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

Chen Jian; Zhou Weidong; Wang Yanhua; Liu Qinfu; Yang Xiaojun

doi:10.3969/j.issn.1002-1949.2024.02.010

Abstract：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 1 000 ng/mL LPS,1 mmol/L SB,10 μmol/L Compound C,and 5 μmol/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-1 β(IL-1 β),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-1 β and 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 1 000 ng/mL LPS,1 mmol/L SB,10 μmol/L Compound C and 5 μmol/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-α and IL-1β significantly 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 LBgrouppromotedtheexpressionof AMPK/Nrf2/HO-1 comparedwith 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.