117.3: A study on the cell death mode of β cells under hypoxia and the protective effect of huc-MSC-CM

Introduction: Hypoxia is one of the major causes of β cell death and dysfunction after islet transplantation. The aim of this study was to explore the hypoxia-induced β cell death mode and to investigate the effect of human cord blood-derived mesenchymal stem cell-conditioned medium (huc-MSCs-CM) on β-cells under hypoxic conditions and the potential underlying mechanisms. Methods: β cells (min6) were cultured in the presence or absence of huc-MSCs-CM under 1% oxygen conditions. Cell viability and apoptosis were analyzed by AO/PI staining and flow cytometry-based Annexin V/ PI apoptosis detection kit, respectively. Proteins related to apoptosis, pyroptosis, necroptosis, parthanatos and ferroptosis were detected by WB method and ELISA. The insulin-secreting function of min6 was determined by ELISA. Min6 was treated with specific inhibitors to reveal the underlying mechanism of MSC action. Results: Hypoxia significantly induced apoptosis and pyroptosis of β cells, while huc-MSCs-CM significantly increased β cells survival. The WB results showed that apoptosis and pyroptosis related proteins, including caspase-3, caspase-8, PARP, NLRP3, caspase-1, GSDMD were upregulated under hypoxic conditions, but were inhibited by huc-MSCs-CM. Moreover, the autophagy pathway was activated by hypoxia and further enhanced by huc-MSCs-CM. ELISA results showed that under hypoxia, pyroptosis-related IL-1β and IL-18 secretion increased, β-cell insulin secretion decreased, and huc-MSCs-CM inhibited IL-1β and IL-18 secretion and improved insulin secretion. At the same time, the formation of autophagosomes increased under hypoxia, and huc-MSCs-CM further promoted its formation. These changes were reversed after treatment of min6 with the autophagy inhibitor CQ. Conclusion: In addition to apoptosis, hypoxia also induces pyroptosis of β cells. Huc-MSCs-CM can protect β cells from hypoxia-induced apoptosis and pyroptosis by inducing autophagy, and enhance cell survival. This result shows that pyroptosis can also be used as a target for β-cell protection, and huc-MSCs-CM can simultaneously target multiple cell death pathways to protect β-cells and may improve the effect of islet transplantation. the National Key Research and Development Program (Grant No. 2019YFA0110703).