*Purpose: Xenotransplantation of porcine islets has been an emerging and effective option for clinicians to treat type 1 diabetes recently. Comparing with other damage, hypoxic damage is one of crucial factors, which leads to apoptosis and reduced beta cell function during the stage of pre-and-post transplantation. Meanwhile, hypoxia is a strong stimulant that induces autophagy, and growing evidence suggests that mesenchymal stem cells (MSCs) may be available to prevent β cells from various extra-/intracellular stimuli through regulating autophagy. However, the mechanisms underlying the MSCs activation of autophagy to promote islets survival in hypoxia-induced injury remain unclear. In this study, the mechanism of MSC-conditioned medium (hu-MSC-CM) in regulating autophagy is investigated when neonatal porcine islet cell clusters (NICCs) were exposed to hypoxia in vitro.

*Methods: Neonatal porcine islet cell clusters were cultured with hu-MSC-CM, and native medium RPMI-1640 (Control) under hypoxic conditions (1%O2) in vitro. Then the effects of hu-MSC-CM on NICCs viability were examined by AO/PI staining, the activity of autophagy was detected by TEM and confocal fluorescence microscopy, and the expression level of protein that is related to cell apoptosis and autophagy was analyzed by western blotting. Besides, to confirm the protective role of hu-MSC-CM on hypoxia-treated NICCs are mediated through autophagy,3-MA and CQ were used to pretreat NICCs.Furthermore, we probed into the role of IL-6 in MSC-conditioned medium. NICCs were pretreated by IL-6 and Sarilumb (IL-6 inhibitor), then cells were cultured with or without MSC-CM under hypoxic conditions. We detected viability and autophagy level of islets by the previously mentioned methods.

*Results: We demonstrated that hu-MSC-CM treatment improve cell viability and help increase autophagy markers (LC3II and Beclin1), as well as help reduce the expression of cleaved Caspase3 that is related to apoptosis. The protective effect of hu-MSC-CM on NICCs was diminish after the use of autophagy inhibitor (3-MA, CQ )and IL-6 inhibitor (Sarilumb).

*Conclusions: Our results demonstrated that MSC-conditioned medium could improve islets viability via elevating autophagy activity through PI3K/Akt/mToR pathway. And we found that IL-6 would take vital effect in the function of MSC-conditioned medium in promoting islet cells survival through regulation of autophagy under hypoxia condition. These findings would enrich the mechanisms related to huc-MSC in protecting islet cells suffered from hypoxia, and provide a new sight into developing new cell-free therapeutic method of mesenchymal stem cells in islets transplantation.

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