Lipopolysaccharide Promotes Proinflammatory Response Via Enhancing IFIT1 Expression In Human Umbilical Vein Enciothelial Cells

BACKGROUNDAtherosclerosis is a chronic inflammatory disease that affects human health seriously,and it is considered that a variety of inflammatory cells and factors play significant roles throughout its pathogenesis.It has been demonstrated that inflammation,implicated in all stages of atherosclerosis,is an integral part of vulnerable plaque development and progression,leading eventually to plaque instability.The vascular wall shows acute exudative inflammation in the early stage and chronic proliferative inflammation in the progressive stage.Inflammatory responses contribute to both pathogenesis and complications of atherosclerosis.Potent proinflammatory cytokines including interleukin(IL)-1?,IL-6,and tumor necrosis factor(TNF)-? and nuclear factor(NF)-?B,are associated with increased risk of atherosclerosis,but the mechanisms involved are still under investigation.Lipopolysaccharide(LPS)is an endotoxin from the outer membrane of Gram-negative bacteria.Absorption of exogenous LPS into the blood lead to endothelial injury and increased secretion of inflammatory cytokines,causes inflammatory responses in immune and non-immune cells;and acts as a critical pathological factor in inflammation.However,it is not clear how LPS contributes to upregulate the expression of inflammatory cytokine.The ISG56/IFIT1 family of genes is clustered on human 10q23.31 and comprises 4 members,ISG56/IFIT1,ISG54/IFIT2,ISG60/IFIT3,and ISG58/IFIT5.While these genes are normally silent in most cell types,their transcription is strongly induced by interferons,virus infection,and molecular patterns such as double-stranded RNA or LPS.Interferon-induced protein with tetratricopeptide repeats 1(IFIT1)mRNA is strongly upregulated in M1 polarized human primary macrophages,and IFIT1 also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery from atherosclerotic ApoE-/-mice.IFIT1 play an important role in inflammation,but its exact mechanism in atherosclerosis remains largely unknown.Therefore,the molecular mechanism of atherosclerosis needs to be further studied for seek new and effective therapeutic targets.The present study aimed to investigate the role of IFIT1 in atherosclerosis in vitro.MATERIALS AND METHODSHuman umbilical vein endothelial cells(HUVECs)were purchased from the American Type Culture Collection(ATCC).1.HUVECs were treated with 0,250,500,1000 ng/mL of LPS,after 24h,qRT-PCR and Western blot(WB)were used to detect the expression of IFIT1.Using 1000ng/mL of LPS to treat HUVECs at 0,6,12,and 24h time points,the mRNA and protein were extracted to detect the expression of IFIT1.2.HUVECs were transfected with IFIT1 plasmid overexpressed vector,after 24h,with or without LPS treatment for another 24h?qRT-PCR and WB were used to detect the transfection efficiency of IFIT1,and the mRNA and protein expression levels of IL-1?,IL-6,TNF-? and NF-?B were detected as well.3.HUVECs were transfected with IFIT1 small interference fragment,after 24h,with or without LPS treatment for another 24h,qRT-PCR and WB were used to detect the transfection efficiency of IFIT1,and the mRNA and protein expression levels of IL-1?,IL-6,TNF-? and NF-?B were detected as well.RESULTS1.LPS Upregulated IFIT1 Expression in HUVECsHUVECs were treated with different concentrations of LPS.The results showed that LPS could increase the expression of IFIT1 in a concentration dependent manner.The effect of 1000ng/mL was more obvious,then 1000ng/mL of LPS was used to treat HUVECs for 0,6,12,24h respectively.The results showed that LPS could upregulate the levels of IFIT1 mRNA and protein expression in a time dependent manner.2.Overexpression of IFIT1 Enhanced LPS-Induced Inflammatory Response in HUVECsWe found that overexpression of IFIT1 with recombinant plasmids upregulated IFIT1 mRNA expression in HUVECs by 1358%and protein expression by 389%.LPS could upregulate IL-1?,IL-6,TNF-? and NF-?B expression in HUVECs and its effect was further enhanced by treatment with recombinant plasmids overexpression of IFIT1.3.Knockdown of IFIT1 Attenuated LPS-Induced Inflammatory Response in HUVECsWe found that treatment with siRNA for IFIT1 significantly downregulated expression of IFIT1 mRNA and protein,treatment with LPS increased mRNA and protein expression of IL-1?,IL-6,TNF-? and NF-?B and LPS-induced upregulation of IL-1 p,IL-6,TNF-? and NF-?B expression was markedly attenuated by treatment with siRNA-IFIT1.ConclusionLPS upregulated IFIT1 expression and induced IL-1?,IL-6,TNF-? and NF-?B expression in HUVECs was likely mediated through the IFIT1 pathway.The results suggested that IFIT1 was involved in LPS-induced inflammatory response,and indicated a molecular mechanism of LPS in the inflammation.Interventions targeting IFIT1 might be useful in reducing inflammation associated with atherosclerosis.