Relationship between nuclear factor-kappaB activation and virulence factors of Helicobacter pylori in Japanese clinical isolates

BACKGROUND: Several factors have been proposed as a possible virulence determinant of Helicobacter pylori infection. The aim of the present study was to evaluate these candidates in nuclear factor (NF)-kappaBeta activation, which is a critical regulator of genes involved in inflammatory reactions. METHODS: We determined the status of cagE, iceA, HP0441 (a virB4 homolog), the s1 signal sequence of vacA and babA2 by polymerase chain reaction, all of which are candidate virulence determinants, in 107 H. pylori strains isolated from Japanese patients. Nuclear factor-kappaBeta activation was evaluated by the luciferase reporter assay. The gastric mucosa of the hosts was examined histologically. RESULTS: The cagE gene was positive in 102 (95.3%) strains, iceA1 was positive in 71 (66.4%) strains, HP0441 was positive in 68 (63.6%) strains, vacA s1 was positive in 105 (98.1%) strains and babA2 was positive in 103 (96.3%) strains. Nuclear factor-kappaBeta was activated by all cagE-positive strains, but was not activated by any of the cagE-negative strains. The status of iceA or HP0441 was not associated with NF-kappaBeta activation. Neutrophil infiltration in gastric mucosa was significantly more severe in patients infected with cagE-positive strains than in patients infected with negative strains. No association was found between the degree of neutrophil infiltration and the status of HP0441 or iceA. Due to very high positivity of vacA s1 and babA2 in Japanese strains, their roles remain to be investigated. CONCLUSIONS: The cag pathogenicity island (PAI) status, as determined by cagE polymerase chain reaction, but not the status of iceA or HP0441, is closely associated with NF-kappaBeta activation and the degree of gastric mucosal inflammation in the hosts.     

Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-kappaB in LPS-stimulated human monocytes

Unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti-inflammatory properties; however, their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)-kappaB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 microg and 1 microg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6 and IL-1beta release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-kappaB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-alpha, IL-8, IL-6 and IL-1beta as well as NF-kappaB translocation. These results indicate equivalent and significant heparin anti-inflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-kappaB activation certainly represents one of the mechanisms by which heparin exerts its anti-inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.     

Effects of nuclear factor-kappaB inhibitors and its implication on natural killer T-cell lymphoma cells

Natural killer/T-cell lymphoma (NKTL) is a highly aggressive disease. Despite the use of various treatment regimens, the prognosis of NKTL is poor, and new treatment strategies need to be determined. Because of the significant survival potential, nuclear factor (NF)-kappaB has become one of the major targets for drug development. In this study, we explored the effect and action mechanism of NF-kappaB inhibitors, BAY 11-7082 and curcumin, on NKTL cell lines (NKL, NK-92 and HANK1). Electrophoretic mobility shift assay showed that NF-kappaB was constitutively active in HANK1, a chemoresistant cell line. BAY 11-7082 and curcumin suppressed NF-kappaB activation in a time- and dose-dependent manner, which finally resulted in cell death. BAY 11-7082- and curcumin-induced cell death was associated with downregulation of Bcl-xL, cyclin D1, XIAP and c-FLIP, followed by caspase-8, poly(ADP-ribose) polymerase cleavage and activation. Given that the chemoresistant NK-92 cells respond to NF-kappaB inhibitors but not to conventional drugs, BAY 11-7082 and curcumin could be potentially useful for achieving improved outcome in chemotherapy-refractory NKTL.     

高血糖促进氧化应激加重大鼠心肌缺血/再灌注损伤

目的:研究高血糖是否可通过增加大鼠急性缺血/再灌注（I/R）心肌氧化应激而加重心肌损伤,并探讨其机制。方法: 将SD大鼠随机分为3组:假手术组(Sham)、生理盐水对照组(Vehicle)和高糖组(HG)。通过缺血30 min再灌注6 h,建立大鼠急性心肌I/R模型。通过静脉输注高浓度葡萄糖溶液,建立大鼠急性心肌I/R并发高血糖动物模型。术中监测血糖水平。再灌注结束后,检测血浆心肌酶谱水平,心肌梗死面积（IS）、心肌细胞凋亡指数（AI）和caspase 3的活性,检测心肌组织中氧化应激指标超氧阴离子、gp91phox、MDA、SOD,以及硫氧还蛋白结合蛋白（Txnip）的水平和硫氧还蛋白（Trx）的活性。结果: 与Vehicle组比较,HG组大鼠血糖水平显著升高,肌酸激酶（CK）、乳酸脱氢酶（LDH）的水平和IS增加,AI和caspase 3的活性升高(P<0.05)。HG组I/R心肌组织氧化应激程度显著升高,超氧阴离子、gp91phox和MDA水平增加(P<0.05)。同时,HG组I/R心肌组织的Txnip表达增加而Trx活性降低(P<0.05)。结论: 高血糖可增加大鼠I/R心肌中Txnip的表达,抑制Trx的活性促进氧化应激,这可能是其加重I/R心肌损伤的机制。     

肿瘤坏死因子受体-抗体融合蛋白对小鼠肺组织氧化损伤的保护作用

[目的]探讨肿瘤坏死因子受体-抗体融合蛋白(TNFR-Fc)通过下调氧化应激减轻急性肺损伤(ALl)小鼠肺组织破坏的可能机制.[方法]SPF级8周龄BALB/c小鼠共54只,雌雄各半,平均体重20g,按随机数字表法分为脂多糖组、干预组和对照组,每组16只.各组均经气管滴入脂多糖复制ALl小鼠模型,TNFR-Fc组在滴入脂多糖前24h腹膜腔注射TNFR-Fc(0.4mg/kg).滴入脂多糖后0h和2h分别处死8只小鼠并收集肺脏组织,测量肺湿/干重比,行肺泡灌洗并通过BCA法检测BALF中蛋白含量,ELISA法检测外周血肿瘤坏死因子(TNF)-α浓度,组织病理半定量评分评价肺损伤程度,比色法测定肺组织匀浆中丙二醛浓度并计算总抗氧化能力,RT-PCR法检测iNOS、Nox1、Nox2、Nox4、XO及SOD等氧化相关基因转录水平.[结果]TNFR-Fc干预组小鼠血清TNF-α浓度[(119±51)mg/L]、BALF蛋白含量[(539±89)mg/L]及肺组织损伤病理评分均低于脂多糖组[(635±89),(793±87)mg/L,均P＜0.01],而肺湿/干重比二者差别无统计学意义(P＞0.05).干预组小鼠肺组织丙二醛水平(22.5±3.9)与脂多糖组(25.4±3.2)相比较低,但差异无统计学意义(P＞0.05),总氧化能力明显高于脂多糖组(P＜0.05).干预组小鼠肺组织Nox1、Nox2、Nox4及XO基因转录强度与脂多糖组相比较低(均P＜0.05),二者SOD基因表达强度差异无统计学意义(P＞0.05).[结论]TNFR-Fc可通过降低TNF-α浓度,控制TNF-α对炎症氧化应激的活化作用,并下调Nox1、Nox2、Nox4及XO等基因表达,减轻组织氧化损伤和ALI的肺组织损害.     

Dl-3-n-butylphthalide attenuates myocardial ischemia reperfusion injury by suppressing oxidative stress and regulating cardiac mitophagy via the PINK1/Parkin pathway in rats

BackgroundAcute myocardial infarction (AMI) is one of the leading causes of mortality worldwide. Undesirable myocardial damage may occur during reperfusion of the ischemic myocardium, and this is known as “ischemic reperfusion injury” (IRI). Currently, there are few effective drugs to alleviate IRI. Dl-3-n-butylphthalide (NBP) is recommended for the treatment of acute ischemic stroke in China. This study investigated the effects of NBP on IRI and its underlying mechanisms.MethodsThe left anterior descending (LAD) coronary arteries of rats were occluded for 30 minutes and reperfused for 6 hours to establish the ischemia/reperfusion (I/R) model. NBP was administered intraperitoneally 2 hours before modeling and immediately after reperfusion. At 6 hours after reperfusion, 2,3,5-triphenyltetrazolium chloride (TTC) staining, enzyme-linked immunosorbent assay (ELISA), oxidative stress index, myocardial injury index, hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), real-time polymerase chain reaction (PCR), immunofluorescence staining, and Western blot analyses were performed to investigate the protective effects of NBP against IRI.ResultsIn the rat I/R model, NBP remarkably reduced the myocardial infarct size, alleviated myocardial injury and oxidative stress, improved the pathological alteration of cardiomyocytes and mitochondria, and upregulated mitophagy. In addition, the study demonstrated that the protective effects of NBP against IRI involved mitophagy mediated by the PTEN-induced putative kinase protein-1 (PINK1)/Parkin signaling pathway.ConclusionsNBP was able to protect the myocardium from IRI in rats through inhibiting oxidative stress and activating mitophagy, mediated by the PINK1/Parkin pathway.     

Glutamine prevents oxidative stress in a model of mesenteric ischemia and reperfusion

AIM: To evaluate preventative effects of glutamine in an animal model of gut ischemia/reperfusion (I/R).METHODS: Male Wistar rats were housed in a controlled environment and allowed access to food and water ad libitum. Twenty male Wistar rats were divided into four experimental groups: (1) control group (control) - rats underwent exploratory laparotomy; (2) control + glutamine group (control-GLU) - rats were subjected to laparotomy and treated intraperitoneally with glutamine 24 and 48 h prior to surgery; (3) I/R group - rats were subjected to occlusion of the superior mesenteric artery for 30 min followed by 15 min of reperfusion; and (4) ischemia/reperfusion + glutamine group (G + I/R) - rats were treated intraperitoneally with glutamine 24 and 48 h before I/R. Local and systemic injuries were determined by evaluating intestinal and lung segments for oxidative stress using lipid peroxidation and the activity of superoxide dismutase (SOD), interleukin-6 (IL-6) and nuclear factor kappa beta (NF-κB) after mesenteric I/R.RESULTS: Lipid peroxidation of the membrane was increased in the animals subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before the I/R procedure showed levels of lipid peroxidation similar to the control groups (P < 0.05). The activity of the antioxidant enzyme SOD was decreased in the gut of animals subjected to I/R when compared with the control group of animals not subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before I/R showed similar SOD activity to both control groups not subjected to I/R (P < 0.05). The mean area of NF-κB staining for each of the control groups was similar. The I/R group showed the largest area of staining for NF-κB. The G + I/R group had the second highest amount of staining, but the mean value was much lower than that of the I/R group (P < 0.05). For IL-6, control and control-GLU groups showed similar areas of staining. The I/R group contained the largest area of IL-6 staining, followed by the G + I/R animals; however, this area was significantly lower than that of the group that underwent I/R without glutamine (P < 0.05).CONCLUSION: These results demonstrate that pretreatment with glutamine prevents mucosal injury and improves gut and lung recovery after I/R injury in rats.     

PPAR agonist rosiglitazone attenuates cardiac dysfunction and oxidative stress in rats after myocardial ischemia/reperfusion injury

Background Whether pre-treatment with peroxisome proliferator activated receptor-γ(PPAR-γ) agonist has beneficial effect on myocardial ischemia / reperfusion(I / R) injury is not well established. In this study,we try to explore the cardioprotective effect of the pre-treatment with PPAR-γ agonist rosiglitazone(Ros) on the hearts suffering I / R injury. Methods Experimental I / R injury was induced by Langendorff heart reperfusion model and left anterior descending artery ligation in rats. Oxidative stress was evaluated by measuring lactate dehydrogenase(LDH), nitric oxide synthase(NOS), superoxide dismutase(SOD) and malonaldehyde(MDA). Bcl-2 and Bax were detected by Western blotting and real-time PCR. Results Ros treatment significantly decreased SOD and inducible nitric oxide synthase and increased creatine kinase, LDH,MDA, and endothelial nitric oxide synthase in-vivo. Both in vitro and in-vivo, Ros treatment increased Bcl-2level and decreased Bax level in a dose-dependent manner. In vitro, Ros treatment significantly increased SOD but lowered MDA and LDH in a dose-dependent manner. Conclusions Pre-treatment with PPARγ agonist Ros has beneficial effect on myocardial I / R injury by attenuating oxidative stress and inhibiting cardiomyocyte apoptosis.     

JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio‐protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up‐regulation of the anti‐apoptotic protein Bcl2, and down‐regulation of the pro‐apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin‐mediated cardio‐protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well‐preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), which indicates that the IR‐induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin‐induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR‐induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.     

Melatonin reduces apoptosis and necrosis induced by ischemia/reperfusion injury of the pancreas

The pancreas is highly susceptible to the oxidative stress induced by ischemia/reperfusion (IR) injury leading to the generation of acute pancreatitis. Melatonin has been shown to be useful in the prevention of the damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney. The aim of the study was to evaluate the cytoprotective properties of melatonin against injury induced by IR in pancreas. The obstruction of gastro-duodenal and inferior splenic arteries induced pancreatic IR in male Wistar rats. Melatonin was intraperitoneally administered before or/and after IR injury. The animals were killed at 24 and 48 hr after reperfusion and there were evaluated parameters of oxidative stress (lipoperoxides, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), glandular endocrine and exocrine function (lipase, amylase, insulin) and cell injury (apoptosis and necrosis). The IR induced a marked enhancement of oxidative stress and impaired pancreatic function. The histological analysis showed that IR induced acute pancreatitis with the accumulation of inflammatory infiltrate, disruption of tissue structure, cell necrosis and hemorrhage. Melatonin administration before or after pancreatic IR prevented all tissue markers of oxidative stress, biochemical and histological signs of apoptosis and necrosis, and restored glandular function. No histological signs of pancreatitis were observed 48 hr after reperfusion in 80% of the animals treated with melatonin, with only a mild edematous pancreatitis being observed in the remaining rats. Preventive or therapeutic administration of melatonin protected against the induction of oxidative stress and tissue injury, and restored cell function in experimental pancreatic IR in rats.