The influence of brain death on liver function.

BACKGROUND: In this study, we investigated the influence of brain death on inflammatory response and the effects of brain death on liver function both directly after explantation and after reoxygenation. METHODS: The influence of brain death on liver function was studied in rats using a brain death model and the liver slice model to mimic reoxygenation. Liver function was assessed by measuring the ATP content and the ATP-driven urea synthesis. The activation of non-parenchymal cells was studied by measuring mRNA levels of IL-10, cytokine production (IL-10 and IL-1 beta) and inducible nitric oxide synthases (iNOS) upregulation (mRNA) and protein level. RESULTS: Brain death had no direct influence on the ATP content of the liver. However, it led to induction of several cytokines because of activation of non-parenchymal cells, which led to upregulation of iNOS and to nitric oxide metabolites production. It is known that cytokine production may influence the drug metabolism capacity; however, no influence of brain death on drug metabolism was observed. An explanation may be the relatively short experimental period. CONCLUSIONS: Kupffer cells seem to be activated during the onset of brain death induction; however, they become quiescent when liver slices of brain dead rats were reoxygenated during incubation. Other non-parenchymal cells, possibly the endothelial cells, remain activated during incubation and reoxygenation in slices from brain death donors but not in slices from control livers. Future experiments in our rat liver transplantation model need to elucidate the implications of these findings.     

Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells

The production of interleukin-8 (CINC: cytokine-induced neutrophil chemo-attractant) from different cell populations in the rat liver was studied and cells related to the initiation of CINC production in lipopolysaccharide (LPS)-injected endotoxaemic rats were characterized. Sinusoidal endothelial cells (16.4 ± 10.6 ng/mL) produced significantly higher amounts of CINC in 24 h primary cultures compared with hepatocytes (0.9 ± 0.9 ng/mL; P < 0.05) and Kupffer cells (6.5 ± 5.1 ng/mL; P < 0.05). Lipopolysaccharide, tumour necrosis factor-α (TNF-α), and interleukin-1α (IL-1α) stimulated different liver cell populations to produce CINC; LPS mainly stimulated Kupffer cells, TNF-α stimulated hepatocytes and IL-1α stimulated all three types of cells. Intraperitoneal injection of LPS (4 mg/kg) caused CINC accumulation in non-parenchymal cells of the rat liver within 1 h of injection, as shown by immunohistochemical staining. In contrast, CINC-positive hepatocytes were not seen until 3 h after injection of LPS. Ethanol was not a direct inducer of CINC production by rat hepatocytes in vitro. These findings strongly suggest that non-parenchymal liver cells, including sinusoidal endothelial cells, are the main source of CINC. Our data also suggest that during endotoxaemia, CINC production is initiated by non-parenchymal cells and this is followed by production from hepatocytes.     

Interleukin 1 beta markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells

To investigate whether a single inflammatory cytokine could stimulate nitric oxide formation in the absence of other cytokines or lipopolysaccharide (LPS), NO was measured by the redox chemiluminescence method in primary cultured rat hepatocytes and in rat Kupffer cells. Interleukin (IL) 1 beta, but neither IL-6, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), nor LPS stimulated NO formation in a dose-dependent manner and induced half-maximal effects at 30 pmol/L. Maximal stimulation was achieved at 12 to 16 hours after the addition of 1 nmol/L of IL-1 beta, and was 50- to 60-fold above basal levels in rat hepatocytes. The combined effect of these cytokines with LPS or IFN-gamma on NO formation was also examined. Neither LPS nor IFN-gamma affected the IL-1 beta-induced NO formation. TNF-alpha, however, stimulated IL-1 beta-induced NO formation, while IL-6 inhibited it, although independently these cytokines had no effect on NO formation. None of the cytokines tested stimulated NO formation in cultured rat Kupffer cells. In hepatocytes, the NO formation induced by IL-l beta was blocked by both the NO synthase (NOS) inhibitor N^G-monomethyl-L-arginine (L-NMMA) and by IL-1 receptor antagonist (IL-1ra). Furthermore, IL-1 beta markedly increased NOS activity, and this increase in activity was accompanied by the expression of inducible NOS (iNOS) messenger RNA (mRNA). This study clearly demonstrated that IL-1 beta markedly stimulates NO formation in hepatocytes, in the absence of other cytokines or LPS. (Hepatology 1996 Apr;23(4):797-802)     

CO liberated from CORM-2 modulates the inflammatory response in the liver of thermally injured mice

AIM： To explore the effects of CO-releasing molecules [tricarbonyldichlororuthenium （Ⅱ） dimer, CORM-2]- liberated CO on attenuation of inflammatory responses in liver of an experimental animal model of thermal injury and to investigate the associated potential mechanisms. METHODS： Thirty-six mice were assigned to three groups in three respective experiments. In each experiment, mice in sham group （n = 4） received sham thermal injury, whereas mice in burn group （n = 4） received a 15% of total body surface area （TBSA） fullthickness thermal injury, and mice in burn ＋ CORM-2 group （n = 4） received the same thermal injury with immediate administration of CORM-2 （8 mg/kg, iv）. Hepatic tissue sections were stained with hematoxylin and eosin and examined under a light microscope. Levels of aminotransferases （ALT and AST） and nitric oxide （NO） were measured by biochemical methods. Tumor necrosis factor-α （TNF-α） and interleukin （IL-1β） activity, and the protein expression of iNOS and HO-1 in serum and tissue homogenates were assessed. In in vitro experiments, Kupffer cells were stimulated with LPS （10 μg/mL） for 4 h in the presence or absence of CORM-2 （10-100 μmol/L）. Subsequently, the expression levels of TNF-α and NO production were assessed. RESULTS： Pro-inflammatory mediators （TNF-α, IL- 1β, NO） in serum and liver homogenates of thermally injured mice were significantly reduced by CORM-2 administration. This was accompanied by a decrease in the expression of iNOS while an increase in the expression of HO-1 in the liver tissue. In parallel, the concentrations of TNF-α and NO in supernatants of LPS-stimulated Kupffer cells co-incubated with CORM-2 （10-100 μmol/L） were also markedly decreased.Histological examination demonstrated that CORM-2 could attenuate the leukocytes infiltration to the liver tissue. CONCLUSION： CORM-released CO modulates liver inflammation and significantly protects liver injury in burn mice by inhibiting the expression     

Acute pancreatitis affects non-parenchymal liver cells by a mechanism dependent on platelet-activating factor.

BACKGROUND/AIM: During acute experimental pancreatitis, inflammatory mediators/cytokines are released by the pancreas and enter the portal venous system, reaching the liver. We investigate some aspects of the liver cell function under conditions of acute pancreatitis and the effect of in vivo treatment with a selective platelet-activating factor (PAF) antagonist. METHODS: Cells were isolated from Wistar rats 24 h after induction of acute pancreatitis by retrograde injection of sodium taurocholate into the main pancreatic duct. The non-parenchymal cell population was separated by Percoll gradient and the adherent cell population (Kupffer cells) obtained. The cells were cultured for 24 h and supernatants assayed for nitrite by Griess reaction and for tumour necrosis factor (TNF) by bioassay in L929 cells. The microbicidal activity was evaluated by killing of Candida albicans. The PAF antagonist WEB2170 (10 mg/kg i.v.) was administered 30 min before induction of pancreatitis. RESULTS: We found that liver cells produce nitric oxide (NO) only under lipopolysaccharide stimulation and that WEB-2170 treatment reduces the NO production by liver cells in the pancreatitis group only. Cells from both groups produced TNF spontaneously, and the levels were further increased after lipopolysaccharide stimulation. WEB-2170 treatment did not affect the TNF levels. Moreover, killing of C. albicans by Kupffer cells wassignificantly increased by the PAF antagonist. CONCLUSION: These results suggest that PAF released during acute pancreatitis upregulates the NO production by non-parenchymal liver cells and inhibits Kupffer cell microbicidal activity which could explain the increased bacterial dissemination observed in acute pancreatitis.     

肝细胞一氧化氮合酶的诱导及动力学研究

目的对内毒素和几种细胞因子诱导肝细胞一氧化氮合酶的协同效应及酶动力学参数进行研究.方法原位预灌流和段原酶循环灌流大鼠肝脏、分离肝实质细胞,观察内毒素、IFN-Y、IFN-α、TNFα、IL-lβ、IL-6及不同组合对肝细胞一氧化氮合酶活性、cGMP及NO2-+NO3的影响,分析酶动力学特征及皮质甾与酶诱导的量效关系.结果内毒素+IFN-v+TNFα+IL-lβ(IL-6)组合诱导酶表达效应最显著;酶参数分析显示Km、Vmax分别为108μmol/L和2632pmol@min1mg-1蛋白质,竞争性抑制剂L-NMMA、L-NNA作用的Ki分别为056μmolL及094μmol/L;诱导时间进程显示iNOS活性表达在9h达到峰值,但cGMP及NO2-NO3-的释放持续增加可维持至l8h;地塞米松和氢化可的松抑制肝细胞酶诱导的IC50分别为35×10-8mol/L和26×10-0mol/L.结论肝细胞诱导性一氧化氮合酶的表达依赖特异多细胞因子协同作用,这种可诱导性特征可能在内毒素血症和败血症休克发病机制中具有重要意义.     

Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells

BACKGROUND: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. METHODS: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100beta. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. RESULTS: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNgamma (100 ng/ml) + TNFalpha (30 ng/ml) + IL-1beta (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. CONCLUSIONS: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.     

Mechanism of neutrophil accumulation in sinusoids after extrahepatic biliary obstruction

BACKGROUND/AIMS: Polymorphonuclear neutrophil (PMN) infiltration represents a potential source of liver injury, but the precise mechanisms of PMN infiltration in cholestatic liver are not fully understood. METHODOLOGY: This study investigated hepatic expression of cytokine-induced neutrophil chemoattractant (CINC) 14 days after bile duct ligation, as well as the number of infiltrated PMNs in livers. Portal venous endotoxin levels were also evaluated. Furthermore, in vitro CINC production by isolated liver cells from obstructive jaundice (OJ) liver or sham-treated liver was evaluated after stimulation with tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta) or LPS. RESULTS: The number of infiltrated PMNs in sinusoids significantly increased in OJ liver, as compared to sham-treated liver. CINC mRNA expression was also increased in OJ liver. Immunohistochemical study revealed that the majority of the CINC-positive cells were hepatocytes. In vitro study proved that CINC production by isolated hepatocytes was markedly enhanced by IL-1beta stimulation in OJ liver. Furthermore, IL-1beta production by LPS-stimulated Kupffer cells isolated from OJ liver was significantly increased, compared to those from sham-treated liver. Portal venous endotoxin was detectable only in OJ rats. CONCLUSIONS: Excessive production of IL-1beta by activated Kupffer cells, as a result of portal endotoxemia, may play an important role for increased CINC release from hepatocytes in cholestatic liver, leading to PMN infiltration.     

Hepatoprotective role of ganoderma lucidum polysaccharide against BCG—induced immune liver injury in mice

AIM：To examine the effect of ganoderma lucidum polysaccharide(GLP)on the immune liver injury induced by BCG infection,and investigate the relationship between degrees of hepatic damage and NO production in mice.METHODS:Immune hepatic injury was markedly induced by BCG-pretreatment(125mg&#183;kg^-1,2-week,iv)or by BCG-pretreatment plus lipopolysaccharide(LPS,125μg&#183;kg^-1,12-hour,iv)in mice in vivo.Hepatocellular damage induced by BCG-pretreated plus inflammatory cytokines mixture(CM).which was includid TNF-α,IL-1β,IFN-γand LPSin culture medium in vitro.Administration of GLP was performed by oral or incubating with culture medium at immune stimuli simultaneity.Liver damage was determined by activity of alaine aminotransferase(ALT)in serum and in hepatocytes cultured supernatant,by liver weight changes and histopathlogical examination.NO production in the cultured supernatant was determined by the Griess reaction.Moreover,inducible nitric oxide synthase(iNOS)protein expression was also examinated by immunohistochemical method.RESULTS:Immune hepatic injury was markedly induced by BCG or BCGplus inflammatory cytokines in BALB/c mice in vivoand in vito.Under BCG-stimulated condition,augment of the liver weight and increase of the serum/supernatant ALTlevel were observed,as well as granuloma forming and inflammatory cells soakage were observed by microscopic analysis within liver tissues.Moreover.NO production was also increased by BCGor/and CM stimuli in the culture supernatant ,and a lot of iNOS positive staining was observed in BCG-prestimulated hepatic sections.Applicatin of GLP significantly mitigated hepatic tunefaction,decreased ALT enzyme release and NO production in serum/supernatant,improved the pathological changes of chronic and acute inflammation induced by BCG-stimuli in mice.Moreover.the immunohistochemical result showed that GLP inhibited iNOS protein expression in BCG-immune hepatic damage model.CONCLUSION:The present study indicates that NO participates in immune liver injury in duced by Mycobacterium bovisBCG infection.The mechanisms of protective roles by GLPfor BCG-induced immune liver injury may be due to influence NO production in mice.     

Protective effect of melatonin against liver injury in mice induced by Bacillus Calmette-Guerin plus lipopolysaccharide

AIM: To investigate the effects and mechanisms of melatonin on immunological liver injury in mice.METHODS: A model of liver injury was induced by tail vein injecton of Bad#us Calmet~ Guenn (BCG) and lipopolysaccharide(LPS) in mice. Kupffer cells and hepatocytes were isolated and cultured according to a modified two-step collagenase perfusion technique. Levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST) and nitric oxide(NO), content of malondiadehyde (MDA), activity of superoxide dismutase (SOD), were measured by biochemical methods.Tumor necrosis factor-α (TNF-α) activity was determined by RIA. Interleukin (IL)-1 activity was measured by thymocyte proliferation bioassay. Hepatic tissue sections were stained with hematoxylin and eosin and examined under a lightmicroscope.RESULTS: Immunological liver injury induced by BCG+LPS was successfully duplicated. Serum transaminase (ALT,AST) activities were significantly decreased by melatonin(0.25, 1.0, 4.0 mg/kg bm). Meanwhile, MDA content was decreased and SOD in liver homogenates was upregulated.Furthermore, pro-inflammatory mediators (TNF-α, IL-1, NO)in serum and liver homogenates were significantly reduced by melatonin. Histological examination demonstrated that melatonin could attenuate the area and extent of necrosis,reduce the immigration of inflammatory cells. In in vitro experiment, TNF-α was inhibited at the concentrations of 10^-8-10^-6 mol/L of melatonin, while IL-1 production of Kupffer cells induced by LPS (5 t~g/mL) was decreased only at the concentration of 10^-6 mol/L of melatonin, but no effect on NO production was observed. Immunological liver injury model in vitro was established by incubating hepatocytes with BCG- and LPS-induced Kupffer cells. Activities of ALT,TNF-α, IL-1, and MDA in supernatant were significantly increased. Melatonin had little effect on the level of ALT,but reduced the content of TNF-α and MDA at concentrations of 10^-7-10^-5 mol/L and decreased the content of IL-1 at concentrations of 10^-6-10^-5 mol/L.CONCLUSION: Melatonin could significantly protect liver injury in mice, which was related to free radical scavenging,increased SOD activity and pro-inflammatory mediators.     

Tumour necrosis factor-alpha and nitric oxide mediate apoptosis by D-galactosamine in a primary culture of rat hepatocytes: exacerbation of cell death by cocultured Kupffer cells.

BACKGROUND: Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. OBJECTIVES: Whether PGE1 protects against D-galactosamine (D-GalN)-associated hepatocyte cell death by the regulation of tumour necrosis factor-alpha (TNF-alpha) and/or nitric oxide (NO) in hepatocytes or cocultured Kupffer cells was examined. METHODS: Anti-TNF-alpha antibodies were used to evaluate the role of TNF-alpha during D-GalN cytotoxicity and its protection by PGE1 in cocultured hepatocytes and Kupffer cells. Cell apoptosis and necrosis were assessed by DNA fragmentation and lactate dehydrogenase release, respectively. Nitrite+nitrate (NOx), as NO end products, and TNF-alpha concentrations were measured in the culture medium. The role of NO was determined by measuring inducible NO synthase (iNOS) expression and the effect of its inhibition during d-GalN cytotoxicity and its protection by PGE1. RESULTS: D-GalN enhanced hepatocyte cell death associated with high TNF-alpha and NOx levels in a culture medium. Anti-TNF-alpha and iNOS inhibition suggested that TNF-alpha was mediating apoptosis, but not necrosis, through the stimulation of NO production. The antiapoptotic activity of PGE1 was associated with a reduction of NO production, but was blocked by iNOS inhibition. This apparent contradiction was explained by the ability of PGE1 to enhance iNOS expression shortly after its administration and inhibit it later during d-GalN treatment. Anti-TNF-alpha antibodies did not reduce the exacerbation of d-GalN-associated cell death in hepatocytes by cocultured Kupffer cells. CONCLUSION: TNF-alpha mediates D-GalN-induced apoptosis via NO production in cultured hepatocytes. The protective effect of PGE1 against D-GalN-induced apoptosis is probably through the induction of low iNOS expression that was followed by a reduction of iNOS expression and NO production induced by the hepatotoxin. The exacerbation of hepatocyte cell death by Kupffer cells was not related to TNF-alpha and NO.     

Suppression of lipopolysaccharide-induced nitric oxide synthase expression by platelet-activating factor receptor antagonists in the rat liver and cultured rat Kupffer cells.

Excessive nitric oxide (NO) generated by hepatic cells in response to lipopolysaccharide (LPS) and inflammatory substances (e.g., platelet-activating factor [PAF]) is a key contributor to the pathophysiological outcomes observed in the liver during sepsis. In rats subjected to liver-focused endotoxemia, inducible nitric oxide synthase (iNOS) levels in the intact liver were elevated by 6 hours; cell-specific expression of iNOS messenger RNA (mRNA) was Kupffer cells (KCs), endothelial cells, and hepatocytes. Elevated serum alanine transaminase (ALT) levels at 6 hours confirmed hepatic damage. Pretreatment of endotoxemic rats with PAF receptor antagonists BN 50739 or WEB 2170 reduced serum ALT and iNOS mRNA levels in the intact liver. Pretreatment of cultured KCs with BN 50739 or WEB 2170 inhibited both LPS and PAF-induced iNOS mRNA formation. In addition, LPS-induced iNOS protein levels in KCs pretreated with BN 50739 or WEB 2170 were decreased. Exposure of KCs to either LPS or PAF caused the translocation of the p65 subunit of nuclear factor kappa B (NF-kappaB) into the nucleus and this process was attenuated by BN 50739 and WEB 2170. There was concomitant inhibition of LPS-dependent degradation of the inhibitory protein IkappaBalpha and increase in intracellular Ca(2+) in KC treated with BN 50739 or WEB 2170. Also, in KCs, LPS was able to induce iNOS mRNA expression independent of CD14. This response was inhibited by pretreatment of KCs with either BN 50739 or WEB 2170. Our findings indicate that PAF receptor antagonists convey protection against hepatocellular injury accompanied by a decrease in nitric oxide (NO) formation in the livers of endotoxemic rats.     

Nitric oxide production by hepatocytes contributes to the inhibitory effect of endotoxin on insulin-like growth factor I gene expression

We tested whether endotoxin (lipopolysaccharide, LPS) inhibits IGF-I gene expression in hepatocytes and the possible role of Kupffer cells and nitric oxide (NO) in this effect. LPS decreased IGF-I mRNA in hepatocyte cultures and increased the nitrite + nitrate levels in the culture medium. Furthermore, there was a negative correlation between the IGF-I mRNA and the nitrite+nitrate levels. When hepatocytes were cocultured with Kupffer cells, the inhibitory effect of LPS on IGF-I mRNA was higher than in hepatocyte cultures, but the stimulatory effect on nitrite+nitrate was similar in both conditions. The exogenous NO donated by S-nitroso-n-acetyl-d,l-penicillamide also decreased the IGF-I gene expression in hepatocyte cultures. In addition, two specific inducible NO synthase (iNOS) inhibitors, l-N6-(1-iminoethyl)lysine (l-NIL) and aminoguanidine, prevented the effect of LPS on nitrite+nitrate levels and on IGF-I gene expression in hepatocyte cultures. These data indicate that iNOS-derived NO may cause downregulation of IGF-I gene expression in hepatocytes. However, in cocultures, the iNOS inhibitor l-NIL prevented the effect of LPS on nitrite+nitrate levels, but only attenuated the LPS-induced decrease in IGF-I gene expression. We conclude that in hepatocytes, LPS-induced decrease in IGF-I is mainly due to induction of iNOS, whereas in the presence of Kupffer cells LPS inhibits IGF-I through NO release and through other inhibitory pathways.     

Altered response to inflammatory cytokines in hepatic energy metabolism in inducible nitric oxide synthase knockout mice

BACKGROUND/AIMS: Production of nitric oxide (NO) in the liver is believed to be a critical factor for carbohydrate and energy metabolism in endotoxin shock. The present study focuses on the involvement of NO produced by inducible nitric oxide synthase (iNOS) in glycogen synthesis and energy metabolism stimulated by insulin.METHODS: Primary hepatocytes prepared from wild-type and iNOS knockout (iNOS(-/-)) mice were employed.RESULTS: Incubation of wild-type hepatocytes with a combination of cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) and lipopolysaccharide (cytokines/LPS) inhibited insulin-stimulated glycogen synthesis and adenosine triphosphate (ATP) increase, and decreased the ketone body ratio (KBR) at 8-12 h, concomitant with expression of iNOS protein and NO production. While the glycogen synthesis was suppressed by cytokines/LPS, reduction of the ATP increase and a decrease in KBR by cytokines/LPS were not observed in iNOS(-/-) hepatocytes. Further, N(G)-monomethyl-L-arginine, a NOS inhibitor, reversed the inhibition of ATP increase and decrease in KBR by cytokines/LPS, but not the inhibition of glycogen synthesis. Conversely, addition of S-nitroso-N-acetylpenicillamine, a NO donor, inhibited the insulin-stimulated ATP increase synthesis in iNOS(-/-) hepatocytes, but not the insulin-stimulated glycogen synthesis.CONCLUSIONS: These results demonstrate that NO mediates the suppression of insulin-stimulated energy metabolism, but not glycogen synthesis, in cytokines/LPS-treated hepatocytes.     

Effect of Chronic Ethanol Feeding on Nitric Oxide Synthesis by Rat Kupffer Cells

Kupffer cells contribute to the important role of the liver defense mechanism through nitric oxide (NO) production. In this study, the effect of chronic ethanol administration on the ability of Kupffer cells to synthesize and release NO was investigated after stimulation with lipopolysaccharide (LPS). Male Wistar rats were chronically fed ethanol for 8 weeks according to the method described by DeCarli and Lieber et al. ( J Nutr. 91:331–336, 1967). Kupffer cells were isolated and cultured with LPS (1 μg/ml) for 24 hr. The levels of nitrite and nitrate, metabolites of NO, were determined in the culture medium. NO synthase (NOS) activity in Kupffer cells was determined by the method that measures conversion of [¹⁴C]arginine into [¹⁴C]citrul-line. In control rats, a significant increase of nitrite and nitrate levels in culture medium was observed after LPS treatment. The magnitude of this increase was significantly smaller in chronic ethanol-fed rats. When the activity of NOS was determined, inducible NOS (iNOS) activity was higher than that of constitutive NOS, and LPS administration produced a significant elevation of iNOS activity in both control and chronic ethanol-fed rats. However, the elevation of iNOS activity by LPS stimulation was diminished by chronic ethanol administration. Distribution of iNOS in Kupffer cells as determined by an immunofluorescence method using a laser scanning confocal image system showed a lower expression of iNOS in chronic ethanol-fed rats even in the presence of LPS. These results demonstrate that the excessive production of NO by increased iNOS activity in Kupffer cells is diminished by chronic ethanol administration.     

Regulation of nitric oxide production in osteoarthritic and rheumatoid cartilage. Role of endogenous IL-1 inhibitors

OBJECTIVE: To investigate the endogenous regulation of interleukin-1 (IL-1) cytokine network in osteoarthritic (OA) and rheumatoid (RA) cartilage in relation to nitric oxide (NO) production. METHODS: Cartilage specimen obtained from OA and RA patients undergoing knee replacement surgery were studied for iNOS expression, NO and IL-1 antagonist production in tissue culture. RESULTS: OA cartilage responded to IL-1beta-stimulation with higher NO production than RA cartilage, whereas there was no difference in NO synthesis between OA and RA samples when stimulated by TNFalpha or LPS. Interleukin-1 receptor antagonist (IL-1Ra) production was higher in RA cartilage than in OA cartilage, and its production was increased by NO synthase inhibitor 1400W. CONCLUSION: IL-1beta is a potent stimulator of NO production by the iNOS pathway in RA and more pronouncedly in OA cartilage. This process is regulated by cartilage derived IL-1 antagonists, and is implicated in cartilage destruction and synovial inflammation in OA and RA joints.     

内毒素诱导产生的一氧化氮对体外肝细胞的损伤作用

目的 探讨NO在LPS所致肝损害中的作用。方法 采用LPS刺激体外肝细胞、Kupffer细胞或两者混合培养,观察NO产生情况及培养肝细胞形态改变。结果 LPS刺激后,各级培养上清中NO产物水平均显著升高,尤其Kupffer细胞单独培养及肝细胞/Kuppffer细胞混合培养组升高更加显著,分别约为对照组的10倍和8倍,并且混合培养组中肝细胞形态出现明显改变,贴壁肝细胞变小、变园,核内染色质致密、结块,部分染色质边积,核膜皱折变形。而加用iNOS抑制剂氨基胍则培养上清中NO产物水平明显降低(约降低60％～65％),肝细胞形态未见明显改变。结论 通过诱导肝细胞、尤其Kupffer细胞产生大量NO而介导肝细胞损伤可能是LPS所致肝损害的重要机理之一。