Objective： To investigate the effects of Panax notoginseng saponins （PNS） on hydrogen peroxide （H2O2）-induced apoptosis in cultured rabbit bone marrow stromal cells （BMSCs）. Methods： The effects of different concentrations of PNS on proliferation and early osteoblast differentiation of BMSCs were determined by the MTT assay and an alkaline phosphatase （ALP） assay. An optimal effective concentration of PNS was determined and used in subsequent experiments. The cultured BMSCs were divided into three groups： untreated control, H2O2 treated, and PNS pretreatment of H2O2 treated. The oxidative stress level was assessed by superoxide dismutase （SOD） and malondialdehyde （MDA） assays. Flow cytometry was used to determine BMSC apoptosis by staining with annexinV-FITC/propidium iodide （PI）. The activity of caspase-3 enzyme was measured by spectrofluorometry. Results： PNS （0.1g/L） significantly increased both BMSC proliferation rate and ALP activity, while it decreased the indicators of oxidative stress, caspase-3 activity, and the apoptosis rate of BMSCs induced by H2O2.. Conclusion： PNS, acting as a biological antioxidant, had a protective effect on H2O2-induced apoptosis in cultured rabbit BMSCs by decreasing oxidative stress and down-regulating caspase-3.
Hui QiangPeiguo GaoChen ZhangZhibin ShiTao WangLei WangKunzheng Wang
Objective： To investigate the effects and possible mechanism of Panax Notoginseng saponins （PNS） on oxidative stress-induced damage and apoptosis in bone marrow stromal cells （BMSCs）.Methods： BMSCs were isolated and cultured from 2-month-old New Zealand rabbits by the density gradient centrifugation combined with adherent method.The third passage cells were used for subsequent experiments.Oxidative stress was induced in cultured BMSCs by H2O2 （0.1 mmol/L）.BMSCs were pretreated with 25-200 μg/mL PNS for 4 h before H2O2 treatment.Proliferation of BMSCs was observed using MTT assay.Alkaline phosphatase （ALP） activity,as an index of early osteoblastic differentiation,was determined with an ALP assay kit.Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/ propidium iodide.Oxidative stress level was examined by reactive oxygen species （ROS） assay.The protein expressions of Bax,Bcl-2 and Caspase-3 in BMSCs were analyzed by Western blotting.Results： PNS had different concentrationdependent effects on proliferation and osteoblast differentiation of BMSCs induced by H2O2.A PNS concentration of 100 μg/mL was determined as the optimal effective concentration.PNS markedly attenuated H2O2-induced apoptosis rate from 41.91% to 14.67% （P〈0.01）.PNS significantly decreased ROS level induced by H2O2 （P〈0.01）.Furthermore,pretreatment with PNS significantly reversed H2O2-induced inhibition of Bcl-2 expression and augmentation of Bax and Caspase-3 expression （P〈0.01）.Conclusion： PNS had a protective effect on oxidative stress-induced damage and apoptosis in cultured rabbit BMSCs through scavenging ROS and regulating the Bcl-2/Bax pathway.
Objective To evaluate the effect of alendronate on osteoprotegerin(OPG)and receptor of activator of nuclear factor κB-ligand(RANKL)expression in human marrow stroma cells(hMSCs)in vitro.Methods hMSCs were isolated from human marrow,cultured in vitro,and randomly divided into two groups:alendronate group,hMSCs culture fluid containing 1×10-7mol/L alendronate;control group,no special treatment but culturing hMSCs in DMEM.Two weeks after treatment,the expressions of OPG and RANKL were evaluated by RT-PCR and Western blot.Results hMSCs became uniform spindle-shaped fibroblasts.As cells proliferated,they formed colonies and showed whirlpool arrangement.After one week’s treatment,hMSCs in alendronate group had reduced processes and gradually showed disc shape,which did not happen in control group but kept fibroblast shape and just increased in density.In RT-PCR,the ratio of OPG/RANKL in alendronate group and control group was 8.77±1.16 and 4.58±1.27,respectively.In Western blot,the ratio of OPG/RANKL in alendronate group and control group was 2.58±0.47 and 1.52±0.32,respectively.The ratio of OPG/RANKL was higher in alendronate group than in control group(P<0.01).Conclusion Alendronate enhances OPG expression and inhibits RANKL expression of hMSCs in vitro.
Jian-zhong Wang,Kun-zheng Wang,Zhi-bin Shi,Yu-qiang Ji,Ming-yu Zhang Department of Orthopedics,the Second Affiliated Hospital,Medical School of Xi’an Jiaotong University,Xi’an 710004,China
