Study on the mechanism of action of short-chain fatty acid in inhibiting M1 type alveolar macrophage polarization
Objective To investigate the effect of short-chain fatty acid(SCFA)sodium butyrate(NaB)on the polarization of lipopolysaccharide(LPS)induced Ml 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 factor α(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-α in 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 flow cytometry.Results(1)qRT-PCR and ELISA results were consistent,M1 type macrophage-associated proinflammatory cytokines IL-6,IL-1β and TNF-α significantly reduced in the LB group after NaB addition compared with the LPS ground(all P＜0.05);(2)The results of qRT-PCR and flow cytometry were consistent,compared with the LPS group,the LB group showed a significant 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 significantly decreased the expression level of iNOS+in the LB group(P＜0.05);compared with the LB group,the addition of Compound C in the LC group reversed the inhibitory effect of NaB on iNOS+(P＜0.05);(4)The qRT-PCR results of MLE-12 cells showed that compared with the LPS group,the LB group showed a significant increase in Z0-1,Claudin-4,and Occludin after the addition of NaB(all P＜0.05).Conclusions SCFA inhibits LPS-induced polarization of M1-type alveolar macrophages and ameliorates the inflammatory response by activating the AMPK/Nrf2/HO-1 signaling pathway.
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