Hypoxia preconditioning protects Ca~(2+)-ATPase activation of intestinal mucosal cells against R/I injury in a rat liver transplantation model

AIM To investigate the effect of ischaemia and reperfusion(I/R) injury on the Ca~(2+)-ATPase activation in the intestinal tissue of a rat autologous orthotopic liver transplantation model and to determine if hypoxia preconditioning(HP) therapy induces HIF-1α to protect rat intestinal tissue against I/R injury.METHODS Rats received non-lethal hypoxic preconditioning therapy to induce HIF-1α expression. We used an autologous orthotopic liver transplantation model to imitate the I/R injury in intestinal tissue. Then, we detected the microstructure changes in small intestinal tissues, Ca~(2+)-ATPase activity, apoptosis, and inflammation within 48 h postoperatively. RESULTS HIF-1α expression was significantly increased in intestinal tissue at 12 h postoperatively in rats that were exposed to a hypoxic environment for 90 min compared with a non-HP group(HP vs AT, P = 0.0177). Pathological analysis was performed on the intestinal mucosa cells, and the cells in the HP group appeared healthier than the cells in the AT group. The Ca~(2+)-ATPase activity in the small intestinal cells in the AT group was significantly lower after the operation, and the Ca~(2+)-ATPase activity in the HP group recovered faster than that in the AT group at 6 h postoperatively(HP vs AT, P = 0.0106). BCL-2 expression in the HP group was significantly higher than that in the AT group at 12 h postoperatively(HP vs AT P = 0.0010). The expression of the inflammatory factors NO, SOD, IL-6, and TNF-α was significantly lower in the HP group than in the AT group.CONCLUSION Hypoxia-induced HIF-1α could protect intestinal mucosal cells against mitochondrial damage after I/R injury. HP could improve hypoxia tolerance in small intestinal mucosal cells and increase Ca~(2+)-ATPase activity to reduce the apoptosis of and pathological damage to intestinal cells. HP could be a useful way to promote the earlier recovery of intestinal function after graft procedure.     

Abdominal paracentesis drainage attenuates intestinal inflammation in rats with severe acute pancreatitis by inhibiting the HMGB1-mediated TLR4 signaling pathway

BACKGROUND Our previous studies confirmed that abdominal paracentesis drainage(APD)attenuates intestinal mucosal injury in rats with severe acute pancreatitis(SAP),and improves administration of enteral nutrition in patients with acute pancreatitis(AP).However,the underlying mechanisms of the beneficial effects of APD remain poorly understood.AIM To evaluate the effect of APD on intestinal inflammation and accompanying apoptosis induced by SAP in rats,and its potential mechanisms.METHODS SAP was induced in male adult Sprague-Dawley rats by 5%sodium taurocholate.Mild AP was induced by intraperitoneal injections of cerulein(20μg/kg body weight,six consecutive injections).Following SAP induction,a drainage tube connected to a vacuum ball was placed into the lower right abdomen of the rats to build APD.Morphological changes,serum inflammatory mediators,serum and ascites high mobility group box protein 1(HMGB1),intestinal barrier function indices,apoptosis and associated proteins,and toll-like receptor 4(TLR4)signaling molecules in intestinal tissue were assessed.RESULTS APD significantly alleviated intestinal mucosal injury induced by SAP,as demonstrated by decreased pathological scores,serum levels of D-lactate,diamine oxidase and endotoxin.APD reduced intestinal inflammation and accompanying apoptosis of mucosal cells,and normalized the expression of apoptosis-associated proteins in intestinal tissues.APD significantly suppressed activation of the intestinal TLR4 signaling pathway mediated by HMGB1,thus exerting protective effects against SAP-associated intestinal injury.CONCLUSION APD improved intestinal barrier function,intestinal inflammatory response and accompanying mucosal cell apoptosis in SAP rats.The beneficial effects are potentially due to inhibition of HMGB1-mediated TLR4 signaling.     

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.     

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.     

Mediated protective effect of electroacupuncture pretreatment by miR-214 on myocardial ischemia/reperfusion injury

Background Electroacupuncture pretreatment plays a protective role in myocardial ischemia/reperfusion （I/R） injury and microRNAs （miRNAs） could act on various facets of cardiac function. However, the role of miRNAs in the cardioprotection by electroacupuncture pre-treatment on myocardial I/R injury remains unknown. The purpose of the study was to examine whether miR-214 was involved in cardio-protection by electroacupuncture. Methods Using rat myocardial I/R model, we examined the role of electroacupuncture pretreatment in myocardial I/R injury and analyzed the changes in the expression of miR-214. In addition, I/R was simulated in vitro by performing oxy-gen-glucose deprivation （OGD） on H9c2 cell cultures, and the effect of electroacupuncture pretreatment on I/R injury as well as expressional level of miR-214 were examined in vitro. Furthermore, the miR-214 mimic was transfected into OGD-treated H9c2 cells, we analyzed the cell apoptosis, lactate dehydrogenase （LDH） and creatine kinase （CK） activities, intracellular free Ca2＋concentration （[Ca2＋]i） as well as the relative protein levels of sodium/calcium exchanger 1（NCX1）, BCL2-like 11 （BIM）, calmodulin-dependent protein kinase IIδ（CaMKIIδ） and Cyclophilin D （CypD）. Results The in vivo results revealed that compared with the I/R group, the electroacupuncture pretreatment group showed significant decreased myocardial infarct size, as well as the increased indices of the cardiac function, including heart rate, mean arterial pressure, left ventricular systolic pressure and maximal rate for left ventricular pressure rising and declining （±dp/dt max）. In addition, electroacupuncture pretreatment could inhibit the elevation of LDH and CK activities induced by I/R injury. The quantitative PCR （qPCR） results demonstrated electroacupuncture pretreatment could provide cardioprotection against myocardial I/R injury in rats with miR-214 up-regulation. In the meanwhile, in vitro, electroacupuncture pretreatment protected     

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-αin 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-αand plasma ALT increased continuously in the I/R group at 1 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-α 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.     

Rosuvastatin reduces rat intestinal ischemia-reperfusion injury associated with the preservation of endothelial nitric oxide synthase protein

AIM: To investigate the protective effect of rosuvastatin on ischemia-reperfusion (I-R)-induced small intestinal injury and inflammation in rats, and to determine the effect of this agent on the expression of endothelial nitric oxide synthase (eNOS) protein. METHODS: Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion for 60 min. Rosuvastatin dissolved in physiological saline was administered intraperitoneally 60 min before ischemia. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers, as well as by histological findings. The protein levels of eNOS were determined by Western blot. RESULTS: The levels of both intraluminal hemoglobin and protein, as indices of mucosal damage, were significantly increased in the I-R group compared with those in the sham-operated group. These increases, however, were significantly inhibited by treatment with rosuvastatin in a dose-dependent manner. The protective ef fects of rosuvastatin were also confirmed by histological findings. Exposure of the small intestine to I-R resulted in mucosal inflammation characterized by significant increases in thiobarbituric acid-reactive substances, tissueassociated myeloperoxidase activity, and the mucosal contents of rat cytokine-induced neutrophil chemoattractant-1 (CINC-1) and tumor necrosis factor-α(TNF-α). These increases in inflammatory parameters after I-R were significantly inhibited by pretreatment with rosuvastatin at a dose of 10 mg/kg. Furthermore, mRNA expression of CINC-1 and TNF-αwas increased after I-R, and this increase was also inhibited by rosuvastatin. The mucosal protein levels of eNOS decreased during I-R, but were preserved in rats treated with rosuvastatin. CONCLUSION: Rosuvastatin inhibits rat intestinal injury and inflammation induced by I-R, and its protection is associated with the preservation of eNOS protein.     

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22(USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIM To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODS An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion.Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22,Cyclin D1, and proliferating cell nuclear antigen(PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival(viability) and cell cycle were evaluated using the Cell Counting Kit-8and flow cytometry, respectively.RESULTS USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group(P < 0.05), while opposite results were observed in the USP22 overexpression group(P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSION USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.     

Lansoprazole ameliorates intestinal mucosal damage induced by ischemia-reperfusion in rats

AIM:To investigate the protective effect of lansoprazoleon ischemia and reperfusion(I/R)-induced rat intestinalmucosal injury in vivo.METHODS:Intestinal damage was induced by clampingboth the superior mesenteric artery and the celiac trunkfor 30 rain followed by reperfusion in male Sprague-Dawleyrats.Lansoprazole was given to rats intraperitoneally 1 hbefore vascular clamping.RESULTS:Both the intraluminal hemoglobin and proteinlevels,as indices of mucosal damage,significantlyincreased in I/R-groups comparion with those of sham-operation groups.These increases in intraluminal hemoglobinand protein levels were significantly inhibited by the treatmentwith lansoprazole at a dose of 1 mg/kg.Small intestineexposed to I/R resulted in mucosal inflammation that wascharacterized by significant increases in thiobarbituric acid-reactive substances(TBARS),tissue-associatedmyeloperoxidase activity(MPO),and mucosal content of ratcytokine-induced neutrophil chemoattractant-1(CINC-1).These increases in TBARS,MPO activities and CINC-1 contentin the intestinal mucosa after I/R were all inhibited bypretreatment with lansoprazole at a dose of 1 mg/kg.Furthermore,the CINC-1 mRNA expression was increasedduring intestinal I/R,and this increase in mRNA expressionwas inhibited by treatment with lansoprazole.CONCLUSION:Lansoprazole inhibits lipid peroxidation andreduces development of intestinal mucosal inflammationinduced by I/R in rats,suggesting that lansoprazole mayhave a therapeutic potential for I/R injury.     

Bone morphogenetic protein 4 mediates myocardial ischemic injury through JNK-dependent signaling pathway

Bone morphogenetic protein (BMP) signaling regulates embryonic development of many organ systems and defective BMP signaling has been implicated in adult disorders of many of these systems. However, its relevance in cardiac disease has not been reported. Here we demonstrate for the first time that Bmp4 activity promotes cellular apoptosis following ischemia-reperfusion (I/R) injury induced myocardial infarction (MI). Bmp4 heterozygous null mice (Bmp4^+/−) demonstrated reduced infarct size, less myocardial apoptosis and down-regulation of pro-apoptotic proteins relative to wild-type mice following I/R injury. This was associated with reduction in I/R induced BMP4 levels in the left ventricular infarcted region. Furthermore, treatment of neonatal cardiomyocytes with BMP4 resulted in time and dose-dependent increase in cellular apoptosis and activation of the JNK MAP kinase pathway. In contrast, while JNK activation was significantly attenuated in Bmp4^+/− mice and following Smad1 inhibition in myocytes, inhibition of JNK with a specific inhibitory peptide, TAT-JBD_20, blocked BMP4 induced apoptosis. In vivo treatment of mice with Noggin, an endogenous extracellular BMP antagonist, or dorsomorphin, a small molecule inhibitor of BMP signaling, reduced infarct size, and inhibited pro-apoptotic signaling accompanied by an inhibition of Smad1 phosphorylation and JNK activation. These studies identify a novel role for Bmp4 in the pathogenesis of myocardial infarction and illustrate the use of a small molecule inhibitor of BMP signaling for treatment of acute I/R injury.     

Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha

BACKGROUND AND AIMS: Both infliximab (chimeric anti-tumor necrosis factor [TNF]-alpha antibody) and etanercept (p75 TNF-alpha receptor/immunoglobulin G fusion protein) are effective against rheumatoid arthritis, but only infliximab induces clinical remission in Crohn's disease. To clarify this difference in clinical efficacy, we investigated reverse signaling through transmembrane TNF-alpha (mTNF) by these 2 anti-TNF agents. METHODS: We stably transfected wild-type and cytoplasmic serine-replaced mutant forms of mTNF in human Jurkat T cells. Cells were stimulated with infliximab and etanercept and then analyzed for E-selectin expression, reactive oxygen species accumulation, apoptosis, and cell cycle distribution by flow cytometry. Interleukin-10 and interferon-gamma were measured by enzyme-linked immunosorbent assay. Phospho-c-Jun NH2-terminal kinase, Bax, Bak, p21(WAF1/CIP1), caspase-8, and caspase-3 were examined by immunoblotting. RESULTS: Both anti-TNF agents induced E-selectin expression, but only infliximab induced interleukin-10 production, apoptosis, and G0/G1 cell cycle arrest. Apoptosis and cell cycle arrest were abolished by substitution of all 3 cytoplasmic serine residues of mTNF by alanine residues. Infliximab induced accumulation of reactive oxygen species and up-regulation of Bax, Bak, and p21(WAF1/CIP1) proteins, suggesting the involvement of p53 activation. Moreover, phosphorylation of c-Jun NH2-terminal kinase was necessary for infliximab-induced apoptosis and cell cycle arrest. CONCLUSIONS: We revealed the mTNF motifs and the downstream intracellular molecular events essential for reverse signaling through mTNF. The biologic effects of mTNF elicited by infliximab should be important action mechanisms of this potent anti-inflammatory agent in addition to the neutralization of soluble TNF-alpha. These observations will provide insight into the novel role of mTNF in inflammation.     

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.     

Lansoprazole ameliorates intestinal mucosal damage induced by ischemia-reperfusion in rats

AIM: To investigate the protective effect of lansoprazole on ischemia and reperfusion (I/R)-induced rat intestinal mucosal injury in vivo. METHODS: Intestinal damage was induced by clamping both the superior mesenteric artery and the celiac trunk for 30 min followed by reperfusion in male Sprague-Dawley rats. Lansoprazole was given to rats intraperitoneally 1 h before vascular clamping. RESULTS: Both the intraluminal hemoglobin and protein levels, as indices of mucosal damage, significantly increased in I/R-groups comparison with those of sham-operation groups. These increases in intraluminal hemoglobin and protein levels were significantly inhibited by the treatment with lansoprazole at a dose of 1 mg/kg. Small intestine exposed to I/R resulted in mucosal inflammation that was characterized by significant increases in thiobarbituric acid-reactive substances (TBARS), tissue-associated myeloperoxidase activity (MPO), and mucosal content of rat cytokine-induced neutrophil chemoattractant-1 (CINC-1). These increases in TBARS, MPO activities and CINC-1 content in the intestinal mucosa after I/R were all inhibited by pretreatment with lansoprazole at a dose of 1 mg/kg. Furthermore, the CINC-1 mRNA expression was increased during intestinal I/R, and this increase in mRNA expression was inhibited by treatment with lansoprazole. CONCLUSION: Lansoprazole inhibits lipid peroxidation and reduces development of intestinal mucosal inflammation induced by I/R in rats, suggesting that lansoprazole may have a therapeutic potential for I/R injury.     

The role of JNK2 in toxic liver injury

Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. In vitro studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis in vivo remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and jnk1-/- mice but markedly decreased in jnk2-/- mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of jnk2, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in jnk2-/- mice Bid cleavage and mitochondrial translocation and cytochrome c release were markedly decreased. This effect was secondary to the failure of jnk2-/- mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in jnk2 null mice after GalN/TNF treatment. Ablation of jnk2 did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in jnk1-/- mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity. [Abstract reproduced by permission of J Biol Chem 2006;281:15258-67].     

Y-27632预处理对心肌缺血再灌注损伤过程中d凋亡的影响研究

目的：探讨Rho激酶抑制剂Y-27623对心肌缺血再灌注损伤(MIRI)中细胞凋亡的影响,以及对丝裂原活化蛋白激酶（Mitogen-activated protein kinase,MAPK）和凋亡相关蛋白表达水平的变化和意义。方法：成年雄性SD大鼠60只, 随机分为4组（n=15）:正常对照组(Sham组) 、缺血再灌注组(ischemia-reperfusion即I/R组) 、Dil组( diltiazem即地尔硫卓组)、Y-27632组。Dil组每日给予地尔硫卓(10mg/kg)灌胃, Y-27632组每日给予Y-27632（5mg/kg）,其余两组给予等体积清水。给药五天后Sham组只穿线,不结扎冠状动脉左前降支,I/R组、Dil组和Y-27632组建立MIRI模型。TUNEL法检测各组心肌凋亡,计算心肌细胞凋亡指数(AI),western blotting 法检测心肌组织中MAPK信号传导途径相关蛋白（p-JNK/ERK/P38）和凋亡相关蛋白（Bcl-2、Bax、Caspase-3和Caspase-9）的表达。结果：相对于Sham组,I/R组AI明显增加（P<0.01）,MAPK信号传导途径和心肌促凋亡相关蛋白（Bax、Caspase-3和Caspase-9）表达显著增加（均为P<0.05）,心肌抗凋亡蛋白Bcl-2表达显著减少（P<0.05）;相对于I/R组,Y-27632治疗组AI明显下降（P<0.01）,与Dil治疗组没有显著差异(P>0.05),Y-27632治疗组MAPK信号传导途径相关蛋白和Bax、Caspase-3和Caspase-9表达均显著降低（P<0.05）,Bcl-2表达显著增加（P<0.05）,与Dil治疗组没有显著差异(P>0.05)。结论：Y-27632通过抑制JNK/ERK/P38的磷酸化,抑制Bax、Caspase-3和Caspase-9的表达,加强Bcl-2的表达,来减少心肌细胞的凋亡,从而减轻心肌缺血再灌注损伤。     

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidative stress-dependent p38MAPK and JNK pathway

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.     

Rapid mitogen－activated protein kinase by basic fibroblast growth factor in rat intestine after ischemia／reperfusion injury

AIM: Previous studies showed that exogenous basic fibroblast growth factor (bFGF or FGF-2) could improve physiological dysfunction after intestinal ischemia/ reperfusion (I/R) injury. However, the mechanisms of this protective effect of bFGF are still unclear. The present study was to detect the effect of bFGF on the activities of mitogen-activated protein kinase (MAPK) signaling pathway in rat intestine after I/R injury, and to investigate the protective mechanisms of bFGF on intestinal ischemia injury. METHODS: Rat intestinal I/R injury was produced by clamping the superior mesenteric artery (SMA) for 45 minutes and followed by reperfusion for 48 hours. Seventy-eight Wistar rats were used and divided randomly into sham-operated group (A), normal saline control group (B), bFGF antibody pre-treated group (C), and bFGF treated group (D). In group A, SMA was separated without occlusion. In groups B, C and D, SMA was separated and occluded for 45 minutes, then, released for reperfusion for 48 hours. After the animals were sacrificed, blood and tissue samples were taken from the intestine 45 minutes after ischemia in group A and 2, 6, 24, and 48 hours after reperfusion in the other groups. Phosphorylated forms of p42/p44 MAPK, p38 MAPK and stress activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) were measured by immunohistochemistry. Plasma levels of D-lactate were examined and histological changes were observed under the light microscope. RESULTS: Intestinal I/R injury induced the expression of p42/p44 MAPK, p38 MAPK, and SAPK/JNK pathways and exogenous bFGF stimulated the early activation of p42/p44 MAPK and p38 MAPK pathways. The expression of phosphorylated forms of p42/p44 MAPK was primarily localized in the nuclei of crypt cells and in the cytoplasm and nuclei of villus cells. The positive expression of p38 MAPK was localized mainly in the nuclei of crypt cells, very few in villus cells. The activities of p42/p44 MAPK and p38 MAPK peaked 6 hours after reperfusion in groups B and C, while SAPK/JNK peaked 24 hours after reperfusion. The activities of p42/p44 MAPK and p38 MAPK peaked 2 hours after reperfusion in group D and those of SAPK/JNK were not changed in group B. D-lactate levels and HE staining showed that the intestinal barrier was damaged severely 6 hours after reperfusion; however, histological structures were much improved 48 hours after reperfusion in group D than in the other groups. CONCLUSION: The results indicate that intestinal I/R injury stimulates the activities of MAPK pathways, and that p42/p44 MAPK and p38MAPK activities are necessary for the protective effect of exogenous bFGF on intestinal I/R injury. The protective effect of bFGF on intestinal dysfunction may be mediated by the early activation of p42/p44 MAPK and p38 MAPK signaling pathways.     

Rapid mitogen-activated protein kinase by basic fibroblast growth factor in rat intestin after ischemia/reperfusion injury

AIM: Previous studies showed that exogenous basic fibroblast growth factor (bFGF or FGF-2) could improve physiological dysfunction after intestinal ischemia/reperfusion (I/R) injury. However, the mechanisms of this protective effect of bFGF are still unclear. The present study was to detect the effect of bFGF on the activities of mitogen-activated protein kinase (MlAPK) signaling pathway in rat intestine after I/R injury, and to investigate the protective mechanisms of bFGF on intestinal ischemia injury. METttODS: Rat intestinal I/R injury was produced by clamping the superior mesenteric artery (SMA) for 45minutes and followed by repeffusion for 48 hours. Seventyeight Wistar rats were used and divided randomly into sham-operated group (A), normal saline control group (B),bFGF antibody pre-treated group (C), and bFGF treated group (D). Tn group A, SMA was separated without occlusion. In groups B, C and D, SMA was separated and occluded for 45 minutes, then, released for reperfusion for 48 hours. After the animals were sacrificed, blood and tissue samples were taken from the intestine 45 minutes after ischemia in group A and 2, 6, 24, and 48 hours after reperfusion in the other groups. Phosphorylated forms of p42/p44 MAPK, p38 MAPK and stress activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) were measured by immunohistochemistry. Plasma levels of D-lactate were examined and histological changes were observed under the light microscope. RESULTS: Intestinal I/R injury induced the expression of p42/p44 MAPK, p38 MAPK, and SAPK/JNK pathways and exogenous bFGF stimulated the early activation of p42/p44 MAPK and p38 MlAPK pathways. The expression of phosphorylated forms of p42/p44 MAPK was primarily localized in the nuclei of crypt cells and in the cytoplasm and nuclei of villus cells. The positive expression of p38MAPK was localized mainly in the nuclei of crypt cells, very few in villus cells. The activities of p42/p44 MAPK and p38MAPK peaked 6 hours after reperfusion in groups B and C,while SAPK/JNK peaked 24 hours after reperfusion. The activities of p42/p44 MAPK and p38 MAPK peaked 2 hours after reperfusion in group D and those of SAPK/JNK were not changed in group B. D-lactate levels and HE staining showed that the intestinal barrier was damaged severely 6hours after reperfusion; however, histological structures were much improved 48 hours after reperfusion in group D than in the other groups. CONCLUSION: The results indicate that intestinal I/R injury stimulates the activities of MAPK pathways, and that p42/p44 MAPK and p38MAPK activities are necessary for the protective effect of exogenous bFGF on intestinal I/R injury.The protective effect of bFGF on intestinal dysfunction may be mediated by the early activation of p42/p44 MAPK and p38 MAPK signaling pathways.