摘要
目的研究1-磷酸鞘氨醇(S1P)后适应对人脐静脉内皮细胞(HUVEC)缺氧/复氧损伤的保护作用,并探讨其作用机制。方法建立HUVEC缺氧/复氧损伤模型,将HUVEC细胞分为5组,正常对照组、S1P低剂量组、S1P中剂量组、S1P高剂量组以及缺氧/复氧损伤(H/R)组。MTT法检测细胞存活率;流式细胞术分析测定细胞凋亡率;比色法测定细胞培养液中总超氧化物歧化酶(total superoxide disnmtase,TSOD)、铜锌超氧化物歧化酶(copper/zinc superoxide dismutase,CuZn-SOD)和锰超氧化物歧化酶(manganese superoxide dismutase,Mn-SOD)活力以及一氧化氮(nitric oxide,NO)、丙二醛(malondialdehyde,MDA)含量;荧光显微镜下观察线粒体膜电位;Western blot测定HUVECs细胞Bcl-2/Bax、eNOS蛋白的表达水平。结果与H/R组相比,S1P低、中、高剂量组可明显增加HUVECs细胞缺氧/复氧损伤后细胞存活率;均明显升高T-SOD活力、CuZn-SOD活力、Mn-SOD活力,降低MDA含量,明显升高NO含量及增加eNOS蛋白表达,降低凋亡率,抑制线粒体膜电位下降。结论 S1P能够使H/R损伤的HUVECs细胞凋亡率降低,且呈一定的浓度依赖性。S1P对H/R损伤的HUVECs细胞凋亡的保护作用可能与降低细胞内MDA含量,提高细胞内SOD活力,提升线粒体膜电位,增强抗凋亡蛋白Bcl-2/Bax的表达有关。
Aim To investigate the protective effects of sphingosine 1-phosphate(S1P) postconditioning on hypoxia/reoxygenation(H/R) injury in human umbilical vein endothelial cells(HUVEC) and its mechanisms.Methods HUVECs cells were divided into five groups:normal(control) group,S1 P low concentration group(L),S1 P medium concentration group(M),S1 P high concentration group(H) and H/R group.MTT method was used to measure cell survival.Using flow cytometric analysis,the rate of cell apoptosis was determined.The activities of total superoxide dismutase(T-SOD),copper/zinc superoxide dismutase(CuZn-SOD),manganese superoxide dismutase(Mn-SOD) activity,nitric oxide(NO) and malondialdehyde(MDA) content in cell culture medium were measured with colorimetry.Mitochondrial membrane potential in cells was observed with fluorescence microscope.Bax/Bcl-2,eNOS protein expression levels in HUVECs cells were observed with Western blot.Results Compared with H/R group,S1 P low,medium and high concentrations in the intervention group could significantly increase the cell survival rate after H/R injury,and increase activity of T-SOD,CuZn-SOD,Mn-SOD and decrease content of MDA.Moreover,S1 P could significantly increase NO content and increase eNOS protein expression,decrease apoptosis rate and inhibit the reduction of mitochondrial membrane potential.Conclusions S1 P can decrease cell apoptosis rate of HUVECs after H/R injury with a certain concentration dependence.The protection of S1 P for cell apoptosis of HUVECs after H/R injury may be related to decreasing the intracellular MDA content and improving intracellular SOD activity,increasing mitochondrial membrane potential and enhancing expression of Bcl-2,anti-apoptotic protein.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2016年第2期184-189,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81173058)
国家自然科学基金青年基金资助项目(No 81502419)
天津市自然科学基金资助项目(No 10JCZDJC20900)
