Anti-aging Effect Of Ginsenoside Rg1 On T-BHP-induced Premature Senescence And Its Possible Mechanism

Objective: To induce WI-38 cells senescence by tertbutyl-hydroperoxide (t-BHP) and establish a model of premature senescence, to investigate the anti-aging effect of the ginsenoside Rgl on the model of premature senescence induced by t-BHP.Method: To induce premature senescence, from 30 population doubling (PD), the WI-38 cells were treated with four times of 100umol/L t-BHP for 1 h at every two PDs. Then the changes of cell shape and ultrastructure were observed by microscope and transmission electron microscopy, and the percent of cells in the G1 phase and SA-β-galactosidase activity were measured with flow cytometry and cytochemistry staining, respectively. So we established a model of premature senescence. After that, the WI-38 cells were divided into fourgroups and different concentrations of Rgl were randomly added into the four groups' MEM. After four stresses of t-BHP, we compared cells in three aspects above. Further more, we analyzed cyclin E, cyclin D1, CDK4, CDK2, p21 and p16 levels by western blot, the length of telomere by southern blot and telomerase activity by PCR for discovering the possible molecular mechanisms of anti-senescence.Result: After four stresses with 100umol/L t-BHP, WI-38 cells presented the character of senescence. The cell proliferation and division was ceased, cell became larger and flat; the number of secondary lysosome rised, the percent of cells in the G1 phase of the cell cycle and activity of SA-β-galactosidase were increased. All these demonstrated that t-BHP could induce WI-38 cells premature senescence. Rgl can against all these changes. It showed Rgl has the effect of anti-aging. After four stresses with lOOumol/L t-BHP, the expression of CDK4, CDK2, cyclin E was diminished and cyclin Dl, p21 and p16 was elevated. Meanwhile, the length of telomere is shorten. However, cells which had been treated by Rgl against the effect of t-BHP. It showed higher expression of CDK4 and cyclin E, lower expression of cyclin D1, p21 and p16, which is compared with cells only treated with t-BHP. Simultaneously, telomere shortening were reduced. It was surprised to detect telomerase activity in cells in Rgl treated groups, while not detecting telomerase activity in other groups.Conclusion: t-BHP can be used to induce WI-38 cells senescence and to establish a model of premature senescence. In senescent WI-38 cells, the expression of CDK4, cyclin E is diminished and cyclin D1, p21 and p16 is elevated. All their expressions are induced by telomere shortening. Surprisingly, Rgl has the anti-senescent effect. It is postulated that Rgl has the effect to activating telomerase, which reduces telomere shortening. So it changes the expression of p21, p16 and so on. Therefore, cells in Rgi treated groups delay the state of irreversible G1 arrest.