Effect Of Homocysteine On Expression Of Interleukin-6 In Cultured Rat Vascular Smooth Muscle Cells And Its Underling Mechanism

Elevated plasma levels of homocysteine (Hey) is an established risk factor for atherosclerotic disease. However, the underlying molecular mechanisms are not fully elucidated. Recent studies suggested that atherosclerosis is an inflammatory process and that cytokines played important roles in this process. Interleukin-6 (IL-6) is a multifunctional proinflammatory cytokine and it could be used as marker of the proinflammatory potential of vascular smooth muscle cells (VSMCs). Nuclear factor- kappa B (NF- K B) is a family of transcription factors that was originally identified in B cells but the novel data showed it was also detectable in VSMCs. NF- K B has been indicated to play a role in the gene expression of inflammatory cytokines and VSMCs proliferation. Abnormal activation of NF- K B is a hallmark of many chronic inflammatory and vascular disease and it also may interfere with initiation of atherosclerotic disease. Hey has been demonstrated to increase IL-6 production in monocyte and induce NF- K B activation in VSMCs, which suggested that Hey may be a inflammatory stimuli. The effect of Hey on the expression of IL-6 in atherognesis and the underling mechanisms are still uncertain till now. We hypothesized that Hey may stimulate IL-6 expression in VSMCs through activation of NF- K B and which may be one of the mechanisms that Hey promote atherogenesis. Part I Effect of homocysteine on IL-6 expression in rat vascular smooth muscle cellsObjective: To determine whether Hey contribute to the pathogenesis of atherosclerosis by increasing the expression of IL-6 mRN A and the production of protein by means of investigating the effect of Hey on rat VSMCs.Methods: Rat VSMCs were stimulated with Hey. Cell ELISA was performed to measure the expression of IL-6 protein. Semiquantitative RT-PCR was used to detect the IL-6 mRN Aexpression.Results: 1. Hey concentration-dependently increased the production of IL-6 protein in rat VSMCs. Compared with control, Hey increased IL-6 production to 2.4-fold at O.lmmol/1 (P<0.05) and 3.4-fold at 0.25mmol/l (P<0.01). 2. Treatment with 0.25mmol/L Hey for 6h began to increase IL-6 production (PO.05), the effect reached maximum at 24h (P<0.01). 3. Hey also significantly enhanced IL-6 mRNA expression with a concentration-dependent and time-dependent pattern. After treating with 0.25mmol/L Hey for 12h, the effect was maximum (PO.01). Hey induced IL-6 mRNA expression in VSMCs to 2.39-fold at O.Olmmol/L (P<0.05), 2.38-fold at 0.05mmol/L (P<0.05^ 3.33-fold at O.lmmol/L (P<0.01) and 3.71-fold at 0.25mmol/LPart II Role of NF- K B on the expression of IL-6 in rat vascular smooth muscle cells induced by homocysteineObjective: To explor the intracellar signalling mechanism, we investigate the effect of Hey on NF- K B activity in VSMCs and role of NF- K B on the expression of IL-6 induced by Hey.Methods: Electrophoretic mobility shift assay(EMSA) was used to examine NF- K B activity. Semiquantitative RT-PCR was used to detect the IL-6 mRNA expression.Results: 1. Hey could rapidly induce the activation of NF- K B in rat VSMCs. Baseline NF- K B nuclear binding in VSMCs increased more than 1.76-fold at 30min(P<0.05^ 1.91-fold at lh(P<0.05) and 1.84-fold at 2h(P<0.05) following stimulation with 0.25mmol/L Hey. 2. PDTC, one of the inhibitor of NF- K B, could significantly inhibit the expression of IL-6 mRNA in VSMCs stimulated by Hcy(P<0.05). Conclution1. Hey significantly increased IL-6 mRNA expression and production in rat VSMCs with a concentration-dependent (0.01-0.25mmol/L) and a time-dependent (0-48h) pattern.2. Hey could induce the activation of NF- K B in VSMCs.3. The effect of Hey on the exprssion of IL-6 in VSMCs is exerted through NF- K B activation pathway.