| Objective:Studies have shown that the risk of coronary heart disease(CHD)is not entirely dependent on the size of atherosclerotic plaque,or the severity of coronary stenosis.It is closely related to the constitution of coronary atherosclerotic plaque,whether it is stable,and whether there is secondary thrombosis or thromboembolism.The instability and vulnerability of atherosclerotic plaques are the result of multiple factors.Studies have shown that extracellular matrix metalloproteinases(MMPs)play an important role in the plaque vulnerability.Our group has previously made a large number of MMPs’ common upstream molecular extracellular matrix metalloproteinase inducer(EMMPRIN,also known as CD 147)involved in the regulation of atherosclerosis(AS)formation and rupture of vulnerable plaque(VP).Research work.This study aimed to investigate and confirm whether EMMPRIN can mediate MMPs in human THP-1 macrophages and the expression of atherosclerosis-related factors such as IL-6,IL-8 and MCP-1 via TLR4 signaling pathway.It is expected to target stable atherosclerotic vulnerable plaque,and provide a new experimental basis and theoretical basis for the comprehensive prevention and treatment of coronary heart disease.Methods:1.A human THP-1 monocyte(THP-1 acute monocytic leukemia cell),purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences,was cultured and passaged as required.The suspended THP-1 monocytes were induced with propylene glycol methyl ether acetate(PMA)(100 ng/ml)to make adherent THP-1 macrophages.2.The cytotoxicity of EMMPRIN stimulated THP-1 macrophages at different time points was examined by Cell Counting Kit-8(CCK-8).Combined with the early experiments of the research group,the time node of IFNMP to stimulate THP-1 macrophages was selected.3.The trials were divided into three groups:blank control group(THP-1 macrophage),EMMPRIN stimulation group(THP-1 macrophage+EMMPRIN stimulation for 6 h),TAK-242 inhibition group(THP-1 macrophage+TAK-242).Inhibition of 4h+EMMPRIN stimulation for 6h).4.Functional tests at the gene and protein levels:1.Three test group cells were collected for RNA extraction and cDNA reversal,and the concentrations of MMP3,MMP7,MMP9 and MMP14 were determined by qRT-PCR assay.2.Three test group cells were collected,proteins were extracted,and the concentrations of MMP 9,MMP14 and TLR4 proteins were detected by Western blotting assay.The supernatants of each test group were collected,and the expression of atherosclerosis-associated inflammatory factors IL-6,IL-8 and MCP-1 in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA)Results:1.Successful modeling of human THP-1 macrophages:PMA(100 ng/ml)was used to stimulate human THP-1 monocytes for 48 h,and the original suspended and round cells were observed to be adherent,spindle or elliptical.And the cell volume becomes larger,and the irregular pseudopod is extended;it indicates that the human THP-1 monocyte has successfully differentiated into THP-1 macrophage,suggesting successful modeling2.Detection of EMMPRIN cytotoxicity:EMMPRIN stimulated THP-1 macrophages to detect cytotoxicity for 5-8 hours by Cell Counting Kit-8(CCK-8),and cytotoxicity increased significantly after 9-10 hours.3.MMP3,MMP7,MMP9 and MMP 14 gene expression levels:Compared with the blank control group,the expression levels of MMP7,MMP9 and MMP 14 were significantly increased in the EMMPRIN-stimulated group,the difference was statistically significant(P<0.05);3 genes did not show up-regulated expression,no statistical significance(P>0.05).Compared with the EMMPRIN-stimulated group,the expression levels of MMP9 and MMP14 were decreased in the TAK-242 inhibitory group(P<0.05),and the MMP7 gene level was not significantly down-regulated(P>0.05).4.Expression of MMP9,MMP14 and TLR4 protein levels:Compared with the blank control group,MMP 9,MMP14 and TLR4 protein levels were significantly increased in the EMMPRIN-stimulated group(P<0.05).Compared with the EMMPRIN-stimulated group,the expression levels of MMP9,MMP 14 and TLR4 in the TAK-242 inhibited group were significantly decreased(P<0.05).In addition,TLR4 protein was significantly inhibited in the TAK-242 inhibition group compared with the blank control group(P<0.05).5.Expression of IL-6,IL-8 and MCP-1 inflammatory factors:Compared with the blank control group,IL-6,IL-8 and MCP-1 were significantly increased in the EMMPRIN-stimulated group(P<0.05),and in the EMMPRIN-stimulated group.In comparison,IL-6,IL-8 and MCP-1 in the TAK-242 inhibition group were significantly inhibited(P<0.05).Conclusions:1.EMMPRIN significantly up-regulated the expression of MMP9,MMP 14,and inflammatory factors IL-6,IL-8,and MCP-1.2.Inhibition of TLR4 signaling pathway can significantly down-regulate the expression of MMP9,MMP 14 and inflammatory factors IL-6,IL-8 and MCP-1,which is expected to provide a theoretical basis for increasing the stability of plaque and targeting blocking vulnerable plaque. |
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