ABSTRACT
BACKGROUND:Numerous basic and clinical trials have confirmed that the low survival rate after bone marrow mesenchymal stem cell transplantation is a serious constraint on its long-term therapeutic effect.Previous studies have shown that apoptosis-related factors play an important role in the apoptosis of bone marrow mesenchymal stem cells,of which apoptosis-inducing factor may be a key factor. OBJECTIVE:Bone marrow mesenchymal stem cells,of which apoptosis-inducing factor was knocked down,were transplanted into infarcted myocardium of mice,aiming to certify the importance of apoptosis-inducing factor in the survival of bone marrow mesenchymal stem cells to further recover cardiac function after infarction. METHODS:Firstly,bone marrow mesenchymal stem cells were infected with LV-AIF-shRNA lentivirus to down-regulate the expression of apoptosis-inducing factor protein.Flow cytometry,western blot assay,and RT-qPCR were used to detect the infection efficiency of lentivirus.CCK-8 assay was used to detect the cell viability of bone marrow mesenchymal stem cells with apoptosis-inducing factor knockdown under hypoxic and ischemic conditions.Then,with the mouse model of acute myocardial infarction constructed,the normal bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were transplanted into the model,respectively.The expression of apoptosis-inducing factor was examined by fluorescence immunoassay.Serum brain natriuretic peptide levels were detected by ELISA.Cardiac ultrasound was used to detect cardiac function.Myocardial fibrosis was observed by Masson staining.The expression of SRY gene was detected by RT-qPCR in apoptosis-inducing factor-knocked bone marrow mesenchymal stem cells after transplantation,reflecting cell survival. RESULTS AND CONCLUSION:(1)Bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were successfully established by LV-AIF-shRNA lentivirus infection,following 97.7%of infection efficiency,and notably decline of the expression of apoptosis-inducing factor(P<0.001).(2)Under ischemia and hypoxia,the cell viability of apoptosis-inducing factor knockdown bone marrow mesenchymal stem cells was significantly increased compared with normal bone marrow mesenchymal stem cells.(3)Compared with normal bone marrow mesenchymal stem cells after transplantation,the survival number of bone marrow mesenchymal stem cells in the infarcted myocardium after apoptosis-inducing factor gene knockdown was significantly increased to 3.71 times(P<0.001),and the apoptosis-inducing factor protein expression and myocardial fibrosis degree in the infarcted area were significantly reduced.(4)Compared with normal bone marrow mesenchymal stem cells,the serum brain natriuretic peptide level of bone marrow stem cells with apoptosis-inducing factor gene knockdown after transplantation was significantly decreased(P<0.05),and left ventricular ejection fraction and left ventricular shortening fraction were significantly improved(P<0.05).(5)These findings confirm that apoptosis-inducing factor gene knockdown can reduce myocardial fibrosis and improve cardiac function after acute myocardial infarction via enhancing the bone marrow mesenchymal stem cell viability and increasing the bone marrow mesenchymal stem cell survival after transplantation in the donor.
ABSTRACT
Objective To observe the impact of alone or combined use of ezetimibe and simvastatin on transient outward potassium current (Ito) in ventricular myocytes of rat model of ischemia and reperfusion (IR). Methods Seventy-five male SD rats were randomly divided into five groups, control group (CON), control-IR group (CIR), ezetimibe treatment group (EIR), simvastatin treatment group (SIR) and combined ezetimibe and simvastatin treatment group (ESIR). After two weeks of treat?ment with intragastic normal saline or drugs (ezetimibe or simvastatin), myocytes were isolated from right ventricular with colla?genaseⅡ, and Ito was recorded by whole-cell patch clamp technique. Results (1) The Ito current density at+60 mV was sig?nificantly decreased in CIR group than that of CON group (P0.05). The Ito current densities were higher in SIR group and ESIR group compared to those of CIR group. There was no significant difference in Ito current density between SIR group and ESIR group (P>0.05). (2) There was a significant increase in the half-inactivation (V1/2) in CIR group than that of CON group, but no significant differ?ence between EIR group and CIR group (P >0.05). There was a significant difference in the half-inactivation (V1/2) in SIR group and ESIR group compared to that of CIR group (P0.05). There was no significant difference in the slope factor (K) between five groups (P>0.05). (3) The time-con?stant (τ) of Ito recovery curves from inactivation was significantly higher in CIR group than that of CON group (P0.05). There was a significant difference in the time-con?stant (τ) of Ito recovery curves from inactivation in SIR group and ESIR group compared to that of CIR group (P0.05). Conclusion Simvastatin pre-treatment or ezetimibe+simvastatin pre-treatment can reverse the effect of IR on Ito of ventricular myocytes, but ezetimibe shows no such effects.