The Role Of Necroptosis In Lung Ischemia Reperfusion Injury In Rats And The Effect Of Nec-1 Pretreatment On P38 MAPK/NF-κB Pathway

Part Ⅰ The role of necroptosis in lung ischemia reperfusion injury in ratsObjective:Necroptosis has been potentially considered as the common mechanism involved in the various inflammatory organ injuries,and it may contribute to ischemia reperfusion injury in several organs or tissues.However,it is still unknown whether necroptosis plays a certain role in lung ischemia reperfusion.Here,the first part of our study was designed to investigate the role of necroptosis in lung ischemia reperfusion injury in rats.Methods:Forty healthy male SD rats were randomized into 5 groups using random number table:sham operation group(SM group),ischemia reperfusion group(IR group),Nec-l+ischemia reperfusion group(NI group),z-VAD-fmk+ischemia reperfusion group(ZI group)and Nec-l+z-VAD-fink+ischemia reperfusion group(NZ group),with 8 rats in each group.The rats in SM group received intraperitoneal injection of DMSo 0.05ml/kg at 1 hour prior toleft thoracotomy without clamping the left hilum of lung,and left thoracic cavity was closed 1 hour later.The rats in IR group received intraperitoneal injection of DMSO 0.05ml/kg at 1 hour prior to ischemia,and then were subjected to left lung ischemia reperfusion by clamping the left hilum of lung for 60 min followed by 3 hour-reperfusion.Nec-1 lmg/kg,z-VAD-fink 3mg/kg and combination of Nec-1 and z-VAD-fink(all the agents were dissolved in 0.05ml/kg DMSO)were intraperitoneally administrated at 1 hour prior lung ischemia in NI group,ZI group and NZ group,respectively,and the remaining operation was identical to IR group.Upon 3-hour reperfusion,microstructure and ultrastructure in the lung tissue were evaluated under light microscopeand transmission electron microscope,cell death manner of lung single-cell suspension was measured using flow cytometry,also the protein expressions of receptor-interacting protein 1(RIP1)and RIP3 were detectedResults:No obvious abnormalities in microstructure and ultrastructure were found in SM group,and significant morphological structure damage,which were evidenced by thickening of alveolar interval,pulmonary interstitial edema,neutrophil infiltration,mitochondrial swelling in alveolar type II epithelial cells,reduced osmiophilic multilamellar body and increased emptying,and damaged membrane integrity.The changes in microstructure and ultrastructure were alleviated in NI group,ZI group and NZ group in varying degrees,where the damages were least in NZ group.Compared to SM group,both the percentage of Annexin V+ PI-and Annexin V+ PI+ cells were increased in IR group,NI group and ZI group;Compared to IR group,the percentage of Annexin V+PI+ cells were decreased in NI group,the percentage of Annexin V+ PI-cells were decreased in ZI group,and both the percentage of Annexin V+ PI-and Annexin V+ PI+cells were decreased in NZ group;The the percentage of Annexin V+ PI+ and Annexin V+PI-cells were decreased in NZ group as compared to NI group and ZI group,respectively(p<0.05).The expressions of RIP1 and RIP3 protein in lung tissue were upregulated in IR group,ZI group and NZ group as compared to SM group;As compared to IR group,RIP 1 and RIP3 protein expressions were downregulated in NI group;Compared to ZI group,the expressions of RIP1 and RIP3 were lower in NZ group(p<0.05).Conclusion:Necroptosis plays a role in morphological structure damage induced by lung ischemia reperfusion in rats;the pretreatment with Nec-1,especially when it was combined with z-VAD-fmk,could significantly attenuated ischemia reperfusion-induced lung injury.Part Ⅱ The effect of Nec-1 pretreament on inflammation induced by lung ischemia reperfusion and p38 MAPK/NF-κB pathway in ratsObjective:Mitogen-activated protein kinases(MAPKs)pathway activation is involved in organ ischemia reperfusion injury,where p38 MAPK/nuclear factor kappa B(NF-κB)pathway is associated with inflammatory response.In the first part of our study,we found that Nec-1 pretreatment could inhibit necroptosis and attenuate ischemia reperfusion-induced lung injury.However,the effect of Nec-1 pretreatment on inflammatory response and p38 MAPK/NF-κB pathway are still not reported.Methods:Thirty-two healthy male SD rats were randomized into 4 groups using random number table:sham operation group(SM group),ischemia reperfusion group(IR group),Nec-1+ischemia reperfusion group(NI group)and p38 MAPK inhibitor SB203580+ischemia reperfusion group(SI group),with 8 rats in each group.The rats in IR group received intraperitoneal injection of DMSO 0.05ml/kg at 1 hour prior to ischemia,and then were subjected to left lung ischemia reperfusion by clamping the left hilum of lung for 60 min followed by 3 hour-reperfusion.The rats in SM group received intraperitoneal injection of DMSO 0.05ml/kg at 1 hour prior toleft thoracotomy without clamping the left hilum of lung,and left thoracic cavity was closed 1 hour later.Nec-1 lmg/kg and SB203580 10mg/kg(all the agents were dissolved in 0.05ml/kg DMSO)were intraperitoneally administrated at 1 hour prior lung ischemia in NI group and SI group,respectively,and the remaining operation was identical to IR group.Upon 3-hour reperfusion,the protein expressions of phosphorylatedp38 MAPK,total p38 MAPK and NF-κB were detected,the levels of high mobility group box-1 protein(HMGB1),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in lung homogenates were measured,also wet/dry weight ratio(W/D)was calculated and blood gases extracted fromaorta abdominalis including arterial partial pressure of oxygen(PaO2)and arterial partial pressure of carbon dioxide(PaCO2)were measured.Results:Compared to SM group,both the expressions of protein p38 MAPK and NF-κB were increased in IR group,NI group and SI group;The expressions of protein p38 MAPK and NF-κB were downregulated in NI group and SI group as compared to IR group(p<0.05).Compared to SM group,the levels of HMGB1,TNF-α,IL-1β and IL-6 in lung homogenates were increased in IR group,NI group and SI group;The levels of HMGB1,TNF-α,IL-1β and IL-6 in lung homogenates were decreased in NI group and SI group as compared to IR group(p<0.05).Compared to SM group,W/D ratio of lung tissue was increased in IR group,NI group and SI group;W/D ratio of lung tissue was decreased in NI group and SI group as compared to IR group(p<0.05).Compared to SM group,PaO2 was lower in IR group,NI group and SI group;PaO2 was higher in both NI group and SI group than that in IR group(p<0.05).No significant differences were found between NI group and SI group as regarding to abovementioned parameters(p>0.05).Conclusion:By modulating p38 MAPK/NF-κB pathway,the pretreatment with Nec-1 could inhibit inflammatory response and release of inflammatory cytokines,decrease pulmonary water content and improve pulmonary oxygenation function in lung ischemia reperfusion injury model in rats.