Chemotactic factors released from hepatocytes exposed to acetaminophen

To clarify the mechanism of neutrophil infiltration in the liver of acetaminophen-induced hepatic injury, chemotactic factor released from hepatocytes exposed to acetaminophen has been investigated. Hepatocytes exposed to acetaminophen release nondialyzable chemotactic factor, although actaminophen in itself inhibits chemotaxis of neutrophils. Chemotactic activity of the nondialyzable chemotactic factor was reduced after treatment with heat (56°C, 30 min) or trypsin. Chemotactic activity was demonstrated at the molecular weights of around 25 and 55 kDa. Chemotactic activity of the conditioned medium was not significantly reduced in the presence of antibody against rat KC/gro protein (interleukin-8-related cytokine in rodent). Chemotactic activity of a 25-kDa factor was reduced by the antibody against KC/gro protein, but that of a 55-kDa factor was not reduced. Immunoblot analysis revealed that the peptide reacted with antibody against rat KC/gro protein was demonstrated at a molecular weight of around 20–25 kDa, but not at around 55kDa, when the conditioned medium of acetaminophen-treated hepatocytes was electrophoresed. These results suggest that hepatocytes exposed to acetaminophen release two types of chemotactic factors for neutrophils and that a major part of the chemotactic factor could be different from a member of interleukin-8 family.     

枯否氏细胞在大鼠非酒精性脂肪性肝炎发病中的作用

目的：探讨枯否氏细胞大鼠非酒精性脂肪性肝炎（non-alcoholic steatohepatitis,NASH)发病中的作用,方法：19只雄性SD大鼠随机分为模型组（10只）和正常组（9只）,分别预高脂肪饮食和标准饮食饲养12周,HE梁色观察肝细胞切片病理学改变,透射电镜和溶菌酶免疫组织化学染色观察枯否氏细胞的数量和形态。结果：模型组大鼠均出现肥胖,高脂血症伴肝细胞大泡性脂肪变,小叶内炎症细胞浸润和坏死,与正常相比,模型组肝小叶内枯否氏细胞数显著增加,并呈活化状态,模型组枯否氏细胞变化与其肝病理学改变相一致,结论：高脂饮食大鼠肝脏枯否氏细胞增多,并可能与其脂肪性肝炎的发病有关。     

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。     

In vitro inhibition of interleukin-2-induced defective polymorphonuclear chemotaxis by TNF inhibitor

Abstract: Patients undergoing immunotherapy with interleukin-2 experience multiple side effects and are highly susceptible to bacteremia. In a previous study, we confirmed that a profound deficiency of neutrophil chemotaxis is induced by interleukin-2 therapy. Migration in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP), being normal before therapy, was markedly impaired after the first cycle and further decreased after the third cycle of treatment. A direct effect of interleukin-2 on neutrophil chemotaxis is controversial. However, peripheral blood cells exposed to interleukin-2 secrete secondary cytokines. In particular, the release of tumor necrosis factor after interleukin-2 injection has been proposed as an important regulatory mechanism. When testing random migration and chemotaxis of neutrophils from normal subjects after incubation with the serum from treated patients, we found that this serum induced a defective chemotaxis similar to that of neutrophils from interleukin-2-treated patients. In order to assess the influence of tumor necrosis factor, we tested the effect of anti-tumor necrosis factor-alpha antibody on the chemotactic response of cells after incubation with the serum, and we observed a dose-dependent reduction of neutrophil chemotaxis deficiency. These data suggest that TNF is counteracting the neutrophil chemotactic deficiency observed during IL-2 treatment.     

Effect of melatonin administration on parameters related to oxidative damage in hepatocytes isolated from old Wistar rats

Aging induces changes in several organs and tissues, such as the liver, and this process might be due to oxidative damage caused by free radicals and inflammatory mediators. Melatonin is a secretory product with well-known antioxidant properties. The aim of this study was to investigate the effect of melatonin administration on age-induced alterations in hepatocytes. Twenty-two-month old male Wistar rats were treated with oral melatonin for 10 wk. At the end of the treatment, hepatocytes were isolated and cultured, and different parameters were measured in both cells and medium. Aging induced a significant increase in lipid peroxidation, nitric oxide, carbon monoxide and cyclic guanosyl-monophosphate, as well as a reduction in adenosine triphosphate content and phosphatidylcholine synthesis when compared to young animals. Melatonin administration significantly ameliorated all these age-related changes in males. Melatonin administration seems to exert beneficial effects against age-induced changes in hepatocytes.     

粒细胞集落刺激因子保护急性损伤人胎肝细胞的实验研究

目的建立D-半乳糖胺(D-galactosamine,D-GalN)对人胎肝细胞急性损伤的模型,观察粒细胞集落刺激因子(Granulocyte-colony stimulating factor,G-CSF)对人胎肝细胞损伤的保护作用。方法分别用梯度浓度的D-GalN和不同的作用时间孵育人胎肝细胞,用四唑盐比色法(MTT法)检测细胞活性,以确定最佳的人胎肝细胞急性损伤造模条件。将胎肝细胞分为4组进行不同处理:第1组为空白对照组,第2组(G组)用G-CSF处理正常细胞,第3组(ND组)和第4组(GD组)都用D-GalN进行损伤造模,但GD组加入G-CSF作为治疗,第3组加入等量的0.9%氯化钠溶液作为实验对照。最后用MTT法和乳酸脱氢酶(LDH)释放量检测各组细胞活性。结果当D-GalN浓度为10 mg/ml,作用时间为12 h时,可以杀伤90%以上的人胎肝细胞,并且可以保证有足够的药物反应时间。空白对照组和G组的细胞活性差异无统计学差异,但GD组细胞活性明显高于ND组(P0.05)。结论 D-GalN对人胎肝细胞急性损伤的造模条件为D-GalN 10 mg/ml作用12 h。G-CSF对D-GalN造成的人胎肝细胞急性损伤具有保护作用。     

尿巨噬细胞趋化蛋白-1和巨噬细胞集落刺激因子对狼疮肾炎复发的诊断作用

目的探讨在成功的泼尼松和环磷酰胺诱导治疗后,引起弥漫增生型(Ⅳ型)狼疮肾炎复发的预测指标。方法收集弥漫增生型狼疮肾炎病例,将泼尼松和环磷酰胺诱导治疗成功的病例纳入研究对象。记录临床和实验室资料,于治疗开始后6个月时检测患者尿巨噬细胞趋化蛋白(MCP)-1和巨噬细胞集落刺激因子(M-CSF)。追踪治疗后的复发情况。结果共收集到64例诱导治疗成功的病例,经平均(27±3)个月随访,18例(28%)患者发生至少一次肾性复发,其复发的平均时间为(14±4)个月。复发组患者尿MCP-1和M-CSF水平明显高于缓解维持组。尿MCP-1和M-CSF升高,及血C3降低和抗dsDNA抗体阳性均是Ⅳ型狼疮肾炎复发的独立预测因子。有7例患者出现血肌酐倍增(CRX2),肾性复发是CRX2的惟一预测因子。结论尿MCP-1和M-CSF持续升高是Ⅳ型狼疮肾炎复发的独立预测因子。该研究提示监测诱导缓解患者肾组织炎症指标可有利于指导狼疮肾炎的治疗。     

Role of naofen in apoptosis of hepatocytes induced by lipopolysaccharide through mitochondrial signaling in rats

Aim: Lipopolysaccharide (LPS) causes apoptosis of hepatocytes, which is probably mediated by inflammatory substances released from Kupffer cells (KCs). Recently, we have reported that naofen, a newly found intracellular WD40-repeat protein, has a role in inducing the apoptosis in HEK293 cells. Hence, the present study was undertaken to investigate a role of naofen in the LPS-induced apoptosis of rat hepatocytes. Methods: Rats were treated with i.v. injections of LPS, and livers were extirpated to evaluate expression of naofen and apoptosis. In in vitro experiments, hepatocytes and KCs were separately isolated from rat livers. The incubation medium for KCs treated with LPS (KC-CM) was used for hepatocyte culture. Results: Intravenous injections of LPS enhanced the expression of naofen in the livers. Livers showed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive staining, and elevated caspase-3 activity. In isolated KCs or hepatocytes, LPS hardly affected naofen expression and caspase-3 activity, whereas incubation of hepatocytes with KC-CM enhanced both naofen expression and caspase-3 activation. Transfection of hepatocyte with naofen siRNA prevented such effects of KC-CM, and clearly eliminated KC-CM-induced reduction of Bcl-2 and Bcl-xL. In contrast, overexpression of naofen in hepatocytes downregulated Bcl-2 and Bcl-xL, released cytochrome c from mitochondria, and activated caspase-3. Conclusion: These results indicate that LPS may induce the hepatic apoptosis in association with enhanced naofen expression, and that naofen may mediate the activation of caspase-3 through downregulating the Bcl-2 and Bcl-xL expression, and releasing cytochrome c from mitochondria to cytoplasm.     

Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes

Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.     

Evidence for an unidentified factor from the pituitary gland which affects the steroid metabolism in isolated hepatocytes and hepatoma cells of the rat

The effects of various extracts obtained from normal rat pituitary tissue (of female origin) and pituitary tumor tissue on the metabolism of 4-androstene-3,17-dione in isolated rat hepatocytes and cultured hepatoma (HTC) cells were investigated. Extracts that increased the 5-reductase/16-hydroxylase ratio in isolated hepatocytes also increased the 5-reductase activity or the 5-reductase/17-hydroxysteroid dehydrogenase ratio in HTC cells. These results indicate that both cell culture methods are suitable for use as in vitro assays for the pituitary principle which affects hepatic steroid metabolism in vivo.

Using the above cell culture technique it has been possible to show that ‘feminizing factor’ activity is located in granules (densities 1.13 and 1.17g/cm³, respectively) in the female rat pituitary and in extracts from the cloned pituitary tumor (C₈11RAP). ‘Feminizing factor’ activity could not be duplicated by single standard anterior or posterior gland hormone preparations over a wide range of concentrations or combinations of such preparations. These results further strengthen the hypothesis put forward in earlier publications that an as yet unidentified pituitary principle may be involved in the control of hepatic steroid metabolism in the rat.     

Collagen synthesis by cultured rat liver cells isolated from chronically alcohol-treated rats

Incorporation rates of 14C-proline into collagen hydroxyproline in cultured Ito cells and hepatocytes isolated from chronically alcohol-treated rats were studied in order to clarify the role of Ito cells in the development of alcoholic liver fibrosis pathogenesis. In the cultured Ito cells isolated from alcohol-treated rats, prolyl hydroxylase activity significantly increased. Total collagen synthesis tended to increase in the alcohol group, and the increase in intracellular intact collagen was statistically significant. More than half of the 14C-radioactivity in intact collagen in cultured Ito cells from control rats was found in collagen other than types I and III collagen (mainly type IV collagen). In Ito cells from alcohol-treated rats, synthesis of collagen other than type I and III significantly increased and type I collagen synthesis tended to be decreased. No significant difference was found in collagen synthesis between the cultured hepatocytes from alcoholic and control rats. These results suggest that chronic alcohol consumption stimulates collagen synthesis in Ito cells, especially type IV collagen. This stimulation of Ito cells may play a role in the development of alcoholic liver fibrosis.     

Aqueous levels of macrophage migration inhibitory factor and monocyte chemotactic protein-1 in patients with diabetic retinopathy.

AIMS: To investigate the relationship of aqueous macrophage migration inhibitory factor (MIF) and monocyte chemotactic protein-1 (MCP-1) levels with the clinical stage of diabetic retinopathy. METHODS: We assayed MIF and MCP-1 levels in aqueous humour samples obtained from 40 diabetic patients (49 eyes) and 24 non-diabetic patients (31 eyes) using enzyme-linked immunosorbent assay. According to the clinical stage of diabetic retinopathy, the diabetic patients were classified into non-diabetic retinopathy (11 eyes), non-proliferative diabetic retinopathy (14 eyes) and proliferative diabetic retinopathy (24 eyes). RESULTS: The aqueous levels of MIF (mean +/- sd) were 6.34 +/- 4.53 ng/ml in proliferative diabetic retinopathy, 3.22 +/- 1.71 ng/ml in non proliferative diabetic retinopathy, 1.25 +/- 0.96 ng/ml in non-diabetic retinopathy and 1.07 +/- 0.94 ng/ml in non-diabetic patients. Significant differences were found among these four groups (P < 0.0001). Aqueous MCP-1 levels were 1668.6 +/- 1442.3 pg/ml in proliferative diabetic retinopathy, 1528.6 +/- 1994.6 pg/ml in non-proliferative diabetic retinopathy, 690.2 +/- 402.1 pg/ml in non-diabetic retinopathy and 622.7 +/- 245.3 pg/ml in non-diabetic patients. Significant differences were also found among these four groups (P < 0.0001). After correcting for total aqueous protein, the ratios of MIF and MCP-1 to total protein remained significantly correlated with the clinical stage of diabetic retinopathy (P < 0.0001, P = 0.0004, respectively). The ratios of MIF to total protein significantly correlated with the ratios of MCP-1 to total protein in diabetic patients (r = 0.680, P < 0.0001). CONCLUSIONS: Aqueous MIF levels significantly correlated with aqueous MCP-1 levels and the clinical stage of diabetic retinopathy. The results suggest that MIF has a co-operative role with MCP-1 in the progression of diabetic retinopathy.     

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM： To determine the platelet-activating factor （PAF） synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis.
METHODS： Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A （ETA） and endothelin B （ETB） receptors were also determined.
RESULTS： A two-fold increase of PAF synthesis （1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA） and a 1.48-fold increase of membrane-bound PAF （1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA） were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [^125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor.
CONCLUSION： Kupffer cells in the course of CCl4- induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.     

内毒素／肿瘤坏死因子所致肝细胞损害机制的体外研究

为了解内毒素（ＬＰＳ）／肿瘤坏死因子（ＴＮＦ）所致肝细胞损害的机制，以乳酸脱氢酶（ＬＤＨ）及噻唑蓝染色（ＭＴＴ）为细胞毒性指标，利用Ｗｉｓｔａｒ大鼠肝细胞进行了ＬＰＳ及其介质ＴＮＦ等的肝细胞损害机制研究。结果发现：ＬＰＳ、ＴＮＦ－α、ＩＬ－１、ＩＬ－６等对肝细胞ＬＤＨ漏出及ＭＴＴ无显著影响（Ｐ＞０．０５），仅在ＬＰＳ、ＬＰＳ／ＴＮＦ－α加入肝细胞－Ｋｕｐｆｆｅｒ细胞混合培养组后有所变化；经ＧａｌＮ／ＬＰＳ体内作用后分离的Ｋｕｐｆｆｅｒ细胞与正常肝细胞混合培养，加入ＬＰＳ、ＴＮＦ及ＬＰＳ／ＴＮＦ－α均可致ＬＤＨ漏出显著增高（Ｐ＜０．０ｌ），多粘菌素Ｂ及抗－ＴＮＦ能分别完全和部分阻断此效应；经ＬＰＳ或ＴＮＦ－α体内作用后抽取的大鼠血清，加入培养肝细胞可致ＬＤＨ漏出显著增高（Ｐ＜０．０１）与ＭＴＴ显著降低（Ｐ＜０．０５），经５６℃灭活后此细胞毒性作用完全消失。上述结果提示，ＬＰＳ及其介质ＴＮＦ无肝细胞直接毒性作用，而经其活化的Ｋｕｐｆｆｅｒ细胞可能引起一系列瀑布效应，导致肝细胞损害。     

Identification of nucleolar organizer regions in non-neoplastic and neoplastic hepatocytes by the silver-staining technique

Abstract— The silver staining technique to demonstrate nucleolar organizer region (NOR)-associated proteins (AgNORs) was applied to a variety of liver tissues, including chronic persistent hepatitis (CPH), chronic active hepatitis (CAH), liver cirrhosis (LC), liver cell dysplasia (LCD), focal nodular hyperplasia (FNH), adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC). In the present study, only discrete, easily counted black dots within nuclei and silver-stained nucleolus were counted under a magnification of X 400 without oil-immersion objectives. The mean AgNOR counts of HCC and LCD were significantly higher than that of normal hepatocytes, and 77% of cases of LCD and 56% of HCC had mean AgNOR counts more than 2, whereas those in CPH, CAH, LC, FNH and AH were always less than 2 and were not different from that of normal hepatocytes. Among HCC, the mean number of AgNORs increased with the grade of the tumor. However, the AgNOR counts of grade I HCC were always less than 2 and overlapped with those of normal hepatocytes and other benign categories. All cases with mean AgNOR counts of more than 2 turned out to be HCC, except LCD which exhibited characteristic histologic appearances easily distinguished from HCC. These findings suggest that AgNORs could be quantitatively useful in evaluating the grade of HCC, even under routine microscopic examination without oil-immersion objectives, and mean AgNOR counts of more than 2 per nucleus are hallmarks of HCC.     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

The digitoxin metabolism in isolated hepatocytes from young and old male Wistar rats

The metabolism of digitoxin (Dt3) in isolated hepatocyte preparations was studied in young (3-mth-old) male and female rats, young castrated (3-mth-old) male rats and old (22- to 28-mth-old) male rats. When hepatocytes were incubated with [3H]Dt3, the predominant metabolite of Dt3 was digitoxigenin-bis-digitoxoside (Dt2) which was 70-90% of the total metabolites in the three male rat groups. Only in the young female rat group was the proportion of Dt2 (40%) slightly exceeded by that of digoxigenin-bis-digitoxoside (Dg2) (50%). A kinetic analysis for Dt3 degradation velocity obtained from studies using different Dt3 concentrations in old male rats yielded a Vmax of only 21% of the young value. In young castrated males, the Vmax also decreased to 13% of the young non-castrated males, which is approaching the young female value. The changes were primarily due to the decline of Dt2 formation velocity in these groups. Apparent Km values also decreased with both castration and aging. The plasma testosterone levels which were much higher in young male rats than in female rats became significantly lower in young castrated rats as well as in old male rats. The results suggest that apparent decreases in Vmax and Km values observed in old male rats for Dt3 metabolic degradation may be at least partly through the steroidal hormone control, presumably, the decline of androgenic induction during aging.     

Non-persisting early foci of altered hepatocytes induced in rats by N-nitrosomorpholine

Summary Male Sprague-Dawley rats were treated for 7 weeks with 120 mg/l N-nitrosomorpholine in their drinking water. At the end of the treatment period there were large numbers of enzyme-altered foci in the liver. During the following 10 weeks, the number of foci decreased significantly. This decrease in the number of enzyme-altered foci was due to the disappearance of a special type of focus. The typical features of these non-persisting foci were distinct enzyme histochemical and striking morphological alterations as well as the localization in or close to the third zone, as defined by Rappaport. In contrast to the simultaneously appearing persisting foci, the non-persisting foci were always glycogen-poor or totally glycogenfree. Signs of cell death were frequently found in or near this type of focus. After these non-persisting foci had disappeared, the total number of pre-neoplastic lesions obviously remained constant. We conclude that this disappearance of early appearing, severely altered foci is due to cell loss caused by the non-specific toxic effect of the carcinogen.Dedicated to Professor Werner Kunz on the occasion of his 65th birthday