPS)induced M1 type alveolar macrophages and the mechanism of action.Methods Mouse alveolar macrophages(MH-S)were randomly(random number)divided into control group(Control group),sodium butyrate group(NaB group),LPS group,LPS+NaB group(LB group),and LPS+NaB+adenylate activated protein kinase(AMPK)inhibitor(Compound C)group(LC group).The mRNA expression levels of interleukin 6(IL-6),interleukin 1β(IL-1β),tumor necrosis factorα(TNF-α),cluster of differentiation 86(CD86),inducible nitric oxide synthase(iNOS)in MH-S cells,and zonula occludens 1(ZO-1),tight junction protein 4(Claudin-4),and closed protein(Occludin)in mouse lung epithelial cells(MLE-12)were detected by qRT-PCR;Protein levels of IL-6,IL-1β,and TNF-αin the supernatant of MH-S cell medium were measured by ELISA;Western blot determed the protein expression of AMPK,P-AMPK,nuclear factor E2-related factor 2(Nrf2),and heme oxygenase 1(HO-1)in MH-S cells;Expression of M1 type macrophage-associated markers CD86 and iNOS were determined by fl ow cytometry.Results(1)qRT-PCR and ELISA results were consistent,M1 type macrophage-associated proinfl ammatory cytokines IL-6,IL-1βand TNF-αsignifi cantly reduced in the LB group after NaB addition compared with the LPS ground(all P<0.05);(2)The results of qRT-PCR and fl ow cytometry were consistent,compared with the LPS group,the LB group showed a signifi cant decrease in M1 type macrophage-related polarization indicators CD86 and iNOS after NaB addition(all P<0.05);(3)Western blot was used to detect the expression of the AMPK/Nrf2/HO-1 signaling pathway,compared with LPS,the addition of NaB in the LB group enhanced the expression of P-AMPK/AMPK,Nrf2(nucleus),and HO-1(all P<0.05);compared with the LB group,the LC group decreased the expression of P-AMPK/AMPK,Nrf2(nucleus),and HO-1(all P<0.05);the results of flow cytometry showed that compared with the LPS group,the addition of NaB signif

关 键 词：短链脂肪酸 巨噬细胞极化 AMPK/Nrf2/HO-1信号通路 炎症 

分 类 号：R563[医药卫生—呼吸系统]