Nrf2-ARE Signaling Pathway Protects Intestinal Ischemia Reperfusion Injury In Rats

Intestinal ischemia reperfusion (ⅡR) injury is one of the most severe clinical disorders, which is caused by many factors, such as acute mesenteric ischemia, severe burn, hemorrhagic, traumatic or septic shock, and some surgical procedures, including small bowel transplantation and abdominal aortic surgery. It is known thatⅡR injury is an important initiating factor in the pathogenesis of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).ⅡR injury which is associated with intestinal transplantation usually lead to the failure of the operation due to the aggravating of immunological rejection. Efforts to prevent and controlⅡR injury remain the challenging and promising for clinical surgery.ⅡR injury is not only limited to the intestine itself, but involves severe distant tissue destruction. The mechanism ofⅡR injury is complicated. AfterⅡR, intestinal barrier function is damaged, bacteria translocation, toxic substances release into the blood, such as inflammatory mediators, bacterial toxins and reactive oxygen species (ROS) is activated, contributing to the oxidative stress. This is considered the key factor. Multiple signaling pathways have involved in this pathologic process which demenstrated by our and others studies, such as nuclear factor kappa B (NFκB), ubiquitin proteasome system (UPS), triggering receptor expressed on myeloid cells 1 (TREM-1), poly(ADP-ribose) polymerase 1 (PARP-1), etc. ROS in various signaling pathways are activated and produce inflammatory amplification, while other proteins may also be activated to produce a protective effect. Transcription factor NF-E2-related factor-2 (Nrf2) regulates a major environmental and oxidative stress response. It is held in the cytoplasm by a cytoskeletal-associated specific inhibitory protein (Kelch-like ECH associating protein 1, Keap1) under circumstances of normal cellular quiescent state. Upon the stimulation of oxidative stress, cysteine residues within the hinge region of Keap1 can be modified and cause a conformational change in KEAP1 with the loss of Nrf2 binding, then Nrf2 translocated into the nucleus, which binds to the antioxidant response element (ARE) in the promoter region of a number of genes, encoding for antioxidative and phase 2 enzymes, such as heme oxygenase 1 (HO-1), glutathione peroxidase (GSH-Px), and NAD(P)H:quinone oxidoreductase 1 (NQO1), which can antagonize oxidative stress induced by ROS. Thus, regulating Nrf2-ARE signaling pathway may be a potent approach against diseases caused by oxidative stress.With the discovery of the Nrf2-ARE signaling pathway can reduce the tissue damage induced by ROS, more and more studies have shown that modulating Nrf2-ARE signaling pathway play a protective role in splanchnic ischemia reperfusion injuries including heart, brain and renal. However, no report addresses a role of Nrf2-ARE signaling pathway in protectingⅡR injury.Sulforaphane (SFN) is an isothiocyanate derived from natural product which is present in cruciferous vegetables such as broccoli and has been used as antioxidants and anti-tumor agents, which drawn many researchers attention. The cytoprotective effect of this compound is exerted by modulating Nrf2-ARE pathway. Epigallocatechin gallate (EGCG), the major component including activity and water-solubility of polyphenols in green tea, which is an effective component of traditional Chinese drug, has recently drawn considerable attention for antioxidative properties.Our aim is to use a known pharmacal inducer of Nrf2 to explore the mechanisms of Nrf2-ARE signaling pathway in IIR by a rat model, then testify the role of the active ingredient of Chinese medicine-EGCG pretreatment on multiple organs dysfunction induced byⅡR, to seek a new target for prevention and treatment onⅡR injury at the molecular level, and provide more favorable theoretical basis on EGCG development and utilization.PartⅠ:Effect of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion injury in ratsObjective:To investigate the role of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion (ⅡR) injury through intervening on the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, and to provide a novel target for prevention and treatment on IIR injury at the molecular level.Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control, IIR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping superior mesenteric artery (SMA) for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Intestinal histology was investigated. Intestinal tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and GSH-Px activity were assayed. The Intestinal Nrf2 and HO-1 were determined by immunohisto-chemical analysis and western blot analysis.Results:Firstly, compared with the control group,ⅡR group:(1) Intestine was obvious edema, hemorrhage, bleed, exudation, and lack of small intestinal mucosa (P<0.01); (2) Intestinal SOD activity was decreased (P<0.01). Intestinal MPO activity was increased (P<0.01); (3) Intestinal GSH level was significantly enhanced (P<0.01); (4) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.01). Secondly, compared with the control group, SFN control group:(1) Intestinal GSH-Px activity increased significantly (P<0.01); (2) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01,P<0.01). Thirdly, compared with theⅡR group, SFN+ⅡR group:(1) Intestine tissue pathological scores of injury significantly were reduced (P<0.05); (2) Intestinal SOD activity was elevated markedly (P<0.05), MPO activity was conspicuously decreased (P<0.05); (3) Intestinal GSH and GSH-Px levels were significantly enhanced (P<0.05,P<0.01); (4) The expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.05).Conclusions:ⅡR induced intestinal injury, characterized by the histological edema, hemorrhage, lack of small intestinal mucosa. The injury of intestine was alleviated through upregulating Nrf2-ARE signaling pathway, which is accompanied with the expressions of Nrf2 and HO-1 in the intestinal tissue. The activation of Nrf2-ARE signaling pathway plays an important role in the protection of intestinal ischemia reperfusion.PartⅡ:SFN protects liver and lung injuries induced by intestinal ischemia reperfusionObjective:To investigate the role of a known inducer of Nrf2-ARE signaling pathway on the liver and lung injuries induced byⅡR through evaluating the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, then clarify the effect of SFN by inducing Nrf2-ARE signaling pathway on liver and lung injuries triggered byⅡR in rats.Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control,ⅡR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia, 5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lung brochia alveolus lung fluid protein (BALFP) was measured. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.01); (2) Lung BALFP was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.05), MPO activities were increased (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.01). Secondly, compared with the control group, SFN control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.05, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.05, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, SFN+IIR group:(1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung BALFP was decreased significantly (P<0.05); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were decreased significantly (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.05, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.05).Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung were alleviated through Nrf2-ARE signaling pathway, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. The activation of Nrf2-ARE signaling pathway induced by SFN plays an important role in the protection of liver and lung injuries induced byⅡR.PartⅢ:EGCG protects liver and lung injuries induced by intestinal ischemia reperfusion in ratsObjective:To investigate EGCG on the protection of liver and lung injuries induced byⅡR, and testify if the role of EGCG pretreatment on multiple organs dysfunction induced by IIR act as SFN through Nrf2-ARE signaling pathway. To provide more favorable theoretical basis on the development and utilization of EGCG.Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, EGCG control,ⅡR, and EGCG+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,50mg/kg EGCG abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of AST, ALT, and lung water content was assayed. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.05); (2) Lung water content was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.01), MPO activities were increased (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.05). Secondly, compared with the control group, EGCG control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.01, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, EGCG+ⅡR group: (1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung water content was decreased significantly (P<0.01); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were, decreased significantly (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.01, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.01,P<0.05).Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung can be alleviated through EGCG pretreatment, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. EGCG, the major component of polyphenols in green tea, can protect liver and lung injuries induced byⅡR through Nrf2-ARE signaling pathway.We use intestinal ischemia reperfusion (ⅡR) rat model to analyze the intestine, liver and lung injuries marker (histology, serum ALT, AST, lung water content and BLAFP), tissue oxidative stress (SOD, MPO), and the expression of Nrf2-ARE pathway (Nrf2, GSH, GSH-Px and HO-1) using biochemical analysis, immunohistochemical analysis and Western blot analysis to evaluate the role of Nrf2-ARE signaling pathway on the protective effect ofⅡR and the effect of SFN, EGCG pretreatment to liver and lung injuries induced byⅡR.The result showed as follows:Intestinal ischemia reperfusion (ⅡR) not only leads to intestinal damage itself, but also causes severe destruction of remote organs, characterized by the histological edema, hemorrhage and infiltration with inflammatory cells, significant increasing of ALT, AST, lung water content, and BLAFP. Intestine, liver and lung SOD and MPO levels changed markedly inⅡR group. Pretreatment with SFN or EGCG, the intestine, liver and lung injuries were improved, which is accompanied with the level of tissue Nrf2. At the meantime, phase 2 antioxidant and detoxification enzymes increased markedly. The Nrf2-ARE signaling pathway has been shown to have beneficial effects on the prevention ofⅡR. This also demonstrate that EGCG, which is the major component of polyphenols in green tea, plays an important role in the induction of Nrf2-ARE signaling pathway, and the protection of liver and lung injuries induced byⅡR.