Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells.

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, while tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 +/- 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In addition, TNF-alpha production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin.     

库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响

目的 研究库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响。方法 封闭和不封闭BALB／c小鼠库普弗细胞(从尾静脉注射GdCl3或生理盐水27ml／kg体重)48h后，夹闭肠系膜上动脉1h后松夹，复制肠缺血再灌注模型。运用流式细胞术和酶联免疫法，分别检测缺血前、缺血60min、再灌注30min、60min肝细胞凋亡情况和血清肿瘤坏死因子的变化。结果 结果表明，肠缺血60min、再灌注30min、60min时，肝细胞凋亡数增多，血清肿瘤坏死因子水平逐渐升高；封闭库普弗细胞后，肝细胞凋亡数增多更显著，血清肿瘤坏死因子水平在同时间点上无显著差异。结论 库普弗细胞功能状态的变化对肠缺血再灌注时肝损伤有重要影响。     

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.     

Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress

BACKGROUND: Apoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms. RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells. CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.     

Liver injury in acute pancreatitis and mitigation by continuous arterial infusion of an antibiotic via the superior mesenteric artery

Macrophage-derived inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), have been shown to play a central role in aggravation of acute pancreatitis (AP), but little is known about their roles in liver injury. We investigated the pathogenesis of the liver injury in AP and assessed the efficacy of arterial infusion of an antibiotic via the superior mesenteric artery (SMA). Infusion of saline (group A) or imipenem (group B) was started 6 hours after induction of AP in dogs by intraductal administration of autologous gallbladder bile. The survival rate in group B was significantly improved compared with group A. Serum alanine aminotransferase levels in peripheral blood and endotoxin levels in portal vein blood were both significantly higher at 18 hours in group A than in group B. When hepatocytes and Kupffer cells were isolated at 18 hours and cultured for 24 hours thereafter, there was significant exacerbation of the hepatocyte injury and significantly greater production of TNF-alpha in the coculture of hepatocytes and Kupffer cells in group A, indicating that the Kupffer cells had been activated. By contrast, both of these manifestations were significantly mitigated in group B. These findings suggest that TNF-alpha secreted by endotoxin-activated Kupffer cells contributes to liver injury in AP, and that SMA infusion of an antibiotic mitigates the liver injury by controlling endotoxin translocation.     

Kupffer cells modulate splenic interleukin-10 production in endotoxin-induced liver injury after partial hepatectomy

BACKGROUND/AIMS: This study was conducted to investigate the implication of Kupffer cells and the spleen in interleukin (IL)-10 production in endotoxin-induced liver injury after hepatectomy. METHODS: Rats were divided into five groups: the S group, sham-operation; the SG group, sham-operation followed by intravenous gadolinium chloride (GdCl(3): 7 mg/kg) administration to inhibit Kupffer cell function; the H group, two-thirds hepatectomy; the HG group, hepatectomy and subsequent GdCl(3) administration; the HGS group, hepatectomy and splenectomy with GdCl(3) administration. Lipopolysaccharide (1.5 mg/kg) was intravenously administered for each group 48 h after surgery. RESULTS: GdCl(3) treatment significantly suppressed the elevation of plasma tumor necrosis factor (TNF)-alpha levels by lipopolysaccharide administration with completely inhibited induction of hepatic TNF-alpha and IL-10 mRNAs. In the HG group, marked increase in plasma IL-10 levels associated with enhanced splenic IL-10 mRNA was observed 1 h after lipopolysaccharide administration when compared to those in the H and HGS groups. Plasma TNF-alpha/IL-10 ratio 1 h after lipopolysaccharide administration was higher in the order of H, HGS and HG groups. Hepatic parenchymal damage and the 24-h mortality were lowest in group HG, followed by groups HGS and H. CONCLUSIONS: Kupffer cells after hepatectomy may aggravate endotoxin-induced liver injury via down-regulation of IL-10 production in the spleen.     

Impaired liver regeneration in mice by lipopolysaccharide via TNF-alpha/kallikrein-mediated activation of latent TGF-beta

BACKGROUND & AIMS: Because impaired regeneration after surgical treatment of the liver is influenced by circulating endotoxin, the underlying molecular mechanism was investigated. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally into mice, followed 24 hours later by 67% partial hepatectomy. We measured serum tumor necrosis factor (TNF) alpha levels as well as proliferating cell nuclear antigen labeling index, transforming growth factor (TGF) beta expression, and plasma kallikrein (PLK) activities in regenerating livers. We also examined the effect of LPS, TNF-alpha, and PLK on latent TGF-beta activation in homotypic and heterotypic cultures of rat or mouse hepatic stellate cells and Kupffer cells. RESULTS: Serum TNF-alpha levels increased after LPS (500 ng/g body wt) injection and after partial hepatectomy, accompanying TGF-beta-mediated suppression of hepatic proliferating cell nuclear antigen labeling index. This suppression was mimicked by a combination of preadministration of 50 ng/g body wt LPS and postoperative administration of 5 ng/g body wt TNF-alpha. In vitro, LPS stimulated Kupffer cells to secrete TNF-alpha, which enhanced PLK activity on the hepatic stellate cell surface through increasing PLK binding, thereby inducing proteolytic activation of latent TGF-beta and its autoinduction. Blockade of TGF-beta, TNF-alpha, or PLK activity prevented LPS-induced impaired regeneration in vivo. CONCLUSIONS: LPS provokes TNF-alpha/PLK-mediated proteolytic activation of latent TGF-beta in hepatic stellate cells, leading to impaired liver regeneration after partial hepatectomy.     

Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats

When rats received endotoxin 48 hours after two-thirds liver resection, 50% of them died within 12 hours with massive hepatic necrosis at a dose that did not affect sham-operated rats. In the hepatic sinusoids, fibrin deposition and endothelial cell destruction occurred 5 hours after endotoxin administration. When antithrombin III concentrate was infused concomitantly with endotoxin administration, all rats survived 12 hours, and the extent of hepatic necrosis and the deranged serum glutamic pyruvic transaminase values were significantly attenuated at 5 hours compared with those in the control rats. Similar improvements in the incidence of mortality and liver injury were observed after treatment with gum arabic before hepatectomy. The stimulatory state of Kupffer cells based on the ability to produce superoxide anions estimated by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was increased between 24 and 72 hours after operation. This increase disappeared after gum arabic treatment. It is concluded that massive hepatic necrosis can occur as a result of sinusoidal fibrin deposition provoked by endotoxin in partially hepatectomized rats. Activated Kupffer cells may contribute to this provocation.     

Kupffer cell inactivation prevents lipopolysaccharide-induced structural changes in the rat liver sinusoid: An electron-microscopic study

Scanning and transmission electron-microscopic examination of the rat liver sinusoid was performed in this study after in vivo treatment of rats with gram-negative bacterial lipopolysaccharide (LPS, 1 mg/Kg(-1) body weight), with or without pretreatment with gadolinium chloride (GdCl3 10 mg(Kg(-1) body weight). Twenty-seven and 48 hours after GdCl3 administration, to inactivate/eliminate part of the Kupffer cell population, a decrease in the number of visualized Kupffer cells was observed, without evident effects on the sinusoidal endothelial cell or on the hepatocyte. Three and 24 hours after its administration, LPS produced ultrastructural changes in the sinusoid characterized by morphological evidence of Kupffer cell activation (i.e., swelling and expanded philopodia anchoring the Kupffer cell to the luminal surface of the sinusoidal wall), and a marked decrease in the population of endothelial cell fenestration. The reduction in the number of fenestrae was associated with a change in the diameter of fenestrae and can be interpreted as a component of the "capillarization" process of the hepatic sinusoid. Such ultrastructural changes were prevented by the administration of GdCl3 24 hours before LPS injection. Hence, these findings suggest that LPS-induced structural changes in the liver sinusoid are mediated by an LPS-induced Kupffer cell activation. Coupled with previous experimental data, showing similar effects of GdCl3 on one of the hepatic sinusoidal endothelial cell (SEC) functions, i.e., hyaluronan scavenging, the data presented in this study strongly support the view that Kupffer cells modulate both the hepatic SEC's functional as well as ultrastructural properties. (Hepatology 1996 Apr;23(4):788-96)     

GdCl_3 abates hepatic ischemia-reperfusion injury by inhibiting apoptosis in rats

BACKGROUND:Gadolinium chloride(GdCl3)is a specific inhibitor of Kupffer cells(KCs),which are important promoters of various liver injuries.It is therefore of interest to explore the role of KCs in liver ischemia-reperfusion injury and their relations with apoptosis caused by ischemia-reperfusion injury. METHODS:One hundred male Wistar rats(190-210 g, 6-7 weeks old)were divided into two groups at random, GdCl3 group and control group.Samples were collected at 0.5,1,6,12,and 24 hours from each group after rep...     

Experimental study on the role of endotoxin in the development of hepatopulmonary syndrome

AIM: To evaluate the role of intestinal endotoxemia in the genesis of hepatopulmonary syndrome. METHODS: A rat model of cirrhosis was prepared with the method of compound factors. At the end of the eighth week, rats with cirrhosis were treated with 300 μg LPS/100 g body weight, and 1 g/rat of glycine about four h prior to LPS. After three h of LPS treatment, blood and tissues were collected for various measurements. Kupffer cells were isolated from male Wistar rats and cultured, and divided into five groups. Supernatant was harvested at 3 h after treatment with LPS for measurement of tumor necrosis factor-alpha (TNF-α). RESULTS: Our results showed that in rats with cirrhosis, slowed and deepened breath with occasional pause was. PaO2, PaCO2 and standard bicarbonate (SB) in arterial blood were decreased. Arterial O2 and actual bicarbonate (AB) were markedly decreased. There was a close correlation between decreased O2 and endotoxin. Metabolic acidosis accompanying respiratory alkalosis was the primary type of acid-base imbalance. The alveolar-arterial oxygen gradient was sharply widened. Massive accumulation of giant macrophages in the alveolar spaces and its wall and widened alveolar wall architecture were observed. The number of bacterial translocations in mesenteric lymph nodes increased. The ratio of TC99M-MAA brain-over-lung radioactivity rose. Endotoxin, and TNF-α, endothelin-1 (ET-1), nitric oxide (NO) in plasma and ET-1, carbon monoxide (CO) in lung homogenates increased. After administration of a given dosage of LPS in rats with cirrhosis, various pathological parameters worsened. Plasma level of endotoxin was related to TNF-α, ET-1, NO in plasma and ET-1, NO, CO in lung homogenates. TNF-α level was related to ET-1 and NO in plasma and lung homogenates and CO in lung homogenate as well. The level of TNF-α increased after infusion of LPS into culture supernatant of Kupffer cells in vitro. However, TNF-α significantly decreased after pretreatment with glycine, PD98059 and SB212850. Glycine could antagonize the effect of LPS in vivo and in vitro. CONCLUSION: Intestinal endotoxemia accompanying by cirrhosis may be an important mechanism in the development of hepatopulmonary syndrome in rats. Overproduction of TNF-α due to endotoxin stimulation of Kupffer cells via mitogen-activated protein kinase (MAPK) signal transduction pathway may be a major mechanism mediating the pathologic alterations of hepatopulmonary syndrome.     

Effect of hepatocyte apoptosis induced by TNF-alpha on acute severe hepatitis in mouse models

AIM:To study the effect of hepatocyte apoptosis and necrosis induced by TNF-alpha on the pathogenesis of acute severe hepatitis (ASH).METHODS:The model of ASH was prepared in D-galactosamine (GalN) sensitized BALB/c mice by injection of either endotoxin (ET) or tumor necrosis factor-alpha(TNF-alpha. Morphological changes of apoptotic hepatocytes were studied by both light and electron microscope and in site end labeling method (ISEL). Molecular biological changes of DNA ladder were observed by electrophoresis of extract from liver tissues. Biochemical changes were measured by alanine aminotransferase (ALT), aspartic aminotransferase (AST) and TNF-alpha. The relation between apoptosis and necrosis was evaluated simultaneously.RESULTS:The sequence of hepatocyte apoptosis, necrosis, and final death from ASH was observed both in GalN/ET and GalN/TNF-alpha group. Apoptosis was prominent at 3.5h and 6h after injection of inducer, while necrosis became dominant at 9h after challenge. The appearance of apoptosis was earlier in GalN/TNF-alpha group than that in GalN/ET group. Pretreatment of mice with antiTNF IgG1 may completely prevent the liver injury induced by GalN/ET.CONCLUSION:TNF-alpha can cause liver amage by inducing hepatic apoptosis and necrosis in mice with endotoxemia.     

Interleukin-6 release by rat liver macrophages

Tissue macrophages of the liver (Kupffer cells) release interleukin-6 (IL-6) in vitro. Since Kupffer cells reside in close proximity to hepatocytes, which are major target cells of IL-6, the regulation of IL-6 release by hepatic macrophages has been investigated in this study. Using the hybridoma growth test to detect IL-6, we found that Kupffer cells already maximally release IL-6 at endotoxin concentrations as low as 1.0 ng/ml. The stimulated secretion of IL-6 was increased 4-8-fold by endotoxin when compared to the control macrophages incubated in serum-containing medium alone. The preincubation of macrophages with interferon-gamma enhanced the capacity of Kupffer cells to respond to endotoxin. The secretion of IL-6 could also be induced by interleukin (IL)-1 beta and tumor necrosis factor (TNF-alpha). The most potent inducers, however, were the paramyxoviruses Newcastle Disease Virus and Sendai Virus. The release of IL-6 by macrophages upon stimulation with endotoxin was almost completely inhibited by 1 microM dexamethasone. Whereas 100 nM of prostaglandin E2 (PGE2) inhibited the release of TNF-alpha in rat Kupffer cells, it did not affect the secretion of IL-6.     

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.     

Dietary glycine blunts liver injury after bile duct ligation in rats

AIM： To investigate the effects of （dietary） glycine against oxidant-induced injury caused by bile duct ligation （BDL）.
METHODS： Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley （SD） rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid （DCA） in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase （LDH） activity was determined in the supernatant of cultivated hepatocytes.
RESULTS： Serum alanine transaminase levels increased to about 600 U/L 1 d alter BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed.
CONCLUSION： These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.     

Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice

AIM: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccha-ride (LPS) -induced liver injury. METHODS: D-galactosamine (GaIN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS. RESULTS: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-α(TIMF-α) and significantly reduced IL-10 levels compared with wild type mice. TIMF-αgene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-αlevels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment. CONCLUSION: Adiponectin supresses TNF-αproduction and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.