| Objective: Stem cell senescence is the key factor and the current theory accounting for organismal aging.It is very important to regulate the aging of stem cells in order to explore the biological mechanism of aging and to search the strategies for prevention and treatment of senile diseases.The main aging characteristics of hematopoietic stem cells(HSCs)are the weakened self-renewal ability and the differentiation of dysfunction.These aging impacts are clinically most evident as an increase in the incidence of myeloproliferative diseases(e.g.,leukemia),a decline in adaptive immunity,a tendency toward anemia,bone marrow hematopoietic inhibition and so on.Ginseng is a well-known traditional Chinese medicine for “invigorating qi”.Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng.The effects of ginsenoside Rg1 include enhanced memory,improved immunity,secondary anti-tumor effects,restoration of cellular function and so on.Our previous work had preliminarily discussed the role of Rg1 in delaying the aging of HSCs,however,the related molecular mechanism and signaling pathway were unclear.In this study,we combined the latest technology of stem cell aging with traditional anti-aging medicine,constructed respectively HSC/HPCs aging model in vitro and in vivo,studied the relationship between ginsenoside Rg1 delaying HSC/HPCs senescence and regulation of Wnt/β-catenin signaling pathway.We will further explain the molecular mechanism of Rg1 about delaying HSC/HPCs senescence,which have important value to promote the "Qi-xue Theory" of traditional Chinese medicine and to find the important molecular targets for the treatment of HSC/HPCs aging.Methods: 1.Isolation and purification of Sca-1+HSC/HPCs from Mouse Bone Marrow: Sca-1 positive HSC/HPCs from mouse bone marrow were obtained by MACS.The labeled cells were processed for flow cytometric analysis and trypan blue staining.2.In vitro aging model of Sca-1+HSC/HPCs was induced by oxidized low density lipoprotein(ox-LDL).There were five groups in the present study: 1)control,2)ox-LDL,3)ox-LDL + Rg1,4)ox-LDL + DKK1,and 5)ox-LDL + NAC.3.In vivo aging model of Sca-1+HSC/HPCs was induced by Dgalactose(D-gal,120 mg/kg·d).Mice were divided into 4 groups at random: 1)control group,2)D-gal model group,3)the D-gal +Rg1 group,4)the D-gal +Vit E group.4.Senescence-related index detection: senescence-β-galactosidase(SA-β-Gal)staining,mixed colony-forming unit(CFU-mix)culture,cell cycle analysis.The aim is to observe the biological characteristics of Sca-1+ HSC/HPCs in vivo and in vitro,and to study the effect of Rg1 on delaying the senescence of Sca-1+ HSC/HPCs.5.Oxidative stress-related index detection: reactive oxygen species(ROS)levels;the activities of SOD,GSH-Px and MDA in the supernatant or in the serum of mice;4-HNE expression by Western blot;AGEs and 8-OHd G concentration by ELISA.The aim is to observe the oxidative stress response of Sca-1+ HSC/HPCs in vivo and in vitro,and to investigate the effect of Rg1 on delaying the aging of Sca-1+ HSC/HPCs and regulating oxidative stress.6.Canonical Wnt signaling pathway-related proteins and genes detection: the expression of GSK-3β,phospho-GSK-3β,β-catenin,TCF-4 by Western blot;the location expression of phospho-GSK-3β and β-catenin by immunofluorescence staining;the m RNA expression of β-catenin and c-myc by RT-q PCR.The aim is to observe the Wnt/β-catenin signaling pathway of Sca-1+ HSC/HPCs in vivo and in vitro,and to investigate the effect of Rg1 on delaying the aging of Sca-1+ HSC/HPCs and regulating Wnt/β-catenin signaling pathway.The use of NAC and Vit E was designed to investigate the relationship between Wnt/β-catenin signaling pathway and oxidative stress.7.Detection of proteins and genes in senescence-associated pathways:the expression of p53、p21Cip1/Waf1、p16Ink4a、Rb、r-H2 A.X by Western blot;the m RNA expression of p21Cip1/Waf1 and p16Ink4 a by RT-q PCR.The aim is to observe the DNA damage responses,p16Ink4a-Rb and p53-p21Cip1/Waf1 pathways of Sca-1+ HSC/HPCs in vivo and in vitro,and to investigate the effect of Rg1 on delaying the aging of Sca-1+ HSC/HPCs and regulating DNA damage responses,p16Ink4a-Rb and p53-p21Cip1/Waf1 pathways.The use of DKK1 was designed to investigate the relationship between Wnt/β-catenin signaling pathway and DNA damage responses,p16Ink4a-Rb,p53-p21Cip1/Waf1 pathways.Results: 1.After the Sca-1+ HSC/HPCs were isolated and purified from mouse bone marrow cells by MACS,the purity of the cells was found to be 90.08 ± 2.7%.The proportion of Sca-1+ HSC/HPCs was much higher than that in collected mononuclear cells before the isolation procedure(7.57 ± 1.38%).The survival of the separated cells was 98~99% by the Trypan blue dye exclusion assay.2.After ox-LDL treatment with Sca-1+ HSC/HPCs,the cells showed the biological characteristics of senescence.The proportion of SA-β-Gal positive Sca-1+HSC/HPCs was significantly increased;the percentage of cells in the G0/G1 phases was increased while it was reduced in the S phases;the number of CFU-mix was markedly decreased.The levels of ROS and MDA rose,while the activities of GSH-Px and SOD declined in the supernatant.The expression of nuclear and cytoplasmic β-catenin was increased,moreover the increase in the inner nucleus was more obvious.The GSK-3β phosphorylation was markedly increased.The expressions of TCF-4,4-HNE,p53,p21Cip1/Waf1,p16Ink4 a,Rb,r-H2 A.X,8-OHd G were upregulated.The m RNA expressions of β-catenin,c-myc,p21Cip1/Waf1,p16Ink4 a were enhanced.3.After the treatment of ginsenoside Rg1 on Sca-1+HSC/HPCs in vitro,the positive proportion of SA-β-Gal was decreased;the percentage of cells in the G0/G1 declined while it rose in the S phases;the number of CFU-mix was increased.The levels of ROS and MDA were reduced,while the activities of GSH-Px and SOD were enhanced in the supernatant.The expression of nuclear and cytoplasmic β-catenin was decreased.The GSK-3β phosphorylation declined.The expressions of TCF-4,4-HNE,p53,p21Cip1/Waf1,p16Ink4 a,Rb,r-H2 A.X,8-OHd G were downregulated.The m RNA expressions of β-catenin,c-myc,p21Cip1/Waf1,p16Ink4 a were attenuated.The addition treatment of DKK1 or NAC could also antagonize ox-LDL-induced changes in signaling pathways in different degrees.4.D-galactose induced mouse model of aging by continuous subcutaneous injection,and mouse Sca-1+HSC/HPCs showed the biological characteristics of senescence.The positive rate of SA-β-Gal staining was significantly increased and the number of CFU-Mix colony formation was markedly decreased.The level of ROS was significantly enhanced and the content of T-AOC was reduced,the content of MDA,8-OHd G and AGEs in mouse serum was markedly rose,while the activity of SOD and GSH-Px declined significantly.The expression of β-catenin was obviously enhanced in cytoplasm and nucleus of Sca-1+HSC/HPCs,and the m RNA expressions of β-catenin and c-myc were upregulated.The level of p GSK-3β was significantly increased and the location expression in cytoplasm was enhanced.The expressions of p16Ink4 a,Rb,p53,p21Cip1/Waf1,TCF-4,4-HNE,r-H2 A.X,p21Cip1/Waf1 m RNA and p16Ink4 am RNA were improved in Sca-1+HSC/HPCs from difference model mice.5.After the treatment of ginsenoside Rg1 on Sca-1+HSC/HPCs in vivo,the positive rate of SA-β-Gal staining was decreased and the number of CFU-Mix colony formation was increased.The level of ROS was reduced and the content of T-AOC was enhanced,the content of MDA,8-OHd G and AGEs in mouse serum declined,while the activity of SOD and GSH-Px rose.The expression of β-catenin was weakened in cytoplasm and nucleus of Sca-1+HSC/HPCs,and the m RNA expressions of β-catenin and c-myc were downregulated.The level of p GSK-3β was reduced and the location expression in cytoplasm was lessened.The expressions of p16Ink4 a,Rb,p53,p21Cip1/Waf1,TCF-4,4-HNE,r-H2 A.X,p21Cip1/Waf1 m RNA and p16Ink4 am RNA were attenuated in Sca-1+HSC/HPCs from difference model mice.The addition treatment of Vit E could also antagonize D-gal-induced changes in multiple signaling pathways in some degrees,but the effect was weaker than Rg1.Conclusion: 1.Ox-LDL can induce Sca-1+HSC/HPCs senescence and establish a model of cell senescence in vitro.Its mechanism is related to oxidative stress and Wnt/β-catenin signaling pathway.2.The continuous subcutaneous injection of D-gal can induce Sca-1+ HSC/HPCs senescence in model mice,which can be used as a model of Sca-1+HSC/HPCs aging in vivo.Its mechanism is related to oxidative stress and Wnt/β-catenin signaling pathway.3.DNA damage response,p16Ink4a-Rb and p53-p21Cip1/Waf1 pathway are the pathways of oxidative stress or Wnt/β-catenin signaling-mediated cell senescence.4.Ginsenoside Rg1 in vitro attenuated ROS-mediated Wnt/β-catenin signaling,to further downregulate DNA damage response,p16Ink4a-Rb and p53-p21Cip1/Waf1 pathway,thereby to delay Sca-1+ HSC/HPCs senescence induced by ox-LDL.5.Ginsenoside Rg1 in vivo regulated Wnt/β-catenin signaling pathway,and repress oxidative stress-mediated DNA damage response,the activation of p16Ink4a-Rb and p53-p21Cip1/Waf1 pathway,thereby to delay Sca-1+ HSC/HPCs senescence in D-gal-induced mouse aging model. |
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