Mechanism Of Ginsenoside RG1 To Delay Hematopoietic Stem Cell And Progenitor Cell Senescence

Objective: New studies suggest that adult stem cell senescence is the root cause of organism aging. So looking for the methods of re-activate the aged stem cell and regulate its differentiation is essential to delay senescence and prevent senile disease. Hematopoietic stem cell and progenitor cell(HSC/HPC) is the most indepth study cell as a kind of adult stem cell. It has been demonstrated that HSC/HPC aging is closely related to organism aging and kinds of senile diseases.Ginseng is an important country medicine be used to replenish qi, Ginsenoside is its main pharmaceutical component, of which Rg1 is an important anti-aging ingredient of ginseng. It has the function of prolonging the organism and cell life. However, until now, It has not seen the report about the anti-aging effect of ginseng on stem cell. In this study, it takes the latest techniques of stem cell closely integrated with the anti-aging theory and the stem cell knowledge of traditional medicine. This study aimed at building aged model through HSC/HPC serial transplantation in vivo and replicate HSC/HPC senescence in vitro and investigating the possible mechanisms of Rg1 to delay HSC/HPC senescence, then it can provid the guidance in theory and experiment to search the methods delaying HSC/HPC senescence.Methods:1. Sca-1⁺HSC/HPC was isolated and purified by magnetic activated cell sorting (MACS). The purity and activity of separated cells was analysed by flow cytometry (FCM), immunofluorescence, trypan blue staining and CFU-Mix.2. Sca-1⁺ HSC/HPC were induced aging by tert-butylhydroperoxide(t-BHP) to establish the aged model in vitro. Study the biology effect of Rg1 to delay aged cells in vitro.3. HSC/HPC replicability aged model in vivo was established through the Sca-1⁺ HSC/HPC serial transplantation to receptor mouse radiated lethal dose. After transplantation, the survival rate, the number of CFU-S, spleen index and the indicator in peripheral blood (WBC, HCT and PLT) were observed in receptor mice. The expression of Sry gene in spleen colny forming cells and bone marrow cells of receptor mice was analyzed by PCR. Study the biology effect of Rg1 to delay aged cells in vivo.4. The biological characteristics of aged Sca-1⁺ HSC/HPC in vitro and in vivo were evaluated by senescence-associatedβ-galactosidase (SA-β-gal) staining, mixed hematopoietic progenitor cell culture and cell cycle assay. Study the effect of Rg1 to delay Sca-1⁺ HSC/HPC.5. The expressions of p16^INK4a, p19^Arf, p53 and p21^Cip1/Waf1 mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR). The expressions of P16^INK4a, P21^Cip1/Waf1, CyclinD1, CyclinE, CDK2 and CDK4 protein was examined by western blotting. To observed the biology mechanism of Sca-1⁺ HSC/HPC aging in aged model. Studying the relationship between the ginsenoside Rgl delay and treat Sca-1⁺ HSC/HPC senescence with the p16^INK4a-Rb and p19^Arf-p53-p21^Cip1/Waf1 signaling pathways.6. Telomere length and telomerase activity were detected by southern blotting and TRAP-PCR to observe the relationship between the Sca-1⁺ HSC/HPC aging and the anti-aging effect of ginsenoside Rgl on Sca-1⁺ HSC/HPC with the changes of telomere length and telomerase activity.Results:1. Flow cytometry showed that the percentage of Sca-1⁺ cells in MNCs was only (1.8±1.2)% before MACS, but the purity of separated Sca-1⁺ cells was(87.33±1.25)% after MACS. Immunofluorescence showed that only (2.7±1.4)% cells expressed Sca-1⁺antigen in MNCs before MACS, but (90.86±1.72)% cells expressed Sca-1⁺antigen after MACS. The survival of Sca-1⁺ cells which detected by trypan blue staining was 96%～99%. These results suggest that the separated Sca-1⁺cells have a higher purity and activity. 2. After being cultured with t-BHP for six hours, the Sca-1⁺ HSC/HPC appeared the bioloy character of senescence. The number of Sca-1⁺ HSC/HPC entered G0/G1 phase and the percentage of SA-β-gal positive cells were increased, the ability to forming CFU-Mix was decreased. After Rg1 delay and treat Sca-1⁺ HSC/HPC senescence in vitro aged model, the number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells were decreased, the ability to forming CFU-Mix was increased. The changes of Rg1 delay aged group was significantly higher than Rg1 treat aged group.3. After three transplantation, the Sry gene was positive expression in spleen colony forming cells and bone marrow cells in receptor mice, indicated that reconstructed hematopoietic cells of receptor mice were derived from male donor. With the increasing of transplantation, the survival rate, the number of CFU-S, the spleen index and the indicator in peripheral blood to the Sca-1⁺ HSC/HPC of receptor mice was decreased. Sca-1⁺ HSC/HPC appeared aging character: the percentage of SA-β-gal positive cells and the number of cells entered G0/G1 phase were increased, the number of CFU-Mix was decreased. After Rg1 delay and treat Sca-1⁺ HSC/HPC senescence in vivo aged model, the survival rate and the indicator of peripheral blood was increased in receptor mice. To Sca-1⁺ HSC/HPC of receptor mice, the number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells were decreased, the number of CFU-Mix was increased. The changes of Rg1 delay aged group was significantly higher than Rg1 treat aged group.4. After being cultured with t-BHP for six hours, the expression of p16^INK4a, p19^Arf, p53, p21^Cip1/Waf1mRNA and P16^INK4a, P21^Cip1/Waf1, CyclinD1 protein was up regulated and the expression of CDK4, CDK2, CyclinE protein was down regulated in Sca-1⁺ HSC/HPC. After Rg1 delay and treat Sca-1⁺ HSC/HPC senescence in vitro aged model, the expression of p16^INK4a, p19^Arf, p53, p21^Cip1/Waf1mRNA and P16^INK4a, P21^Cip1/Waf1, CyclinD1 protein was down regulated and the expression of CDK4, CDK2, CyclinE protein was up regulated.5. With the increasing of transplantation, the expression of p16^INK4a, p19^Arf, p53, p21^Cip1/Waf1 mRNA and P16^INK4a, P21^Cip1/Waf1, CyclinD1 protein was up regulated and the expression of CDK4, CDK2, CyclinE protein was down regulated in Sca-1⁺ HSC/HPC of receptor mice. After Rg1 delay and treat Sca-1⁺ HSC/HPC senescence in vivo aged model, the expression of p16^INK4a, p19^Arf, p53, p21^Cip1/Waf1mRNA and P16^INK4a, P21^Cip1/Waf1, CyclinD1 protein was down regulated and the expression of CDK4, CDK2, CyclinE protein was up regulated in Sca-1⁺ HSC/HPC.6. To aged model in vitro and in vivo, the telomere length was shorten and the telomerase activity was decreased in Sca-1⁺ HSC/HPC. After Rg1 delay and treat aged model, the telomere length shorten was reduced and the telomerase activity was increased in Sca-1⁺ HSC/HPC. Conclusion:1. It can successfully isolated and purified Sca-1⁺HSC/HPC from mice by MACS, and the cells after MACS have high purity and activity.2. The t-BHP can induce Sca-1⁺ HSC/HPC senescence in vitro to establish a model of premature senescence. Rg1 could delay and treat Sca-1⁺ HSC/HPC senescence induced by t-BHP. The effect of Rg1 delay aging is better than treatment.3. It is successful to establish the replicating HSC/HPC aged model in vivo through the Sca-1⁺ HSC/HPC serial transplantation. Rg1 could delay and treat Sca-1⁺ HSC/HPC senescence during transplantation. The effect of Rg1 delay aging is better than treatment.4. The signal pathway of p16^INK4a-Rb and p19^Arf-p53-p21^Cip1/Waf1 may be play a key role in the Sca-1⁺ HSC/HPC senescence and in the antiaging effect of Rg1 to Sca-1⁺ HSC/HPC senescence.5. One of the mechanism to Sca-1⁺ HSC/HPC senescence may be the telomere shorten and telomeras activity decreased. Activation of telomerase and prolonging of telomere length might be involved in the process of Rg1 delay and treat Sca-1⁺ HSC/HPC senescence.