Astragaloside Combined With 5-azacytidine Induce The Differentiation Of HUCB-MSCs Into Cardiomyocytes And Inhibit Their Apoptosis

Objective: 5-azacytidine(5-aza) is an effective inducer that can promote the differentiation of mesenchymal stem cells(MSCs), but has bigger toxicity. Astragaloside(AST) is the major active component of Radix Astragali Mongolici, and can also induce MSC differentiation. The aim of this study was to clarify the effects of AST in combination with 5-aza on the differentiation of human Umbilical cord blood mesenchymal stem cells(h UCB-MSCs) into cardiomyocytes, and to explore their protection against cardiomyocyte apoptosis.Methods: 1. MSCs were isolated from Human umbilical cord blood,and then were cultured and cloned by density gradient method and adhesive cultivation. The cell surface antigens of MSCs including CD34, CD44 and CD29 were identified using flow cytometer. 2. The third generation of h UCB-MSCs were randomly divided into four groups: blank control group(cultured medium), low-dose 5-aza group(5 μmol/L 5-aza), high-dose 5-aza group(10 μmol/L 5-aza), and AST+5-aza group(200 mg/L AST combined with5 μmol/L 5-aza. After four weeks of induction, changes of cell morphology were observed under the microscope. The expression of cardiac troponin T(c Tn T) and Desmin in cardiomyocytes was detected by immunocytochemical staining. The m RNA and protein levels of atrial natriuretic peptide(ANP) were measured through Fluorescence real-time PCR and western blot, respectively. Enzyme linked immunosorbent assay(ELISA) was used to analyzed the intracellular content of cyclic adenosine monophosphate(c AMP). 3. in MSCs were examined by RT-PCR respectively. 3. After four weeks of induction, cells were treated with high glucose(33 mmol/L) and glucose oxidase(2 m U/m L) for 12 h to induce apoptosis. Apoptotic rate was measured by flow cytometer. Additionally, the expresions of Bcl-2 and Bax was detected using western blot.Results: 1. The expression of CD44 and CD29 in h UC-MSCs was positive while CD34 expression was negative. 2. After induction with AST plus 5-aza, h UCB-MSCs showed significantly morphological alternations. There was no expression of c Tn T and Desmin in blank control group. The levels of c Tn T and Desmin were significantly increased in high-dose 5-aza group and AST+5-aza group compared with low-dose 5-aza group(P<0.01). There was no significant difference in c Tn T and Desmin levels between AST+5-aza group and high-dose 5-aza group(P>0.05). In comparison with low-dose 5-aza group, ANP expression and intracellular content of c AMP were higher in high-dose 5-aza group and AST+5-aza group(P<0.01). However, there was no significant difference in above indicators between AST+5-aza group and high-dose 5-aza group(P>0.05). Three drug-treated groups showed a marked increase in ANP expression and intracellular content of c AMP compared to blank control group(P<0.01). The expression of Bcl-2 protein was lower(P<0.05 or 0.01) but apoptotic rate and Bax protein expression were higher(P<0.05 or 0.01) in low- and high-dose 5-aza groups than those of blank control group. In addition, in comparison with blank control group, Bcl-2 protein expression was significantly increased(P<0.05) but apoptotic rate and Bax protein expression were significantly decreased(P<0.05) in AST+5-aza group.Conclusion: 1. AST in combination with 5-aza can induce the differentiation of h UCB-MSCs into cardiomyocytes in vitro and the effects are similar to 10 μmol/L 5-aza. 2. AST in combination with 5-aza markedly suppress cardiomyocyte apoptosis through upregulation of Bcl-2 protein and downregulation of Bax protein.