丁酸钠经AMPK/Nrf2/HO-1信号通路调节脂多糖诱导肺泡巨噬细胞极化的作用机制  

Study on the mechanism of sodium butyrate in regulating alveolar macrophage polarization induced by lipopolysaccharide via AMPK/Nrf2/HO-1 signaling pathway

作　　者：陈健 周卫东 王艳华 刘勤富 杨晓军[4] Chen Jian;Zhou Weidong;Wang Yanhua;Liu Qinfu;Yang Xiaojun(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;不详)

机构地区：[1]宁夏医科大学临床医学院,宁夏银川750004 [2]宁夏回族自治区干细胞与再生医学重点实验室,宁夏银川750004 [3]宁夏医科大学总医院妇科宫颈病变中心实验室,宁夏银川750004 [4]宁夏医科大学总医院重症医学科,宁夏银川750004

出　　处：《中国急救医学》2024年第2期156-163,共8页Chinese Journal of Critical Care Medicine

基　　金：宁夏回族自治区自然科学基金项目(2023AAC03559)。

摘　　要：目的探讨丁酸钠(sodium butyrate,SB)对脂多糖(lipopolysaccharide,LPS)诱导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细胞)随机分为对照(Control)组、LPS组、SB组、LPS+SB(LB)组、LPS+SB+腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)(LC)组、LPS+SB+核因子E2相关因子2(Nrf2)抑制剂(ML385)(LM)组。通过CCK8检测MH-S细胞活力,筛选出最佳的1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C、5μmol/L ML385药物浓度进行后续实验;实时荧光定量(qRT-PCR)检测MH-S细胞白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)、白细胞分化抗原86(CD86)、巨噬细胞甘露糖受体(CD206)、AMPK、Nrf2和血红素加氧酶1(HO-1)的mRNA表达水平;酶联免疫吸附试验(ELISA)检测培养基上清IL-6、TNF-α、IL-1β和IL-10蛋白含量;流式细胞术测定M1和M2型巨噬细胞相关标记物CD86和CD206的表达。各组数据通过单因素方差分析和Tukey法进行检验。结果通过CCK8选取了1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C和5μmol/L ML385进行造模和干预。qRT-PCR和ELISA结果一致显示,与LPS组比较,LB组M1型巨噬细胞相关促炎细胞因子IL-6、TNF-α、IL-1β显著降低(均P<0.01),但M2型巨噬细胞相关抑炎细胞因子IL-10显著升高(均P<0.01)。qRT-PCR和流式细胞术结果一致显示,与Control组比较,LPS组CD86水平显著升高(均P<0.01),SB组差异无统计学意义;与LPS组比较,LB组CD86表达水平显著降低(均P<0.01),但M2型巨噬细胞标记物CD206的变化趋势与CD86相反。qRT-PCR结果显示,与LPS组比较,LB组促进AMPK/Nrf2/HO-1的表达(均P<0.05);与LB组比较,LC组降低了AMPK/Nrf2/HO-1的表达(均P<0.05),LM组降低了Nrf2/HO-1的表达(均P<0.05)。流式细胞术结果显示,与LB组比较,LC组和LM组逆转了SB对CD86水平的抑制作用(均P<0.01);M2型巨噬细胞标记物CD206的表达趋势与M1型巨噬细胞标记物CD86相反。结论SB�Objective To investigate the effect of sodium butyrate(SB)on the polarization of lipopolysaccharide(LPS)-induced alveolar macrophages and the mechanism of action.Methods Mouse alveolar macrophages(MH-S cells)were randomly divided into Control group,LPS group,SB group,LPS+SB group(LB),LPS+SB+adenosine monophosphate-activated protein kinase(AMPK)inhibitor(Compound C)group(LC),LPS+SB+nuclear factor erythroid 2-related factor 2(Nrf2)inhibitor(ML385)group(LM).MH-S cell viability was measured by CCK8,and the best drug concentrations of 1000 ng/mL LPS,1 mmol/L SB,10μmol/L Compound C,and 5μmol/L ML385 were selected for subsequent experiments.The quantitative real-time PCR(qRT-PCR)was used to detect mRNA expression levels of interleukin-6(IL-6),tumor-necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-10(IL-10),leukocyte differentiation antigen 86(CD86),macrophage mannose receptor(CD206),AMPK,Nrf2,and heme oxygenase-1(HO-1).IL-6,TNF-α,IL-1βand IL-10 were measured by the enzyme-linked immunosorbent assay(ELISA);expression of M1 and M2 type macrophage-associated markers CD86 and CD206 were determined by flow cytometry.Results 1000 ng/mL LPS,1 mmol/L SB,10μmol/L Compound C and 5μmol/L ML385 were selected for model making and intervention through CCK8.The qRT-PCR and ELISA results indicated that the levels of the M1 macrophage-associated pro-inflammatory cytokines IL-6,TNF-αand IL-1βsignificantly decreased in the LB group(all P<0.01),but there were significant increases in the level of M2 macrophage-related anti-inflammatory cytokine IL-10 compared with the LPS group(all P<0.01).The results of qRT-PCR and flow cytometry showed that CD86 increased in LPS group compared with the Control group(all P<0.01),and there was no difference between SB group and the Control group;compared with the LPS group,the CD86 expression level was significantly reduced in the LB group(P<0.01),but the trend of M2 macrophage marker CD206 was opposite to that of CD86.The expression of AMPK/Nrf2/HO-1 was detected by qRT-PCR,the LB gr

关 键 词：丁酸钠 巨噬细胞极化 炎症 白细胞分化抗原86 巨噬细胞甘露糖受体 腺苷酸活化蛋白激酶 核因子E2相关因子2 血红素加氧酶1 

分 类 号：R459.7[医药卫生—急诊医学] R563[医药卫生—治疗学]