María Isabel Torres

Experimental Ulcerative Colitis Impairs Antioxidant Defense System in Rat Intestine

Increasing attention has been given recently to the role of free radicals in the pathogenesis of ulcerative colitis, since the inflamed intestine is exposed to oxidative stress generated by infiltrating macrophages and neutrophils within the lamina propia. The overall goal of this study was to evaluate whether experimental ulcerative colitis induces significant changes in the antioxidant defense system in an experimental model induced by the intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid. Twenty rats were treated with 80 mg/kg body weight of trinitrobenzenesulfonic acid and 20 with the same volume of 0.9% NaCl. Rats were killed at one and two weeks after treatment to evaluate colon damage by light and electron transmission microscopy. The degree of tissue injury and inflammation was determined by measuring alkaline phosphatase, γ-glutamyltranspeptidase, and myeloperoxidase activities and prostaglandin E2 and leukotriene B4. Glutathione levels and the activity of the enzymes of the antioxidant defense system were determined. Enzymatic markers of colon injury showed higher activities in rats with ulcerative colitis. Concentrations of prostaglandin E2 and leukotriene B4 were higher in the groups treated for one week with trinitrobenzenesulfonic acid and markers decreased after two weeks of treatment. All antioxidant enzyme activities were higher at one and two weeks after treatment; however, a significant decrease in total glutathione content was also observed. In conclusion, ulcerative colitis induced by trinitrobenzenesulfonic acid damages the intestinal mucosa and is accompanied by a shift in the antioxidant enzyme activities, and low levels of glutathione. This deficiency in glutathione could be a target for new therapies to treat ulcerative colitis.

The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

Dietary Monounsaturated n-3 and n-6 Long-Chain Polyunsaturated Fatty Acids Affect Cellular Antioxidant Defense System in Rats with Experimental Ulcerative Colitis Induced by Trinitrobenzene Sulfonic Acid

The intrarectal administration oftrinitrobenzene sulfonic acid in rats induces ulcerativecolitis, which results in histological alterations ofcolonic mucosa, severe modification of the cellularantioxidant defense system, and enhanced production ofinflammatory eicosanoids. This study evaluated theinfluence of different dietary fatty acids, ie,monounsaturated, n-3, and n-3 + n-6 polyunsaturatedfatty acids, on the recovery of the colonic mucosahistological pattern, the cellular antioxidant defensesystem of colon, and PGE2 and LTB4colonic mucosa contents in a model of ulcerative colitisinduced by intrarectal administration of trinitrobenzene sulfonicacid. Administration of dietary n-3 polyunsaturatedfatty acids led to a minimum stenosis score, a higherhistological recovery, lower colon alkaline phosphatase and gamma-glutamyltranspeptidase activities,and lower mucosal levels of PGE2 andLTB4 compared with the other two experimentalgroups. However, glutathione transferase, glutathionereductase, glutathione peroxidase, and catalase activities were lowerin the group treated with n-3 polyunsaturated fattyacids than in the groups fed with either themonounsaturated or the n-6 + n-3 polyunsaturatedenriched diet. We conclude that n-3 polyunsaturatedfatty acids can be administered to prevent inflammationin ulcerative colitis, but they cause a decrease in thecolonic antioxidant defense system, promoting oxidative injury at the site of inflammation.

Serum amino acid changes in rats with thioacetamide-induced liver cirrhosis

To date, no attempt has been made to study alterations occurring in the amino acid profile in chronic models of thioacetamide-induced liver cirrhosis. In this work, changes in serum amino acids and proteins in rats with thioacetamide-induced liver cirrhosis are reported, together with changes in enzyme activities in the liver and serum. Seventeen female Wistar rats were used. Eight rats were given 300 mg thioacetamide/l in drinking water for 4 months and nine rats were given water ad libitum during the same time-period. Significant increases in glycine, alanine, serine, methionine, glutamate, ornithine, phenylalanine, tyrosine, histidine and proline were observed in rats with the resulting experimental liver cirrhosis. Threonine, taurine, glutamine, lysine and citrulline tended to increase while isoleucine, leucine, aspartate, arginine and tryptophan tended to decrease. Total and nonessential amino acids increased significantly in cirrhotic animals. Total essential and aromatic amino acids tended to increase in the thioacetamide-treated group, whereas branched chain amino acids tended to decrease in the same group. Regarding serum proteins, a decrease in albumin concentration in the thioacetamide-treated animals was the only change detected. The liver enzyme activities under observation (aspartate and alanine aminotransferases, glutamate dehydrogenase and threonine deaminase) were lower in the thioacetamide group. Decreases were significant for both transaminases and threonine deaminase. Results for serum activities showed that transaminases did not change in thioacetamide-treated rats in comparison with controls. In contrast, alkaline phosphatase rose dramatically in cirrhotic rats. We conclude that the serum amino acid pattern in this chronic model of liver cirrhosis resembles in part that of the corresponding human disease.

QUANTITATIVE AND ULTRASTRUCTURAL CHANGES IN GLIA AND PERICYTES IN THE PARIETAL CORTEX OF THE AGING RAT

The frequency of astrocytes, microglia plus oligodendrocytes, and pericytes displaying nuclei was analyzed and quantified in 160‐μm‐wide strips of the parietal cortex (Par1 region) from young and aged Wistar rats. The study was performed on two groups of rats aged 3–4 and 32–36 months. Quantifications of the glial cell types and pericytes were made in 1‐μm‐thick sections stained with toluidine blue. Ultrathin sections were also made to analyze the ultrastructural features of these cells during aging. Astrocytes and pericytes increased in number by about 20% and 22%, respectively, with age. These increases were most significant in layers II–IV and V for both cellular types. Clusters of astrocytes were common in these layers of aging rats. The ultrastructural analysis also indicated changes in all cell types that stored inclusions and vacuoles with age, which were particularly abundant in microglial cells. End‐feet astrocytes and pericytes surrounding the vascular wall also contained vacuoles and inclusions, and consequently the vascular wall increased in thickness. In conclusion, the aging process increased astrocyte and pericyte populations, but not microglia plus oligodendrocyte populations, in the rat parietal cortex. Although no significant change in nuclear size could be observed in any cell type, all glial cells as well as pericytes underwent morphological ultrastructural changes. These modifications may result from the need to correct possible homeostatic imbalances during aging. Microsc. Res. Tech. 43:34–42, 1998.

Intestinal anti-inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat

The present study was undertaken to investigate the intestinal anti‐inflammatory effects of UR‐12746 on the acute and chronic stages of a trinitrobenzene sulphonic acid (TNBS) experimental model of inflammatory bowel disease (IBD) in the rat. UR‐12746 is a novel, locally‐acting compound which combines, through an azo bond, 5‐aminosalicylic (5‐ASA) and UR‐12715, a potent platelet activating factor (PAF)‐antagonist. UR‐12746 oral pretreatment of colitic rats (50 and 100 mg kg−1) reduced acute colonic damage when evaluated 2 days after colonic insult. Postreatment for 4 weeks with UR‐12746 (50 and 100 mg kg−1) resulted in a faster recovery of the damaged colonic mucosa, which was macroscopically significant from the third week. The intestinal anti‐inflammatory effect of UR‐12746 was associated with a decrease in leukocyte infiltration in the colonic mucosa, which was evidenced both biochemically, by a reduction in myeloperoxidase activity, and histologically, by a lower leukocyte count after morphometric analysis. This effect was higher than that seen with sulphasalazine, when assayed at the same doses and in the same experimental conditions. Several mechanisms can be involved in the beneficial effects showed by UR‐12746: inhibition of leukotriene B4 synthesis in the inflamed colon, improvement of the altered colonic oxidative status, and reduction of colonic interleukin‐1β production. The results suggest that the intestinal anti‐inflammatory activity of UR‐12746 can be attributed to the additive effects exerted by 5‐ASA and UR‐12715, the PAF antagonist compound, that are released in the colonic lumen after reduction of the azo bond by the intestinal bacteria.

Dietary nucleotides correct plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by oral intake of thioacetamide

BACKGROUND/AIMS Dietary nucleotides modulate a number of metabolic processes, including long-chain polyunsaturated fatty acid metabolism. In this study, we evaluated the effect of dietary nucleotides on plasma and liver microsomal fatty acid profiles in a rat model of liver cirrhosis induced by oral intake of thioacetamide. METHODS Fifty-four female Wistar rats were assigned to one of the following groups: rats in the thioacetamide group (n=45) were given 300 mg thioacetamide/l in their drinking water for 4 months, and rats in the control group (n=9) received water during the same period. After 4 months of treatment, 9 rats in each group were killed. The remaining rats in the thioacetamide group were divided into two new groups, and the animals in each were allowed to recover for 1 or 2 weeks on either a nucleotide-free diet or the same diet supplemented with 50 mg of each of the following: AMP, GMP, CMP, IMP and UMP per 100 g diet. RESULTS Saturated (mainly stearic acid), monounsaturated, and n-6 long-chain polyunsaturated fatty acids (mainly arachidonic acid), and also the unsaturation index decreased in plasma of rats with experimental cirrhosis. Administration of the diet supplemented with nucleotides to thioacetamide-treated rats corrected plasma levels of saturated, n-6 long-chain polyunsaturated fatty acids and the unsaturation index. In liver microsomes, the cirrhotic rats showed lower levels of protein and higher levels of palmitic, oleic, linoleic and arachidonic acids. Protein concentrations and levels of all the above-mentioned fatty acids were corrected with the nucleotide-enriched diet. CONCLUSIONS Dietary nucleotides contribute to correcting plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by chronic oral administration of thioacetamide.

Chronic Diarrhea Impairs Intestinal Antioxidant Defense System in Rats at Weaning

The aim of the present study was to evaluate the influence of severe protein–energy malnutrition on the antioxidant defense system in the small and large intestine in rats at weaning. Chronic diarrhea and the subsequent malnutrition were induced by oral intake of a lactose-enriched diet. Twenty rats were weaned at 21 days of age, and the control group was fed a semipurified synthetic diet for two weeks. The malnourished group was fed the same diet but carbohydrates were replaced by lactose, and they developed diarrhea one day after. Rats were killed, and macroscopic and histological features were analyzed, DNA content was measured, and alkaline phosphatase, myeloperoxidase, and γ-glutamyltranspeptidase activities were determined to assess the degree of intestinal injury. Glutathione levels as well as the activities of intestinal glutathione transferase, glutathione reductase, total glutathione peroxidase, selenium-dependent glutathione peroxidase, superoxide dismutase, and catalase were measured to study the antioxidant defense system. Malnourished rats showed loss of body weight and an increase in length and weight in jejunum and ileum, while no significant changes were observed in colon. Epithelial cells showed fewer and shorter microvilli, larger mitochondria with low inner density and loss of cristae, dilated endoplasmic reticulum, and Golgi apparatus. The protein-to-DNA ratio was higher in the jejunum, ileum, and colon of malnourished rats. Glutathione levels decreased 40% in jejunum and 50% in colon of malnourished rats. A 40–50% decrease in the activity of all the enzymes of the antioxidant defense system was observed in the jejunum and ileum of malnourished rats, while only catalase and glutathione transferase activities decreased 50% in colon. These results suggest that early chronic diarrhea and severe protein–energy malnutrition impair the antioxidant defense system in both the small and large intestine, which may have a role in the pathogenesis and maintenance of the vicious circle of malabsorption–diarrhea–malnutrition in infancy.

Steatosis and Collagen Content in Experimental Liver Cirrhosis Are Affected by Dietary Monounsaturated and Polyunsaturated Fatty Acids

BACKGROUND AND METHODS We used thioacetamide administered orally to induce cirrhosis in rats, and after these had recovered for 1 and 2 weeks we examined the effects of dietary supplementation with monounsaturated and n-3 polyunsaturated fatty acids, or with a combination of n-3 and n-6 polyunsaturated fatty acids, on the extent of steatosis and collagen content in the liver. RESULTS Nodular cirrhosis, increased collagen content, and lipid accumulation were established after 4 months of treatment with thioacetamide. When the animals were fed a diet rich in oleic acid for 2 weeks, the steatosis and fibrosis decreased. Supplementation with n-3 polyunsaturated fatty acids favored reductions in collagen content but did not reduce the fat accumulation. With a diet supplemented with a mixture of n-3 and n-6 fatty acids we found no reduction in either lipid accumulation or collagen content. CONCLUSIONS Fibrosis and steatosis may be influenced by dietary fat, and monounsaturated fat appears to influence favorably the histologic recovery of the damaged liver.

Dietary nucleotides have cytoprotective properties in rat liver damaged by thioacetamide

Liver cirrhosis has been induced with thioacetamide administered via different routes in rats and other species. The oral intake of thioacetamide causes nodular liver cirrhosis in rats characterized by extensive fibrosis occupying most of the hepatic parenchyma. To characterize the cytological features of cirrhosis induced by thioacetamide, and the degree of recovery obtained with dietary nucleotides, we made a morphometric study of the hepatocytes in rats administered 300 mg/l of thioacetamide for 4 months, and in rats receiving the same hepatotoxic treatment but allowed a 2-weeks recovery period on a nucleotide-free diet or a 250 mg/100 g nucleotide-supplemented diet. Thioacetamide caused to cell damage and affected the ultrastructure of hepatocytes leading to a decrease in cytoplasmic area together with increased nuclear and nucleolar size. Dietary supplementation with nucleotides favoured recovery, restoring the cytoplasmic (TN=491.7+/-9.6 vs TAA=305.1+/-3.7), nuclear (73.6+/-2.8 vs 97.4+/-2.9), and nucleolar area of damaged hepatocytes (5.6+/-0.3 vs 14.0+/-0.9). The injury from thioacetamide intake increased liver collagen, but dietary nucleotides prevented hepatic deposition of this protein. This study supports the hypothesis that dietary supplementation with nucleotides is decisive in ensuring hepatocyte recovery after thioacetamide-induced liver damage, and that dietary nucleotides have antifibrotic properties.