Hepatoprotective effect of pinostrobin against thioacetamide-induced liver cirrhosis in rats

The study in vivo assessed the effect of pinostrobin on the histology, immunohistochemistry, and biochemical parameters of thioacetamide (TAA) induced liver cirrhosis in Sprague Dawley rats. The rats were noticeably gavaged with two doses of pinostrobin (30 mg/kg and 60 mg/kg) with TAA and exhibited a substantial decrease in the liver index and hepatocyte propagation with much minor cell injury. These groups meaningfully down-regulated the proliferation of cellular nucleus antigen (PCNA) and alpha-smooth muscle actin (α-SMA). The liver homogenate displayed augmented antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities escorted with reducing in malondialdehyde (MDA) level. The serum level of bilirubin, total protein, albumin, and liver enzymes (ALP, ALT, and AST) returned to normal and was similar to that of normal control and silymarin with TAA-treated groups. pinostrobin-fed groups also decreased the level of Tumor necrosis factor-alpha (TNF-α), Interleukin-6 (IL-6), and increased the level of Interleukin-10 (IL-10). Acute toxicity with a higher dose of 500 mg/kg of pinostrobin did not manifest any toxicological signs in rats. The hepatoprotective effect of pinostrobin could be due to potentially inhibited the progression of liver cirrhosis, down-regulation of PCNA and α-SMA proliferation, prevented oxidation of hepatocytes, improved SOD and CAT enzymes, condensed MDA, repairs of liver biomarkers, reduced cellular inflammation and modulation of inflammatory cytokines.     

Metabolic zonation in thioacetamide-induced liver cirrhosis

After TAA administration to rats a central part may be distinguished histochemically from a marginal part in most of the cirrhotic nodules. The centre is characterized by a high glycogen content and by high activity of phosphorylase, G6Pase and SDH; the maxima of which are situated around the larger blood vessels. The vasculatory periphery, however, shows moderate G6PDH-activity. The marginal parts of the nodules are poor in glycogen and possess only weak G6Pase and phosphorylase activity, whereas high SDH- and G6PDH-activity can be demonstrated here. This distribution pattern leads to the conclusion that the larger blood vessels in the centre of the nodules are themselves the terminal afferent vessels. Thus the centre of the nodule corresponds to periportal zone 1, while G6PDH-activity marks the area corresponding to zone 3. The fact that the marginal parts of the nodules are marked by high SDH- but weak G6Pase-activity is interpreted as the result of a preferential arterial supply to this parenchymal part. The high G6PDH-activity of the marginal part is seen in context with the regeneration processes. In all animals single nodules could be found with a high glycogen content and extremely high G6PDH-activity. This loss of heterogeneity is interpreted as a first step in the direction of malignancy.     

Metabolic zonation in thioacetamide-induced liver cirrhosis

Summary After TAA administration to rats a central part may be distinguished histochemically from a marginal part in most of the cirrhotic nodules. The centre is characterized by a high glycogen content and by high activity of phosphorylase, G6Pase and SDH; the maxima of which are situated around the larger blood yessels. The vasculatory periphery, however, shows moderate G6PDH-activity. The marginal parts of the nodules are poor in glycogen and possess only weak G6Pase and phosphorylase activity, whereas high SDH-and G6PDH-activity can be demonstrated here. This distribution pattern leads to the conclusion that the larger blood vessels in the centre of the nodules are themselves the terminal afferent vessels. Thus the centre of the nodule corresponds to periportal zone l, while G6PDH-activity marks the area corresponding to zone 3. The fact that the marginal parts of the nodules are marked by high SDH-but weak G6Pase-activity is interpreted as the result of a preferential arterial supply to this parenchymal part. The high G6PDH-activity of the marginal part is seen in context with the regeneration processes. In all animals single nodules could be found with a high glycogen content and extremely high G6PDH-activity. This loss of heterogeneity is interpreted as a first step in the direction of malignancy.Dedicated to Prof. Dr. W. Graumann on the occasion of his 65th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Sa 127/7)The essential part of this study was presented as an Inaugural Dissertation to the Medical Faculty of the University of Freiburg by R. Nuber     

Experimental thioacetamide-induced cirrhosis of the liver.

Hepatic cirrhosis is a complex disease in which several biological, biochemical and chemical alterations are combined, none of these alone being sufficient for diagnosis. The morphological characteristics of the final stages of cirrhosis are well known, but the initial lesions and intermediate stages still have not been fully clarified. An experimental model of hepatic cirrhosis by chronic administration over 30 weeks of thioacetamide (50 mg/kg twice weekly) to female Wistar rats has been produced. In a macroscopic, microscopic and ultrastructural study. The different lesions that appeared were evaluated according to the dose of the toxic agent administered up, until hepatic cirrhosis was finally installed; this was after 60 doses of the toxic agent (30 weeks). Discussion is made of the different types of administration and the doses employed to obtain a suitable survival rate for these cases; in our experiments this was 95%. It has been demonstrated in both human and experimental pathology that once the disease itself has been installed, currently there is no rational or useful treatment for it. A beneficial effect has been demonstrated for certain substances, improving the initial and intermediate lesions, so we conclude by stating that it is necessary to further study the hepatic lesions preceeding cirrhosis. Knowledge of these lesions could form the basis for establishing a useful and rational therapy for such cases.     

In vitro effects of glycosylated insulin in perfused liver and hindquarter in rats.

Using the perfused liver and hindquarter of the rat, the uptake of glycosylated insulin and its effect on glucose output were investigated. Insulin was glycosylated in ambient high glucose concentration, and glycosylated insulin GI80 (insulin incubated with 0.08% glucose), GI350 (incubated with 0.35% glucose), and GI1000 (incubated with 1% glucose) were prepared. The liver and hindquarter were perfused with nonglycosylated insulin (N-GI) or glycosylated insulin at a concentration of 100 or 1000 microU/ml. There were no significant differences in the fractional uptake of insulin by perfused liver and hindquarter despite glycosylation. Insulin-induced decrement in glucose output was significantly lower in the liver perfused with GI1000 than that in the liver perfused with N-GI, GI80, and GI350 at an insulin concentration of 100 microU/ml. There were no significant differences in insulin-induced decrement in glucose output between the hindquarter perfused with N-GI, GI80, GI350, and GI1000. These results suggest that when insulin (100 microU/ml) is incubated with a markedly elevated concentration of glucose (1000 mg/dl) its biological activity is reduced in the liver, but not in the hindquarter.     

Attenuated anomeric difference of glucose-induced insulin release in the perfused pancreas of diazoxide-treated rats

Mild hyperglycemia was induced in normal rats by oral administration of both diazoxide and D-glucose. After 48 hours of such a treatment, the insulin and glucagon secretory responses of the perfused pancreas to alpha- and beta-D-glucose (3.3 mM) were examined in the presence of 10.0 mM L-leucine. The output of insulin, but not that of glucagon, and the perfusion pressure were higher in treated than control rats. The alpha-anomer of D-glucose was a more potent insulin secretagogue than beta-D-glucose in both control and treated rats. However, the alpha/beta ratio in insulin output was twice higher in control than treated rats. By analogy with other experimental models of diabetes, the attenuation in the anomeric difference of glucose-stimulated insulin output in the treated rats could reflect an altered secretory response to alpha- rather than beta-D-glucose. These findings suggest that hyperglycemia provokes, as a function of its severity and duration, first attenuation and then suppression, if not inversion, of the anomeric preference for alpha-D-glucose in insulin release. They are also compatible with the hypothesis that the anomeric malaise, associated with B-cell glucotoxicity, is caused by a progressive accumulation of glycogen in this cell.     

Protective effects of Rosmarinus tomentosus ethanol extract on thioacetamide-induced liver cirrhosis in rats

The capability of an ethanol extract of Rosmarinus tomentosus to protect rat liver in an experimental model of cirrhosis induced by thioacetamide (TAA) has been evaluated. Four groups of rats were used: Two of them received 300 mg TAA/l in the drinking water for 3 months while the other two, which served as controls, were given water ad libitum. During the same period and for each one of the treatments, one group received a semi-purified (SP) diet and the other one was fed the same diet supplemented with 1% of the dry residue obtained from R. tomentosus ethanol extract (SP+E). There was a significant reduction of TAA toxicity in rats fed the SP+E diet, as assessed by plasma and liver biochemical markers, and by liver histopathology. Plasma total protein concentration was restored, urea concentration and plasma alkaline phosphatase and gamma-glutamyl-transferase activities were reduced. A significant correction of plasma fatty acids concentrations was also evident. Hepatic alkaline phosphatase and gamma-glutamyl-transferase activities were significantly reduced in animals fed SP+E diet and glucose-6-phosphatase activity was significantly enhanced. The results suggest that R. tomentosus ethanol extract administered in the diet affords protection against TAA-induced cirrhosis, preventing most of the histological changes and functionality alterations own to this experimental pathology.     

Eightfold induction of nicotine elimination in perfused rat liver by pretreatment with phenobarbital

Elimination of nicotine by isolated rat livers was increased eightfold after pretreatment with phenobarbital (PB) as an inducer of cytochrome P-450 while it was only marginally influenced after pretreatment with 5,6-benzoflavone (BF) as an inducer of cytochrome P-448. Initial rates of cotinine formation were enhanced in the same order of magnitude in PB-induced livers. The 14C-nicotine-derived radioactivity excreted into bile within 2 h ranged between 6 -17% of the dose with only 2.7 fold higher values after PB pretreatment compared to controls.     

Impaired glucose priming of insulin secretion from perfused pancreas in aged female rats.

Effects of age and glucose levels on insulin secretion and synthesis were studied in the perfused pancreas of young (2-month-old) and older (10-month-old) female Wistar rats. Insulin secretion induced by 16.7 mM glucose showed a triphasic pattern: an early spike and fall (first phase, 0-6 min), followed by a sustained gradual increase (second phase, 7-120 min) and a gradual decreased release thereafter (third phase, 121-360 min) during the perfusion period of 360 min. First and second phase insulin secretion, but not third phase, were lower in older rats than in young rats. Insulin synthesis in old rat pancreas perfused with 16.7 nM glucose for 360 min was much greater than that of young rats. Second phase insulin secretion was restored to comparable levels by 28 mM glucose in older rats. Repeated pulses of 28 mM glucose potentiated subsequent insulin secretion in young rats, but not in older rats. These findings provide further evidence that sensitivity to glucose in pancreatic B cells is altered by aging.     

In vitro effects of glycosylated insulin and glucagon in perfused liver of the rat.

Using perfused liver of the rat, the hepatic uptake of glycosylated insulin (GI) and glucagon (GG) and its effects on hepatic glucose output were investigated. Insulin and glucagon were glycosylated in ambient high glucose concentration, and GI80 or GG80 (insulin or glucagon incubated with 0.08% glucose), GI350 or GG350 (incubated with 0.35% glucose), and GI1000 or GG1000 (incubated with 1% glucose) were prepared. The liver was perfused with the medium containing 1000 microU/ml insulin and 200 pg/ml glucagon or 200 microU/ml insulin and 1000 pg/ml glucagon. The fractional uptake of insulin or glucagon by perfused liver was not significantly altered by the glycosylation. In the liver perfused with 1000 microU/ml insulin and 200 pg/ml glucagon, glucose output was not changed by the glycosylation of the hormones, while in the liver perfused with 200 microU/ml insulin and 1000 pg/ml glucagon, GI1000 reduced its biological activity, as reflected by insulin-mediated decrease in glucose output. These results suggest that in the liver insulin incubated with markedly high concentration of glucose reduces its biological activity at a physiological concentration in the presence of high concentration of glucagon.     

Polaprezinc prevents ongoing thioacetamide-induced liver fibrosis in rats

AimsCirrhotic patients commonly have a liver zinc deficiency, which may aggravate liver fibrosis due to the lack of antioxidative effects of zinc. This study examined the ability of polaprezinc, N-(3-aminopropionyl)-l-histidinato zinc, to prevent fibrosis in a rat model of thioacetamide (TAA)-induced hepatic fibrosis.Main methodsLiver cirrhosis was induced by orally administering TAA for 20 weeks. The rats were cotreated with one of the following for the last 10 weeks of TAA treatment: (1) polaprezinc (50 or 200 mg/kg/day); (2) l-carnosine (155 mg/kg/day), which contained equal amounts of l-carnosine as 200 mg/kg/day polaprezinc; (3) zinc sulfate (112 mg/kg/day) or (4) zinc-l-aspartic complex (317.8 mg/kg/day). Both zinc supplementations contained equal amounts of zinc as high-dose polaprezinc.Key findingsHepatic zinc levels fell significantly in rats treated with TAA for 20 weeks. Cotreating with high-dose polaprezinc and zinc-l-aspartic complex for 10 weeks prevented hepatic zinc loss. Hepatic hydroxyproline and tissue inhibitor of metalloproteinases-1 (TIMP-1) were significantly higher in rats treated with TAA for 20 weeks than 10 weeks, whereas polaprezinc prevented changes in these fibrosis markers and reduced hepatic transforming growth factor-β1 protein concentration, macroscopic and histologic changes. TAA caused oxidative stress-related changes in the liver that were prevented by high-dose polaprezinc and partially by zinc-l-aspartic complex. Treatment with l-carnosine, low-dose polaprezinc or zinc sulfate for 10 weeks did not affect liver fibrosis progression or oxidative stress-related changes.SignificancePolaprezinc may prevent ongoing fibrosis by preventing zinc depletion, oxidative stress and fibrosis markers in cirrhotic livers.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Amylin secretion from the perfused pancreas: dissociation from insulin and abnormal elevation in insulin-resistant diabetic rats

Amylin has been co-secreted from pancreatic islet beta-cells in constant proportion with insulin in some studies. We measured basal and glucose-stimulated amylin and insulin secretion from isolated perfused pancreases of normal and diabetic fatty Zucker rats. Glucose concentrations in the perfusion buffer were increased then decreased in small steps to mimic physiologic changes occurring after a meal. The absolute rate of amylin secretion and the molar ratio of amylin to insulin secreted from diabetic pancreases increased dramatically when infused glucose concentrations fell. Similar changes also occurred in normal pancreases, although the absolute change in amylin secretion was smaller. These studies provide the first evidence that (i) there is a mechanism within the pancreas whereby independent secretion of amylin and insulin can occur; (ii) the molar ratio of amylin to insulin secreted from both normal and diabetic pancreases can vary over a wide range; and (iii) there are important differences in the kinetics of amylin and insulin secretion or their coupling to stimulation by glucose between the isolated pancreases of normal rats and those with genetically transmitted insulin resistance and diabetes mellitus.     

Somatostatin, insulin and glucagon secretion by the perfused pancreas from the cysteamine-treated rat

In rats, administration of a single dose of cysteamine (300 mg/kg, intragastrically) induces a depletion of pancreatic somatostatin content (approximately 60%) without modifying pancreatic insulin or glucagon content. In perfused pancreases from cysteamine-treated rats, there was a lack of somatostatin response to glucose, arginine or tolbutamide. In the absence of stimulated somatostatin release, the secretory responses of insulin and glucagon to glucose, to arginine, and to tolbutamide were not significantly different from those observed in pancreases from control rats. Our data do not support the concept that pancreatic somatostatin plays a major role in the control of insulin and glucagon release.