鹿茸多肽对过氧化氢所致血管内皮细胞损伤的保护作用

Protective effect of velvet antler polypeptides on hydrogen peroxide-induced injury to endothelial cells

目的观察鹿茸多肽对H2O2诱导的血管内皮细胞损伤的保护作用及其作用机制。方法体外培养人静脉血管内皮细胞EVC-304加入鹿茸多肽20,40和80 mg·L-1,培养24 h后再加入H2O2100μmol·L-1作用12 h。MTT法检测细胞存活率,Hoechst333258染色法观察EVC-304细胞形态,用试剂盒测定超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,Western蛋白质印迹法检测细胞内胱天蛋白酶3和热激蛋白70(HSP70)的表达。结果同正常组相比,H2O2使细胞存活率明显降低(P<0.01),细胞染色质固缩,细胞核致密浓染,且胞核变小浓集,SOD活性降低,MDA含量升高(P<0.01),胱天蛋白酶3和HSP70表达升高(P<0.01)。与H2O2组相比,鹿茸多肽20,40和80 mg·L-1组细胞存活率明显增加(P<0.01),凋亡率由(25.3±1.0)%下降至(15.2±1.2)%,(10.3±0.9)%和(7.9±1.4)%(P<0.01),SOD活性升至19.2±0.5,22.3±1.7和(24.9±0.6)kU·g-1蛋白(P<0.01),MDA含量降至1.51±0.2,1.48±0.3和(1.02±0.1)μmol·g-1蛋白(P<0.01),细胞内胱天蛋白酶3和HSP70表达显著下降(P<0.01)。结论鹿茸多肽对H2O2诱导的血管内皮细胞损伤具有明显的保护作用,其机制可能与改善细胞内的氧化应激水平有关。

OBJECTIVE To investigate the protective effect of velvet antler polypeptides（VAP）on hydrogen peroxide（ H2 O2 ）-induced injury in vascular endothelial cells and the possible mechanism.METHODS The EVC-304 cells cultured invitrowere incubated with H2 O2 for another 12 h after pretreat-ment with VAP 20,40 and 80 mg·L-1 for 24 h. Cell viability was determined by MTT assay, Hoechst333258 staining was used to observe cell morphology,the activity of superoxide dismutase （SOD）and the level of malondialdehyde（ MDA）were detected with kits and the expression of heat shock protein（HSP70）and caspase 3 was detected by Western blotting. RESULTS Compared with the normal control group,the cell survival rate was decreased significantly in H2 O2 injury group（ P ﹤0.01）,cell shrinkage,chromatin condensation,and nuclear fragmentation were seen,the intracellular SOD activity decreased while MDA content increased（P﹤0.01）,and caspase 3 and HSP70 expression increased（P﹤0.01）. Compared with H2 O2 group,the cell survival rate in VAP 20,40 and 80 mg·L-1 pre-treatment groups increased significantly（P﹤0.01）,the apoptosis ratio declined from（25.3±1.0）% to （15.2±1.2）%,（10.3±0.9）% and（7.9±1.4）%（P﹤0.01）,the SOD activity increased to 19.2±0.5,22.3± 1.7 and（24.9±0.6）kU·g-1 protein（P﹤0.01）,MDA concentration decreased to 1.51±0.2,1.48±0.3 and （1.02±0.1）μmol·g-1 protein（P﹤0.01）,and the expression of caspase 3 and HSP70 declined significant-ly（P﹤0.01）. CONCLUSION VAP has exert protective effect on H2 O2-induced injury in vascular endothe-lial cells. The possible mechanism might be related to improvement of intracellular oxidative stress level.