激活腺苷酸活化蛋白激酶α在苯扎贝特抑制氧化低密度脂蛋白诱导血管内皮细胞炎症中的作用

Bezafibrate inhibits oxidized low-density lipoprotein-induced endothelial cell inflammation via activation of AMP-activated protein kinase α

目的探索激活腺苷酸活化蛋白激酶α(AMPKα)在苯扎贝特(BZF)抑制氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVEC)炎症中的作用。方法将HUVEC与ox-LDL 50 mg·L^(-1)和(或)BZF(25～100μmol·L^(-1))共孵育24 h,荧光定量PCR检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)m RNA的表达。Western蛋白印迹法检测TNF-α,IL-6,ICAM-1,VCAM-1和NF-κB(P65)的磷酸化水平。采用分子对接软件Autodock探索AMPK用于进行分子对接的蛋白结构是AMPKα与β复合物,与BZF之间的相互关系。将原代HUVEC与BZF 25～100μmol·L^(-1)孵育24 h后,Western蛋白印迹法检测p-AMPKα和AMPKα表达水平。BZF与AMPKα抑制剂Compound C5μmol·L^(-1)共孵育HUVEC 24 h,观察AMPKα对BZF抑制ox-LDL诱导HUVEC炎症的影响。结果与细胞对照组比较,ox-LDL刺激24 h,HUVEC的TNF-α,IL-6,ICAM-1,VCAM-1和p-P65的表达明显升高(P<0.01);不同浓度BZF显著抑制ox-LDL诱导的HUVEC中TNF-α,IL-6,ICAM-1,VCAM-1和p-P65表达的升高(P<0.01)。分子对接模拟显示,BZF与AMPK结合力为-8.66 kcal·mol-1。与细胞对照组比较,BZF能显著升高HUVEC中p-AMPKα的表达(P<0.01)。与ox-LDL+BZF处理组比较,AMPKα抑制剂ox-LDL+BZF+Compound C组TNF-α,IL-6,ICAM-1和VCAM-1表达显著升高(P<0.01)。结论 AMPKα的激活在BZF抑制ox-LDL诱导HUVEC炎症中发挥重要作用。

OBJECTIVE To investigate the involvement of AMP-activated protein kinase α(AMPKα) in the inhibitory effect of bezafibrate(BZF) on oxidized low-density lipoprotein(ox-LDL)-induced human umbilical vein endothelial cell(HUVEC) inflammation. METHODS HUVECs were incubated with ox-LDL 50 mg·L^-1 with or without BZF(25-100 μmol·L^-1) for 24 h. The expressions of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were detected by quantitative PCR(q PCR). The expression levels of TNF-α, IL-6,ICAM-1, VCAM-1 and the phosphorylation level of NF-κB(P65) were measured by Western blotting.The interaction of BZF and AMPKα was detected by Autodock Vina. After treatment with BZF(25-100 μmol·L^-1) for 24 h, the protein expression levels of p-AMPKα and AMPKα were determined by Western blotting. The HUVECs were exposed to BZF 100 μmol·L-1 and AMPKα inhibitor Compound C(CC) 5 μmol·L^-1 for 24 h, and then the inhibitory effects of BZF on ox-LDL induced HUVECs inflammation and dysfunction were analyzed. RESULTS Treatment with ox-LDL significantly enhanced the expression levels of TNF-α, IL-6, ICAM-1, VCAM-1 and p-P65(P<0.01), while BZF(25-100 μmol·L^-1)significantly reduced and inhibited the expressions of TNF-α, IL-6, ICAM-1 and VCAM-1(P<0.01). The binding energy between AMPKα and BZF was-8.66 kcal·mol-1. Treatment with BZF(25-100 μmol·L^-1)significantly enhanced the expression level of p-AMPKα in HUVECs(P<0.01). Co-treatment with CC significantly increased the expression levels of TNF-α, IL-6, ICAM-1 and VCAM-1 which were inhibited by BZF(P<0.01). CONCLUSION Activation of AMPKα might play an important role in the inhibitory effect of BZF on ox-LDL-induced HUVEC inflammation.