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目的:研究内脏脂肪素(visfatin)参与血管内皮细胞损伤的机制及丹参酮ⅡA磺酸钠保护作用的机制。方法:培养人脐静脉血管内皮细胞(HUVEC,1×105/mL),用visfatin(250μg·L-1)刺激HUVEC 4 h,以丹参酮ⅡA(30,60,120 mg·L-1))干预24 h,酶联免疫吸附法(ELISA)检测细胞上清液中高敏C反应蛋白(hs-CRP)、肿瘤坏死因子-α(TNF-α)、金属基质蛋白-9(MMP-9)水平,用Western blotting方法观察丝裂原激活蛋白激酶(MAPKs)信号转导通路中p38丝裂原激活蛋白激酶(p38MAPK)、细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)的活化情况。分别用p38 MAPK,ERK,JNK特异性抑制剂进行预处理细胞,再给予visfatin(250μg·L-1)刺激4 h,检测细胞上清液中hs-CRP,TNF-α,MMP-9水平。结果:与正常组比较,模型组细胞活力显著降低,细胞上清炎症因子hs-CRP,TNF-α,MMP-9显著增高,细胞内p38 MAPK,ERK,JNK磷酸化蛋白表达显著增高。与模型组比较,丹参酮ⅡA呈剂量依赖性地降低visfatin导致的hs-CRP,TNF-α,MMP-9高表达,并能抑制p38MAPK,ERK1/2磷酸化活化,但对JNK无显著抑制作用。用p38 MAPK,JNK,ERK1/2特异性抑制剂预刺激,可阻止visfatin诱导的hs-CRP,TNF-α,MMP-9细胞因子大量表达。结论:visfatin可能通过激活MAPK磷酸化信号通路,诱导炎症细胞因子高表达,促使血管内皮细胞炎症反应,丹参酮ⅡA通过抑制MAPK信号通路p38,JNK的激活,抑制visfatin诱导的炎症因子高表达,从而减少血管内皮细胞损伤。
OBJECTIVE: To study the mechanism of visfatin involved in vascular endothelial cell injury and the protective effect of tanshinone Ⅱ A sulfonate. Methods: Human umbilical vein endothelial cells (HUVEC, 1 × 105 / mL) were cultured with HUVEC for 24 h with 250 μg · L -1 of visfatin and treated with tanshinone Ⅱ A (30, 60, 120 mg · L -1) for 24 h The levels of hs-CRP, TNF-α and MMP-9 in supernatants were detected by enzyme-linked immunosorbent assay (ELISA) blotting was used to observe the activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in mitogen-activated protein kinase (MAPKs) signal transduction pathway. The cells were pretreated with specific inhibitor of p38 MAPK, ERK and JNK respectively, and then stimulated with visfatin (250μg · L-1) for 4 h. The levels of hs-CRP, TNF-α and MMP-9 in the cell supernatant were measured. Results: Compared with the normal group, the cell viability of the model group was significantly decreased. The expressions of hs-CRP, TNF-α and MMP-9 in the supernatant of the model group were significantly increased. The phosphorylation of p38 MAPK, ERK and JNK in the model group was significantly increased. Compared with the model group, tanshinone ⅡA reduced the visfatin-induced high expression of hs-CRP, TNF-α and MMP-9 in a dose-dependent manner and inhibited the phosphorylation of p38MAPK and ERK1 / 2, but had no significant inhibitory effect on JNK. Pre-stimulation with p38 MAPK, JNK and ERK1 / 2 specific inhibitors could prevent visfatin-induced large-scale expression of hs-CRP, TNF-α and MMP-9 cytokines. CONCLUSION: Visfatin can induce the inflammatory cytokines to be highly expressed by activating MAPK phosphorylation signal pathway and promote the inflammatory reaction of vascular endothelial cells. Tanshinone IIA can inhibit visfatin-induced high expression of inflammatory cytokines by inhibiting the activation of MAPK signal pathway p38 and JNK, thereby reducing Vascular endothelial cell injury.