Kaempferol Attenuates Apoptosis Of BMSCs Against Oxidative Stress By Altering FoxO Signaling

Objective: To explore the mechanism of kaempferol(KA)on the apoptosis inhibition to mesenchymal stem cells(BMSCs)under oxidative stress environment in vitro,and to further investigate the role of ROS/ Fox O signaling pathways in the course.Methods: Bone mesenchymal stem cells were extracted from the bone marrow of Specific pathogen Free(SPF)Sprague Dawley(SD)healthy rat,using the extracted bone marrow adherent,cultured,purified and amplified BMSCs,inverted the morphological changes through phase contrast microscopic observation,Flow cytometry instrument were used to detect cells surface markers of BMSCs.P3 generation cells were randomly divided into several groups: A: normal control group,B: H2O2-treated group,C: kaempferol treatment group,D: kaempferol + H2O2 group,CCK8 assay was adopted to detect cell cell viability of different concentration of H2O2 and KA stimulation group;each group was measured by flow cytometry to test the cell apoptosis and intracellular levels of reactive oxygen species;The expression level of antioxidant genes(CAT)and DNA damage repair genes(Gadd45a)in each group were determined by RT-PCR,and the expressions of Fox O3 a were examined by Western blot.Results: The morphology of three generations cultured cells were observed,the morphology of completely adherent cells were spindle-shaped or flat,with uniform in colonies,closely spaced growth,in line with the BMSCs feature of SD rats;The results show that H2O2 increased cellular oxidative stress level on BMSCs which can induced apoptosis,cell viability and apoptosis rate decrease with H2O2 concentration increse in a H2O2 concentration-dependent manner dependencies,Kaempferol can promote cell growth and protect cell from apoptosis without inhibition effects within certain ranges of concentration pretreatment(2?M-10?M),but fail to protect BMSCs from apoptosis beyond this concentration,its reversal effect has no obvious change.conversely,high concentrations' Kaempferol exert toxic effect to BMSCs,indicating KA could protect BMSCs from apoptosiscaused by oxidative stress.At the same time,compared with the normal group,the BMSCs cell viability decreased and the apoptosis rate increased which were induced by H2O2,along with the level of reactive oxygen free radicals,Fox O3 a content regulation,the expression of the CAT gene and Gadd45 a gene in cells increased significantly.It showed growth retardation;KA added which significantly reduce the expression of said gene and protein levels,and inhibits apoptosis and toxic effects of reactive oxygen species(ROS)on BMSCs cells.Conclusion: To set up oxidative stress environment of BMSCs induced by H2O2,BMSCs showed apoptosis-like changes with a H2O2 concentration-dependent manner;KA could protect BMSCs from apoptosis caused by oxidative stress;kaempferol can down-regulate the expression of ROS / Fox O signaling pathways,which lead to the transcription of antioxidant genes(CAT)and DNA repair gene(Gadd45a)levels decreased,thereby protecting the BMSCs from oxidative shock-induced apoptosis.The present study was designed to investigate the mechanism of KA on againsting apoptosis caused by oxidative stress which provided some experimental reference for stem cell transplantation and stem cell drugs to improve survival,improving the efficiency of its restoration to provide some experimental basis.