The Effect Of Astragaloside Ⅳ On Hypoxia/serum Deprivation-induced Cell Apoptosis And MAPK Signaling Pathway Of Bone Marrow Mesenchymal Stem Cells In Vitro

Objective:To observe the influence of Astragaloside Ⅳ on the hypoxia/serum deprivationinduced cell apoptosis and cell viability;to investigate the effect and mechanisms of AS-Ⅳagainst apoptosis mediated by possible MAPK signaling pathway and endoplasmic reticulum apoptosis pathway.Methods:Primary BMMSCs were isolated,cultured and identified in vitro,Cell Counting Kit-8 assessed the influence of AS-Ⅳ pretreatment on BMMSCs viability on the basis of successful establishment of apoptosis model induced by H/SD.The BMMSCs cell death was visualized by measuring nuclear morphology using fluorescence microscopy after Hoechst33342 staining and the rate of cell apoptosis was measured by flow cytometry analysis after staining with Annexin V-FITC and propidium iodide(PI).The protein levels of MAPK signaling pathway and endoplasmic reticulum apoptosis pathway,such as p-P38/p38,p-JNK/JNK,p-ERK/ERK,CHOP and Procaspase-12/Caspase-12,were examined by Western blot in the presence or absence of SB202190 and SP600125 to evaluate the specific target of AS-Ⅳ against apoptosis figuring out the exact signal transduction mechanisms.Results:① BMMSCs from P5to P6 exhibit homogeneous,spindle-shaped,fusiform and whorled arrangement.Flow cytometry showed that cells were negative for CD45(11.32%)and positive for CD44(69.33%)&CD90(99.56%).These results were consistent with biological characteristics of BMMSCs.②BMMSCs exposed to H/SD for 6h showed the highest rate of apoptosis in the assessment of Hoechst33342 staining and flow cytometry analysis(P<0.05).③The protein expression of p-P38,p-JNK,Procaspase-12/Caspase-12 of BMMSCs exposed to H/SD for 4h were remarkably increased in contrast to those exposed to H/SD for 2h or 6h(P<0.01);The protein level of CHOP of BMMSCs exposed to H/SD for 6h significantly increased(P<0.01);The protein expression of p-ERK also showed great increase in different time of H/SD pretreatment(P>0.05).④The rate of BMMSCs apoptosis was decreased in the examination of Hoechst33342 and flow cytometry analysis after p38 and JNK signaling pathways were blocked by SB202190 and SP600125(P<0.05).⑤The protein expression of Caspase-12 was suppressed significantly after p38 signaling pathways were blocked by SB202190(P<0.05).⑥The viability of BMMSCs pretreated to 80μg/ml AS-Ⅳ for 72h showed significant change(P<0.05).⑦The rate of apoptosis was decreased after BMMSCs was exposed to 80μg/ml AS-IV for 12h(P<0.05).⑧The expression levels of p-P38、p-JNK and Caspase-12 were inhibited after BMMSCs pretreated to 80μg/ml AS-Ⅳ were exposed to H/SD for 6h,instead,the expression of p-ERK was increased(P<0.05).Conclusion:①MAPK signaling pathway and endoplasmic reticulum apoptosis pathway mediated hypoxia/serum deprivation-induced cell apoptosis of BMMSCs.②AS-IV has the protective effect on BMMSCs apoptosis-induced by hypoxia/serum deprivation.③To a certain degree,AS-IV down-regulate Caspase-12,which is the key protein of endoplasmic reticulum apoptosis pathway,by means of decreasing the protein levels of p-P38 and p-JNK.Moreover,AS-IV is able to increase p-ERK level.In this way,AS-IV reduced the number of apoptotic BMMSCs and increased the survival rate of BMMSCs exposed to hypoxia/serum deprivation.