内脏脂肪素诱导血管内皮细胞损伤的MAPK信号通路及丹参酮Ⅱ_A磺酸钠的干预作用

Sodium Tanshinone Ⅱ_A Sulfonate Attenuates Visfatin-induced Inflammatory Injury on Human Umbilical Vein Endothelial Cell Through MAPK Pathway

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 effect and mechanism of sodium tanshinone ⅡAsulfonate（STS） on human umbilical vein endothelial cell（HUVEC） inflammatory injury by visfatin. Method： The human umbilical vein endothelial cells（HUVECs） were cultured in vitro,after the cells were treated with visfatin（250 μg·L^-1）for 4 hours,STS was added to the cells in different concentration of（30,60,120 mg·L^-1） for 24 hours. The levels of inflammatory cytokines as high sensitivity C reactive protein（hs-CRP）,tumor necrosis factor α（TNF-α）and metal matrix protein（MMP）-9 were determined by enzyme-linked immunosorbent assay（ELISA）. The activation of mitogen-activated protein kinases（MAPKs） was assayed by Western blotting. In order to evaluate the role of MAPKs in the inflammatory injury by visfatin,HUVECs were pretreated with MAPKs inhibitor,and visfatin was added after the pretreatment,then the levels of hs-CRP,TNF-α and MMP-9 was measured. Result： Visfatin led to an increase level of hs-CRP,TNF-α and MMP-9,while the increase of above mentioned cytokines induced by visfatin could be inhibited by STS in a dose dependent way. STS could also inhibit the activation of p38 MAPK and extracellular signalregulated kinase（ERK）,but no significant inhibition of Jun N-terminal kinase（JNK）.The p38 MAPK inhibitor SB203580（25 μmol·L^-1）,ERK inhibitor PD98059（25 μmol·L^-1） and JNK inhibitor SP600125（25 μmol ·L^-1） markedly decreased visfatin-induced enhance in hs-CRP,TNF-α and MMP-9expression. Conclusion： Inflammatory response of HUVEC could be induced by visfatin,the mechanism may be related to high expression of inflammatory cytokines,which may be mediated by MAPK phosphorylation signaling pathway. STS attenuates visfatin-induced inflammatory injury by interfering with the modulation of MAPK signal pathway.