The Protective Effect Of Tetramethylpyrazine On Hydrogen Peroxide-induced Bone Marrow-mesenchymal Stem Cells Apoptosis And Underlying Mechanisms

Objective To observe the effect of Tetramethylpyrazine(TMP)on hydrogen peroxide-induced rat bone marrow-mesenchymal stem cells(BMSCs)apoptosis and explore the possible mechanisms via the expression of Bax/Bcl-2 and the pathways of PI3K/Akt and ERK1/2.Methods ①Isolate and culture the BMSCs by the whole bone marrow adherent method,the morphological changes were observed under the inverted microscope.Different concentrations of H2O2(50,100,200,300,400,500 μmol·L-1)was added to BMSCs and the viability of BMSCs was detected by MTT method at different time(3、6、12、24、48 h),the best dose effect and time effect of H2O2 were defined.②MTT method was used to detect the cell viability after pretreatment with different concentrations of TMP(10,25,50,100,200 μmol·L-1)on BMSCs,observed the dose-effect relationship of TMP.③The flow cytometry was used to detect the content of intracellular reactive oxygen species,and define hydrogen peroxide induces the damage of BMSCs.④The Annexin V-FITC/PI method and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay were used to detect the apoptosis of BMSCs and the effect of TMP pretreatment on BMSCs.⑤The PI3K/Akt and ERK1/2 phosphorylation inhibitors wortmannin and PD98059 were interfered BMSCs to observe whether the effect of TMP pretreatment on BMSCs survival was decreased.⑥qRT-PCR and Western blot assay were used to detect the expression of Bax/Bcl-2,Akt and p-Akt,ERK1/2 and p-ERK1/2and to primarily explore the possible mechanisms.Results ①The whole bone marrow adherent culture method could obtain considerable amount of rat BMSCs in vitro.The BMSCs were well-growing and morphological stable,and had strong activity and high purity.②The MTT assay showed that different concentrations of H2O2 have different damage to BMSCs,the effect was in a dose and time dependent manner,and 500μmol·L-1 at 24 h was the best.③Compared with control group,incubated with different concentrations of TMP could improve BMSCs viability significantly(P<0.01 or P<0.05),and 25～100μmol·L-1 at 24 h was the best(P<0.01).④The detect of flow cytometry showed that compared with control group,the H2O2 group increase the content of intracellular reactive oxygen species significantly thus induce BMSCs apoptosis(P<0.01 or P<0.05).⑤The Annexin V-FITC/PI staining and TUNEL assay showed that compared with control group,the TMP pretreatment(50,100 μmol·L-1)have the protective effect on hydrogen peroxide-induced BMSCs apoptosis(P<0.01 orP<0.05).⑥The qRT-PCR and Western blot assay showed that compared with control group,TMP pretreatment(50,100 μmol·L-1)down-regulated the mRNA and protein expression of Bax(P<0.01 or P<0.05),up-regulated the mRNA and protein expression of Bcl-2(P<0.01 or P<0.05);the PI3K/Akt and ERK1/2 phosphorylation inhibitors wortmannin and PD98059 inhibited the protective effect of TMP pretreatment(P<0.01 or P<0.05).Conclusion TMP protects hydrogen peroxide-induced apoptosis on BMSCs,it’s possible mechanisms are related to down-regulate the express of Bax,up-regulate the express of Bax and promote the phosphorylation of Akt、ERK1/2.