Trimethylamine N-Oxide Promotes Macrophage-Myofibroblast Transformation By Upregulating The NF-κB Signaling Pathway

Objective:Fibrosis is formed due to excessive deposition of extracellular matrix(ECM),involving the proliferation and activation of myofibroblasts,which eventually leads to the destruction of tissue structure and organ dysfunction.Organ fibrosis leads to high morbidity and mortality worldwide.There are no effective therapeutic measures to block or delay the progression of progressive fibrosis.Intestinal flora is closely related to human health and disease.Trimethylamine N-oxide(TMAO)is one of the most widely studied microbial metabolites.Studies have found that TMAO is closely related to cardiac and renal fibrosis,the mechanism involves TGF-βRI/Smad2,TGF-β1/Smad3,NLRP3,NFκB signaling pathway,macrophage-myofibroblast transition(MMT)can exacerbate cardiac fibrosis,renal fibrosis and lung fibrosis.Studies have shown that elevated TMAO levels can cause inflammation,TMAO can significantly activate the p65 NF-κB signaling pathway to promote vascular inflammation,and the NF-κB signaling pathway can promote inflammation and fibrosis,but whether TMAO can promote the transformation of macrophages into myofibroblasts and the The mechanism is still unclear.The purpose of this study is to investigate that TMAO promotes macrophage-myofibroblast transformation by up-regulating NF-κB signaling pathwaMethods:In this study,bone marrow-derived macrophages were isolated and cultured,and flow cytometry was used to identify whether the bone marrow-derived macrophages were isolated and cultured successfully.Subsequently,the bone marrow-derived macrophages were intervened with different concentrations of TMAO and NFκB inhibitor(BAY 11-7082).The bone marrow-derived macrophages were divided into 5 groups,and each group was the control group,TMAO 150μmol/L,TMAO 300μmol/L,TMAO 150μmol/L and BAY 11-7082 5μmol/L,TMAO 300μmol/L and BAY 11-7082 5μmol/L,respectively.after 24 hours of intervention,total cell protein was extracted,and Western blot was performed to observe the Effects of TMAO and BAY 11-7082 on the expression levels of α-smooth muscle actin(α-SMA)and type I collagen(Collagen I)after intervention of bone marrow-derived macrophages.Results:More than 90%of the double-positive cell population expressing the bone marrow-derived macrophage marker F4/80+CD11b+ was identified by flow cytometry,which proved that the bone marrow-derived macrophages were isolated and cultured successfully.The results of Western blot experiments showed that compared with the control group,TMAO 150μmol/L could significantly increase the expression level of Collagen I,the difference was statistically significant(p<0.01),TMAO 150 μmol/L treatment had no significant effect on the expression of α-SMA,and TMAO 300 μmol/L could significantly increase the expression level of α-SMA and Collagen I,The difference was statistically significant(p<0.05);compared with the TMAO 300 gmol/L group,the expression levels of α-SMA and Collagen I in the TMAO 300μmol/L and BAY 11-7082 5 μmol/L group were significantly down-regulated,and the difference was statistically significant(p<0.05).Conclusion:TMAO can promote the transformation of bone marrow-derived macrophages into myofibroblasts and collagen deposition,and the mechanism is hypothesized to be the NF-κB signaling pathway.