p38MAPK在ox-LDL诱导的血管内皮细胞损伤中的作用研究

Effect of p38MAPK in vascular endothelial cell injury induced by oxidized low density lipoprotein

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)在氧化低密度脂蛋白(ox-LDL)诱导的血管内皮细胞损伤中的作用。方法人脐带静脉血管内皮细胞HUVEC-12分为对照组、ox-LDL组、实验1组、实验2组、实验3组。ox-LDL组、实验1组、实验2组、实验3组细胞分别用ox-LDL诱导处理,实验1组、实验2组、实验3组用不同浓度(5μmol/L、15μmol/L、30μmol/L)的p38MAPK抑制剂预处理。Western blot测定各组细胞中p38MAPK磷酸化水平,二硝基苯肼法、硫代巴比妥酸法、黄嘌呤氧化法分别测定培养液上清中乳酸脱氢酶(LDH)、丙二醛(MDA)和匀浆液中超氧化物歧化酶(SOD)含量,流式细胞术测定各组细胞凋亡情况,Western blot法测定各组细胞中Cleaved Caspase-3蛋白水平,比色法测定培养液中一氧化氮(NO)含量,ELISA法测定培养液中细胞间黏附分子-1(ICAM-1)、白细胞介素-1β(IL-1β)含量。结果 ox-LDL组细胞中p38MAPK磷酸化水平明显高于对照组(P<0.05)。实验1组、实验2组、实验3组细胞中p38MAPK磷酸化水平明显低于ox-LDL组(P<0.05)。ox-LDL组培养液中LDH、MDA含量明显升高,匀浆液中SOD水平明显降低,Caspase-3介导的细胞凋亡明显增多,培养液中NO含量明显降低,同时培养液中ICAM-1、IL-1β含量明显升高,与对照组相比,差异均具有统计学意义(P<0.05)。实验1组、实验2组、实验3组细胞培养液中LDH、MDA含量明显降低,匀浆液中SOD水平明显升高,Caspase-3介导的细胞凋亡明显减少,培养液中NO含量明显升高,同时培养液中ICAM-1、IL-1β含量明显降低,与ox-LDL组相比,差异均具有统计学意义(P<0.05)。结论 ox-LDL诱导血管内皮细胞p38MAPK磷酸化,而抑制p38MAPK后可以减少ox-LDL诱导的血管内皮细胞氧化损伤,减少细胞凋亡。

Objective To investigate the effect of p38mitogen-activated protein kinases(p38MAPK)in vascular endothelial cell injury induced by oxidized low density lipoprotein(ox-LDL).Methods Human umbilical vein endothelial cells12(HUVEC-12)were divided into control group,ox-LDL group,and test groups1,2and3.The ox-LDL group and test groups1,2and3were given inducing treatment with ox-LDL,and then test groups1,2and3were given pretreatment with p38MAPK inhibitor in different doses respectively(5μmol/L,15μmol/L,30μmol/L).The phosphorylation level of p38MAPK was determined by using Western blotting assay in all groups.The levels of lactic dehydrogenase(LDH),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected respectively by using dinitrophenylhydrazine(DNPH)method,thiobarbituric acid(TBA)method and xanthineoxidation(XTO)method.The apoptosis of HUVEC-12was determined by using flow cytometry(FCM),and level of Cleaved Caspase-3was detected by using Western blotting assay in all groups.The content of nitric oxide(NO)was detected by using chromatoptometry,and content of intercellular adhesion molecule-1(ICAM-1)and interleukin-1β(IL-1β)were detected by using ELISA.Results The phosphorylation level of p38MAPK was significantly higher in ox-LDL group than that in control group(P<0.05),and was significantly lower in test groups1,2and3than that in ox-LDL group(P<0.05).The content of LDH and MDA increases significantly,SOD level decreased significantly,HUVEC-12apoptosis mediated by Caspase-3increased significantly,NO content decreased significantly,and content of ICAM-1and IL-1βincreased significantly in ox-LDL group compared with control group(P<0.05).The content of LDH and MDA decreases significantly,SOD level increased significantly,HUVEC-12apoptosis mediated by Caspase-3decreased significantly,NO content increased significantly,and content of ICAM-1and IL-1βdecreased significantly in test groups1,2and3compared with ox-LDL group(P<0.05).Conclusion The phosphorylation of p38MAPKin vascular endothelial cells can be induced by ox-LDL,and oxidative damage of vascular endothelial cells induced by ox-LDL can be relieved and apoptosis can be decreased after inhibiting p38MAPK.