| PURPOSE:Stem cell aging theory believes that the occurrence and development of human aging and age-related disease has close relationship with somatic stem cell aging. Especially, hematopoietic stem/progenitor cells(HSC/HPC) aging is closely related to a variety of age-related diseases. It has been carried on the thorough research that the mechanism of ginseng saponin Rg1 deters hematopoietic stem/progenitor cells(HSC/HPC) senescence via our early study, but the key targets in the signaling pathways of Rg1 regulating HSCs aging are still unclear. It has not been reported that the relationship between Ginseng saponin Rg1 detering HSC/HPC senescence with the Wnt/beta-catenin signaling pathway and the targets of Rg1 regulating Wnt/beta-catenin signaling pathway. This article will combine the latest research on stem cells with traditional Chinese medicine, on the basis of previous research, to discusses the scientific problems of ginsenoside Rg1 delaying HSC/HPC senescence and regulating aging cell signaling pathways, and the research aims to provide theory and laboratory basis on ginseng saponin Rg1 regulating the targets of HSC/HPC aging.METHODS:1. Sca-1~+HSC/HPCs were separated and purified by immune magnetic-activated cell sorting(MACS) technology. The cell purity was measured by immunofluorescence.2.The separated and purificated Sca-1~+HSC/HPCs were randomly divided into five groups: control group, Rg1 group, D-gal group, Licl group and Rg1+Licl group.(1) Detection of related biological indicators of aging:(1) the ability of proliferation was measured by CCK-8;(2)the capability of colony formation was examined by CFU-Mix cultivation;(3) the ratio of the SA-β-Gal staining positive cell were counted to evaluate the level of senescence.(2) Detection of Wnt/ beta-catenin signaling pathway related proteins and genes: the protein expression of β-catenin, GSK-3β and TCF-4 were detected and The mRNA expression of β-catenin, c-myc and CyclinD1.(3)Discussion on mechanism:(1)the distribution of ROS levels was analyzed;(2)The content of DNA oxdative damage marker 8-OH-d G were detected;(3)The level of DNA damage marker γ-H2 AX were examined;(4)the protein expression of P53, P21;(5)The mRNA expression of p53,RESULTS:1.The activity of Sca-1~+HSC/HPCs was 95%-97%. The purity of Sca-1~+HSC/HPCs was(83.32±2.57)% after MACS. These results suggest that the separated Sca-1~+HSC/HPCs have a higher activity and purity.2.Compare with the control group, Licl group: the proliferation of Sca-1~+HSC/HPCs was blocked; the colony formation of CFU-Mix was decreased; the percentage of SA-β-Gal was increased; The expression of β-catenin, TCF-4, P53 and P21 was increased; The expression of GSK-3βwas decreased; The expression of β-catenin, C-myc, CyclinD1, P53 and P21 mRNA were up-regulated. The product of ROS、8-OH-dG and γ-H2 AX were increased.3.Compare with the Licl group, Rg1+Licl group: the proliferation of Sca-1~+HSC/HPCs was enhanced; the colony formation of CFU-Mix was increased; the percentage of SA-β-Gal was decreased; The expression of β-catenin, TCF-4, P53 and P21 were decreased; The expression of GSK-3βwas increased; The expression of β-catenin, C-myc, CyclinD1, P53 and P21 mRNA were down-regulated. The product of ROS、8-OH-dG and γ-H2 AX were decreased.CONCLUSIONS:1.Wnt/β-catenin signaling pathway can induce the Sca-1~+HSC/HPCs age-related biological performance in active state.2.Rg1 ginsenosides can inhibit the Sca-1~+HSC/HPCs senescence via activating Wnt/β-catenin signaling pathway.3.P53 / P21 pathway, cell DNA oxidative damage and oxidative stress may be the new mechanisms in Rg1 detering Sca-1~+HSC/HPCs senescence via regulating Wnt/β- catenin signaling pathway. |
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