Mehdi Hassanpour
1,2* 
, Omid Cheraghi
3 , Reza Rahbarghazi
4* , Mohammad Nouri
2 1 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Clinical Biochemistry and Laboratory Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran
4 Department of Applied Cell Science, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Introduction: Cardiovascular disease (CVD) is a type of disease that affects the function of cardiac-vascular tissues. This study aimed to consider the possible effects of autophagy, as an intrinsic catabolic pathway of cells, on the differentiation and aging process of mesenchymal stem cells (MSCs).
Methods: In this study, bone marrow-derived MSCs were obtained from rabbit bone marrow aspirates. The stemness feature was confirmed by using flow cytometry analysis Cells at passage three were treated with 50 μM Metformin and 15μM hydroxychloroquine (HCQ) for 72 hours. The intracellular accumulation of autophagolysosomes was imaged using LysoTracker staining. Protein levels of autophagy (LC3II/I ratio), aging (Klotho, PARP-1, and Sirt-1) effectors, and cardiomyocyte-like phenotype (α-actinin) were studied by western blotting.
Results: Based on our findings, flow cytometry analysis showed that the obtained cells expressed CD44 and CD133 strongly, and CD31 and CD34 dimly, showing a typical characteristic of MSCs. Our data confirmed an increased LC3II/I ratio in the metformin-received group compared to the untreated and HCQ-treated cells (P < 0.05). Besides, we showed that the incubation of rabbit MSCs with HCQ increased cellular aging by induction of PARP-1 while Metformin increased rejuvenating factor Sirt-1 comparing with the normal group (P < 0.05). Western blotting data showed that the autophagy stimulation response in rabbit MSCs postponed the biological aging and decreased the differentiation potential to the cardiac cells by diminishing α-actinin comparing with control cells (P < 0.05).
Conclusion: In summary, for the informants in this study, it could be noted that autophagy inhibition/stimulation could alter rabbit MSCs aging and differentiation capacity.