Heat Stress-induced Intestinal Epithelial Cells Necroptosis Via TLR3-TRIF-RIP3 Pathway Was Dependent On P53

Background and objective:Heatstroke is a life-threatening disease,characterized by increased core temperature over and central nervous system dysfunction.Intestine was not only the first damaged organ of heat stress,but also the initiation for further systemic inflammation and multi-organ dysfunction.Necroptosis was a recently identified regulated necrosis with passive and active proinflammatory functions.Since systemic inflammatory response was the core physiopathologic mechanism,necroptosis might involve in this process.Present study was aimed to investigate the mechanism in heat induced intestinal epithelial cell death,which might help develop treatment for heatstroke.Method and results:Part 1:Heat stress in vitro and in vivo model was establised on IEC and C57BL/6J mice.The necroptosis related proteins and inflammatory cytokines were measured.The cell viability decreased significantly and cell death increased in the heat stress group after 6 hours recovery,and the pro-inflammatory cytokines,TNF-α,MCP-1,IL-1β,IL-6 were increased in the supernatant culture medium after heat stress.Those genes that involve in TNF-α signaling pathway exhibited remarkable changes in their expression in response to heat stress in the intestine of mice.Immunoblotting analyses showed a significantly increased in the expression of RIP1,p-RIP1,RIP3,p-RIP3,p-Mlk1,caspase-8 and a decreased expression of p-Drpl in the intestine of mice in heat stress group.Together,heat stress could induce the cell death of intestine epithelial cells,secretion of inflammatory cytokines and activation of necroptosis signaling.Part 2:IEC cells were treated with caspase-8 inhibitor Ac-IETD-CHO,caspase-3 inhibitor Ac-DEVD-CMK and RIP3 inhibitor GSK’872 for 12 hours before heat stress.The analysis of cell viability revealed that the treatment of caspase-8 or caspase-3 inhibitor could only partially reverse the heat stress-induced reduction of cell viability,while treatment of RIP3 inhibitor could almost normalized the heat stress-induced decrease of cell viability and decrease the phosphorylation of MLKL.After overexpression of RIP3,the phosphorylation of MLKL was increased and cell viabilities were decreased.TLR3 agonist treatment could increase the phosphorylation of RIP3 and MLKL and promote the formation of TRIF-RIP3 complex.Taken together,our data show that necroptosis was the main cell death way for intestine epithelial cells under heat stress.Upregulation TLR3 and trigger TRIF-RIP3 signal activation underlies induction of necroptosis by heat stress.Part 3:We established in vitro and in vivo model by p53 inhibitor and p53 knockout mice,The expression and phosphorylation of p53 and its target genes were measured.Immunoblotting analysis showed that p53 phosphorylation increased significantly after heat stress.Inhibition or knockout p53 would diminish the expression of TLR3 in IEC cells,which blocked the formation of TRIF-RIP3 complex.There was no effect on heat stress-induced induction of necroptosis by upregulation of TLR3 when p53 was deficiency.These data indicate that heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on the activation of p53.Conclusion:Heat stress promoted p53 phosphorylation,then upregulated TLR3 and enhanced the interaction of TRIF-RIP3,which would activate the RIP3-MLKL signaling pathway to mediate necroptosis in intestinal epithelial cells.