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Zhong, Zhi, Matthias Froh, Mark Lehnert, Robert Schoonhoven, Liu Yang, Henrik Lind, John J. Lemasters, and Ronald G. Thurman. Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats. Am J Physiol Gastrointest Liver Physiol 285: G1004--G1013, 2003. First published June 4, 2003; 10.1152/ ajpgi.00008.2003.--Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-[alpha]1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of [alpha]-smooth muscle actin and transforming growth factor-[beta] and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis. bile duct ligation; fibrosis; oxidative stress; transforming growth factor-[beta]

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPAR[alpha] in cell proliferation after PH was tested. After PH, an increase in PPAR[alpha] DNA binding was observed in wild-type mice, correlating with an increase in the PPAR[alpha]-regulated enzyme acyl-CoA oxidase. In addition, the PPAR[alpha]-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPAR[alpha] knockout (PPAR[alpha] -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by ~60% in PPAR[alpha] -/- mice, despite increases in TNF-[alpha] and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPAR[alpha] -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPAR[alpha] -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPAR[alpha] -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPAR[alpha] blunted the activation of cyclindependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPAR[alpha] is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA. hepatocyte proliferation; cell cycle regulation; RhoA; peroxisome proliferation-activated receptor-[alpha]

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PG[E.sub.2], which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in hepatic parenchymal cells by agonists released during stress, such as with PG[E.sub.2] and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [[[Ca.sup.2+]].sub.i] in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PG[E.sub.2] caused a dose-dependent increase in [[[Ca.sup.2+]].sub.i] from basal values of 130 [+ or -] 10 to maximal levels of 434 [+ or -] 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is [Ca.sup.2+] dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of [Ca.sup.2+] from intracellular stores, also partially blocked the increase in [[[Ca.sup.2+]].sub.i] caused by PG[E.sub.2], suggesting that intracellular [Ca.sup.2+] pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular [Ca.sup.2+] pools are involved in the increase in [[[Ca.sup.2+]].sub.i] caused by PG[E.sub.2]. Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [[[Ca.sup.2+]].sub.i] due to PG[E.sub.2] in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular [Cl.sup.-] was omitted, glycine was much less effective in preventing the increase in [[[Ca.sup.2+]].sub.i] due to PG[E.sub.2]. Phenylephrine, an [[alpha].sub.1]-type adrenergic receptor agonist, also increased [[[Ca.sup.2+]].sub.i], as expected, from 159 [+ or -] 20 to 432 [+ or -] 43 nM. Glycine also blocked the increase in [[[Ca.sup.2+]].sub.i] due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [[[Ca.sup.2+]].sub.i] in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection. intracellular calcium; prostaglandin [E.sub.2]; strychnine

These experiments were designed to determine whether green tea extract (GTE), which contains polyphenolic free radical scavengers, prevents ischemia-reperfusion injury to the liver. Rats were fed a powdered diet containing 0-0.3% GTE starting 5 days before hepatic warm ischemia and reperfusion. Free radicals in bile were trapped with the spin-trapping reagent [alpha]-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and measured using electron spin resonance spectroscopy. Hepatic ischemia-reperfusion increased transaminase release and caused pathological changes including focal necrosis and hepatic leukocyte infiltration in the liver. Transaminase release was diminished by over 85% and pathological changes were almost totally blocked by 0.1% dietary GTE. Ischemia-reperfusion increased 4-POBN/radical adducts in bile nearly twofold, an effect largely blocked by GTE. Epicatechin, one of the major green tea polyphenols, gave similar protection as GTE. In addition, hepatic ischemia-reperfusion activated NF-KB and increased TNF-[alpha] mRNA and protein expression. These effects were all blocked by GTE. Taken together, these results demonstrate that GTE scavenges free radicals in the liver after ischemia-reoxygenation, thus preventing formation of toxic cytokines. Therefore, GTE could prove to be effective in decreasing hepatic injury in disease states where ischemia-reperfusion occurs. ischemia; free radicals; liver; nuclear factor-kappaB; cytokines

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both [alpha]- and [beta]-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the [beta]-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycinegated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system. membrane potential; intracellular calcium; lipopolysaccharide

Although glycine prevents renal tubular cell injury in vitro, its effect in vivo is not clear. The purpose of this study was to investigate whether a bolus injection of glycine given before reperfusion plus continuous dietary supplementation afterward would reduce renal injury caused by ischemia-reperfusion. Female Sprague-Dawley rats received a semisynthetic powdered diet containing 5% glycine and 15% casein (glycine group) or 20% casein (control group). Two days later, renal ischemia was produced by cross-clamping the left renal vessels for 15 min, followed by reperfusion. The right kidney was removed before reperfusion. The postischemic glomerular filtration rate (GFR) showed that renal function was less impaired and recovered more quickly in rats receiving glycine. For example, at day 7, GFR in controls (0.31 [+ or -] 0.03 ml * [min.sup.-1] * 100 [g.sup.-1]) was about one-half that of glycine-treated rats (0.61 [+ or -] 0.06 ml * [min.sup.-1] * 100 [g.sup.-1], P < 0.05). Furthermore, tubular injury and cast formation observed in controls was minimized by glycine (pathology score, 3.2 [+ or -] 0.4 vs. 1.0 [+ or -] 0.4, P < 0.05). Urinary lactate dehydrogenase (LDH) concentration was elevated by ischemia-reperfusion in the control group (260 [+ or -] 22 U/l), but values were significantly lower by about fourfold (60 [+ or -] 30 U/l) in glycine-fed rats. Similarly, free radical production in urine was significantly lower in glycine-treated animals. Importantly, on postischemic day 1, binding of pimonidazole, an in vivo hypoxia marker, was increased in the outer medulla in controls; however, this phenomenon was prevented by glycine. Two weeks later, mild leukocyte infiltration and interstitial fibrosis were still observed in controls, but not in kidneys from glycine-treated rats. In conclusion, these results indicate that administration of glycine indeed reduces mild ischemia-reperfusion injury in the kidney in vivo, in part by decreasing initial damage and preventing chronic hypoxia. kidney; hypoxia; rat

Intravenous infusion of glycine significantly improves survival in liver transplantation in rats. This was gleaned from the results of an experiment, which revealed that minimal dissection of the rat liver for 12 minutes plus 13 minutes without manipulation did not affect survival. Thus, pretreatment of donors with intravenous glycine minimizes reperfusion injury caused by organ manipulation during harvest and after transplantation.

Research was conducted to find out if various techniques employed to isolate Kupffer cells have an impact on CD14, a lipopolysaccharide receptor found mostly in macrophages. Kupffer cells were isolated by collagenase or collagenase-Pronase perfusion and differential centrifugation. Results showed that Pronase cleaves CD14 from Kupffer cells, while collagenase perfusion does not, elucidating on why Kupffer cells do not show a CD14-mediated pathway when prepared with processes utilizing Pronase.

The function of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure was studied. The specific Kupffer cell toxicant GdCl3 totally prevented increases in oxygen uptake in female Sprague-Dawley rats because of endotoxin. The results illustrate that endotoxin has an important function in SIAM, most probably due to the stimulation of eicosanoid release from Kupffer cells.

The ability of glycine to minimize cyclosporin A-induced nephrotoxicity by preventing hypoxia-reperfusion injury in the kidney was investigated. It was determined that cyclosporin A can cause hypoxia via vasoconstriction during increased blood pressure. It was also observed that cyclosporin A can cause increases in free radical formation that can attack lipid components and result in lipid peroxidation. The production of free radicals and a hypoxic condition result in renal injuries such as proximal tubule cell swelling, necrosis and infiltration of macrophages.

A study was conducted to determine whether dietary choline can prevent endotoxin shock in rats. Choline was found to increase survival of the rats in a dose-dependent manner. It also improved the microscopic appearance of the lungs and reduced increases in serum aspartate aminotransferase levels. Feeding choline blunted tumor necrosis factor-alpha production. These data suggested that choline diminishes endotoxin shock by preventing macrophage activation.

A study involving Kupffer cells was conducted to determine if prostaglandin E2 (PGE2) stimulates oxygen uptake in an oxygen-dependent manner in the isolated perfused rat liver and if non-parenchymal cells participate in this phenomenon. Results show that high oxygen tension stimulates Kupffer cells to release mediators such as PGE2 which increases oxygen consumption in parenchymal cells. PGE2 was also found to stimulate oxygen uptake in parenchymal cells through oxygen-dependent pathways involving cAMP.

Sublethal injection of bacterial endotoxin resulted in mortality among rats pretreated with estrogen but not among subjects treated with gadolinium chloride, a substance toxic to Kupffer cells. Investigations on the mechanisms by which estrogen enhances the sensitivity of rats to endotoxin revealed that estrogen enhances the sensitivity of Kupffer cells to endotoxin leading to an overproduction of toxic mediators.

The effects of glycine on lipopolysaccharide (LPS)-induced elevations in [Ca2+]i in Kupffer cels were analyzed to determine the presence of glycine-gate chloride channels. Kupffer cell preparations that were exposed to strychnine reversed the inhibitory effects of glycine on LPS-induced elevations in [Ca2+]i. Furthermore, glycine blunted the levation of [Ca2+]i in the LPS-stimulated Kupffer cells through the actions of the chloride channels.

Alcohol ingestion results in increases in the release of endotoxin from gut bacteria or membrane permeability of the gut to endotoxin, or both. Female rats are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, tumor necrosis factor-[Alpha] and free radicals. Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver. The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, [Alpha]-hydroxyethyl free radicals are formed that lead to tissue damage in oxygen-poor pericentral regions of the liver lobule. KEY WORDS: alcohol-induced liver injury endotoxin free radicals sex differences rats

An in vivo enteral ethanol feeding model was utilized to determine the mechanisms responsible in the high susceptibility of females to alcohol-induced liver injury. Analysis of female susceptibility to alcohol-induced injury based on a novel ethanol feeding model indicated the presence of higher plasma endotoxin levels in females compared to males after exposure to ethanol. Higher endotoxin levels in female also induced a more extensive Kupffer cell activation in females compared to males.

Rats showed a marked increase in hepatic oxygen uptake and ethanol metabolism two to three hours after exposure to acute alcohol and treatment with pimonidazole. Pericentral regions of liver lobules also showed a significant accumulation of pimonidazole, a hypoxia marker, suggesting that exposure to alcohol causes hypoxia. Pimonidazole accumulation was blocked by treatment with a Kupferr cell-specific toxicant, indicating that the increase in oxygen uptake involved Kupffer cells.

The role of glycine in the treatment of lipopolysaccharide (LPS)-induced endotoxin shock was investigated in a model using rats fed a glycine diet. Rats fed control diet died 24 hrs. after injection of LPS, whereas rats fed a glycine diet survived endotoxin shock. It is suggested that glycine feeding reduced the deleterious effects of LPS such as elevation of serum transaminase levels, hepatic necrosis, and lung and liver injury.

Liver cells signal neighboring cells by releasing prostaglandin E2 to increase oxygen uptake when exposed to ethanol. This signalling mechanism was observed in parenchymal cells which were treated with conditioned medium derived from ethanol-exposed Kupffer cells. Elevated levels of oxygen consumption were observed in the parenchymal cells and a similar reaction was observed when the cells were directly treated with prostaglandin E2.

The effect of glycine on reperfusion injury of rat livers was investigated in a low-flow, reflow liver perfusion model. Livers were perfused at low flow rates to turn the cells anoxic. An oxygen-dependent reperfusion injury occurred upon restoration of flow rates, and lactate dehydrogenase was released into the effluent perfusate. Glycine regulated LDH concentrations, which caused cell death when present in maximal amounts. Infusion of glycine reduced cell death by minimizing reperfusion injury.

Experimental studies of the inactivation of Kupffer cells by GdCl3 reveal the vital role of Kupffer cell activation in the pathogenesis of hepatic reperfusion injury. Lactate dehydrogenase (LDH) and malondialdehyde (MDA) release and oxygen uptake reach a maximum within the first 10 minutes of reperfusion. Inactivation of Kupffer cells by GdCl3 decreases the release of LDH and MDA. Electron paramagnetic resonance spectroscopic studies reveal the involvement of a carbon-centered radical during reflow and that GdCl3 inhibits the formation of the radical.

Oxygen uptake by parenchymal cells in the liver is regulated by eicosanoids produced by non-parenchymal cells, through a hepatic O2 sensor mechanism involving Ca2+ and nonparenchymal cells. This mechanism leads to the production of eicosanoids in reactions which depend on O2. Eicosanoids further increase O2 utilization in regions downstream of the liver lobule. This causes a rise in the rate of O2 uptake and in the production of intracellular Ca2+ in parenchymal cells.

ATP secretion from parenchymal cells and activation of eicosanoid secretion from Kupffer cells that trigger glycogenolysis in parenchymal cells are part of the activation of parenchymal cell glycogenolysis by adenosine. Prostaglandin secretion from cultured Kupffer cells results from ATP action. ATP concentrations in cultured media of parenchymal cells are enhanced by adenosine.