Mechanisms And Role Of Necroptosis In Intestinal Injury Induced By Heaptic Cold Ischemia Reperfusion

Background For more than 30 years liver transplantation has been an effective treatment procedure for end-stage liver diseases.Five-year survival rate following liver transplantation are 70%.During liver transplantation,the more challenges are from the organ injury,the worse early recovery in recipients.During the procedure,the anhepatic phase can induce ischemic injury of the liver and therefore cause dysfunction of remote organs,especially gut,lung,and kidney,which is also called extra-hepatic multiple organ damage.Occlusion of portal vein and inferior vena cava,can lead to intestine congestive ischemia and gastrointestinal congestion,which lead to intestinal injury.The epithelial cells of the intestinal mucosa are sensitive to the hypoxia-ischemia upon intestinal congestion,and they are often subjected to apoptosis and necrosis after the surgery.These factors are causatively associated with postoperative complications including the damage to intestinal barrier and barrier dysfunction,and the increase of the postoperative mortality rate.This study has investigated the intestinal injury during perioperative of liver transplantation.The clinical trial demonstrated significant occurrence of intestinal injury in patients undergoing liver transplantation.Therefore,this research investigated intestinal injury induced by hepatic cold ischemia reperfusion,identified the mechanisms of necroptosis and the protective effects of dexmedetomidine in intestinal injury.Part one: Necroptosis is a key mediator of enterocytes loss in intestinal injury induced by heaptic cold ischemia-reperfusionObjective Cell death is an important biological process that is believed to have a central role in intestinal ischemia/reperfusion(I/R)injury.While the apoptosis inhibition is pivotal in preventing intestinal I/R,how necrotic cell death is regulated remains unknown.Necroptosis represents a newly discovered form of programed cell death that combines the features of both apoptosis and necrosis.Receptor interaction protein kinase 1/3(RIPK1/3)and mixed lineage kinase domain like protein(MLKL)recruitment mediates necroptosis in a model of intestinal injury.Herein we aimed to demonstrate the functional relevance of necroptosis in regulating intestinal injury induced by hepatic cold I/R.We identified that the inhibition of necroptosis using necrostatin-1(Nec-1)during intestinal I/R confers enhanced intestinal protection.Methods: Adult male Sprague-Dawley rats weighing 250-300 g were anesthetized with choral hydrate.Rats were randomly assigned to the following three groups(n=16):(1)Sham group in which animals just underwent dissection of the heaptic vessels and ligaments,but without cold ischemia/reperfusion;(2)I/R group in which rats received blocking the portal vein(PV)and the infrahepatic vena cava(IHVC)aboved the right renal vein and superior mesenteric vein.Meanwhile vascular clamps were used at the suprahepatic vena cava(SVC)and 1-mm incision was made in the wall of the IHVC as an outflow tract.Ringer lactate solution(4 ?)was injected for reperfusion of liver as an inflow tract.The total anhepatic phase lasted 30 minutes;(3)Nec group in which rats received nec-1(1.0 mg/kg,dissolved in normal saline,intraperitoneally)30 min before ischemia and dissection of vascular for I/R animals.The above cohort of rats(n=8 per group)received 6 hrs of reperfusion.The other cohort of rats(sham group,I/R group and Nec group,n=8 per group)received identical procedures as described above for each group but followed 24 hrs of reperfusion.Blood samples were taken from inferior vena cava and centrifuged,and supernatant was stored at-80? for subsequent measurements.The blood samples was obtained to detect the levels of D-lactic acid,Diamine oxidase(DAO),Intestinal fatty acid binding protein(I-FABP)and endotoxin.The ileum tissues were obtained for determination of intestinal histopathological changes and scores by HE staining,and wet/dry(W/D)ratio,to detect the intestinal proinflammatory cytokines(TNF-?,IL-6,and HMGB1),anti-inflammatory(IL-10)level;intestinal tissue homogenate was to detect myeloperoxidase(MPO)activity,antioxidases superoxide dismutase(SOD)and CAT activity,malondialdehyde(MDA)content,and active caspase3 expression at 6h,24 h after sham or reperfusion.Western blotting detected the protein expression levels of RIPK1,RIPK3 and MLKL.The expression level of caspase-3 was analyzed by the immunochemistry methods.We brought TUNEL method to measure the count of cell apoptosis of intestinal tissues.Results: 1.The intestinal histopathological changes: Light microscopy demonstrated that histological tissue damage was reduced in the Nec group than the I/R group,while no injury was evident in the sham group.Compared with sham group,the Chiu's score of intestinal injury and W/D ratio were significantly increased followed by 6hrs/24 hrs of reperfusion in the I/R and Nec group(P<0.05).Compared with I/R group,the Chiu's score of intestinal injury and W/D ratio were significantly decreased followed by 6hrs/24 hrs of reperfusion in the Nec group(P<0.05).2 The serum and intestinal inflammatory cytokines changes: Compared with sham group,the serum and intestinal proinflammatory cytokines TNF-?,IL-6,HMGB1 level and anti-inflammatory IL-10 level were significantly increased followed by 6hrs/24 hrs of reperfusion in the I/R and Nec group(P<0.05&<0.01),serum LPS level was increased(P<0.05).RIPK1 inhibitor nec-1 treatment could significantly decreased the serum and intestinal proinflammatory cytokines TNF-?,IL-6,HMGB1 level;increased anti-inflammatory IL-10 level;decreased serum LPS level followed by 6hrs/24 hrs of reperfusion(P<0.05&<0.01).3.The markers of intestinal injury changes: Compared with sham group,the intestinal DAO,D-lactic acid,and I-FABP level were significantly increased followed by 6hrs/24 hrs of reperfusion in the I/R and Nec group(P<0.05).RIPK1 inhibitor nec-1 treatment could significantly decreased the intestinal DAO,D-lactic,and I-FABP level followed by 6hrs/24 hrs of reperfusion(P<0.05).4.The intestinal oxidases and antioxidases marker changes: Compared with sham group,the intestinal oxidases(MDA,MPO)activity were significantly increased,and the intestinal antioxidases(SOD,CAT)activity were significantly decreased followed by 6hrs/24 hrs of reperfusion in the I/R and Nec group(P<0.01).RIPK1 inhibitor nec-1 treatment could significantly decreased the intestinal oxidases(MDA,MPO)activity,and increased he intestinal antioxidases(SOD,CAT)activity followed by 6hrs/24 hrs of reperfusion(P<0.05).5.The count of cell apoptosis in the intestinal tissue: Compared with sham group,the activated caspase3 expression was significantly increased followed by 6hrs/24 hrs of reperfusion,and the count of cell apoptosis was significantly increased in the I/R and Nec group(P<0.05).Compared with I/R group,the activated caspase3 expression was significantly decreased followed by 6hrs/24 hrs of reperfusion,and the count of cell apoptosis was significantly decreased(P<0.05).6.The RIPK1,RIPk3,and MLKL protein expression: Compared with sham group,the RIPK1,RIPk3,and MLKL protein expression were upregulated followed by 6hrs/24 hrs of reperfusion in the I/R and Nec group(P<0.05&<0.01);RIPK1 inhibitor nec-1 treatment could significantly down-regulated the RIPK1,RIPk3,and MLKL protein expression(P<0.05&<0.01).Conclusion: The inflammatory cytokines and the oxidative stress were the main factors leading intestinal injury induced by hepatic cold I/R,and pre-treatment with the specific inhibitor Nec-1 reduces necroptosis via down-regulation of RIPK1/RIPK3-MLKL.Part two: Protective effects and mechanisms of Dexmedetomidine on intestinal injury induced by hepatic cold ischemia reperfusionObjective: To investigate the effects of Dexmedetomidine(Dex)on the intestinal injury induced by liver cold I/R in rats,and explain the Dex's protective effects on intestinal injury based on inflammatory response,oxidative stress and necrotic cell death.Methods: Forty SPF healthy adult male SD rats,aged 10-12 weeks and weighing 250-300 g,were randomly divided into 5 groups(n=8): Sham group,I/R group,Dex group,Nec group,Dex+Nec group.The sham group in which animals just underwent dissection of the heaptic vessels and ligaments;I/R group in which rats received anhepatic for 30 minutes and followed by 6hrs of reperfusion.Dex group in which rats received Dex(50?g/kg,dissolved in normal saline,intraperitoneally)30 min before ischemia and other manipulation for I/R animals;Nec group in which rats received nec-1(1.0 mg/kg,dissolved in normal saline,intraperitoneally)30 min before ischemia and other manipulations for I/R animals;Dex+Nec group in which rats received Dex(50?g/kg)and nec-1(1.0 mg/kg)respectively before ischemia.At 6 hr of reperfusion,blood samples were collected from the IHVC for determination of serum TNF-?,IL-6,IL-10,HMGB1,DAO,D-lactic,I-FABP,and LPS level.The rats were sacrificed,and intestines were harvested for determination of histopathological changes(with light microscope)and W/D ratio;and for examination of MDA content(using thiobarbituric acid method),MPO activity,SOD activity(by xanthine oxidase method),and CAT activity;and for detecting activated caspase3 expression(by immunohistochemistry),and apoptotic cells(using TUNEL).Apoptotic rate was calculated.The expression of phosphorylations of RIPK1,RIPK3 and MLKL were assessed by Western blot.Results: Compared with sham group,the serum TNF-?,IL-6,IL-10,HMGB1,LPS,DAO,D-lactic and I-FABP levels were increased;the intestinal tissue MDA content and MPO activity were increased,SOD activity and CAT activity were decreased;Caspase3 protein expression and the count of cell apoptosis were increased;the Chiu's scores and W/D ratio were increased;the RIPK1,RIPk3,and MLKL protein expression were upregulated followed by 6 hr of reperfusion in the I/R,Dex,Nec,and Dex+NEC group.Dex treatment and/or RIPK1 inhibitor nec-1 treatment could significantly decreased serum TNF-?,IL-6,HMGB1,DAO,D-latic,LPS,and I-FABP level;increased serum IL-10 level;decreased intestinal MDA and MPO content,increased intestinal SOD and CAT activity;decreased the Chiu's scores and W/D ratio;downregulated the RIPK1,RIPK3,and MLKL protein expression followed by 6 hr of reperfusion.Compared with Dex group,Nec-1 treatment combined with or without Dex could significantly decrease the serum proinflammatory cytokines and increased the anti-inflammatory cytokines;improved the markers of intestinal injury,oxidative stress,histopathological scores,ultrastructure changes;downregulated intestinal RIPK1,RIPK3,and MLKL protein expression.Conclusion: Dexmedetomidine can attenuate intestinal injury by liver cold ischemia-reperfusion in rats,and the mechanism is related to its anti-inflammatory,antioxidant and partly inhibition of activation of programed cell death signal pathway.