Over-expression of BMP4 inhibits experimental choroidal neovascularization by modulating VEGF and MMP-9

Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.     

Deletion of macrophage migration inhibitory factor protects the heart from severe ischemia-reperfusion injury: a predominant role of anti-inflammation

Inflammation plays an important role in mediating and exacerbating myocardial ischemia–reperfusion (I/R) injury. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, facilitates inflammation and modulates metabolism. However, the role of MIF in mediating local inflammation subsequent to ischemic myocardial injury has not been established. We hypothesized that genetic deletion of MIF protects the heart against severe I/R injury by suppressing inflammation and/or modulating energy metabolism. We showed in the mouse I/R model that duration of both ischemia and reperfusion is a determinant for the degree of regional inflammation and tissue damage. Following a prolonged cardiac I/R (60 min/24 h) MIF KO mice had a significant reduction in both infarct size (26 ± 3% vs. 45 ± 4%, P < 0.05) and cardiomyocyte apoptosis (1.4 ± 0.2% vs. 5.4 ± 0.4%, P < 0.05) and preserved contractile function compared with WT. MIF KO mice with I/R had reduced expression of various inflammatory cytokines and mediators (P < 0.05), suppressed infiltration of neutrophils (− 40%) and macrophages (− 33%, both P < 0.05), and increased macrophage apoptosis (14.4 ± 1.4% vs. 5.2 ± 0.6%, P < 0.05). Expression of toll-like receptor-4 (TLR-4), phosphorylation of c-Jun N-terminal kinase (JNK), and nuclear fraction of NF-κB p65 were also significantly lower in MIF KO hearts with I/R. Further, MIF KO mice exhibited a lower glucose uptake but higher fatty acid oxidation rate than that in WT (both P < 0.05). In conclusion, MIF deficiency protected the heart from prolonged/severe I/R injury by suppressing inflammatory responses. We have identified a critical role of MIF in mediating severe I/R injury. Thus, MIF inhibitory therapy may be a novel strategy to protect the heart against severe I/R injury.     

Cyclin-dependent kinase 2 signaling regulates myocardial ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury to the heart is accompanied by the upregulation and posttranslational modification of a number of proteins normally involved in regulating cell cycle progression. Two such proteins, cyclin-dependent kinase-2 (Cdk2) and its downstream target, the retinoblastoma gene product (Rb), also play a critical role in the control of apoptosis. Myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size (IFS), when compared to control mice, associated with a reduction in apoptotic myocytes. To confirm that Rb was the critical target in Cdk2-mediated I/R injury, we determined the consequences of I/R injury in cardiac-specific Rb-deficient mice (CRb^L/L). IFS was increased 140% in CRb^L/L mice compared to CRb^+/+ controls. TUNEL positive nuclei and caspase-3 activity were augmented by 92% and 36%, respectively, following injury in the CRb^L/L mice demonstrating that loss of Rb in the heart significantly exacerbates I/R injury. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo and support a cardioprotective role for Rb.     

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice

The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. CONCLUSION: Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.     

Vitamin E attenuates myocardial ischemia-reperfusion injury in murine AIDS

The incidence of myocardial infarction in patients who have the aquired immunodeficiency syndrome (AIDS) is increasing. However, no effective therapeutic agents have been discovered to reduce myocardial ischemia-reperfusion (I/R) injury in pathologies associated with AIDS. The aim of this study was to determine if infarct size is increased in murine AIDS after I/R injury and if I/R injury could be attenuated with vitamin E supplementation. Three groups of mice were studied: control, murine AIDS, and murine AIDS with vitamin E supplementation. Anesthetized mice were subjected to 30 min of left anterior descending coronary artery occlusion and 120 min of reperfusion. The hearts in mice that had murine AIDS had a larger infarct size compared to controls after I/R injury. Vitamin E supplementation significantly reduced infarct size and inhibited polymorphonuclear neutrophil (PMN) CD11b expression (p<0.05). However, vitamin E supplementation did not affect PMN reactive oxygen species (ROS) production and platelet CD62p expression. These results suggest that the reduction of myocardial I/R injury with vitamin E supplementation may be the result of the inhibition of PMN CD11b expression. Vitamin E may be a promising prophylactic agent for the reduction of the severity of myocardial I/R injury in patients who have AIDS.     

Cardiomyocyte-specific overexpression of human stem cell factor protects against myocardial ischemia and reperfusion injury

Background

Cardiomyocyte-specific overexpression of human membrane-associated stem cell factor (hSCF) improves cardiac function post-myocardial infarction. However, whether hSCF overexpression protects the heart from ischemia and reperfusion (I/R) injury is unknown. We aimed to investigate the effects of cardiomyocyte-specific overexpression of hSCF on cardiac injury after acute myocardial I/R and related cellular and molecular signaling mechanisms.

Methods and results

Wild-type (WT) and hSCF/tetracycline transactivator (tTA) transgenic mice (hSCF/tTA) were subjected to myocardial ischemia for 45 min followed by 3 h of reperfusion. Infarct size and myocardial apoptosis were decreased in hSCF/tTA compared to WT mice (P < 0.05). Furthermore, these cardioprotective effects in the hSCF/tTA mice were abrogated by doxycycline, which turned off hSCF overexpression, and by a PI3 kinase inhibitor LY294002. Myocardial expression of insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF), which are upstream activators of Akt signaling, was significantly increased in hSCF/tTA compared to WT mice after I/R (P < 0.05), and was associated with higher number of c-kit⁺ cardiac stem cells (CSCs) (P < 0.05). Inhibition of c-kit signaling by ACK2 treatment abolished these protective effects in hSCF/tTA mice.

Conclusions

Cardiomyocyte-specific overexpression of hSCF protects the heart from I/R injury. The cardioprotective effects of hSCF overexpression are mediated by increased c-kit⁺ CSCs, enhanced growth factor expression and activation of Akt signaling pathway.     

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb^th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.     

Tmprss6 is a genetic modifier of the Hfe-hemochromatosis phenotype in mice

The hereditary hemochromatosis protein HFE promotes the expression of hepcidin, a circulating hormone produced by the liver that inhibits dietary iron absorption and macrophage iron release. HFE mutations are associated with impaired hepatic bone morphogenetic protein (BMP)/SMAD signaling for hepcidin production. TMPRSS6, a transmembrane serine protease mutated in iron-refractory iron deficiency anemia, inhibits hepcidin expression by dampening BMP/SMAD signaling. In the present study, we used genetic approaches in mice to examine the relationship between Hfe and Tmprss6 in the regulation of systemic iron homeostasis. Heterozygous loss of Tmprss6 in Hfe(-/-) mice reduced systemic iron overload, whereas homozygous loss caused systemic iron deficiency and elevated hepatic expression of hepcidin and other Bmp/Smad target genes. In contrast, neither genetic loss of Hfe nor hepatic Hfe overexpression modulated the hepcidin elevation and systemic iron deficiency of Tmprss6(-/-) mice. These results indicate that genetic loss of Tmprss6 increases Bmp/Smad signaling in an Hfe-independent manner that can restore Bmp/Smad signaling in Hfe(-/-) mice. Furthermore, these results suggest that natural genetic variation in the human ortholog TMPRSS6 might modify the clinical penetrance of HFE-associated hereditary hemochromatosis, raising the possibility that pharmacologic inhibition of TMPRSS6 could attenuate iron loading in this disorder.     

Histamine H2 receptor activation exacerbates myocardial ischemia/reperfusion injury by disturbing mitochondrial and endothelial function

There is evidence that H2R blockade improves ischemia/reperfusion (I/R) injury, but the underlying cellular mechanisms remain unclear. Histamine is known to increase vascular permeability and induce apoptosis, and these effects are closely associated with endothelial and mitochondrial dysfunction, respectively. Here, we investigated whether activation of the histamine H2 receptor (H2R) exacerbates myocardial I/R injury by increasing mitochondrial and endothelial permeability. Serum histamine levels were measured in patients with coronary heart disease, while the influence of H2R activation was assessed on mitochondrial and endothelial function in cultured cardiomyocytes or vascular endothelial cells, and myocardial I/R injury in mice. The serum histamine level was more than twofold higher in patients with acute myocardial infarction than in patients with angina or healthy controls. In neonatal rat cardiomyocytes, histamine dose-dependently reduced viability and induced apoptosis. Mitochondrial permeability and the levels of p-ERK1/2, Bax, p-DAPK2, and caspase 3 were increased by H2R agonists. In cultured human umbilical vein endothelial cells (HUVECs), H2R activation increased p-ERK1/2 and p-moesin levels and also enhanced permeability of HUVEC monolayer. All of these effects were abolished by the H2R blocker famotidine or the ERK inhibitor U0126. After I/R injury or permanent ischemia, the infarct size was reduced by famotidine and increased by an H2R agonist in wild-type mice. In H2R KO mice, the infarct size was smaller; myocardial p-ERK1/2, p-DAPK2, and mitochondrial Bax were downregulated. These findings indicate that H2R activation exaggerates myocardial I/R injury by promoting myocardial mitochondrial dysfunction and by increasing cardiac vascular endothelial permeability.     

The role of TNF-α receptors p55 and p75 in acute myocardial ischemia/reperfusion injury and late preconditioning

The specific role of TNF-α receptors I (TNFR-I, p55) and II (TNFR-II, p75) in myocardial ischemic injury remains unclear. Using genetically engineered mice, we examined the relative effects of TNF-α signaling via p55 and p75 in acute myocardial ischemia/reperfusion injury under basal conditions and in late preconditioning (PC). Wild-type (WT) (C57BL/6 and B6,129) mice and mice lacking TNF-α (TNF-α^−/−), p55 (p55^−/−), p75 (p75^−/−), or both receptors (p55^−/−/p75^−/−) underwent 30 min of coronary occlusion and 24 h of reperfusion with or without six cycles of 4-min coronary occlusion/4-min reperfusion (O/R) 24 h earlier (ischemic PC). Six cycles of O/R reduced infarct size 24 h later in WT mice, indicating a late PC effect. This late PC-induced infarct-sparing effect was abolished not only in TNF-α^−/− and p55^−/−/p75^−/− mice, but also in p55^−/− and p75^−/− mice, indicating that TNF-α signaling via both p55 and p75 is necessary for the development of protection. In nonpreconditioned TNF-α^−/−, p55^−/−/p75^−/−, and p75^−/− mice, infarct size was similar to strain-matched WT mice. In contrast, infarct size in nonpreconditioned p55^−/− mice was reduced compared with nonpreconditioned WT mice. We conclude that (i) unopposed p75 signaling (in the absence of p55) reduces infarct size following acute ischemia/reperfusion injury in naive myocardium, whereas unopposed p55 signaling (in the absence of p75) has no effect; and (ii) the development of the infarct-sparing effects of the late phase of PC requires nonredundant signaling via both p55 and p75 receptors. These findings reveal a fundamental, heretofore unrecognized, difference between the two TNF-α receptors in the setting of myocardial ischemia/reperfusion injury: that is, both p55 and p75 are necessary for the development of protection during late PC, but only signaling via p75 is protective in nonpreconditioned myocardium.     

FoxO1变化对糖尿病小鼠心肌缺血/再灌注损伤的影响

目的:观察心肌中插头转录因子O1（FoxO1）在糖尿病（DM）小鼠心肌中表达量变化及对小鼠心肌缺血/再灌注（I/R）损伤的影响。方法: 将90只健康雄性Swiss小鼠随机分为5组:假手术（Sham）组、I/R组、DM+Sham组、DM+I/R组及DM+FoxO1SiRNA+I/R组,每组18只。采用高糖高脂饮食加链脲菌素(Streptozocin,STZ)腹腔注射诱导建立DM小鼠模型。采用FoxO1SiRNA心肌点注射下调心肌FoxO1表达。心肌I/R损伤模型的建立,采用结扎心脏冠状动脉左前降支30 min后再灌注方案实施。心肌再灌注3 h后,用原位缺口末端标法(TUNEL)检测心肌细胞凋亡。用ELISA法检测心肌中 Caspase-3的活性。用Western blot法检测心肌中FoxO1的表达量。心肌再灌注24 h后,用2,3,5-三苯基氯化四氮唑（TTC）染色法检测心肌梗死（MI）的面积。结果: 与Sham组比较,DM+Sham组心肌中FoxO1的表达量明显增高（P<0.01）。与I/R组比,DM+I/R组MI的面积增大（P<0.05）,心肌细胞凋亡数量及Caspase-3活性明显增加（P<0.01）。与DM+I/R组相比,DM+FoxO1SiRNA+I/R组心肌FoxO1的表达量下调（P<0.05）,MI面积及Caspase-3的活性减小（P<0.05）,心肌细胞凋亡数量减少（P<0.01）。结论: DM小鼠心肌中FoxO1表达量的增加可加重心肌I/R损伤;而下调心肌中FoxO1的表达量后,心肌I/R损伤减轻。     

Context-dependent signaling defines roles of BMP9 and BMP10 in embryonic and postnatal development

Many important signaling pathways rely on multiple ligands. It is unclear if this is a mechanism of safeguard via redundancy or if it serves other functional purposes. In this study, we report unique insight into this question by studying the activin receptor-like kinase 1 (ALK1) pathway. Despite its functional importance in vascular development, the physiological ligand or ligands for ALK1 remain to be determined. Using conventional knockout and specific antibodies against bone morphogenetic protein 9 (BMP9) or BMP10, we showed that BMP9 and BMP10 are the physiological, functionally equivalent ligands of ALK1 in vascular development. Timing of expression dictates the in vivo requisite role of each ligand, and concurrent expression results in redundancy. We generated mice (Bmp10^9/9) in which the coding sequence of Bmp9 replaces that of Bmp10. Surprisingly, analysis of Bmp10^9/9 mice demonstrated that BMP10 has an exclusive function in cardiac development, which cannot be substituted by BMP9. Our study reveals context-dependent significance in having multiple ligands in a signaling pathway.     

Depletion of PHD3 protects heart from ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis

PHD3, a member of a family of Prolyl-4 Hydroxylase Domain (PHD) proteins, has long been considered a pro-apoptotic protein. Although the pro-apoptotic effect of PHD3 requires its prolyl hydroxylase activity, it may be independent of HIF-1α, the common substrate of PHDs. PHD3 is highly expressed in the heart, however, its role in cardiomyocyte apoptosis remains unclear. This study was undertaken to determine whether inhibition or depletion of PHD3 inhibits cardiomyocyte apoptosis and attenuates myocardial injury induced by ischemia–reperfusion (I/R). PHD3 knockout mice and littermate controls were subjected to left anterior descending (LAD) coronary artery ligation for 40 min followed by reperfusion. Histochemical analysis using Evan's Blue, triphenyl-tetrazolium chloride and TUNEL staining, demonstrated that myocardial injury and cardiomyocyte apoptosis induced I/R injury were significantly attenuated in PHD3 knockout mice. PHD3 knockout mice exhibited no changes in HIF-1α protein level, the expression of some HIF target genes or the myocardium capillary density at physiological condition. However, depletion of PHD3 further enhanced the induction of HIF-1α protein at hypoxic condition and increased expression of HIF-1α inhibited cardiomyocyte apoptosis induced by hypoxia. In addition, it has been demonstrated that PHD3 plays an important role in ATR/Chk1/p53 pathway. Consistently, a prolyl hydroxylase inhibitor or depletion of PHD3 significantly inhibits the activation of Chk1 and p53 in cardiomyocytes and the subsequent apoptosis induced by doxorubicin, hydrogen peroxide or hypoxia/reoxygenation. Taken together, these data suggest that depletion of PHD3 leads to increased stabilization of HIF-1α and inhibition of DNA damage response, both of which may contribute to the cardioprotective effect seen with depletion of PHD3.     

JNK mediates hepatic ischemia reperfusion injury

BACKGROUND/AIMS: Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-kappaB and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors. METHODS: Male Wistar rats (200+/-25 g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90 min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90 min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation. RESULTS: In the I/R survival model, vehicle-treated rats have a 7-day survival of 20-40%, while rats treated with the three different JNK inhibitors have survival rates of 60-100% (P<0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid. CONCLUSIONS: Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways.     

丙酮酸对缺血/再灌注大鼠心肌的保护作用

目的:探讨丙酮酸(Pyr)对缺血/再灌注(I/R)大鼠心肌的影响及其可能的机制。方法:30只SD成年大鼠随机分为假手术(Sham)组、I/R组及I/R+Pyr组,每组10只。I/R+Pyr组大鼠于再灌前5 min,开始持续性静脉灌注Pyr 2 h[25 mg/(kg·h)]。再灌注2 h后,利用多道生理记录仪检测大鼠在体血流动力学指标:平均动脉压(MABP)、左室收缩压(LVSP)及左室最大收缩、舒张末压微分(±LV dP/dt max)。采用Western blot方法检测磷酸化-JNK(p-JNK)和总的JNK(t-JNK)表达。用原位缺口末端标记法(TUNEL)评价心肌细胞的凋亡。结果:I/R组的MABP、LVSP、±LV dP/dt max显著低于Sham组(P0.01),Pyr干预可增加I/R后MABP、LVSP及±LV dP/dt max的水平(P0.05)。与Sham组相比,I/R组大鼠左心室p-JNK的水平明显增高(P0.01);而Pyr可降低大鼠左心室p-JNK的水平(P0.01),并抑制心肌细胞凋亡(P0.05)。结论:Pyr可改善I/R大鼠心肌的功能,抑制心肌细胞凋亡,其机制可能与抑制JNK信号的激活有关。     

Tumor necrosis factor-α mediates JNK activation response to intestinal ischemia-reperfusion injury

AIM:To investigate whether tumor necrosis factor-α(TNF-α)mediates ischemia-reperfusion(I/R)-induced intestinal mucosal injury through c-Jun N-terminal kinase(JNK)activation.METHODS:In this study,intestinal I/R was induced by 60-min occlusion of the superior mesenteric artery in rats followed by 60-min reperfusion,and the rats were pretreated with a TNF-α inhibitor,pentoxifylline,or the TNF-α antibody infliximab.After surgery,part of the intestine was collected for histological analysis.The mucosal layer was harvested for RNA and protein extraction,which were used for further real-time polymerase chain reaction,enzyme-linked immunosorbent assay and Western blotting analyses.The TNF-α expression,intestinal mucosal injury,cell apoptosis,activation of apoptotic protein and JNK signaling pathway were analyzed.RESULTS:I/R significantly enhanced expression of mucosal TNF-α at both the mRNA and protein levels,induced severe mucosal injury and cell apoptosis,activated caspase-9/caspase-3,and activated the JNK signaling pathway.Pretreatment with pentoxifylline markedly downregulated TNF-α at both the mRNA and protein levels,whereas infliximab pretreatment did not affect the expression of TNF-α induced by I/R.However,pretreatment with pentoxifylline or infliximab dramatically suppressed I/R-induced mucosal injury and cell apoptosis and significantly inhibited the activation of caspase-9/3 and JNK signaling.CONCLUSION:The results indicate there was a TNFα-mediated JNK activation response to intestinal I/R injury.