| OBJECTIVE:Mesenchymal stem cells(MSCs)have attracted much attention as seed cells of tissue engineering and cell engineering.In recent years,mesenchymal stem cell transplantation has made a breakthrough in the field of regenerative medicine,and has been applied in the treatment of many diseases.Studies have shown that the rapid aging of mesenchymal stem cells in the process of transplantation leads to gradual loss of stem cells,and the transplanted cells cannot proliferate and differentiate into functional cells,which ultimately fails to achieve the desired therapeutic effect.Therefore,it is not only of great theoretical significance,but also of potential value in clinical transplantation to explore the ways of regulating the aging of mesenchymal stem cells.Ginseng is a good prescription of traditional Chinese medicine to replenishing "qi".Ginsenoside is the main medicinal ingredient of ginseng,with many monomer saponins and different effects.Studies have shown that ginsenosides have many pharmacological functions,including antitumor,anti-oxidation,anti-inflammation,promoting metabolism,regulating cell proliferation and differentiation,etc.Previous studies by our group have shown that ginsenoside Rg1 is an important anti-aging monomer in ginseng,which can antagonize the damage of oxidative senile agents to various tissues,cells,and organs,and significantly delay the aging of organs and cells.It is speculated that the mechanism is closely related to the reduction of oxidative stress damage.Our latest study shows that ginsenoside Rg1 can promote the proliferation and differentiation of bone marrow mesenchymal stem cells and delay their senescence,but the mechanism remains to be interpreted.Stem cells are not "immortal".Studies have proved that with the increase of age,stem cells will gradually become senescent,which will lead to tissue cell renewal disorder,or even abnormal proliferation and differentiation,and then accompanied by the occurrence and development of related age-related diseases.Therefore,we should attach great importance to the latest research results of stem cells in the study of aging biology and anti-aging.Recent stem cell aging theories suggest that the KEAP1-NRF2-ARE pathway is a key pathway to regulate the oxidative stress aging of stem cells.In this study,Bone marrow mesenchymal stem cells(BM-MSCs)were used as research objects to investigate the mechanism of ginsenoside Rg1 in regulating KEAP1-NRF2-ARE antioxidant signaling pathway and delaying the aging of BM-MSCs.The purpose of this study is to provide a new theoretical basis for explaining "Qi and blood theory" and stem cell senescence theory of Chinese medicine.To provide new technical support for building a new platform for stem cell aging research;To provide theoretical and laboratory basis for ginsenoside Rg1 in the prevention and treatment of senile diseases.METHODS:1.BM-MSCs culture and identification: Bone marrow was collected from mice,bone marrow mononuclear cells were isolated,adherent culture was conducted in vitro,and the adherent cells were passed and purified.Determination of BM-MSCs surface antigen markers by flow cytometry(including: CD34,CD45,HLA-DR,CD29 and CD44).2.To study the aging effect and mechanism of ginsenoside Rg1 in vitro against D-Gal on BM-MSCs: D-Gal constructed the aging model of BM-MSCs in vitro,and ginsenoside Rg1 was added to intervene the aging process.The DNA damage of BM-MSCs was detected by immunofluorescence and Western blot.The expressions of senescence specific enzymes and senescence related proteins(P53,P21)in BM-MSCs were detected by senescence related-?-galactosidase staining and Western blot.The senescence related secretion phenotype(SASP)of BM-MSCs secretion was detected by real-time fluorescence quantitative PCR(RTPCR).The cell proliferation was detected by EDU method.Cell apoptosis was detected by flow cytometry(FCM).CFU-F assay was used to determine the viability of cell cloners.3.Study ginseng saponin Rg1 injected antagonism D-gal protection to vital organs in mice: the experiment selects the wild type C57 BL / 6 j mice and NRF2-/-type C57 BL / 6 j mice,were randomly divided into four groups(normal group,Rg1 groups,group D-gal,Rg1 + D-gal group),with D-gal injected construction of aging model mice,intraperitoneal injection of Rg1 ginsenosides in the process of modeling,Rg1 on D-gal aging mice organs,including heart,liver,lungs,protection;Aging related?-galactosidase staining(SA-?-gal)was used to detect the aging level of organs.The expression of organs age-related proteins(p-P53,P53,P21,P16)was detected by Western blot.The expression of ?-H2 AX,a marker of DNA damage response,was detected by immunofluorescence and Western blot.The expression of SASP in organs was detected by RT-PCR.4.D-gal induced aging models were established in vitro and in vivo,and relevant indicators were detected: NRF2 protein expression in cells and tissues was detected by immunofluorescence and Western blot.The expression levels of NRF2 downstream targets(GCLC,GCLM,HO-1 and NQO1)were detected by Western blot.The relative expression levels of NRF2 downstream target genes(SRX,NQO1 and GSTA1)were detected by RT-PCR.The proportion of senescent cells in BM-MSCs was detected by SA-?-Gal staining.The expression levels of cell senescence related proteins(P21 and P16)were detected by Western blot.ROS content in BMMSCs was determined by DCFH-DA fluorescence staining.Serum oxidation and antioxidant indices(including CAT,SOD,8-OHd G,MDA and 4-HNE)were determined by ELISA.5.D-gal induced senescence models in vitro and in vivo were established.Ginsenoside Rg1 was used to intervene the process of senescence,and relevant indicators were detected: the expression of Keap1 protein in cells and organs was detected by immunofluorescence and Western blot.The intracellular autophagy and the regulation of Keap1 degradation by P62-dependent autophagy pathway were detected by Western blot.DCFH-DA fluorescence staining,Western blot,immunofluorescence,and fibroblast colony formation assay were used to detect the role of PI3K-Akt signaling pathway in ginsenoside Rg1 activating NRF2 activity.RESULT:1.BM-MSCs were successfully isolated,cultured and identified: the cells expressed BM-MSCs antigen markers CD29 and CD44,but did not express CD34,CD45 and HLA-DR.It was proved that BM-MSCs was successfully isolated and obtained in this study.2.Ginsenoside Rg1 can antagonize the aging effect of D-Gal on BMMSCs in vitro: Ginsenoside Rg1 can effectively reduce the expression levels of ?-H2 AX,the DNA damage response marker of BM-MSCs,and P16 and P21,the aging marker of BM-MSCs.Ginsenoside Rg1 can reduce the number of SA-?-Gal staining positive cells in D-Gal-induced senescence BM-MSCs and decrease the secretion capacity of SASP.Ginsenoside Rg1 can improve the CFU-F generation ability of senile BMMSCs.Ginsenoside Rg1 can reduce ROS content of BM-MSCs.Ginsenoside Rg1 can increase the proliferation ability of BM-MSCs and the expression of antioxidant enzymes SOD2,catalase and HO-1.3.Ginsenoside Rg1 can alleviate D-Gal-induced organ damage and senescence: Ginsenoside Rg1 can reduce D-Gal-induced damage to the heart,liver,and lung,and delay the senescence process.Ginsenoside Rg1 can decrease the protein expression levels of DNA damage response markers ?-H2 AX and aging markers P16 and P21 in heart,liver,and lung.Ginsenoside Rg1 can reduce the number of SA-?-Gal positive cells and the secretion capacity of SASP in organs.Ginsenoside Rg1 decreased the accumulation of oxidative damage products MDA,8-OHd G and 4-HNE in serum of senile mice,and increased the activities of antioxidant substances SOD and TAC.4.Ginsenoside Rg1 reduces the aging effect of D-gal by activating NRF2 transcription factor:(1)Ginsenoside Rg1 can increase the expression of NRF2 in BMMSCs,activate the activity of NRF2 transcription factor,and then promote the translocation of NRF2 from cytoplasm to nucleus.Ginsenoside Rg1 can increase the expression of NRF2 downstream target proteins(GCLC,GCLM,HO-1 and NQO1)and target genes(SRX,NQO1 and GSTA1).NRF2 inhibitor(ML385,Brusatol)blocked the aging effect of Rg1 antagonistic D-gal on BM-MSCs.The effect of Rg1 on reducing SA-?-gal activity was blocked.Antagonistic effect of Rg1 on the decrease of agerelated proteins(P16,P21);The inhibitory effect of Rg1 on the secretion of SASP was decreased.The inhibition effect of Rg1 on ROS production was also reduced.(2)NRF2-/-mice validated the antagonistic effect of NRF2 transcription factor on aging induced by Rg1: NRF2 gene deletion blocked the protective effect of Rg1 on DNA damage: increased expression of ?-H2 AX,a DNA damage response marker;The aging effect of Rg1 antagonistic D-gal on BM-MSCs was blocked,and the number of SA-?-gal positive cells was increased.The expression levels of antioxidant proteins(GCLC,GCLM,HO-1 and NQO1)were decreased.The expression of age-related proteins(P16 and P21)was increased.5.Ginsenoside Rg1 regulates P62-dependent autophagy through the PI3K-Akt signaling pathway to enhance Keap1 degradation,promote the expression of NRF2 and enhance its activity:(1)Ginsenoside Rg1 enhanced Keap1 degradation through P62protein-dependent autophagy.Ginsenoside Rg1 can reduce the expression of Keap1 protein in BM-MSCs.The proteasome inhibitor(MG-132)could not block the inhibition of Keap1 expression by Rg1.Autophagy inhibitors(3-MA,CQ)can inhibit the down-regulation of Keap1 expression by Rg1,suggesting that the down-regulation of Keap1 expression by ginsenoside Rg1 may be mediated by autophagy degradation.The P62 protein expression level of BM-MSCs was increased after ginsenoside Rg1 intervention.The P62 inhibitor(XRK3F2)could block the regulation effect of ginsenoside Rg1 on NRF2 and Keap1 proteins,that is,it increased the expression of Keap1 protein and decreased the expression of NRF2 protein.The P62 inhibitor(XRK3F2)antagonized the inhibitory effect of ginsenoside Rg1 on the expression of age-related proteins by increasing the expression of P16 and P21 proteins.(2)Ginsenoside Rg1 intervention decreased the expression of phosphorylated S6 protein in BM-MSCs and increased the activity of phosphorylated Akt(T380,S473)in BM-MSCs.Combined treatment of BM-MSCs by LY294002(PI3K inhibitor)and RG1 reduced the conversion of LC3-?to LC3-II.The inhibiting effect of ginsenoside Rg1 on ROS production of BM-MSCs;The protective effect of ginsenoside Rg1 on BMMSCs clone formation ability was inhibited.Reversing ginsenoside Rg1 intervention reduced the expression of senescence protein(P16,P21)in BM-MSCs and reduced the number of SA-?-gal staining positive cells.CONCLUSIONS:1.Mice BM-MSCs conforming to international standards were successfully isolated and identified;2.Ginsenoside Rg1 can antagonize the aging effect of oxidative senile agent on BM-MSCs;3.Ginsenoside Rg1 can antagonize the senile effects of oxidative senile agents on various organs of the body;4.Ginsenoside Rg1 antagonizing oxidative senile agent on BMMSCs and organs is closely related to alleviating oxidative stress injury;5.Ginsenoside Rg1 can reduce the oxidative stress injury of BMMSCs by regulating the NRF2 and PI3K/Akt antioxidant signaling pathway,and then delay cell senescence. |
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