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

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通过激活AMPK/Nrf2/HO-1信号通路,抑制LPS诱导的M1型、促进M2型肺泡巨噬细胞极化,改善了炎症反应。

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 group promoted the expression of AMPK/Nrf2/HO-1 compared with LPS(all P<0.05);compared with the LB group,AMPK/Nrf2/HO-1 expression was decreased in the LC group(all P<0.05),the expression of Nrf2/HO-1 was reduced in the LM group(all P<0.05);the flow cytometry results showed that compared with the LB group,Compound C and ML385 reversed the inhibition of SB to CD86 in the LC and LM groups(all P<0.01);the trend of the M2 macrophage marker CD206 expression was exactly opposite to that of the M1 macrophage marker CD86.Conclusions SB improves inflammation by activating AMPK/Nrf2/HO-1 signaling pathway,inhibiting LPS-induced M1 and promoting M2 alveolar macrophage polarization.