Role Of PAR1 In The Apoptosis And Necroptosis Of Endothelial Cells Of Severe Heatstroke-induced Acute Lung Injury

BackgroundSevere heatstroke is a life-threatening illness,which always complicated with multiple organ dysfunctions.The lung is regarded as the most vulnerable remote organ injured after severe heatstroke.The lung tissues after severe heatstroke present changes,including pulmonary edema,hemorrhage,respiratory failure and even ARDS.The apoptosis and necroptosis of pulmonary vascular endothelial cells play important roles in heat stress induced-acute lung injury.Protein-activated receptor 1(PAR1)is a G-protein coupled transmembrane receptor,which was activated by thrombin and a main receptor of inflammatory response.Previous studies have suggested that PAR1 was significantly increased after the heat stress and induced hyper-permeability of vascular endothelial cells which leaded to the development of acute lung injury.However,the mechanisms remain unclear.In this study,two heat stress models were established.Light and moderate heat stress were apoptosis predominant,and severe heat stress model was cell necroptosis predominant.We aimed to investigate the effect of apoptosis and necroptosis on actue lung injury,the role of PARI and its downstream NF-?B/c-Jun in the heat stress induced-apoptosis,and the relationship of PARI with downstream RIP1/RIP3 and MLKL/HMGB1.Methods and results1.Adult male C57BL/6 mouse was placed in 39? and 65%humidity atmosphere until the Tc reached 42.7? and then heatstroke mice model was established.The mice were divided into normal control(NC)group,severe heatstroke(HS)group,PAR1 antibody pretreatment and heatstroke group(anti-PAR1),and PAR1 inhibitors RWJ56110 pretreatment and heatstroke group(RWJ + HS).Heat tolerance was significantly improved in the mice of RWJ + HS group and anti-PAR1 group compared with HS group.The hypothermia duration and the lowest core body temperature were the independent risk factors of poor outcome in mice.There were significant differences in time of hypothermia arrival,heat exposure time,duration of the depth of the low temperature,and the water take off between the RWJ+HS group and HS group.PAR1 inhibitor can significantly prolong the survival time and improve survival rate of severe heatstroke mice.TNF-alpha,IL-6 and HMGB1 levels in serum were decrease in mice pretreated with PARI inhibitor.The white blood cell count,neutrophil count,protein content in BALF,lung water and dry wet weight ratio were all decreased.2.PMVECs were incubated at 43? for 90 minutes.At each rewarming time point(0,2,6,12 h),the cell viability was measured with fluorescent microscope.Mcl,Bax,PAR1,and c-jun expression levels was determined by Western blot.Caspase-3 and NF-?B activities were measured by ELISA.Flow cytometry analysis was used to detect the cell apoptosis.The results showed that the apoptosis cell number of PMVECs was significantly elvated,and the expressions of Mcl-1,Bax c-Jun and p-c-Jun,and the activity of caspase-3 increased significantly after rewarming.The PMVECs were transfected with NC siRNA,PAR1 siRNA,Ad-empty or Ad-PARI and rewarmed.Twelve hours later,the apoptosis and protein were determined.In the cells transfected with Ad-PAR1 or pretreated with PAR1 agonist TFLLR-NH2,apoptosis was increased and Mcl-1 expression was significantly reduced.The expression of Bax and the activity of Caspase 3 were also increased significantly.In the cells transfected with PAR1 siRNA or pretreated with PAR1 inhibitor SCH79797,apoptosis was decreased and Mcl-1 expression was significantly increased.The expressions of Bax,p-c-Jun,c-Jun and the activity of Caspase 3 were decreased significantly and NF-?B DNA binding was enhanced.The cells were transfected with NC siRNA or c-Jun siRNA,and rewarmed.Twelve hours later,the cell apoptosis was detected.With the knockout of c-Jun,the apoptosis was reduced significantly.3.Heatstroke mice model was established as before.At rewarming 2h,HMGB1 and RIP3 activities were measured by ELISA.Compared with the control group,HMGB1 and RIP3 levels in the alveolar lavage were decreased in mice pretreated with PAR1 inhibitor RWJ56110.The formation of RIP 1 and RIP3 complexes in lung tissues were inhibited.PMVECs were incubated at 43? for 180 minutes.At each rewarming time point(0,2,6,12 h),RIP1 and MLKL protein expression levels increased significantly.At 6 h rewarming,Ripl-rip3 complex formation was increased.In the cells pretreated with RIP1 inhibitor Nec-1,RIP 1 siRNA,and RIP3 siRNA,procedural necrosis developed.The pretreatment of PAR1 inhibitor can reduce the HMGB1 shift caused by heat stress and decrease the cell necroptosis.ConclusionPAR1 inhibits the activation of NF-kappa B and acitvatiate c-Jun which increase the apoptosis of PMVECs induced by heat stress.PAR1 increases the formation of Ripl-rip3 complex which involves in the necroptosis of PMVECs.These effects both involve in the pathophysiology of severe heatstroke induced lung injury.PAR1 maybe the effective target to reduce the severe heatstroke induced lung injury and improve survival rate.