TNFalpha is required for cholestasis-induced liver fibrosis in the mouse.

TNFalpha, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFalpha knockout mice (TNFalpha-/-). Survival after BDL was 60% in wild-type mice (TNFalpha+/+) and 90% in TNFalpha-/- mice. Body weight loss and liver to body weight ratios were reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFalpha+/+ mice (268.6+/-28.2U/L) compared to TNFalpha-/- mice (105.9U/L+/-24.4). TNFalpha-/- mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, alpha-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-beta mRNA, a profibrogenic cytokine, were markedly reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Thus, our data indicate that TNFalpha induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Heme oxygenase-1 overexpression increases liver injury after bile duct ligation in rats.

AIM: To investigate the effects of heme oxygenase-1 (HO-1) against oxidant-induced injury caused by bile duct ligation (BDL). METHODS: Either cobalt protoporphyrin (CoPP), a HO-1 inducer, or saline were injected intraperitoneally in male SD-rats. Three days later, BDL or sham-operations were performed. Rats were sacrificed 3 wk after BDL and livers were harvested for histology. Fibrosis was evaluated by sirius red staining and image analysis. Alpha-smooth muscular actin, which indicates activation of stellate cells, was detected by immunohistochemical staining, and cytokine and collagen-Ialpha (Col-Ialpha) mRNA expression was detected using RNase protection assays. RESULTS: Serum alanine transaminase increased 8-fold above normal levels one day after BDL. Surprisingly, enzyme release was not reduced in rats receiving CoPP. Liver fibrosis was evaluated 3 wk after BDL and the sirius red-positive area was found to be increased to about 7.8%. However, in CoPP pretreated rats sirius red-positive areas were increased to about 11.7% after BDL. Collagen-Ialpha and TGF-beta mRNA increased significantly by BDL. Again, this effect was increased by HO-1 overexpression. CONCLUSION: Hepatic fibrosis due to BDL is not reduced by the HO-1 inducer CoPP. In contrast, HO-1 overexpression increases liver injury in rats under conditions of experimental chronic cholestasis.

Hypolipidemic action of the soybean isoflavones genistein and genistin in glomerulonephritic rats.

Effects of genistein and its glycoside genistin were studied in nephritic rats with endogenous hyperlipidemia. Male Wistar rats with glomerulonephritis caused by a single intravenous injection of nephrotoxic serum were orally given 5 mg of genistein or 8 mg of genistin/d/100 g body weight for 12 d. These isoflavones suppressed nephritis-induced severe hypercholesterolemia and hypertriglyceridemia, and their hypolipidemic action was almost identical. Fecal steroid excretion was unchanged by administration of the two isoflavones. Genistein inhibited the incorporation of [1-14C]acetate into cholesterol and FA in liver slices from nephritic rats when added to an incubation buffer, whereas genistin did not. These results suggest that genistin may be hydrolyzed to genistein and that genistein itself and/or its metabolite(s) may be intracorporal entities suppressing hepatic lipid syntheses. They also suggest that the suppression of hepatic lipid synthesis may be one mechanism of the hypolipidemic action of genistein.

Beneficial effects of soybean isoflavone supplementation on bone metabolism and serum lipids in postmenopausal japanese women: a four-week study.

OBJECTIVE: The aim of this study was to determine the effects of soy isoflavones with weak estrogen-like activities both on bone metabolism and on serum lipids in perimenopausal women. METHODS: Twenty-three healthy perimenopausal women were assigned randomly to either isoflavone or placebo groups. The isoflavone group (n = 12) received daily capsules of soy isoflavone extract (61.8 mg of isoflavones) and the placebo group (n = 11) received daily placebo capsules for four weeks. Urinary excretion of isoflavone was measured at weeks 0, 2 and 4. Urinary excretion of pyridinoline and deoxypyridinoline, bone stiffness and levels of serum cholesterol, triglyceride and cholesterol fractions were measured at weeks 0 and 4. RESULTS: As compared to the placebo group, urinary isoflavone, primarily daidzein, excretion was increased at weeks 2 and 4 in the isoflavone group. Excretion of bone resorption markers was reduced significantly in the isoflavone group. Both total serum cholesterol and LDL cholesterol were decreased significantly in the isoflavone group. Other serum biochemical parameters were not changed in either group. CONCLUSION: Soy isoflavone supplementation for four weeks showed potentially beneficial effects on bone metabolism and on serum lipids in perimenopausal women. These effects could have the potential to reduce the risks of postmenopausal osteoporosis and of cardiovascular diseases in such women.

Evidence of estrogenic effect by the three-month-intervention of isoflavone on vaginal maturation and bone metabolism in early postmenopausal women.

Objective of the present study is to determine the estrogenic effect of isoflavone on vaginal epithelia and bone metabolism in early postmenopausal women. Twenty-two postmenopausal women were randomly assigned to either a group that was given isoflavone extract (61.8 mg) for three months or a control group that was given placebo. We measured the L2-4 bone mineral density (BMD) before and 3 months after treatment by dual X-ray absorptiometry (DXA). Blood and urine samples were obtained from the women before and 3 months after treatment. We measured FSH using radioimmunoassay and, urinary pyridinoline and deoxypyridinoline levels by HPLC. For endocrine cytology, vaginal smears were collected before and 3 months after the treatment. Three months after the treatment, the serum FSH levels and the BMD values did not significantly differ between the two groups. Urinary excretion of isoflavone was significantly higher in the group given isoflavone compared with that given placebo (p<0.03). Numbers of parabasal and intermediate types of cells were significantly decreased (58.2 +/- 12.4% to 25.0 +/- 10.7%; p<0.05) and increased (24.1 +/- 8.7% to 63.7 +/- 10.7%; p<0.05), respectively in the isoflavone group, but remained unchanged in the control group. Urinary pyridinoline excretion was significantly decreased (49.6% vs. before, p<0.01 by paired t-test) in the isoflavone group. The intake of 60 mg of isoflavone daily for 3 months produced maturational changes of vaginal epithelia without affecting serum FSH levels, and could possibly slow down bone turnover rates as judged by decreased urinary pyridinoline excretion.

Evaluation of a soybean product fujiflavone P40 as an antiosteoporotic agent in rats.

The preventive effects of Fujiflavone P40 (a soybean isoflavone product) against both bone loss and periodontal alteration were evaluated using an ovariectomized rat model. Rats were divided into five groups: sham-operated (Sham), ovariectomized (OVX), OVX given Fujiflavone P40, OVX given 17beta-oestradiol, and OVX given the vehicle for 17beta-oestradiol, respectively. Fujiflavone P40 contains 46.6% isoflavones which consist of 24.1% daidzin, 16.5% glycitin and 5.9% genistin. Administration of Fujiflavone P40 to OVX rats suppressed the body weight gain until 5 weeks. Fujiflavone P40 also decreased total and high-density lipoprotein (HDL) cholesterols and triglyceride level of OVX rats, significantly. After 7 weeks, Fujiflavone P40 did not recover the coarsened fibre of the periodontal ligament. The ovariectomy decreased the uterine weight by 78%. The administration of 17beta -oestradiol recovered the weight loss by 99%, while Fujiflavone P40 restored it by 33%. The ovariectomy decreased the tibial bone mineral density (BMD) by 22%. The administration of 17beta-oestradiol to OVX rats recovered the tibial BMD decrease by 100%, while Fujiflavone P40 recovered it by 78%. The results suggest that Fujiflavone P40 may be useful as a preventive agent for osteoporosis.

Effectiveness of endoscopic nasobiliary drainage for postoperative bile leakage after hepatic resection.

The effectiveness of endoscopic nasobiliary drainage (ENBD) for postoperative bile leakage after hepatic resection was investigated retrospectively. Between 1997 and 2002 a series of 486 hepatectomies without biliary reconstruction were performed. Bile leakage was divided into two categories. Type A was defined as bile leakage communicating with the main bile tree fistulographically or endoscopic cholangiographically, and type B was bile leakage without such a patency of bile flow. Bile leakage developed in 31 patients (6.4%) (types A/B = 16/15). Type A frequently occurred at the major Glisson's sheath. In contrast, most type B cases occurred at the peripheral bile duct at the cut surface of the liver. Among the type A patients, 10 of 11 were effectively treated with ENBD. For the type B patients, 12 of 15 patients were successfully treated with intraabdominal drainage via surgical drains inserted during the operation or percutaneous tubes newly inserted for biliary fluid collection. ENBD was effective in two of three type B patients. The duration of bile leakage significantly shortened after initiation of ENBD in type A patients (15.3 +/- 6.9 vs. 25.8 +/- 13.2 days, p < 0.05). The classification based on communication with the main bile tree is useful for determining therapeutic strategy. Type A leakage has a good indication for ENBD, whereas type B can be treated with intraabdominal drainage in most cases, although ENBD may be effective in some intractable type B cases. It is preferable to initiate ENBD as early as possible to shorten the duration of bile leakage and the subsequent hospital stay.

Chronic intragastric alcohol exposure causes hypoxia and oxidative stress in the rat pancreas.

The effect of chronic enteral ethanol on pancreatic hypoxia was investigated using the hypoxia marker, pimonidazole. Male Wistar rats were fed an ethanol-containing diet for 3 weeks using an enteral model shown to cause pancreatic damage; pimonidazole (120 mg/kg i.v.) was injected 1h before sacrifice. Pimonidazole and 4-hydroxynonenal (an index of lipid peroxidation) adducts were detected immunochemically. Breathing air with low oxygen content (8% O(2)) for 1h increased pimonidazole adduct accumulation approximately 2-fold in pancreata of nai;ve rats, confirming that this technique will detect increases in hypoxia in pancreata. Pancreata of rats fed ethanol began to show signs of damage after 3 weeks. Ethanol feeding also significantly increased pimonidazole adducts in pancreas approximately 2-fold (1 or 3 weeks of ethanol produced similar values). Concomitant with increasing hypoxia in the pancreas, alcohol also caused a significant increase in 4-hydroxynonenal adducts, indicative of increased oxidative stress. These results indicate that chronic ethanol causes hypoxia at the cellular level in the pancreas in vivo; further, the data support the hypothesis that hypoxia is involved in mechanisms of chronic alcoholic pancreatitis.

Contribution of angiotensin II to alcohol-induced pancreatic fibrosis in rats.

The mechanisms by which alcohol causes pancreatic fibrosis remain unknown. Recent studies have demonstrated that angiotensin II contributes to the development of fibrosis in liver, kidney, and heart injury. Here, the effects of angiotensin-converting enzyme inhibitor (captopril) and angiotensin II receptor antagonist (losartan) on alcohol-induced pancreatic fibrosis were examined in an intragastric ethanol-feeding model. Male rats were fed a high-fat liquid diet with either ethanol (16-20 g/kg/day) or isocaloric maltose-dextrin (control) for 4 weeks. Subgroups daily received captopril (60 mg/kg/day), losartan (3 mg/kg/day), or no additional agent included in liquid diets. Mean urine alcohol concentrations in all groups fed ethanol were more than 270 mg/dl and not significantly different. Dietary alcohol caused diffuse gland atrophy and interlobular and intralobular fibrosis with mild structural distortion in the pancreas, an effect that was blunted by captopril or losartan treatment. Alcohol also increased the number of alpha-smooth muscle actin-positive cells and transforming growth factor-beta mRNA expression in the pancreas. These increases were blunted significantly by captopril or losartan treatment. These data suggest that angiotensin II contributes to the development of chronic alcohol-induced pancreatic fibrosis through its stimulation of transforming growth factor-beta expression.

Green tea extract protects against early alcohol-induced liver injury in rats.

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.

Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice.

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-alpha participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 +/- 31 U/L) over high-fat controls (24 +/- 7 U/L), effects which were blunted significantly in LBP knockout mice (60 +/- 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 +/- 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 +/- 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-alpha mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.

Cocoa extract protects against early alcohol-induced liver injury in the rat.

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.