Objectives Senescence of endothelial cells has been proposed to play an important role in endothelial dysfunction andatherogenesis. In the present study we aimed to investigate whether Tongxinluo (TXL) capsule protects human umbilical vein endothelial cells (HUVECs) from H2O2-induced endothelial senescence.
Methods Solation, culture and identification of HUVECs: With the informed consents of puerperants, the normal fetal umbilical cords were obtained through uterine-incision delivery in Third Affiliated Hospital, Sun Yat-sun University. HUVECs were isolated by Percoll density gradient centrifugation from fetal umbilical cords with digestion of collagenase type I perfusion, and then suspended in Medium 199 cultured in 0.05 volume fraction of CO2 incubator at 37°C. After the HUVECs were identified by flow cytometry with the cell marker CD31, The second or third passage was used for study. The exponentially growing HUVECs were plated at a cell density of 1×105/well in 6-well plate and cultured overnight at 37 °C. The next day, the medium was changed with M199 supplemented with 2% FBS for at least 8 h to starve the cells. Then the cells were exposed to various concentrations of H2O2 to induce premature senescence. After 1 h, the medium was replaced with normal medium. Different concentration of TXL (0.1, 0.5, 1.0, and 2.0 mg/ml) was added in the media 30 min before the induction of senescence by addition of H2O2. Finally the cells were harvested in indicated time for Western blot analysis and real-time polymerase chain reaction (PCR). The MDA level and SOD activity were determined using commercially available kits following the manufacturer's instructions. Intracellular ROS generation was monitored by flow cytometry using peroxide sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (H2DCFDA, Invitrogen).
Results (1) Treatment with H2O2 caused significant increase in intracellular thiobarbituric acid reactive substances (TBARS) level (p<0.01 vs untreated control), while pre-incubation with TXL (0.5, 1.0 mg/ml) markedly attenuated the increase (p<0.05). Compared to control group, treatment with H2O2 decreased the activity of SOD to 50.4±6.9%. However, pre-incubation with TXL (0.1, 0.5, 1.0, and 2.0 mg/ml) significantly increased SOD activity compared with H2O2 alone treated group (p<0.01).
(2) Real-time PCR analysis showed that at the time point of 24 h, SOD1 mRNA in the 60mmol/l H2O2 treated group decreased by 1.99 fold, compared with untreated group (p<0.01). However, SOD1 mRNA increased by 1.54, 1.77, 1.75 fold in 0.1, 0.5, 1.0mg/ml TXL pretreated group respectively, compared with H2O2 alone treated group (p<0.01).
(3) Western blotting results demonstrated that the level of SOD1 protein reduced in H2O2 treated group compared to untreated group after 24, 48 and 72 h incubation. However, TXL reversed the decreased expression of SOD1 protein in HUVECs treated with H2O2 in a dose-dependent manner As the control, TXL alone did not significantly change the expression of SOD1 at either mRNA or protein level.
(4) TXL alone did not change the ROS generation. However, compared to the nontreated control group, 60 mmol/l H2O2 significantly increased DCF fluorescence whereas pre-treatment with 1.0 mg/ml TXL markedly inhibited the production of ROS induced by H2O2.
Conclusions our data demonstrate that ginseno side TXL modulates redox status such as upregulating SOD1 expression, scavenging ROS, and decreasing the peroxidation to prevent the cellular senescence in HUVECs.