Research On The Protective Role Of Bone Marrow-derived Mesenchymal Stem Cells In High Glucose-induced β-cell Injury And Its Underlying Mechanisms

Objective:To investigate the cytoprotective effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on chronic high glucose-treated (3 cells and the possible mechanisms involved.Methods:Initially, in our in vitro study, INS-1 cells were exposed to high glucose (33.3 mmol/L) medium for 48 h, and then co-cultured with BM-MSCs or 208F cells for another 12 h before the following measurements. (1) The viability of INS-1 cells was assessed by CCK8 assay. (2) The cell apoptosis incidence was detected using Annexin V/ Propidium iodine (PI) staining. (3) The insulin secretion of INS-1 cells was measured by a Ratio Immunity Assay (RIA) kit. (4) Western blotting was applied to examine the expression of cleaved caspase 3 and two widely used autophagic markers, Beclinl and microtubule-associated protein 1 light chain 3 (LC3). (5) Green fluorescent protein (GFP)-LC3 plasmid was transiently transfected into INS-1 cells to quantify autophagy. (6) Immunofluorescent staining and transmission electronic microscope (TEM) analysis were performed to examine the morphological and ultrastructural changes in INS-1 cells. (7) Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production in INS-1 cells were detected by a flow cytometer. Additionally, in our in vivo study, we performed BM-MSC infusion into rats with type 2 diabetes (T2D) induced by a high-fat diet and streptozotocin (STZ) administration. The levels of blood glucose, serum insulin and serum C-peptide were carefully monitored. Histological and immunofluorescent staining as well as TEM analysis were performed to observe morphological and ultrastructural changes in pancreatic islets and β cells of T2D rats.Results:Prolonged exposure to high glucose decreased cell viability, increased cell apoptosis and impaired basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) of INS-1 cells. BM-MSC co-culture significantly alleviated these glucotoxic alternations, while 208F cells did not cause any significant change in those parameters mentioned above. In addition, western blotting displayed upregulated expression of Beclinl and LC3-Ⅱ in INS-1 cells co-cultured with BM-MSCs. Results from immunofluorescent staining and TEM analysis also revealed that BM-MSCs promoted autophagosomes and autolysosomes formation in high glucose-treated INS-1 cells. However, it should be noted that inhibition of autophagy significantly diminished the protective effects of BM-MSCs on high glucose-treated INS-1 cells, suggesting that the improvement of β-cell function and survival induced by BM-MSCs was at least partially mediated through autophagy enhancement. Furthermore, our results demonstrated that compared to INS-1 cells treated with high glucose alone, BM-MSCs improved mitochondrial function in high glucose-treated INS-1 cells, as reflected by the recovery of MMP and the reduction in intracellular ROS levels, which were largely attributed to autophagic clearance of impaired mitochondria. In vivo study revealed that BM-MSC infusion not only ameliorated hyperglycemia, but also promoted restoration of pancreatic islets and β cells in T2D rats. What’s more, BM-MSC infusion upregulated lysosome-associated membrane protein 2 (LAMP2) expression and enhanced formation of autophagosomes and autolysosomes in pancreatic β cells in T2D rats, accompanied by reduced P-cell apoptosis and increased number of insulin granules.Conclusions:(1) BM-MSCs co-culture enhanced autophagy and thereby protected INS-1 cells against chronic high glucose-induced injury in vitro. (2) Infusion of BM-MSC promoted restoration of pancreatic β cells through modulation of autophagy in T2D rats. The present study provides new insights into our understanding of MSC-based therapeutic mechanisms and establishes an important theoretical foundation for future clinical use of MSCs in type 2 diabetes therapy.