Protective Effect Of Inula Britannica Flower Tatal Flavonoids On Aging Bone Marrow Mesenchymal Stem Cells

Part oneObject: The methods of isolate,culture and purify rat bone marrow mesenchymal stem cells(BMSCs).Morphology of BMSCs was observed,cell surface markers and the ability of differentiation were identified.Methods: BMSCs were isolated in vitro by adherence screening method.Morphology of BMSCs was observated.Flow cytometry was used to detect the cell surface markers and the cell cycle distribution.BMSCs was induce into osteogenic differentiation and adipogenic differentiation.Results: BMSCs were mainly composed of spindle cells,and the cell colonies emerged in a radial array.BMSCs could stable passage over 3 generations.CD29,CD44 and CD90 were positive in 3rd generation of BMSCs,while CD11 b was negative.Oil Red O staining was positive after adipogenic differentiation,and Alizarin Red staining was positive after osteogenic differentiation.Cell cycle analysis showed that BMSCs were consistent with normal cell growth characteristics,in addition its with a high growth activity.Conclusions: The adherence screening method is a simple way to isolate,purify and amplify BMSCs.The BMSCs obtained from this method plays an important significance in subsequent experiment.Part twoObject:To explore the optimal concentration for D-gal to induce bone mesenchymal stem cells(BMSCs)aging.Methods: The experimental groups were as follows: normal control group(Control group);low dose D-gal group(L group,BMSCs+10g/L D-gal);medium dose D-gal group(M group,BMSCs+20g/L D-gal);high dose D-gal group(H group,BMSCs+40g/L D-gal).CCK-8 method was used to detect the cell proliferation rate.The comercial kits were used to measure the activity of SOD and the content of MDA.Western blot was used to measure the proteins related to apoptosis such as Bax,Bcl2 and cleaved-caspase-3.Results: The proliferation rate in L,M and H groups were statistically lower than those in control group(P <0.01,respectively),and the proliferation rate decreased gradually with the increase of the concentrations,the differences in M and H groups were statistically significant compared with the L group(P <0.05,respectively).The contents of ROS and MDA in diferent concentrations of D-gal induced groups were statistically higher than those in control group(P <0.01,respectively),and the contents of ROS and MDA increased gradually with the increase of the concentrations,the differences in M and H groups were statistically significant compared with the L group(P <0.05,respectively).While the activity of SOD in different concentrations of D-gal induced groups was statistically lower than that in control group(P <0.01,respectively),and there was no significant difference between M group and L group(P =0.07),but the difference between H group and L group was statistically significant(P <0.01).The apoptosis in diferent concentrations of D-gal induced groups were statistically higher than those in control groups,and the apoptosis decreased gradually with the increase of the concentrations,but there was no significant difference between M group and L group(P =0.105),the difference between H group and L group was statistically significant(P <0.05).In addition,the medium and high concentration of D-gal induced groups mainly cause cells' late phase of apoptosis and necrosis.The expression of cleaved-caspase-3 and Bax / Bcl2 in diferent concentrations of D-gal induced groups were statistically higher than those in control group(P <0.01,respectively),but there was no statistically difference between those three concentrations of D-gal induced groups(P >0.05,respectively).Conclusions: All three concentrations of D-gal could induce the senescence of BMSCs,which may cause by the increasing oxidative stress induced by D-gal in BMSCs.However,the medium and high concentration of D-gal mainly induce the late phase apoptosis and necrosis of the cells,therefore we considered 10 g / L is the appropriate concentration of D-gal to induce BMSCs aging.Part threeObject: To investigate the protective effect on aging bone mesenchymal stem cells(BMSCs)induced by D-galactose(D-gal).Methods: IBFTF was extracted using the alcohol refluxing method,and the concentration of the extract was determined by spectrophotometry.The experimental groups were as follows: The normal group(N group,BMSCs).The high dose group(H group,BMSCs+50?g / ml IBFTF).The aging group(D group,BMSCs+10g/L D-gal),treat the aging cells with three concentrations of IBFTF: low concentration of IBFTF group(DL,BMSCs+10g/L D-gal+12.5?g/ml IBFTF),medium concentration of IBFTF group(DM groups,BMSCs+10g/L D-gal+25?g/ml IBFTF),high concentration of IBFTF group(BMSCs+10g/L D-gal+50?g/ml IBFTF),and the positive group(DT group,BMSCs+10g/L D-gal+50?g/ml TP).Expression of the longevity gene SIRT1,cell apoptosis,cell cycle distribution,and oxidative stress level were examined in each group..Results:The concentration of the extract was 82.6% which was determined by spectrophotometry.The cell viability,SOD and CAT activities,the expression of m RNA and protein related to cell cycle and SIRT1 in D group were significantly lower than those in N group(P <0.01),while different doses of IBFTF promoted aging cells viability,increased SOD and CAT activities,enhanced SIRT1 and cell cycle-related m RNA and protein expression(P <0.01,respectively).The apoptotic cells,ROS content and MDA level in the D group were significantly higher than those in N group(P <0.01).Treatment with different concentrations of IBFTF could significantly decrease the apoptotic rate,ROS production,MDA level and m RNA and protein expression related to apoptotic pathway in aging cells(P <0.05,respectively).Conclusions: IBFTF exerts protective effects on D-gal-induced aging BMSCs by regulating the longevity gene SIRT1 to reduce oxidative damage and control cell signaling pathways related to cell cycle and apoptosis.