Effect And Mechanisms Of N-acetyl-L-cysteine On H₂O₂-induced Apoptosis In BMSCs

Purpose: To simulate the ischemia hypoxia microenvironment in organism andestablish oxidative stress environment in vitro. To observe the injury of rat BMSCscaused by oxidative stress. To observe the protection effect of n-acetyl cysteine (NAC)via the pretreatment of bone marrow mesenchymal stem cells.Method: Bone mesenchymal stem cells were isolated from4weeks SD rat andcultured by whole bone marrow adherent cultivation method. Flow cytometryinstrument were used to detect P3generation cells surface marker CD90of bonemarrow stromal cells and CD45of hematopoietic cells.P3generation cells wererandomly divided into four groups,normal control group,H2O2treatment group, theNAC group and NAC+H2O2,Then the cellular morphology was observed underinverted phase contrast microscope,MTT method was adopted to detect cell survivalrate of different concentration of H2O2and NAC stimulation group,Flow cytometryto detect each group’s cell apoptosis rateResult: p3generation cell morphology was uniform,presenting spindle or flatshape growth,growing like typical fish, vortex or fence type;H2O2can induceapoptosis of BMSCs, the survival rate of BMSCs changed depend on the H2O2concentration,and its shape presents typical apoptotic changes;when BMSCs waspretreated with100μM NAC,reslut showed that NAC have weak protection effect,and with the concentration increasing, its protection effect becomed even moreobvious,but with the concentration of NAC increasing, its reversal effect has noobvious change.on the other hand, high concentration of NAC has toxic effect onBMSCs;NAC can inhibit apoptosis of BMSCs caused by oxidative stress and play aprotective role.Conclusion: Under the oxidative stress induced by H2O2, BMSCs presentapoptosis change and correlate with concentration, NAC work through inhibiting apoptosis of BMSCs caused by oxidative stress.This experiment providesexperimental basis for further research the anti-apoptosis mechanism of NAC inducedby oxidation stress. Purpose: To set up apoptosis model of rBMSCs induced by oxidative stress,andresearch the molecular mechanisms of N-acetyl cysteine protecting rBMSCs fromapoptosis induced by oxidative stress based on PI3K/Akt signal pathway.Methods BMSCs isolated from SD rats by whole bone marrow adherenceculture method were randomly divided into normal control group, H2O2group, NACgroup, H2O2plus NAC group，H2O2and NAC plus LY294002group. Both apoptosisof rBMSCs and ROS production were detected by flow cytometry.The mRNA levelsof FoxO1, FoxO3and FoxO4in rBMSCs were determined by RT-PCR, and theexpressions of p-Akt、Bcl-2and Akt were examined by Western blot.Results Compared with the control group, the production of ROS and thenumber of cell apoptosis in H2O2group significantly increased and the expression ofFoxO1,FoxO3and FoxO4mRNA levels decreased in rBMSCs; NAC treatmentinhibited H2O2-induced ROS production, cell apoptosis, but increased p-Akt andBcl-2expression, and upregulated FoxO1,FoxO3and FoxO4mRNAlevels.LY294002,a PI-3Kinse inhibitor,not only decreased the expression ofFoxO1,FoxO3and FoxO4mRNA levels and the expression of p-Akt、Bcl-2,but alsomarkedly increased ROS production and cell apoptosis. Conclusion: N-acetyl-L-cysteine can protect BMSCs against apoptosis inducedby oxidative stress through decreasing reactive oxygen species productionandincreasing the expression of FoxO1, FoxO3and FoxO4mRNA levels andanti-apoptotic protein Bcl-2expression in BMSCs by activation of PI3K-Aktsignaling pathways.This experiment for further research of mechanism on oxidationstress induced apoptosis by n-acetyl cysteine provides a certain experimentalbasis,providing some experimental basis on improving stem cell survival and theefficiency of the repair in stem cell transplantation combination with drugs.