Ginsenoside Rg1 Attenuates Oxidative Damage In Human Umbilical Cord Blood-derived Stromal Cells: Involvement Of The FoxO3a Signal Pathway

Hematopoietic inductive microenvironment(HIM),the internal environment for sustaining and regulating the growth and development of hematopoietic stem cells(HSCs),acts as an important role in normal hematogenesis function.As the main component of HIM,stromal cells have a close relationship not only with the self-renewal and differentiation of HSCs,but also with the occurrence,development,and prognosis of hematologic diseases.Besides bone marrow,there are many studies concentrating on stromal cells from other different sources.Human umbilical cord blood-derived stromal cells(h UCBDSCs)were isolated and cultured in our previous study,and their potential to support HSCs proliferation in vitro has been identified.Moreover,co-transplantion of HSCs with h UCBDSCs could promote hematopoietic reconstitution,repair impaired HIM and treat GVHD(graft-versus-host disease)in animal studies.Despite promising results,attempts to use h UCBDSCs into human subjects were hampered mainly due to their poor survival in response to various stressful stimuli during cell expansion in vitro and injection in vivo.h UCBDSCs may face with dangerous challenges,such as nutrient deprivation,inflammatory reaction and oxidative stress,which lead to increased free radicals and reactive oxygen species(ROS)formation,and unavoidably cause oxidation-induced injury even cell death and apoptosis.The mitochondria is the main source of ROS intracellularly and the homeostatic regulation of mitochondrial ROS(mt ROS)could be crucial for supporting normal cellular function.Mitochondrial oxidative stress,which is defined as an imbalance between mt ROS production and antioxidant defenses,results in mitochondrial dysfunction and associated diseases.Therefore,development of drugs,which stimulate endogenous antioxidant pathway and maintain mt ROS homeostasis could be vital to prevent mitochondrial oxidative injury and enhance the survival of h UCBDSCs.Accumulating evidence indicate that Ginsenoside Rg1(G-Rg1),one of the most important pharmacological components of Panax ginseng,confers a wide range of health benefits such as anti-cancer,anti-inflammation,anti-senescence,anti-diabetic and neuroprotective effects.Recent evidence focused on a novel and protective property of G-Rg1 against oxidative damage and apoptosis in various cell types.Our previous study has proved that G-Rg1 can not only promote the proliferation and differentiation of bone marrow stromal cells(BMSCs)and the secretion of hematopoietic growth factors,but also enhance its anti-oxidant and anti-inflammatory ability under D-Galactose-induced stress condition to delay cell senescence.However,little information is available regarding the effects of G-Rg1 on oxidation-induced cell injury and apoptosis in h UCBDSCs and related molecular mechanisms.Aim: Herein this study was designed to test whether G-Rg1 could promote the survival of h UCBDSCs and inhibit oxidative injury induced by tert-Butylhydroperoxide(t-BHP),a well-known pro-oxidant,and further explored Fox O3 a related pathway might involved in this process.The clinical significance of these findings is the possible benefit of G-Rg1 in cancer patients undergoing HSCs transplantation in terms of improving the survival and engraftment of transplanted h UCBDSCs and further supporting hematopoietic reconstitution.Methods: 1.h UCBDSCs were isolated and expanded in vitro,and then exposed to a series of concentrations of t-BHP for 6 h to establish the oxidative injury model.Afer that,cells were incubated with G-Rg1 at different concentrations for an additional 24 h.The effects of G-Rg1 on cell viability and proliferation were detected by CCK-8 assay,and the CFU-F(colony-forming unit of fibroblast)were counted under an inverted phase-contrast microscope.The level of(malondialdehyde,MDA)and the enzymatic activities of(lactate dehydrogenase,LDH)and(superoxide dismutase,SOD)were determined by commercial assay kits according to the manufacturer's instructions,respectively.2.h UCBDSCs were exposed to t-BHP(80 ?M)for 6 h,and then were incubated with G-Rg1 at different concentrations for an additional 24 h.Cell apoptosis were analyzed on a FACScan Flow Cytometer.The expression of apoptotic-related proteins(Caspase-3,Bim,Bax and Bcl-2)were determined by Western blot assay,respectively.The effect of Akt-Fox O3 a signal on the downregulation of pro-apoptotic protein Bim was determined by Western blot assay,and the effect of G-Rg1 on the nucleus-to-cytoplasm translocation of Fox O3 a was also determined by Western blot assay.3.h UCBDSCs were exposed to t-BHP(80 ?M)for 6 h,and then were incubated with G-Rg1 at different concentrations for an additional 24 h.The level of intracellular and mitochondrial ROS were determined using laser scanning confocal microscope(LSCM),and the effect of G-Rg1 on the ??m in t-BHP-induced h UCBDSCs were also determined using LSCM.The enzymatic activities of Mn-SOD and Catalase were measured by commercial assay kits according to the manufacturer's instructions,respectively.The role of Sirt1 on G-Rg1-induced anti-oxitavie effect was determined by CCK-8 assay,LSCM and commercial assay kits.The effect of AMPK-Sirt1 signal on G-Rg1-induced Fox O3 a deacetylation was also determined by Western blot assay.Results: 1.Establishment of cell model of t-BHP-induced oxidative injury To evaluate t-BHP-induced toxicity in h UCBDSCs,cells were treated with series of concentrations of t-BHP for 6 h and then cell viability was measured by CCK-8 assay.We found that cell viability of h UCBDSCs gradually lost with the increased concentration of t-BHP.The viability rate was reduced to(52±3.10)% when treated with 80 ?M of t-BHP for 6 h,and this concentration was employed in the subsequent experiments.2.G-Rg1 attenuated oxidative injury and apoptosis in t-BHP-induced h UCBDSCs(1)G-Rg1 concentration-dependently inhibited cell viability loss in t-BHP-induced h UCBDSCs,and markedly attenuated the detrimental effect of t-BHP on cell viability at the concentrations of 0.1,1,10 and 50 ?M.G-Rg1 treatment attenuated t-BHP-impaired cell growth capacity,and the cell proliferation and CFU-F formation were promoted compared with t-BHP-treated cells.In addition,G-Rg1 alone(50 ?M)could also support the growth of h UCBDSCs.(2)There was a dramatic increase of MDA production and LDH release in h UCBDSCs after exposure to t-BHP,compared to the control group.However,incubation with different concentrations(1,10 and 50 ?M)of G-Rg1 significantly ameliorated t-BHP-induced increase levels of MDA and LDH.Treatment of h UCBDSCs with t-BHP markedly decreased the activity of SOD,whereas G-Rg1 at the concentrations of 10 and 50 ?M significantly attenuated the loss of SOD activity.(3)Exposure to t-BHP led to increased cell apoptosis,whereas incubation with G-Rg1 dose-dependently reduced the apoptotic percentage in comparison to the t-BHP-treated group.G-Rg1(50 ?M)inhibited the activation of pro-apoptotic protein Caspase-3,decreased the expression of pro-apoptotic Bim and Bax and increased the expression of anti-apoptotic Bcl-2.3.Role of Akt-Fox O3a-Bim signaling pathway in the G-Rg1-induced inhibitive effect on cell apoptosis G-Rg1(50 ?M)activated Akt and Fox O3 a phosphorylation,which promoted the translocation of Fox O3 a from the nucleus to the cytoplasm.This change inhibited Fox O3a-induced expression of downstream pro-apoptotic protein Bim,then down-regulated the expression of pro-apoptotic Bim and Bax,and up-regulated the expression of anti-apoptotic Bcl-2.These effects of G-Rg1 could be inhibited by PI3 K inhibitor LY29004,which indicated that Akt-Fox O3-Bim signaling pathway plays an important role in the G-Rg1-induced inhibitive effect on cell apoptosis.4.G-Rg1 attenuated mitochondrial oxidative injury in t-BHP-induced h UCBDSCs(1)The intracellular ROS concentration was determined by measuring the intensity of DCFH fluorescence in h UCBDSCs using LSCM.We found that the production of DCFH fluorescence in t-BHP-treated h UCBDSCs was significantly increased compared to the control group.However,incubation with G-Rg1 significantly reduced the fluorescence of DCFH in a concentration-dependent manner,suggesting that t-BHP-induced ROS generation was inhibited by G-Rg1.(2)The effect of G-Rg1 on mt ROS generation in h UCBDSCs was evaluated using Mito SOX Red,a fluorescent probe for detection of O2·-generated within the mitochondria.We showed that t-BHP caused a significantly increased mitochondrial O2·-generation compared to the control group.After treated with G-Rg1 at the concentrations of 10 ?M and 50 ?M,the level of mitochondrial O2·-was notably decreased,which suggested that G-Rg1 could attenuate t-BHP-induced mitochondrial oxidative stress by inhibiting mt ROS production in h UCBDSCs.(3)We performed and quantified JC-1 staining to assess the mitochondrial membrane potential(??m)in h UCBDSCs using LCSM.Exposure to t-BHP led to a significantly increased green fluorescence in h UCBDSCs,which suggested the decrease in ??m.However,treatment with G-Rg1 restored the decline of ??m induced by t-BHP and preserved mitochondrial function,as monitored by a reduction of green fluorescence and increase of red fluorescence.(4)The levels of Mn-SOD and Catalase were dramatically decreased after exposure to t-BHP,whereas incubation with G-Rg1 resulted in up-regulation of these two detoxification enzymes in a dose-dependent manner.These results showed that G-Rg1-induced mitochondrial protection may be through stimulating mitochondrial antioxidant enzymes to scavenge excessive mt ROS.5.Role of AMPK-Sirt1-Fox O3 a signaling pathway in the G-Rg1-induced downregulation of mitochondrial ROS in h UCBDSCs(1)Western blot analysis revealed that the level of Sirt1 increased in response to G-Rg1 single-treatment,and similar result was found in G-Rg1 and t-BHP co-treated cells.We demonstrated that G-Rg1 treatment alleviated mt ROS accumulation induced by t-BHP and recoverd cell viability loss in h UCBDSCs.The levels of Mn-SOD and Catalase were also increased significantly by G-Rg1 treatment compared with cells treated with t-BHP only.However,all these effects of G-Rg1 were blocked when Sirt1 was suppressed by Sirt1 si RNA transfection.Our findings indicated a key role of Sirt1 in anti-oxidative action of G-Rg1 in t-BHP-induced h UCBDSCs.(2)Treatment of h UCBDSCs with t-BHP resulted in decreased levels of phosphorylated AMPK,Sirt1 expression and deacetylated Fox O3 a.However,G-Rg1 treatment promoted AMPK-Sirt1 activation(AMPK phosphorylation and Sirt1 up-expression)and then enhanced deacetylation of Fox O3 a.These effects of G-Rg1 were diminished by AMPK si RNA and Sirt1 si RNA transfection,indicating that G-Rg1-mediated up-expression of deacetylated Fox O3 a and then mt ROS downregulation may be due to the stimulation of AMPK-Sirt1 signal pathway.Conclusion: 1.G-Rg1 enhances the survival and proliferation of t-BHP-induced h UCBDSCs,and protects them against apoptosis partially through Akt-Fox O3a-Bim signaling pathway.2.G-Rg1 protects h UCBDSCs against t-BHP-induced mitochondrial oxidative damage through decreasing mt ROS accumulation,in which the AMPK-Sirt1-Fox O3 a signaling pathway plays a key role.