| Perioperative hemorrhage affects the mortality and morbidity rates after liver surgery. In order to reduce hemorrhage of liver resection, temporary clamping of portal triad (Pringle maneuver) is widely used. Not only can this operation lead to hepatic ischemia/reperfusion (I/R) injury, but also result in intestinal congestion/reperfusion (C/R) injury. Liver transplantation also causes hepatic I/R injury and intestinal C/R injury.Heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified initially as a product of cultured human macrophages and later found to be a member of the epidermal growth factor (EGF) family. HB-EGF is a potent mitogen and chemotactic factor for various cell types, such as gastrointestinal epithelial cell, hepatocytes, smooth muscle cells, fibroblasts and keratinocytes.Previous studies have demonstrated the ability of HB-EGF in protecting the intestine from injury caused by superior mesenteric artery occlusion followed by reperfusion. Administration of exogenous HB-EGF protects the intestines from injury in animal models of hemorrhagic shock and resuscitation and necrotizing enterocolitis. Recent studies have shown that HB-EGF suppresses experimental liver fibrosis in mice and plays a protective role in CC14-induced acute liver injury. HB-EGF, given intraluminally, also reduces the severity of acute lung injury after intestinal I/R in mice.There are many research studies about intestinal I/R injury. However, there is little research about intestinal C/R injury. Congestion injury is different from ischemia injury. A previous study found that the degree of intestinal damage due to venous occlusion was greater than the damage resulting from aterial occlusion in rats. So we used a rat model of portal triad clamping to show that HB-EGF protects the intestine from C/R injury. And then we used a rat model of orthotopic autologous liver transplantation to show that HB-EGF protects the liver from total hepatic I/R injury with intestinal congestion.Part1. Protective effects of HB-EGF on intestinal congestion/reperfusion injury in ratsAimThe current study examined whether HB-EGF has protective effects in intestinal congestion/reperfusion injury, which is caused by portal triad clamping.MethodsAnimals and materialsA total of30male Sprague-Dawley rats aged7to9weeks and weighing213-256g were obtained from the Southern Medical University Animal Center, Guangzhou, China. All animals received humane care in compliance with the European Convention on Animal Care. The following experimental protocol was approved by the Southern Medical University Animal Care and Use Committee. The rats were fasted for12hours with access to water only before the procedure.The HB-EGF used in all experiments was a highly purified form of recombinant human HB-EGF which was purchased from R&D Systems, USA. Surgical procedure and experimental designUnder inhalation anesthesia using diethyl ether, the animal was fixed with a self-made operation cushion, placed supinely. The incision was sterilized by iodine tincture and alcohol, and then a midline laparotomy was made.Rats were randomly divided into three equally-sized groups:Ⅰ. sham-operated (Sham group); Ⅱ. Pringle maneuver (PM), all structures in the portal triad (hepatic artery, portal vein, and bile duct) were occluded with an atraumatic microvascular clamp for30min and then the clamp was removed, the abdomen was closed (C/R group);Ⅲ. PM+intraluminal administration of HB-EGF15min after the initiation of portal triad clamping (C/R+HB-EGF group).Rats in the group III received a1mL intraluminal injection of HB-EGF (600μg/kg) diluted in0.1%bovine serum albumin (BSA) in PBS at the jejuno-ileal junction to ensure that the entire small bowel was filled with the HB-EGF solution. Animals in group II received1mL of0.1%BSA in PBS solution only.Animals underwent re-laparotomy after6h of reperfusion.Detection indicatorsBlood and ileum tissue samples were obtained. Blood was collected from the inferior vena cava. Blood was allowed to clot for15min at room temperature and then centrifuged (3,000rpm) for15min to isolate the serum. Serum levels of TNF-a and IL-1β were measured. The ileum samples were sampled for histopathological examination and TUNEL assays. Myeloperoxidase (MPO) activity and Malonaldehyde (MDA) levels in the ileum samples were also measured.Statistical analysisData was analyzed using SPSS13.0for Windows software. Data were expressed as mean and standard deviation. The statistical significance of differences between groups was analyzed using one-way analysis of variance (ANOVA). Ap value<0.05 was considered to be statistically significant.ResultsIntestine pathologic changesThe color of the intestine was dark reddish brown and the intestinal wall was thick and enlarged after portal triad clamping for30min. HE staining was carried out to determine the histological changes in the ileum tissue. As expected, no mucosal injury was observed in the sham group. However, hemorrhage on the mucosa, loss of villous epithelium and mucosal neutrophil infiltration infiltration were observed in the C/R group. HB-EGF administration significantly attenuated the injury.Serum TNF-α and IL-1β levelsThe clamping of portal triad caused significant increases in serum levels of proinflammatory cytokines, TNF-α and IL-1β (P=0.000, P=0.000). HB-EGF administration significantly decreased both of these cytokine levels (P=0.031, P=0.038).MPO activityWhen compared with the sham group (0.49±0.10U/g), intestinal MPO activity was increased significantly in the C/R group (1.28±0.18U/g). This elevation in MPO activity induced by C/R was reversed with HB-EGF administration (0.89±0.15U/g, P=0.000vs the C/R group).MDA levelsThe intestine MDA levels were found to be significantly higher in the C/R group than in the sham group (3.05±0.39vs1.32±0.23nmol/mg protein, P=0.000). However, administration with HB-EGF decreased the elevated MDA levels significantly (2.39±0.27nmol/mg protein, P=0.000).Enterocyte apoptosisTUNEL-positive enterocyte were revealed by brown staining. The mean apoptosis index (AI) of enterocytes in the C/R group were significantly increased compared with the sham group (P=0.000). The AI in the C/R+HB-GEF group was lower than that in the C/R group (P=0.014).ConclusionOur results show that Pringle manoeuvre of30min duration results in intestinal I/R injury. HB-EGF attenuates intestinal C/R injury induced by portal triad clamping. The protective effects are probably associated with inhibition of inflammation responses, enhancement of antioxidant capacities and reduction of enterocyte apoptosis.Part2. Protective effects of HB-EGF on hepatic ischemia/reperfusion injury in ratsAimThe current study examined whether intraluminal administration of HB-EGF has protective effects in total hepatic ischemia-reperfusion (I/R) injury induced by liver transplantation in rats and explored the possible mechanism.MethodsAnimals and materialsA total of30male Sprague-Dawley rats aged7to9weeks and weighing217-255g were obtained from the Southern Medical University Animal Center, Guangzhou, China.The HB-EGF used in all experiments was a highly purified form of recombinant human HB-EGF which was purchased from R&D Systems, USA.Surgical procedure and experimental designThe rats were fasted for12hours with access to water only before the procedure. Under inhalation anesthesia using diethyl ether, the animal was fixed with a self-made operation cushion, placed supinely. The incision was sterilized by iodine tincture and alcohol, and then a midline laparotomy was made.The ligaments around the liver were dissociated and severed. The left diaphragm vein, hepatoesophageal ligament vein and right adrenal vein were separated, ligated and severed. The liver was turned to the left and the suprahepatic venae cavae (SVC) was anatomized. Then the common bile duct, hepatic artery (HA), and portal vein (PV) were anatomized over the margin of the duodenal bulb. The infrahepatic venae cavae (IVC) was dissociated downwards about6-8mm. The liver was dissociated completely, except for the hepatic blood vessels traversing in and out and the common bile duct. The pyloric vein was occluded by a clamp. The PV was picked with transfixion pin and fixed by a clamp. Then heparin saline (35U/ml,2ml) was injected into the PV to drive the blood of the liver into the systematic circulation. The rat was heparinized. Subsequently, the HA was occluded by a clamp and another clamp was placed on the crossing of the cranial mesenteric vein and splenic vein. The countdown to reperfusion was initiated. The clamp was placed on the IVC and SVC. An incision of about1mm was made in the IVC above the right renal vein as an outflow tract. Lactated Ringer’s solution (4℃, including heparin12.5U/ml,20ml) was perfused at2.5ml/min through the PV by an infusion pump. The liver surface was covered with lactated Ringer’s solution (4℃) to reduce the temperature. Finally, the color of the liver faded.The transfixion pin was removed after the perfusion, the PV was repaired with a9-0prolene suture, and the incision in the IVC was oversewn with an8-0suture. The PV, SVC, IVC, HA and pyloric vein were unclamped after30min for an anhepatic phase. The surface of the liver was irrigated with20ml normal saline of38℃for rapid rewarming. The liver was filled with blood and turned red.The abdomen was closed, and the animals were then allowed to recover in individual cages.Experimental designMale Sprague-Dawley rats were randomly divided into three groups:Ⅰ) sham-operated (n=10), Ⅱ) an orthotopic autologous liver transplantation model with an anhepatic phase for30min followed by reperfusion for6h (I/R model, n=10), and Ⅲ) I/R with intraluminal administration of HB-EGF15min after the initiation of anhepatic phase (I/R+HB-EGF model, n=10).Rats in the group Ⅲ received a1ml intraluminal injection of HB-EGF (600μg/kg) diluted in0.1%bovine serum albumin (BSA) in PBS at the jejuno-ileal junction. Timing, route, and dose of administration of HB-EGF in our experiment were referred to Martin’s description. Animals in group II received1ml of0.1%BSA in PBS solution only.Animals underwent re-laparotomy after6h of reperfusion.Detection indicatorsBlood was collected from the inferior vena cava. Blood was allowed to clot for15min at room temperature and then centrifuged (3,000rpm) for15min to isolate the serum. Liver specimens were rapidly excised.The serum level of alanine aminotransferase (ALT) was measured to assess the extent of hepatocyte damage using commercial kits, according to the manufacturers’ protocols with an biochemistry analyzer.The liver tissue specimens were cut into4μm sections, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). All microscopic slides were analyzed by a pathologist blinded to the group assignments of the animals. Each section was scored for congestion, vacuolization and necrosis described by Suzuki et al.Localization of apoptotic cells in liver samples was assessed by a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)- biotin nick end labeling (TUNEL) technique using an in situ apoptosis detection kit. The apoptosis index (AI) was defined as the proportion of TUNEL-positive cells in the total number of hepatocytes.TNF-α and IL-1β levels in serum samples and liver samples were assayed using a commercial enzyme-linked immunosorbent assay (ELISA) kit. MPO activity was measured and calculated from the absorbance (at460nm) changes resulting from decomposition of H2O2in the presence of o-dianisidine. And the expression of ICAM-1in liver tissues was analyzed by immunohistochemical analysis.The expression of NF-kB p65in the nuclear area and IkBα were analyzed by western blot analysis. Protein bands were visualized with an enhanced chemiluminescence assay kit and then exposed to film.Statistical analysis of dataAll data were expressed as mean±standard deviation. Data were analyzed using SPSS13.0for Windows software. Data were compared by a one-way analysis of variance. Ap value<0.05was considered to be statistically significant.ResultsSerum ALT levelsIn our study, the ALT serum levels were greatly elevated in the I/R group compared with the sham group (P=0.000). The levels of ALT in the I/R+HB-GEF group were lower than those in the I/R group (P=0.034).Pathologic changesAs expected, no hepatocellular injury was observed in the sham group. However, swelling of the hepatocytes, cytoplasmic vacuolization, sinusoidal congestion, and cellular infiltration were observed in the I/R group. HB-EGF administration attenuated the injury.Hepatocyte apoptosis TUNEL-positive hepatocytes were revealed by brown staining. The mean AI of hepatocytes in the I/R group were significantly increased compared with the sham group (P=0.000). The AI in the I/R+HB-GEF group was lower than that in the I/R group (P=0.023).Expression of TNF-a and IL-1βThe total hepatic I/R injury caused significant increases in serum levels of proinflammatory cytokines, TNF-a and IL-1β (P=0.000, P=0.000). HB-EGF administration significantly decreased both of these cytokine levels (P=0.013, P=0.027).TNF-a and IL-1β levels in the liver samples were significantly increased in the I/R group (P=0.000, P=0.000), which can be markedly reduced by HB-EGF administration (P=0.045, P=0.023).MPO activityMPO is used as a marker of neutrophil accumulation and activation in liver tissues. When compared with the sham group (0.88±0.10U/g), liver MPO activity was increased significantly in the I/R group (1.87±0.13U/g). This elevation in MPO activity was reversed with HB-EGF administration (1.72±0.11U/g, P=0.005versus the I/R group).Expression of ICAM-1The expression of ICAM-1in the Sham group was little. However, ICAM-1was increased in the I/R group significantly, especially in the sinusoidal endothelial cells and small vein endothelial cells. HB-EGF administration significantly decreased ICAM-1expression in the liver.NF-kB activityNF-kB is the upstream mediator of pro-inflammatory cytokine transcription. The expression of NF-kB p65in the I/R group was significantly elevated compared to the sham group, whereas HB-EGF remarkably inhibited the activation of NF-κb.IkBα levelsThe degradation of IκBα in I/R rats was significantly increased at6h. HB-EGF administration was found to suppress the degradation of IκBα.ConclusionIn summary, our results showed that intraluminal administration of HB-EGF attenuates total hepatic I/R injury induced by liver transplantation in rats. We also investigated the mechanism from the perspective of the inflammatory response. HB-EGF administration inhibited NF-κB activation and decreased pro-inflammatory cytokine levels and neutrophil accumulation. The inhibition of inflammation responses may be one of the protection mechanisms. However, the precise mechanisms need to be further investigated. Our current study may represent a promising therapeutic method for total hepatic I/R injury. |
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