Protective Effects Of Gastrin-releasing Peptide Receptor Antagonist In A Murine Model Of Hepatic Ischemia And Reperfusion Injury

ObjectiveHepatic ischemia and reperfusion(I/R)injury affects the prognosis of patients,which is usually happened in the following clinical cases,including liver transplantation,liver resection surgery,trauma,hemorrhagic shock and cardiopulmonary resuscitation,etc.However,due to hepatic I/R injury of inner molecular mechanism has not yet fully understood,research evidence suggests that complex internal factors,such as reactive oxygen species(ROS),chemokines and inflammatory cytokines lead to liver injury including hepatocellular damage,leukocyte accumulation and inflammation process during hepatic I/R.And treatment strategy used to prevent liver I/R injury so far is not optimal.Gastrin-releasing peptide(GRP),a bioactive neuropeptide,interacts with it's receptor(gastrin-releasing peptide receptor,GRPR)promoting cell growth and participating in a variety of biological functions such as immune function regulation.The existing research shows that GRP-GRPR plays an important role in inflammatory diseases(such as sepsis),Blocking the GRPR can inhibit the inflammation development and improve immune function disorder.However,what role does GRP play in mice hepatic I/R model,whether blocking the GRPR can improve liver injury remains unknown.Therefore,this research intends to set up mice liver I/R injury model and dectect GRP and GRPR expression in hepatic I/R injury mice.Furthermore,we will determine the protective effect and explore the possible mechanisms of RC-3095 in mice with liver I/R injury.Methods1.The 70% hepatic ischemia/reperfusion(I/R)injury mice model was established in this experiment.C57BL/6 mice were randomly divided into two groups: sham group(sham,n=6)and I/R group(I/R,n=32).The surgery was administrated in I/R group with ischemia 60 mins,while mice in the sham group didn't experience the hepatic ischemia portal vein ligation processing.After ischemia 60 min and 3h,6h,12 h,24h reperfusion,blood and liver tissue were collected for detecting GRP and GRPR expression with sevoflurane anesthesia.GRP expression level in blood and liver tissue was determined by enzyme-linked immunosorbent assay(ELISA),and to determineGRPR protein expression in liver tissue by Western-blotting(WB)2.C57BL/6 mice were randomly divided into three groups: sham group,I/R group and RC-3095 group.After ischemia 60 mins,mice in RC-3095 group were subcutaneously injected RC-3095(3 mg/kg,150ul)before reperfusion happened,while mice in the sham group and the I/R group were treated with150 ul saline.After 6h reperfusion,blood and liver tissue were collected with sevoflurane anesthesia.Determining liver enzyme(ALT/AST)levels in serum was assessed by biochemical detector in changhai hospital clinical laboratory.H&E staining was used to assess liver tissue pathological.MPO activity was conducted to assess neutrophils accumulation in the liver tissue.Inflammatory cytokines(TNF-?,IL-1?,IL-6 and IL-10)expression levels in serum and liver tissue detected by ELISA.TUNEL was used to judge hepatocytes apoptosis.3.32 male C57BL/6 mice were randomly divided into three groups as same as the second point.After ischemia 60 mins and 6h,12 h,24h reperfusion,collected mice ischemic liver tissues were harvested to detected NF-?B protein activation and the phosphorylation levels of MAPK(JNK,ERK,p38)by Western-blotting.Results1.GRP and GRPR expression: GRP expression levels in both serum and liver tissue began to increase as early as 3h after reperfusion,peaked at 6h after reperfusion(P<0.01),and then decreased subsequently.However,compared with sham group,in the level of GRP in liver tissue was lower at 12 h and 24 h after reperfusion while there was no difference in blood.The same as the GRP,GRPR expression in liver tissue upregulated at 3h,peaked 6h after reperfusion(P<0.05),and then began to decrease.And there is no statistical difference compared with sham group after 24 h reperfusion.2.Compared with I/R group,changes in RC-3095 group are following: serum ALT/AST levels was significantly lower(P<0.01);H&E and TUNEL showed that liver injury was less severe and the percentage of hepatocytes apoptosis markedly decreased(P<0.01);FCM and the MPO activity showed RC-3095 inhibited neutrophil infiltration(P<0.01).The levels of IL-6,IL-1? and TNF-? levels in the liver tissue and blood were significantly downregulated with RC-3095 treatment(P<0.01),while the level of IL-10 showed no significantly difference.All these results indicated that RC-3095 inhibited hepatic I/R injury and inflammation response.3.The mechanism of GRP-GRPR involved in the liver I/R injury: The phospho-MAPK(JNK/SAPK,ERK,p38)and the NF-?B protein expression levels in RC-3095 group was significantly lower than that in the I/R group between 3h and 24 h after reperfusion(P<0.01).Conclusion1.GRP-GRPR expression in hepatic I/R injury significantly upregulated.2.RC-3095 significantly inhibited liver inflammation,hepatocellular necrosis and liver damage after I/R,consequently improve liver injury.3.RC-3095 inhibited NF-?B activity and phosphorylation of MAPKs in hepatic I/R injury,which suggested that GRP regulated inflammation of the liver I/R injury may related with the MAPK(JNK/SAPK,ERK,p38)and NF-?B pathway.