Objective: Xenotransplantation of pancreatic islets represents a promising therapeutic alternative for the treatment of type 1 diabetes mellitus. However, hypoxia-induced injury or death of islets graft was often observed after transplantation, and the underlying molecular mechanism has not yet been completely elucidated. Programmed necrosis (necroptosis) is a caspase-independent form of regulated cell death which implicated in the development of a range of diseases, including inflammatory, autoimmune and neurodegenerative. It’s widely accepted that apoptosis and necroptosis are the main manners involved in cell death regulation. In this study we investigated the role of necroptosis in hypoxia-induced islets cells death.

Methods: 3-to 5-old new born pigs were supplied by Xeno company. The NICCs were isolated from the donor pancreas and divided into four groups followed by pre-treated with vehicle, Nec-1, zVAD and Nec-1/zVAD in a hypoxia incubator with 5% CO2 and 1% O2, respectively. Cell viability was assessed by MTS assay at various time points, and protein markers involved in necroptosis were tested via immunoblotting. The variation of inflammation factors was evaluated by q-PCR array and ELISA. Treatment with Etanercept, a specific inhibitor for TNFR, was conducted to further confirm the effect of TNFα in hypoxia-induce necroptosis and inflammation.

Results: We identified that necroptosis plays a crucial role in hypoxia-induced islets cell death in vitro. We demonstated that the activation of RIPK1/RIPK3/MLKL signaling pathway was involved in islets cells necroptosis. More importantly, TNFα and other inflammation factors were dramatically up-regulated in hypoxia-induced necroptosis, pre-treated with Nec-1 or Etanercept substantially inhibited necroptosis and TNFα secretion, which implied hypoxia-induced necroptosis was inflammation-dependent.

Conclusions: Taken together, our results suggest necroptosis was the main manner of hypoxia-induced islets cell death in vitro, and we unexpectedly found TNFα and other inflammatory factors plays pivotal role in this procedure.