Macrophage Activation Induced By TMAO Through CD147/MMPs

BackgroundIn the progression of atherosclerosis(AS),unstable plaque ruptured,followed by thrombosis,which blocked blood vessels,leading to acute coronary syndrome.Clinically,stabilizing plaque and delaying plaque progression are important therapeutic methods to prevent cardiovascular events.Unstable plaques have the following characteristics:larger lipid core,thinner fibrous cap,plaque microcalcification,intraplaque neovascularization and hemorrhage,expression of inflammatory factors in plaque,plaque surface thrombosis and so on.Plaque stability involves the interaction of a variety of cells including macrophages and neutrophils.Macrophages can participate in AS process and promote plaque progression in inflammation,oxidative stress and other aspects,and play an important regulatory role in plaque stability.Activated macrophages can degrade collagen,the main component of plaque fibrous caps,by secreting matrix metalloproteinases(MMPs),making the fibrous caps thinner.The thinner fibrous caps are more likely to rupture under the action of shear force,resulting in cardiovascular adverse events.Therefore,MMPs and its upstream regulator,extracellular matrix metalloproteinase inducer(CD 147),are also considered to be an important pathway that can effectively regulate collagen synthesis and degradation.Nowadays,more and more studies have confirmed that intestinal flora plays an important role in cardiovascular diseases.AS a metabolite of intestinal flora,trimethylamine n-oxide(TMAO)can promote the progress of AS through a variety of ways.Clinical observation showed that the plasma TMAO concentration in patients with acute myocardial infarction was significantly higher than that in normal controls.Observations of plaques by optical coherence tomography also found that patients with high plasma TMAO levels were more likely to have plaque rupture than those with normal levels.These studies suggest that TMAO may be closely related to plaque instability.By referring to relevant literature,it is found that the studies on TMAO’s plaque stability are mainly focused on clinical studies,while the studies on its molecular mechanism are rarely reported.ObjectiveBy establishing mouse macrophage RAW264.7 cell model,the effect of TMAO on secretion of plaque stability related factors CD147,MMP2 and MMP9 by macrophages and its mechanism were investigated.MethodsMouse macrophage RAW264.7 was stimulated with different concentrations of TMAO(0,150 and 300μmol/L)to establish cell model.The expression of CD 147,MMP2 and MMP9 was observed by real-time quantitative PCR(RT-QPCR)and Western blot.SiRNA transfection was further used to construct CD 147 gene silencing model,which was divided into normal control group and 0μmol/L,150μmol/L and 300μmol/L experimental groups.The control group was normal RAW264.7 cells treated with 300μmol/L TMAO,and the experimental group was RAW264.7 cells treated with CD 147 siRNA.Rt-qPCR and Western blot were used.To explore the interaction between CD147 and MMP2 and MMP9.ResultsRt-qPCR and Western blot results showed that there was no significant change in the expression level of CD147 gene in mouse macrophage RAW264.7(P>0.05),but the expression level of CD 147 gene was significantly increased(P<0.05).The mRNA and protein levels of MMP2 and MMP9 increased(P<0.05).The expression of CD 147,MMP2 and MMP9 was significantly inhibited in CD 147 siRNA transfected cells(P<0.05).ConclusionsIn this study,the expression of MMP2 and MMP9 in RAW264.7 cells was significantly increased at the gene and protein levels by co-culture with mouse macrophages using TMAO.For CD147,the expression of MMP2 and MMP9 was only upregulated at the protein level,but no significant effect was observed at the gene level.These results indicate that TMAO can induce macrophages to express MMP2 and MMP9,while the increased expression of CD 147 only occurs at the post-transcriptional level.These results suggest that the expression of MMPs in macrophages increased after TMAO stimulation,which may lead to the thinning of plaque fibrous caps and promote the development of plaque instability.Further studies showed that RAW264.7 cells were transfected with CD147 siRNA and co-cultured with TMAO for 24 h,and the expression of CD 147,MMP2 and MMP9 in mouse macrophages were inhibited.These results indicated that TMAO up-regulated the expression of MMP2 and MMP9 in macrophages partly by inducing the expression of CD 147,which also suggested that TMAO could regulate the degradation of extracellular matrix by regulating the expression of CD147/MMPs.In conclusion,it is speculated that TMAO can promote the secretion of MMP2 and MMP9 by macrophages at the gene and protein levels,thus promoting plaque instability,and this effect may be partially realized through the CD147/MMPs pathway.