Hypoxia Induces Inflammatory Response and Cell Death by Promoting NLRP3 Inflammsome Activation via ROS-TXNIP Axis in Pancreatic β cells

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IntroductionHypoxia is the major obstruction in improving the efficiency of islet transplantation and it is involved in the pathogenesis of type 2 diabetes. Activation of inflammasomes in ischemic condition is identified in various tissues. Here we investigated whether hypoxia activates the inflammasome in β cells and the role of inflammsome plays in hypoxic stress.MethodsNeonatal porcine islet cell cluster (NICC) and mouse insulinoma cell line 6 (MIN6) cells were exposed to hypoxia(1% O2) for 8-48hrs. MTS and Annexin V/PI FCA was performed to assess the β cell viability and apoptosis. NICC cell death was measured by AO/PI staining. IL-1β and HMGB1 releasing was detected by ELISA. Protein level of inflammsome component NLRP3, ASC, cleaved caspase-1, TXNIP and precursor/mature form of IL-1β and IL-18 was measured by Western blot. NLRP3, ASC and TXNIP were knocked down using shRNA. NAC were used as a ROS scavenger to investigate the underlying mechanism.ResultsBoth in NICC and MIN6, hypoxia(1% O2) time dependently increased NLRP3 and ASC protein level associated with up-regulation of NF-κB signaling. In NICC, hypoxia directly activated NLRP3 inflammasome characterized by increasing protein level of cleaved caspase-1 and mature form of IL-1β and IL-18, while in MIN6, it worked only in the presence of a small dose of LPS(10ng/ml). Besides, hypoxia increased extracellular level of IL-1β and HMGB1 in MIN6. Knockdown of NLRP3 and ASC markedly reduced mature form of IL-1β and decreased HMGB1 releasing after exposure to hypoxia, and it partially protected against hypoxia-induced decrease of β cell viability. Additionally, NLRP3 inflammasome inhibitor parthenolide significantly increased β cell viability in hypoxia. Finally, in response to hypoxia reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) were up-regulated. Pretreated with ROS scavenger NAC significantly decreased TXNIP and inhibited NLRP3 inflammasome activation. The important role of TXNIP was highlighted by the rescue of hypoxic MIN6 from apoptosis in TXNIP-knockdown MIN6 cells.ConclusionOur data indicate for the first time that inflammasome is involved in hypoxia-induced β cell death, and suggest that ROS-TXNIP-NLRP3 axis could be a potential target for improving islet transplantation.NSFC 81201171. NSFC 81471715. NSFC 81671752.

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