| Senescence is a universal phenomenon, and anti-senescence is a popular project in scientific research. According to the free radical theory for aging, degenerative change in the aging process is mainly caused by the harmful effects of free radicals from cell metabolism. Accordingly, antioxidant is the first step to prevent the free radical damage. Therefore, finding a natural antioxidant medicine to achieve anti-senescence effect by scavenging free radicals has become hot. Tricholoma lobayense Heim is a kind of precious edible fungus in development. Based on the previous work, we found that the polysaccharide fraction (TLH-3) extracted from Tricholoma lobayense owned strong scavenging ability on free radicals with IC50values similar to Vc, which significantly inhibited erythrocyte hemolysis and MDA in vivo. Therefore, whether the polysaccharide TLH-3possessed considerable anti-senescence activity remains to be further studied. In this study, the effects of TLH-3on the cell cycle and aging related proteins were explored through the establishment of cellular and animal aging models. The effect and mechanism of TLH-3were studied in vivo and in vitro. The significance of this research is to provide a scientific basis for human longevity and development of polysaccharide drugs.(1) The protection effect of TLH-3on t-BHP induced cell apoptosis were detected. The human embryonic lung fibroblast (HELF) cells were used in this study. The effect of TLH-3on the growth of HELF cells cultured with t-BHP was detected by MTT assay. The effect of TLH-3on the morphology of HELF cells cultured with t-BHP were observed by confocal laser scanning microscope with Hoechst33342/PI staining method. The protection of TLH-3on t-BHP induced cell apoptosis were determined by Annexin V-FITC/PI staining with flow cytometry. According to the results, compared with t-BHP induced group, the viability of cells treated with TLH-3was higher, and the cell survival increased with the concentration of TLH-3arising. The apoptotic cells and necrotic cells of t-BHP induced group increased significantly, in contrast to the control group. The cell growth treated with TLH-3improved and was positively correlated with the dosage of TLH-3. These indicated that TLH-3could protect cells from apoptosis.(2) To study the effects of TLH-3on the cell senescence, the HELF cell aging model was set up firstly by t-BHP induction. And different concentrations of TLH-3were added to interfere in the process at the same time. The cell cycle and the expression of p21were detected by flow cytometry and western blot respectively. Contrast with aging model group, TLH-3could decrease the content of G1phase of cell cycle, which indicated that TLH-3could slow down the cellular senescence caused by oxidative damage.(3) To study the effects of TLH on the aging mice, the aging model mice were set up firstly by D-Gal induction. Meanwhile, the effects of different concentrations of TLH on the aging model mice were detected by intragastric administration. The hydroxyproline content of the skin, the SOD activity and MDA content of serum, heart and liver were detected. HE staining method was used to observe the pathological changes in mice. The expression of p21protein of kidney tissue was analyzed by the method of immunohistochemistry. The results showed that, compared with the aging mice, hydroxyproline content increased, the p21expression levels of mice in each group has no significant difference. The activity of SOD increased and the content of MDA reduced in mice significantly. |
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