Role of Endotoxin in NF-κB Activation by Ethanol in Rat Hepatocytes

Background Endotoxin plays an important role in the progression of alcoholic liver injury. However, the role of endotoxin in acute activation of NF-κB by ethanol remains unclear.
Methods In primary rat hepatocyte cultures treated with ethanol and/or endotoxin, the DNA-binding activity of NF-κB in the nuclear extract was estimated by electrophoretic mobility shift assay. After pretreatment of a CYP2E1 inhibitor, 4-methyl pyrazole or diallyl sulfide, NF-κB activity was also measured in the same manner.
Results Ethanol or endotoxin caused activation of NF-κB in primary rat hepatocytes. Taking 50 mM of ethanol and endotoxin together raised the rapid increase in NF-κB activation, but both 100 mM of ethanol and endotoxin treatment reduced the increase. Addition of diallyl sulfide decreased the activity at rapid phase but increased it after 60 min, whereas addition of 4-methyl pyrazole caused no change of the activity at rapid phase but raised it after 60 min. Pretreatment with both endotoxin and a CYP2E1 inhibitor raised the activation of NF-κB constantly after ethanol addition. These findings suggest that endotoxin plays a critical role in the metabolism-independent activation of NF-κB by ethanol.
Conclusions Endotoxin raised metabolism-independent activation of NF-κB by high ethanol in rat hepatocytes.     

Ethanol Upregulates iNOS Expression in Colon Through Activation of Nuclear Factor-kappa B in Rats

Background:  Alcohol inhibits colonic motility but the mechanism is unknown. The goal of this study was to test the possibility that nuclear factor-kappa B (NF-κB) is involved in the upregulation of inducible nitric oxide synthase (iNOS) expression induced by ethanol in colon.
Methods:  The isometric contraction of longitudinal muscle strips of proximal colon (LP) was monitored by polygraph. Western blot analysis was used to measure the amount of iNOS and I-κB in the cytoplasm and P65 in the nucleus. Immunohistochemistry was applied to locate iNOS in colon.
Results:  Ethanol (87mM) inhibited the contraction of LP. Pretreatment of S-methylisothioure (SMT) (1 mM), a specific iNOS inhibitor, Pyrrolidine dithiocarbamate (PDTC) (10 mM) and BAY11-7082(10 mM), specific inhibitors of NF-κB significantly reversed the inhibitory effect of ethanol on LP contraction. Ethanol increased the amount of iNOS and content of NO in colon, and these effects were attenuated by pretreatment of PDTC. Following ethanol administration, the amount of I-κB in the cytoplasm decreased, but that of P65, the subunit of NF-κB in the nucleus, increased. The iNOS was located in the cell body of myenteric plexus in colon.
Conclusion:  Ethanol inhibited the contraction of LP in colon mainly through activation of NF-κB, the subsequent upregulation of iNOS expression and increase of NO release in myenteric plexus.     

Signalling pathways involved in retinal endothelial cell proliferation induced by advanced glycation end products: inhibitory effect of gliclazide

Aim:  We have previously demonstrated that advanced glycation end products (AGEs) stimulate bovine retinal endothelial cell (BREC) proliferation through induction of vascular endothelial growth factor (VEGF) production by these cells. We have also shown that gliclazide, a sulfonylurea which decreases oxidative stress, inhibits this effect. The aim of the present study was to characterize the signalling pathways involved in AGE-induced BREC proliferation and VEGF production and mediating the inhibitory effect of gliclazide on these biological events.
Methods:  BRECs were treated or not treated with AGEs in the presence or absence of gliclazide, antioxidants, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) or nuclear factor-κB (NF-κB) inhibitors. BREC proliferation was assessed by measuring [³H]-thymidine incorporation into DNA. Activation of PKC, MAPK and NF-κB signal transduction pathways and determination of VEGF expression were assessed by Western blot analysis using specific antibodies. MAPK activity was also determined by an in vitro kinase assay.
Results:  Treatment of BRECs with AGEs significantly increased cell proliferation and VEGF expression. AGEs induced PKC-β translocation, extracellular signal-regulated protein kinase 1/2 and NF-κB activation in these cells. Pharmacological inhibition of these signalling pathways abolished AGE effects on cell proliferation and VEGF expression. Exposure of BRECs to gliclazide or antioxidants such as vitamin E or N -acetyl- l -cysteine resulted in a significant decrease in AGE-induced activation of PKC-, MAPK- and NF-κB-signalling pathways.
Conclusions:  Our results demonstrate the involvement of PKC, MAPK and NF-κB in AGE-induced BREC proliferation and VEGF expression. Gliclazide inhibits BREC proliferation by interfering with these intracellular signal transduction pathways.     

An insight into the protection of rat liver against ischemia/reperfusion injury by 2-selenium-bridged β-cyclodextrin

Aim:  The reperfusion following liver ischemia results in the damage and apoptosis of hepatocytes. The aim of this study was to investigate the possible effects and mechanism of a new synthesized glutathione peroxidase (GPX) mimic, 2-selenium-bridged β-cyclodextrin (2-SeCD), on rat liver ischemia-reperfusion (I/R) injury.
Methods:  Male Wistar rats ( n  = 32) were randomly divided into four groups: I. sham-operated group, II. I/R group, III. I/R +2-SeCD group, IV. I/R + Ebselen group. Hepatic I/R was administered by 90 min of ischemia and 12 h of reperfusion. Liver tissues were collected at the end of reperfusion period for measurement of various biochemical parameters.
Results:  The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and tissue malondialdehyde, myeloperoxidase levels were increased in I/R group, while the increase was significantly reduced by 2-SeCD treatment. The glutathione level, depressed by I/R, was elevated back to normal levels by treatment with 2-SeCD. Severe hepatic damage were observed by light and transmission electron microscopy whilst pretreatment with 2-SeCD resulted in tissue and cellular preservation. Furthermore, 2-SeCD reduced cytochrome c release from mitochondria and subsequent DNA fragmentation by regulating Bcl-2/Bax expression ratio. Results suggested that 2-SeCD was more effective than ebselen in the reversal of the alteration in tissue structural and biochemical parameters caused by I/R injury.
Conclusion:  2-selenium-bridged β-cyclodextrin playes an important role in the protection of liver against I/R injury and this treatment may be a novel pharmacological agent for liver surgery.     

Liver-lung interactions following Escherichia coli bacteremic sepsis and secondary hepatic ischemia/reperfusion injury

We hypothesized that ischemia/reperfusion (I/R) injury of the liver during normotensive gram-negative bacteremic sepsis alters the kinetics of circulating endotoxin, tumor necrosis factor-alpha (TNF-alpha), and coinduced mediators, thereby exacerbating sepsis-induced lung inflammation. Liver and lung dysfunction were studied after hematogenous infection of Sprague-Dawley rats with 10(9) Escherichia coli serotype O55:B5 (EC) and 90 min of secondary hepatic ischemia in EC + I/R and saline-infused (normal saline NS) x I/R rats, followed by brief (1 h) or longer reperfusion (24 h). TNF- alpha:leukotriene interactions in this model were examined using the 5-lipoxygenase-activating protein inhibitor MK-886. Compared with sham-operated EC + Sham animals, peak serum endotoxin, TNF-alpha, alanine aminotransferase, interleukin-6 (IL-6), and hepatic neutrophil (PMN) influx were higher in EC + I/R rats through 24 h (p < 0.05) despite comparable arterial pressure. Lung PMN influx and wet/dry weight ratios were likewise enhanced in EC + I/R versus EC + Sham or NS + I/R rats. MK-886 attenuated TNF-alpha concentrations and ischemic liver injury but not mortality. Thus, focal hepatic I/R augments circulating endotoxin, TNF-alpha, and postbacteremic lung inflammation early after normotensive E. coli bacteremic sepsis.     

Endotoxin tolerance protects against local hepatic ischemia/reperfusion injury in the rat

Liver surgery and liver transplantation as well as circulatory shock are often associated with hepatic ischemia/reperfusion (I/R) injury. Recent evidence suggests that TNF-alpha plays a central role in I/R injury and, therefore, down-regulation of TNF-alpha seems to be a promising way to protect against the deleterious consequences of I/R. Endotoxin tolerance represents a state of unresponsiveness to endotoxin and is associated with diminished TNF-alpha production. Thus, the effect of endotoxin tolerance on hepatic I/R injury of the liver was investigated in a rat model. I/R injury was induced by temporary ischemia of the left lateral liver lobe for 90 min followed by a 3 h observation period of reperfusion. I/R injury resulted in functional hepatic disorder characterized by a decrease both in bile flow and bile acid concentration and 50% mortality. This was prevented by induction of endotoxin tolerance. Hepatic TNF-alpha mRNA expression after I/R of the liver was determined by RT-PCR. In untreated rats, TNF-alpha mRNA was induced in the liver 60 min after reperfusion and further increased until 3 h after reperfusion. In contrast, in endotoxin-tolerant rats, no increases in TNF-alpha mRNA expression were detected. This suggests that induction of endotoxin tolerance protects against hepatic I/R injury possibly via down-regulation of intra-organ TNF-alpha expression.     

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma

Objectives:  Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and – among other mechanisms – results in a reduced nuclear factor-kappa B (NF-κB) activity. HDACi promote histone acetylation, and also interfere with NF-κB signaling.
Methods:  Human MCL cell lines (JeKo-1, Granta-519 and Hbl-2) were exposed to bortezomib and/or SAHA. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. Reactive oxygen species (ROS) were analyzed using the fluorophore H₂DCFDA. In addition, activated caspases, proteasome- and NF-κB activity were quantified.
Results:  Combined incubation with bortezomib and SAHA resulted in synergistic cytotoxic effects, as indicated by combination index values <1 using the median effect method of Chou and Talalay. The combination of both inhibitors led to a strong increase in apoptosis as compared to single agents and was accompanied by enhanced ROS generation, while each agent alone only modestly induced ROS. The free radical scavenger N -acetyl- l -cysteine blocked the ROS generation and reduced the apoptosis significantly. In addition, coexposure of bortezomib and SAHA led to increased caspase-3, -8 and -9 activity, marked reduction of proteasome activity and decrease of NF-κB activity.
Conclusions:  This is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in MCL cells. These data build the framework for clinical trials using combined proteasome and histone deacetylase inhibition in the treatment of MCL.     

库普弗细胞Toll样受体2在小鼠肝脏缺血再灌注损伤中的表达及意义

目的探索库普弗细胞Toll样受体2(TLR2)在肝脏缺血再灌注损伤中的表达变化,分析库普弗细胞TLR2信号通路参与肝脏缺血再灌注损伤的机制。方法动物分假手术对照(SH)组、缺血再灌注(I/R)组及氯化钆处理(Gd)组,复制小鼠肝脏部分缺血1 h再灌注4 h损伤模型,结束再灌注后,取缺血叶肝脏组织及其库普弗细胞,提取总RNA及膜蛋白质分析TLR 2 mRNA及蛋白的表达,同时提取缺血肝叶组织核蛋白分析核因子(NF—κB),并检测门静脉血中肿瘤坏死因子(TNF α)、丙氨酸氨基转移酶(ALT) 及内毒素水平。结果再灌注4 h,(1)I/R组缺血肝叶TLR2 mRNA及蛋白表达水平较SH组高,△Ct值分别为1.06±0.91和5.08±1.32,t=7.80,P<0.01;A值分别为433.91±25.53和102.86±13.58,t=28.04, P<0.01。Gd组缺血肝叶TLR2 mRNA及蛋白表达水平较I/R组下降,△Ct值分别为4.22±0.84和1.06±0.91,t=7.56,P<0.01;A值分别为125.89±15.49和433.91±25.53,t=25.27,P<0.01。(2)I/R组缺血肝叶库普弗细胞中TLR2 mRNA及蛋白表达水平较SH组高,△Ct值分别为0.52±0.23和2.61±0.1 8, t=17.47,P<0.01;A值分别为379.70±34.16和114.98±21.90,t=15.98,P<0.01。Gd组缺血肝叶库普弗细胞中TLR2 mRNA及蛋白表达水平则较I/R组下降,△Ct值分别为1.90±0.14和0.52±0.23,t= 12.     

Inhibition of Fas/FasL mRNA expression and TNF-α release in concanavalin A-induced liver injury in mice by bicyclol

AIM: Bicyclol, 4,4'-dimethoxy-5,6,5',6'-dimethylene-dioxy-2-hydroxymethyl-2'-carbonyl biphenyl, is a new anti-hepatitis drug. The aim of the present study was to investigate the protective effect of bicyclol on concanavalin A (Con A)-induced immunological liver injury in mice and its mechanism.METHODS: Liver injury was induced by injection of Con A via tail vein of mice and assessed biochemically and histologically. Serum transaminase and tumor necrosis factor alpha (TNF-α) were determined. Liver lesions were observed by light microscope. Expressions of TNF-α, interferon gamma (IFN-γ), Fas and Fas ligand (FasL) mRNA in the livers were measured by RT-PCR.RESULTS: Serum transaminase level and liver lesions in Con A-induced mice were markedly reduced by oral administration of 100, 200 mg/kg of bicyclol. TNF-α level in serum was also reduced by bicyclol. Con A injection induced up-regulation of TNF-α, IFN-γ, Fas and FasL mRNA expression in liver tissues. Bicyclol significantly down-regulated the expression of IFN-γ, Fas and FasL mRNA, but only slightly affected TNF-α mRNA expression in liver tissues.CONCLUSION: Bicyclol protects against Con A-induced liver injury mainly through inhibition of Fas/FasL mRNA expression in liver tissues and TNF-α release in mice.     

Inhibition of Fas／FasL mRNA expression and TNF-a release in concanavalin A-induced liver injury in mice by bicyclol

AIM: Bicyclol, 4,4‘-dimethoxy-5,6,5‘,6‘-dimethylene-dioxy-2-hydroxymethy1-2‘-carbonyl biphenyl, is a new anti-hepatitis drug. The aim of the present study was to investigate the protective effect of bicyclol on concanavalin A (Con A)-induced immunological liver injury in mice and its mechanism. METHODS: Liver injury was induced by injection of Con A via tail vein of mice and assessed biochemically and histologically. Serum transaminase and tumor necrosis factor alpha (TNF-a were determined. Liver lesions were observed by light microscope. Expressions of TNF-a, interferon gamma (IFN-y), Fas and Fas ligand (FasL) mRNA in the livers were measured by RT-PCR. RESULTS: Serum transaminase level and liver lesions in Con A-induced mice were markedly reduced by oral administration of 100, 200 mg/kg of bicyclol. TNF-a level inserum was also reduced by bicyclol. Con A injection in ducedup-regulation of TNF-a, IFN-7, Fas and FasL mRNA expression in liver tissues. Bicyclol significantly down-regulated the expression of IFN-y, Fas and FasL mRNA, but only slightly affected TNF-a mRNA expression in liver tissues. CONCLUSION: Bicyclol protects against Con A-induced liver injury mainly through inhibition of Fas/FasL mRNA expression in liver tissues and TNF-a release in mice.     

Protective effect of bicyclol on lipopolysaccharide-induced acute lung injury in mice

Bicyclol is synthesized based on schisandrin, which is one of the main active components of Chinese herb Fructus Schisandrae. The purpose of this study is to investigate whether bicyclol has a beneficial effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Bicyclol was given to mice by gavage for three times. ALI was induced by vena caudalis injection of LPS. The last dose of bicyclol was administrated 1 h before LPS given. Mice in each group were sacrificed at different time point after LPS administration. As revealed by survival study, pretreatment with high doses of bicyclol reduced the mortality of mice from ALI. Bicyclol pretreatment significantly improved LPS-induced lung pathological changes, inhibited myeloperoxidase (MPO) activity, and reduced lung/body and lung wet/dry weight ratios. Bicyclol also inhibited the release of TNF-α, IL-1β and HMGB1, whereas simultaneously increased the expression of IL-10. Furthermore, the phosphorylation level of NF-κB p65 was markedly decreased by bicyclol. Taken together, our study showed that bicyclol improves survival rate and attenuates LPS-induced ALI. The protective mechanism may be due to the inhibition of NF-κB activation and regulation of cytokine secretion.     

双环醇片治疗肾移植术后药物性肝损伤的疗效观察

目的探讨双环醇片治疗肾移植术后药物性肝损伤的安全性和有效性。方法将80例肾移植术后出现药物性肝损伤的患者按照随机原则分为两组,双环醇治疗组和硫普罗宁治疗组各40例,在应用常规抗免疫排斥药物同时采用双环醇片或硫普罗宁片进行保肝治疗,连续用药4周。结果双环醇治疗组在肝功能与综合疗效改善方面明显优于硫普罗宁治疗组(P<0.01或0.05),未发现与研究药物明显相关的不良事件。结论双环醇片可有效治疗肾移植术后由抗免疫排斥药物引起的肝损伤,疗效确切,安全性好。     

Augmentation of Ethanol-Induced Cell Damage and Activation of Nuclear Factor-κB by Annexin IV in Cultured Cells

Background: We previously reported that the amount of annexin IV expression was increased in culture cells by exposure to ethanol. Here, to investigate the physiologic role of annexin IV, we analyzed ethanol-induced cytotoxicity and nuclear factor (NF)-κB activity by using annexin IV-overexpressed cells.
Methods: Annexin IV overexpression was performed by transfection of expression vector, in which annexin IV complementary DNA was ligated, to culture cells (rat glioma C6 cells and human neuroblastoma SH-SY5Y cells). Ethanol-induced cytotoxicity was assayed by measuring the mitochondrial enzyme (dehydrogenase) activity or trypan blue exclusion. NF-κB activity was measured by electrophoretic mobility shift assay with a κB-oligonucleotide probe.
Results: Ethanol-induced cytotoxicity was increased by overexpression of annexin IV in both C6 cells and SH-SY5Y cells. Annexin IV overexpression augmented ethanol-induced NF-κB activation.
Conclusions: Ethanol-induced increase in annexin IV expression might amplify ethanol-induced cytotoxicity via NF-κB activation.     

Protective effect of sulfhydryl-containing antioxidants against ischemia/reperfusion injury of prepubertal rat intestine

Background and Aim:  Reactive oxygen species generated during reperfusion of the tissue are known to play an important role in the basic pathophysiology of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate and compare the protective effects of three sulfide-based antioxidants, N -acetylcysteine (NAC), erdosteine (ERD), and α-lipoic acid (LA), on I/R injury of the small intestine tissue.
Methods:  Forty male Sprague–Dawley rats weighing between 100–150 g were divided into five groups ( n  = 8 for each): control (sham operated), I/R, I/R + NAC, I/R + ERD, and I/R + LA. Intestinal ischemia was provided by occluding the superior mesenteric artery via a special microvascular clamp; ischemia for 30 min and reperfusion for 3 days were carried out. Ileal specimens were obtained to determine the tissue levels of malondialdehyde (MDA), protein carbonyl contents (PCO), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and histological changes.
Results:  The rats subjected to intestinal I/R exhibited an increase in tissue MDA and PCO; the levels could hardly be ameliorated in the treatment groups. SOD and GPx activities were significantly decreased in the I/R group, whereas their reduction was less expressed in the treatment groups. Additionally, the histopathological injury scores of the disulfide-treated groups were lower than those of the I/R group.
Conclusion:  All of the sulfhydryl-containing antioxidants used in this study exhibited a significant role in attenuating intestinal I/R injury; however, the outcome of the LA-treated group was significantly marked than that of the others.     

Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells

Aims:  Thiazolidinediones (TZDs), ligands for peroxisome proliferator–activated receptor γ, are antidiabetic agents that improve hyperglycemia by decreasing insulin resistance in obese diabetic animal models and patients with type 2 diabetes. We have studied whether pioglitazone, a TZD, can exert a direct effect against pancreatic β-cell lipoapoptosis.
Methods:  MIN6 cells were cultured in medium containing either 5.6 (low glucose) or 25 mM glucose (high glucose) in the presence or absence of 0.5 mM palmitate for 48 h. We examined the effect of 10 μM pioglitazone on MIN6 cells on glucose-stimulated insulin secretion, cellular ATP, uncoupling protein-2 (UCP-2) mRNA expression, intracellular triglyceride content, reactive oxygen species production, the number of apoptotic cells and nuclear factor-κB (NF-κB) activity.
Results:  Pioglitazone recovered partly impaired glucose-stimulated insulin secretion and cellular ATP in MIN6 cell exposed to high glucose with 0.5 mM palmitate. Pioglitazone suppressed intracellular triglyceride accumulation in cells exposed to high glucose with 0.5 mM palmitate. Palmitate-induced upregulation of UCP-2 mRNA levels was suppressed by pioglitazone in a dose-dependent manner. Pioglitazone decreased palmitate-induced reactive oxygen species production in MIN6 cells by 24% and in mouse islet cells by 53%. Pioglitazone also decreased palmitate-induced NF-κB activity by 40% and protected β-cells from palmitate-induced apoptosis by 22% in MIN6 cell.
Conclusions:  Pioglitazone attenuated fatty acid–induced oxidative stress and apoptosis in pancreatic β-cells. TZDs might be used as a mean for maintaining β-cell survival and preserving capacity of insulin secretion in patients with diabetes mellitus.     

Glimepiride upregulates eNOS activity and inhibits cytokine-induced NF-κB activation through a phosphoinoside 3-kinase–Akt-dependent pathway

Aims:  Several studies suggest increased mortality postcoronary angioplasty in patients on sulphonylureas. However, a theoretical reduction in cardiac risk has been suggested with the newer sulphonylurea agents, which differ from the first-generation agents. In the present study, we investigated whether a third generation of sulphonylurea, glimepiride, might stimulate nitric oxide (NO) production and thereby inhibit cytokine-induced nuclear factor (NF)-κB activation in endothelial cells compared with the classical sulphonylurea glibenclamide.
Methods and Results:  We demonstrated that glimepiride, but not glibenclamide, induces NO production in human umbilical vein endothelial cells (HUVEC). A significant increase in endothelial NO synthase (eNOS) activity, measured in terms of citrulline production, was observed with glimepiride treatment. Akt phosphorylation followed by phosphorylation of eNOS (Ser1177) was observed with glimepiride treatment in HUVEC. Moreover, two phosphoinoside 3-kinase inhibitors, wortmannin and LY294002, significantly inhibited glimepiride-induced NO production. We also demonstrated inhibition of tumour necrosis factor-alpha (TNFα)-induced NF-κB activation in HUVEC treated with glimepiride, which was attenuated by pretreatment with N^ω-nitro- l -arginine methyl ester. We also demonstrated a marked increase in p65 in nuclear extracts from untreated HUVEC following stimulation with TNFα, which was dose dependently inhibited by glimepiride, but not by glibenclimide in association with NF-κB levels.
Conclusion:  These data suggest that glimepiride might be a preferable sulphonylurea agent in the setting of type 2 diabetes and vascular disease because it may have protective effects on vascular endothelial cells.