Mechanism Of Ginsenoside Rg1to Delay Hematopoietic Stem Cell And Progenitorcellaging Induced By Radiation Damage

Objective:Aging related research is the hotspot in life science inrecent years, senile disease adt and accelerating social population agingprocess, makes the study of mechanism of age-related and anti-agingresearch is no longer just a medical problem, more become a socialproblem. The body is the most basic structure and functional unit cells, andstem cells maintain more mature and has the functions of tissues andorgans of the steady state key. Because stem cells are highly self-renewaland proliferation differentiation ability, so it is considered to be immortal intraditional concept can’t aging. However when hit by the body under theaction of internal or external damage factors, reduce tissue repair capacityand cancer susceptibility rate is higher, shows that stem cells may occurwith a variety of internal and external factors causing the damage relatedfunction decline and even aging.Hematopoietic stem cells (hematopoietic stem cell, HSC) isresponsible for the lifelong production of all types of blood cells, so it islocated in hematopoiesis system to the top, it’s aging is not only relationship with the steady-state level of hemopoietic system and immunesystem, more experiments prove that HSC aging and the aging of the bodyare closely linked. Recent studies have reported that DNA damage isrecession lead to the decrease in the number of adult stem cells andfunction of one of the main mechanisms. In fact biological genomeintegrity affected by endogenous and exogenous multiple damage factors,including radiation the accumulation of DNA damage to cells, cell agingand disease is an important factor.We usethe way of using total bodyirradiation (TBI) to built the hematopoietic stem/progenitor cells aginginduced by radiation damage model, to explore the biologicalcharacteristics of HSCs aging.Methods:1.Ionizing radiation induced hematopoietic stem cell aging modelconstruction: model group, C57BL/6mice body, one-time χ irradiationdose was6.5GY; Control group: a false light, take the same treatment inradiation group, but not to illuminate.2.Mouse model of age-related indicators detection: observed for up to28days after irradiation, the mice in each group were observed behavior;Peripheral blood detection in peripheral blood; remove the spleendetermination spleen index, the mice were sacrificedtake both sides of thefemur and tibia, counted after the marrow out of the bone marrow mononuclear cells and hematopoietic stem cells.3.HSC/HPC age-related biological detection: SA-β-gal staining ofcell senescence. Proliferation and differentiation capacity through a mixedset down (CFU-Mix) culture assays; flow cytometry to determine thepercentage of cells in G1arrest.4.Single cell gel electrophoresis (comet assay) detection ofhematopoietic stem cell DNA damage.5.The age-related genes and its protein expression testing: PCR detectthe aging-related genes, including p16INK4a、p19Arf、p53、p21Cip1/Waf1.Theexpressions of p16INK4a、p19Arf、p53、p21Cip1/Waf1protein was examined bywestern blotting.Results1.The experimental groups of C57mice show apathetic and sluggish;Peripheral blood like decreased significantly after irradiation. Sublethaldoses of irradiation of mice after irradiation the individual number offemoral bone marrow mononuclear cells and after separation the number ofSca-1+HSC/HPC have dropped sharply in the sham-irradiation group.Spleen index decreased obviously.2.After immune magnetic beads purification, flow cytometry resultsshowed that the Sca-1+cells for93.660.83%. Separation of the Sca-1+cellactivity is96%~99%. 3.Irradiated group mice Sca-1+HSC/HPC of beta-galactosidase(SA-β-gal) staining percentage compared with sham significantly increased;irradiation Rg1group of positive cells was significantly lower than theirradiated group. The irradiation group formed mixed hematopoieticprogenitor cell colonies (CFU-Mix) reduce the number of colony-cell count,the number of irradiation Rg1group formed CFU-Mix significantlyincreased significantly compared to the irradiated group. Flow cytometryanalysis showed that irradiation group HSC cell G1arrest in the G0/G1phase was significantly increased, decrease in S phase fraction, of whichthe first3-7d cell cycle arrest in the G1phase of the most serious28dgradually recovered after and close to the level of sham irradiation group;reduce the proportion of cells in G1phase after irradiation,3and7daysirradiation Rg1group as compared to the irradiated group.4.Under a fluorescence microscope observation showed that mostcellular DNA in the irradiated group in addition to showing a brightorange-red head, there is still a comet-like tail, irradiation Rg1group ofcells can also be found a comet-like tail, but small compared to theirradiated group. The tail length and Olive tail moment of the irradiatedgroup were significantly greater than the sham-irradiated group andirradiation Rg1group.5.Compared with the sham-irradiated group, irradiated group Sca-1+HSC/HPC SOD activity was significantly lower，but MDA contentwas significantly higher; compared to the irradiated group, Rg1groupexposure Sca-1+HSC/HPC SOD activity rose significantly MDAdecreased significantly.6.Agarose gel electrophoresis can clearly see the28S,18S and5S3bands, extracted RNA integrity. PCR results showed that, the irradiatedgroup Sca-1+HSC/HPC aging-related genes of p16INK4a and p19Arf,P53, p21Cip1/Waf1mRNA expression level was significantly higher thanthe sham-irradiated group; irradiation Rg1group p16INK4a the p19Arf,p53, p21Cip1/Waf1mRNA expression level lower than the irradiated group7.Western blot results show the irradiation group hematopoietic stemprogenitor cells senescence-associated gene of p16INK4a p19Arf, p53,p21CIP1/WAF1, protein levels compared with sham-irradiated group wassignificantly higher;Rg1group protein expression level lower than theirradiated group.Conclusions1.X-ray irradiation-induced aging of HSC/HPC2.MACS sorting can be purified high activity of the Sca-1+HSC/HPC.3.Rg1could delay Sca-1+HSC/HPCaging induced by radiationdamage 4.p16INK4a-Rb and p19Arf-p53-p21Cip1/Waf1signaling pathway inSca-1+HSC/HPC Rg1delay and treatment of radiation-inducedsenescence may play an important role.