Ying Xie1, Zhi-Hua Liu1, Xiao-Yun Li1, Yan-de Zhou1, Xingshun Xu2, Li-Fang Hu3, Yan-Lin Zhang4, Chun-Feng Liu5. 1. Department of Endocrinology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China. 2. Department of Neurology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China. 3. Institute of Neuroscience, Soochow University, Suzhou 215123, China. 4. Department of Neurology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China. 5. Department of Neurology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China; Institute of Neuroscience, Soochow University, Suzhou 215123, China. Electronic address: liucf@suda.edu.cn.
Abstract
AIMS: Humanin (HN) is known for its anti-apoptotic functions in neuronal cells. In this study, we sought to investigate the protective effect of [Gly14]-Humanin (HNG) in high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability, DNA chromatin morphology was assessed using Hoechst 33342 staining, and the generation of intracellular reactive oxygen species (ROS) was assessed using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). The expression of poly ADP-ribose polymerase (PARP), the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2 were examined using western blot analysis. The mRNA level of bax and bcl-2 were detected by quantitative Real-Time PCR. RESULTS: Compared with treatment with HG 72h, pretreatment with HNG for 3h significantly increased cell viability (P<0.001), reduced nuclear fluorescence of HUVECs (P<0.05), the levels of cleaved PARP (P<0.05), ROS formation (P<0.05) and the ratio of bax/bcl-2 (P<0.05) compared with treatment with HG for 72h. Quantitative Real-Time PCR showed that mRNA level of bax reduced (P<0.05) and mRNA level of bcl-2 increased (P<0.05) after pretreatment with HNG. CONCLUSIONS: Our results imply that HNG can protect HUVECs from apoptosis induced by HG through the bax/bcl-2 pathway.
AIMS: Humanin (HN) is known for its anti-apoptotic functions in neuronal cells. In this study, we sought to investigate the protective effect of [Gly14]-Humanin (HNG) in high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability, DNA chromatin morphology was assessed using Hoechst 33342 staining, and the generation of intracellular reactive oxygen species (ROS) was assessed using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). The expression of poly ADP-ribose polymerase (PARP), the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2 were examined using western blot analysis. The mRNA level of bax and bcl-2 were detected by quantitative Real-Time PCR. RESULTS: Compared with treatment with HG 72h, pretreatment with HNG for 3h significantly increased cell viability (P<0.001), reduced nuclear fluorescence of HUVECs (P<0.05), the levels of cleaved PARP (P<0.05), ROS formation (P<0.05) and the ratio of bax/bcl-2 (P<0.05) compared with treatment with HG for 72h. Quantitative Real-Time PCR showed that mRNA level of bax reduced (P<0.05) and mRNA level of bcl-2 increased (P<0.05) after pretreatment with HNG. CONCLUSIONS: Our results imply that HNG can protect HUVECs from apoptosis induced by HG through the bax/bcl-2 pathway.