Pretreatment of cromolyn sodium prior to reperfusion attenuates early reperfusion injury after the small intestine ischemia in rats

AIM: To investigate the effects of Cromolyn Sodium (CS) pretreated prior to reperfusion on the activity of intestinal mucosal mast cells (IMMC) and mucous membrane of the small intestine in ischemia-reperfusion (IR) injury of rats. METHODS: Thirty-two Sprague-Dawley (SD) rats were randomly divided into four groups: sham group (group S), model group (group M), high and low dosage of CS groups, (treated with CS 50 mg/kg or 25 mg/kg, group C1 and C2). Intestinal IR damage was induced by clamping the superior mesenteric artery for 45 min followed by reperfusion for 60 min. CS was intravenouly administrated 15 min before reperfusion. Ultrastructure and counts of IMMC, intestinal structure, the expression of tryptase, levels of malondisldehyde (MDA), TNF-α, histamine and superoxide dismutase (SOD) activity of the small intestine were detected at the end of experiment. RESULTS: The degranulation of IMMC was seen in group M and was attenuated by CS treatment. Chiu’s score of group M was higher than the other groups. CS could attenuate the up-regulation of the Chiu’s score, the levels of MDA, TNF-α, and expression of tryptase and the down-regulation of SOD activity and histamine concentration. The Chiu’s score and MDA content were negatively correlated, while SOD activity was positively correlated to the histamine concentration respectively in the IR groups. CONCLUSION: Pretreated of CS prior to reperfusion protects the small intestine mucous from ischemia- reperfusion damage, the mechanism is inhibited IMMC from degranulation.

Effect of nuclear factor kappa B on intercellular adhesion molecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM： To investigate the role of nuclear factor kappa B （NF-κB） in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion （I/R）, and its effect on intercellular adhesion molecule-1 （ICAM-1） expression and neutrophil infiltration. METHODS： Twenty-four Wistar rats were divided randomly into control, I/R and pyrrolidine dithiocarbamate （PDTC） treatment groups, n = 8 in each. I/R group and PDTC treatment group received superior mysenteric artery （SMA） occluding for 1 h and reperfusion for 2 h. PDTC group was administrated with intraperitoneal injection of 2% 100 mg/kg PDTC 1 h before surgery. Lung histology and bronchia alveolus lung fluid （BALF） protein were assayed. Serum IL-6, lung malondialdehyde （MDA） and myeloperoxidase （MPO） as well as the expression level of NF-κB and ICAM-1 were measured.RESULTS： Lung injury induced by intestinal I/R, was characterized by edema, hemorrhage and neutrophil infiltration as well as by the significant rising of BALF protein. Compared to control group, the levels of serum IL-6 and lung MDA and MPO increased significantly in I/R group （P=0.001）. Strong positive expression of NF-κB p65 and ICAM-1 was observed. After the administration of PDTC, the level of serum IL-6, lung MDA and MPO as well as NF-κB and ICAM-1 decreased significantly （P〈 0.05） when compared to I/R group.CONCLUSION： The activation of NF-κB plays an important role in the pathogenesis of lung injury induced by intestinal I/R through upregulating the neutrophil infiltration and lung ICAM-1 expression. PDTC as an inhibitor of NF-κB can prevent lung injury induced by intestinal I/R through inhibiting the activity of NF-κB.

Ginkgo biloba extract(EGb 761) attenuates lung injury induced by intestinal ischemia/reperfusion in rats:Roles of oxidative stress and nitric oxide

AIM： To investigate the effect of ginkgo biloba extract （EGb 761） on lung injury induced by intestinal ischemia/ reperfusion （ Ⅱ/R）. METHODS： The rat model of Ⅱ/R injury was produced by damping the superior mesenteric artery for 60 min followed by reperfusion for 180 min. The rats were randomly allocated into sham, Ⅱ/R, and EGb ＋Ⅱ/R groups. In EGb ＋Ⅱ/R group, EGb 761 （100 mg/kg per day） was given via a gastric tube for 7 consecutive days prior to surgery. Rats in Ⅱ/R and sham groups were treated with equal volumes of the vehicle of EGb 761. Lung injury was assessed by light microscopy, wet-todry lung weight ratio （W/D） and pulmonary permeability index （PPT）. The levels of malondialdehyde （MDA） and nitrite/nitrate （NO2/NO3）, as well as the activities of superoxide dismutase （SOD） and myeloperoxidase （MPO） were examined. Western blot was used to determine the expression of inducible nitric oxide synthase （iNOS）. RESULTS： EGb 761 markedly improved mean arterial pressure and attenuated lung injury, manifested by the improvement of histological changes and significant decreases of pulmonary W/D and PPT （P 〈 0.05 or 0.01）.Moreover, EGb 761 markedly increased SOD activity, reduced MDA levels and MPO activity, and suppressed NO generation accompanied by down-regulation of iNOS expression （P 〈 0.05 or 0.01）. CONCLUSION： The results indicate that EGb 761 has a protective effect on lung injury induced by Ⅱ /R, which may be related to its antioxidant property and suppressions of neutrophil accumulation and iNOS- induced NO generation. EGb 761 seems to be an effective therapeutic agent for critically ill patients with respiratory failure related to Ⅱ/R.

Protective effect and mechanism of clemastine fumarate on acute lung injury in mice with intestinal ischemia-reperfusion

Objective:To evaluate the protective effect and mechanism of clemastine fumarate(CLE)on acute lung injury(ALI)in intestinal ischemia-reperfusion(I/R)mice.Methods:Twenty-four SPF Balb/c mice were randomly divided into sham operation group(sham group),ischemia-reperfusion group(I/R group),and clemastine fumarate pretreatment group(I/R+C group).In the I/R group,an intestinal ischemia-reperfusion model was established(ischemia for 40 minutes,reperfusion for 2 hours).In the I/R+C group,CLE 5 mg/kg was intraperitoneally injected before the operation.Lung tissue morphology was observed and scored by HE staining;and the ratios of wet weight to dry weight(W/D)were recorded.the levels of MDA,SOD,GSH-px,NF-κB and TNF-αin lung tissue of each group were determined by ELISA;Western blot method was used to determine the expression of TLR4 protein in lung tissue.Results:Compared with the Sham group,the I/R group had significantly higher lung tissue injury score and wet/dry ratio(P<0.05),increased lung tissue MDA level(P<0.05),decreased SOD and GSH-px levels(P<0.05),and increased NF-κB and TNF-αlevels,the expression of TLR4 protein in lung tissue increased(P<0.05);compared with the I/R group,the lung tissue injury score and wet/dry ratio of the I/R+C group decreased(P<0.05),the level of MDA in lung tissue decreased(P<0.05),the levels of SOD and GSH-px increased(P<0.05),and the levels of NF-κB and TNF-毩decreased(P<0.05),the expression of TLR4 protein in lung tissue decreased(P<0.05).Conclusion:Clemastine fumarate can alleviate acute lung injury after intestinal ischemia-reperfusion in mice,and the mechanism may be related to the inhibition of oxidative stress and inflammatory response in lung tissue.

Resveratrol inhibits the P38MAPK pathway as well as downstream apoptosis, inflammation and oxidative stress molecule expression in secondary lung injury in intestinal ischemia-reperfusion

Objective:To study the protective effect of resveratrol on secondary lung injury in intestinal ischemia reperfusion and its effect on P38MAPK pathway.Methods:Adult male SPF SD rats were selected and divided into control group, I/R group, Res-L group, Res-M group and Res-H group, the small intestinal ischemia reperfusion model was made, and Res-L group, Res-M group and Res-H group were given 5.0 mg/kg, 10.0 mg/kg and 15.0 mg/kg resveratrol for intervention. The contents of P38MAPK pathway molecules as well as downstream apoptotic molecules, inflammatory factors and oxidative stress products in the lung were detected.Results:P38MAPK, MAPKK, MAPKKK, Fas, FasL, caspase-8, caspase-9, NF-kB, TNF- and IL-1β expression as well as ROS and MDA contents in lung tissue of I/R group were significantly higher than those of control group;P38MAPK, MAPKK, MAPKKK, Fas, FasL, caspase-8, caspase-9, NF-kB, TNF-α and IL-1β expression as well as ROS and MDA contents in lung tissue of Res-L group, Res-M group and Res-H group were significantly lower than those of I/R group.Conclusion: Resveratrol can inhibit the function of P38MAPK pathway to reduce apoptosis, inflammation and oxidative stress, and then protect the secondary lung injury induced by intestinal ischemia reperfusion.

Plasma D(-)-lactate as a new marker for diagnosis of acute intestinal injury following ischemia-reperfusion

IM To observe the kinetics of D()lactate alteration in both portal and systemic circulations, and its relationship with intestinal injury in rats subjected to acute intestinal ischemiareperfusion.METHODS Anesthetized rats underwent 75min superior mesenteric artery occlusion followed by 6hour reperfusion. Plasma D()lactate levels were measured by an enzymatic spectrophotometric assay.RESULTS Intestinal ischemia for 75 min resulted in a significant elevation of D()lactate levels in portal vein as compared with the baseline values (P<005). Plasma D()lactate levels had a tendency to further increase after reperfusion up to 6 hours. Similar alterations in D()lactate were also found in systemic circulation, there were no significant differences between the portal and systemic circulations at any time point. Moreover, the macropathological evaluation scores were significantly correlated to the portal D()lactate levels in animals at various time points (r=0415, P<001). In addition,there was a remarkable rise of endotoxin concentration within the portal vein at the end of 75min ischemia (P<005), reaching a peak at 2 hours postreperfusion.CONCLUSION Acute intestinal ischemia is associated with failure of mucosal barrier resulting in increased plasma D()lactate levels in both portal and systemic blood. The subsequent reperfusion might further increase D()lactate levels, which are correlated to the macropathological alterations. Plasma D()lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insults.

Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antiox-idant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four ex-perimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfu-sion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the op-eration. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a signif icant increase in serum AST and ALT levels (AST: 260.13 ± 40.17 U/L vs 186.00 ± 24.21 U/L, P < 0.01; ALT: 139.63 ± 11.35 U/L vs 48.38 ± 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 ± 40.17 U/L vs 216.63 ± 22.65 U/L, P < 0.05; ALT: 139.63 ± 11.35 U/L vs 97.63 ± 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In ad-dition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevat-ed liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Fish oil alleviates liver injury induced by intestinal ischemia/reperfusion via AMPK/SIRT-1/autophagy pathway

AIM To evaluate whether fish oil(FO) can protect liver injury induced by intestinal ischemia/reperfusion(I/R) via the AMPK/SIRT-1/autophagy pathway.METHODS Ischemia in wistar rats was induced by superior mesenteric artery occlusion for 60 min and reperfusion for 240 min. One milliliter per day of FO emulsion or normal saline was administered by intraperitoneal injection for 5 consecutive days to each animal. Animals were sacrificed at the end of reperfusion. Blood andtissue samples were collected for analyses. AMPK, SIRT-1, and Beclin-1 expression was determined in lipopolysaccharide(LPS)-stimulated HepG2 cells with or without FO emulsion treatment.RESULTS Intestinal I/R induced significant liver morphological changes and increased serum alanine aminotransferase and aspartate aminotransferase levels. Expression of p-AMPK/AMPK, SIRT-1, and autophagy markers was decreased whereas tumor necrosis factor-α(TNF-α) and malonaldehyde(MDA) were increased. FO emulsion blocked the changes of the above indicators effectively. Besides, in LPS-stimulated HepG2 cells, small interfering RNA(siRNA) targeting AMPK impaired the FO induced increase of p-AMPK, SIRT-1, and Beclin-1 and decrease of TNF-α and MDA. SIRT-1 siRNA impaired the increase of SIRT-1 and Beclin-1 and the decrease of TNF-α and MDA.CONCLUSION Our study indicates that FO may protect the liver against intestinal I/R induced injury through the AMPK/SIRT-1/autophagy pathway.

Protective effect of pyrrolidine dithiocarbamate on liver injury induced by intestinal ischemia-reperfusion in rats

BACKGROUND: The nuclear translocation of transcription factors may be a critical factor in the intracellular pathway involved in ischemia/reperfusion(I/R) injury. The aim of the study was to evaluate the role of nuclear factor-kappa B (NF-κB) in the pathogenesis of liver injury induced by intestinal ischemia/reperfusion (IIR) and to investigate the effect of pyrrolidine dithiocarbamate (PDTC) on this liver injury. METHODS: Male Wistar rats were divided randomly into three experimental groups (8 rats in each): sham operation group (control group); intestinal/reperfusion group(I/R group): animals received 1-hour of intestinal ischemia and 2-hour reperfusion; and PDTC treatment group (PDTC group): animals that received I/R subject to PDTC treatment (100 mg/kg). The histological changes in the liver and intestine were observed, and the serum levels of tumor necrosis factor-α (TNF-α), alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver superoxide dismutase (SOD), and nitrite/nitrate (NO) were measured. The immunohistochemical expression and Western blot analysis of liver NF-κB and intercellular adhesion molecule-1(ICAM-1) were observed. RESULTS: IIR induced liver injury characterized by the histological changes of liver edema, hemorrhage, polymorphonuclear neutrophil (PMN) infiltration, and elevated serum levels of AST and ALT. The serum TNF-α level was significantly higher than that of the control group(P<0.01) and a high level of liver oxidant product was observed (P<0.01). These changes were parallel to the positive expression of NF-κB and ICAM-1. After the administration of PDTC, the histological changes after liver injury were improved; the levels of SOD and NO in the liver were elevated and reduced, respectively (P<0.01). The expressions of ICAM-1 and NF-κB in the liver were weakened (P<0.01). CONCLUSION: NF-κB plays an important role in the pathogenesis of liver injury induced by HR. PDTC, an agent known to inhibit the activation of NF-κB, can reduce and prevent this injury.

Attenuation of graft ischemia-reperfusion injury by urinary trypsin inhibitor in mouse intestinal transplantation

AIM: Ischemia/reperfusion (I/R) injury is one of the major obstacles for intestinal transplantation (ITx). Urinary trypsin inhibitor (Ulinastatin, UTI) suppresses proteases and stabilizes lysosomal membranes. We supposed that Ulinastatin would diminish I/R injury of intestinal graft.METHODS: UTI- treated group and untreated control group were investigated by histological assessment at 1.5, 4, 24, and 72 h after ITx. Myeloperoxidase (MPO)activity was used as the activity of neutrophils, and malondialdehyde (MDA) was used as an index of lipid peroxidation. TNFα and i-NOS mRNA expression in graft tissue were measured by semi-quantitative RT-PCR.CD11b+ Gr1+ cells in graft lamina propria were analyzed by flow cytometry.RESULTS: Histological scores of the graft showed that the tissue injury was markedly attenuated by UTI treatment at different time points after ITx, with reduced MPO and MDA value in the grafts. The expression of TNFα and i-NOS mRNA was profoundly inhibited, while the infiltration of CD11b+ Gr1+ cells into the intestinal graft was decreased in UTI group.CONCLUSION: Urinary trypsin inhibitor attenuates I/R injury in mouse intestinal transplantation by reducing monocytes infiltration and down-regulation of TNFα and i-NOS mRNA expression.

Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury

AIM: To determine the potential protective role of adiponectin in intestinal ischemia reperfusion(I/R) injury.METHODS: A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzymelinked immunosorbent assay(ELISA). The serum levels of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase d UTP nick end labeling assay. The production of malondialdehyde(MDA) and superoxide dismutase(SOD) and villous injury scores were also measured.RESULTS: Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines,including IL-6, IL-1β, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase(AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1(HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. CONCLUSION: Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway.

Protective effect of Astragalus membranaceus on intestinal mucosa reperfusion injury after hemorrhagic shock in rats

AIM： To study the protective effect of Astragalus rnernbranaceus on intestinal mucosa reperfusion injury and its mechanism after hemorrhagic shock in rats. METHODS： A total of 32 SD rats were randomly divided into four groups （n = 8, each group）： normal group, model group, low dosage group （treated with 10 g/kg Astragalus membranaceus） and high dosage group （treated with 20 g/kg Astragalus membranaceus）. The model of hemorrhagic shock for 60 min and reperfusion for 90 min was established. Therapeutic solution （3 mL） was administrated before reperfusion. At the end of the study, the observed intestinal pathology was analyzed. The blood concentrations of lactic acid （LD）, nitric oxide （NO）, endothelin-1 （ET-1）, malondialdehyde （MDA） and the activity of superoxide dismutase （SOD）, glutathione peroxidase （GSH-PX） in intestinal mucosa were determined. RESULTS： The intestinal mucosa pathology showed severe damage in model group and low dosage group, slight damage in high dosage group and no obvious damage in normal group. The Chiu＇s score in low dose group and high dose group was significantly lower than that in model group. The content of MDA in model group was higher than that in low and high dose groups, while that in high dose group was almost the same as in normal group. The activity of SOD and GSH-PX was the lowest in model group and significantly higher in high dose group than in normal and low dose groups. The concentrations of LD and ET-1 in model group were the highest. The concentrations of NO in model group and low dose group were significantly lower than those in high dose group and normal group. CONCLUSION： High dose Astraga/us membranaeus has much better protective effect on hemorrhagic shockreperfusion injury of intestinal mucosa than low dose Astragalus membranaceus. The mechanism may be that Astragalus membranaceus can improve antioxidative effect and regulate NO/ET level during hemorrhagic reperfusion.

Ischemic post-conditioning to counteract intestinal ischemia/reperfusion injury

Intestinal ischemia is a severe disorder with a variety of causes.Reperfusion is a common occurrence during treatment of acute intestinal ischemia but the injury resulting from ischemia/reperfusion(IR)may lead toeven more serious complications from intestinal atrophy to multiple organ failure and death.The susceptibility of the intestine to IR-induced injury(IRI)appears from various experimental studies and clinical settings such as cardiac and major vascular surgery and organ transplantation.Where as oxygen free radicals,activation of leukocytes,failure of microvascular perfusion,cellular acidosis and disturbance of intracellular homeo-stasis have been implicated as important factors inthe pathogenesis of intestinal IRI,the mechanisms underlying this disorder are not well known.To date,increasing attention is being paid in animal studies to potential pre-and post-ischemia treatments that protect against intestinal IRI such as drug interference with IR-induced apoptosis and inflammation processes and ischemic pre-conditioning.However,better insight is needed into the molecular and cellular events associated with reperfusion-induced damage to develop effective clinical protection protocols to combat this disorder.In this respect,the use of ischemic post-conditioning in combination with experimentally prolonged acidosis blocking deleterious reperfusion actions may turn out to have particular clinical relevance.

PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury.PTEN-induced putative kinase 1(PINK1)and phosphorylation of dynamin-related protein 1(DRP1)are critical regulators of ROS and apoptosis.However,the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated.Thus,examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion.Chiu’s score was used to evaluate intestinal mucosa damage.The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection.Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions.Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression.The protein expression levels of PINK1,DRP1,p-DRP1 and cleaved caspase 3 were measured by Western blotting.Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining.Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637.Pretreatment with mdivi-1 inhibited mitochondrial fission,ROS generation,and apoptosis and ameliorated cell injury in intestinal I/R.Upon PINK1 knockdown or overexpression in vitro,we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1.Furthermore,we verified the physical combination of PINK1 and p-DRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R.These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury,and provide a new approach for prevention and treatment.

Protective Effects of Epigallocatechin-3-gallate on Intestinal Ischemia Reperfusion Injury through Enhanced Activation of PI3K/Akt Pathway in Rats

Inflammation plays a critical role in intestinal ischemia reperfusion injury（IRI）. Epigallocatechin-3-gallate（EGCG） has been demonstrated to possess anti-inflammatory effect. This study examined the effect of EGCG on intestinal IRI and explored the possible mechanisms. Male Wistar rats were randomly divided into three groups： sham-operated group（Sham）, IRI control group（IRI） and IRI-EGCG group（EGCG）. Rats in IRI-EGCG group were administered dissolved EGCG in drinking water（0.4 mg/m L） for 14 days prior to IRI induction. A rat model of intestinal IRI was established by ligating the superior mesenteric artery（SMA） for 30 min, followed by reperfusion for 1 h. Intestinal histology, pro-inflammatory cytokines and mediators were examined and the effect of EGCG on PI3K/Akt signalling was assessed. EGCG significantly alleviated the pathological changes of the intestine and suppressed the IRI-induced up-regulation of TNF-α, IL-1 and IL-6 m RNA and protein expression in the serum and intestine. The mechanism might be that EGCG enhanced the activation of PI3K/Akt signalling pathway. In conclusion, the administration of EGCG can significantly mitigate the acute intestinal IRI in rats by enhancing the activation of PI3K/Akt signalling pathway to suppress inflammatory response and might be a promising alternative for the prevention or treatment of intestinal IRI in the clinical practice.

N-acetylcysteine inhibits activation of toll-like receptor 2 and 4 gene expression in the liver and lung after partial hepatic ischemia-reperfusion injury in mice

BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R injury. This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group (SH), a hepatic ischemia/reperfusion group (I/R) or a NAC pretreated, hepatic I/R group (I/R-NAC). The levels of TNF-alpha in the portal vein and plasma alanine aminotransferase (ALT) were measured at 1 and 3 hours after reperfusion. The lung wet-to-dry ratio was measured, and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS: Compared with the I/R group, the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05). The levels of portal vein TNF-a and plasma ALT increased continuously in the l/R group at I and 3 hours of reperfusion compared with the SH group; however, they declined significantly in the group pretreated with NAC (P<0.05). The extent of lung edema was relieved in the I/R-NAC group compared with the I/R group (P<0.05). CONCLUSIONS: TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury. NAC inhibited the activation of TLR2/4 and the induction of TNF-alpha resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.

Bone-marrow mesenchymal stem cells reduce rat intestinal ischemia-reperfusion injury, ZO-1 downregulation and tight junction disruption via a TNF-α-regulated mechanism

AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. I/R injury was induced by occlusion of the superior mesenteric artery for 30 min. Rats were treated with saline, BM MSCs (via intramucosal injection) or tumor necrosis factor (TNF)-α blocking antibodies (via the tail vein). I/R injury was assessed using transmission electron microscopy, hematoxylin and eosin (HE) staining, immunohistochemistry, western blotting and enzyme linked immunosorbent assay.RESULTS: Intestinal permeability increased, tight junctions (TJs) were disrupted, and zona occludens 1 (ZO-1) was downregulated after I/R injury. BM MSCs reduced intestinal mucosal barrier destruction, ZO-1 downregulation, and TJ disruption. The morphological abnormalities after intestinal I/R injury positively correlated with serum TNF-α levels. Administration of anti-TNF-α IgG or anti-TNF-α receptor 1 antibodies attenuated the intestinal ultrastructural changes, ZO-1 downregulation, and TJ disruption. CONCLUSION: Altered serum TNF-α levels play an important role in the ability of BM MSCs to protect against intestinal I/R injury.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, or anti-TRIF antibodies(all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Gene Expression Profile of Pulmonary Tissues in Different Phases of Lung Ischemia-reperfusion Injury in Rats

In order to provide us new clues to induce some endogenous protective molecular mechanisms, the changes in gene expression profile induced by ischemia-reperfusion in pulmonary tissues of rats were investigated and the dynamic mechanism of pulmonary ischemia-reperfusion injury was elucidated. Thirty male Wistar rats were randomly divided into 6 groups： 5 ischemia-reperfusion （I/R） groups （I/R 0-h, I/R 1-h, I/R 3-h, I/R 6-h, I/R 24-h） and control group （n=5 in each）. An in situ ischemia-reperfusion lung injury rat model was established by occluded hilus of lung. The RatRef-12 Expression Beadchip （22 226 gene probes per array） was used to analyze the pattern of gene expression in all groups. The results showed that 648, 340, 711, 1279 and 641 genes were differentially expressed in I/R 0-, 1-, 3-, 6- and 24-h groups respectively. The differentially expressed genes were classified as following 7 functional categories： cytokine, adhesion molecule, growth factor and apoptosis-related factor, oxidation and antioxidation molecule, metabolic enzyme, ion channel and aquaporin, signal transduction molecule. It was suggested that gene chip technology was an effective and quick method for screening differentially expressed genes. Many differentially expressed genes with different functions interacted each other to result in pulmonary ischemia-reperfusion injury.

Expression of bradykinin as a substrate of CD26 /DPP IV in rats ischemia/reperfusion injury following lung transplantation

Objective To investigate the expression of bradykinin as a substrate of CD26 /DPP IV in rats with ischemia/reperfusion injury following lung transplantation ( LTx) . Methods Thirty - six syngeneic male SD rats were randomly allocated into control group and experimental group ( n = 18 each) ,and 36 rats served as do-