Effects of protein deficiency on liver trace elements and antioxidant activity in carbon tetrachloride-induced liver cirrhosis

In liver cirrhosis, liver tissue becomes progressively substituted by fibrosis, ultimately leading to architectural distortion, liver circulatory changes, and liver failure. Some data support the hypothesis that protein undernutrition may play a role in the development and progression of nonalcoholic liver cirrhosis and that this progression is at least partially mediated by changes in glutathione peroxidase (GPX), superoxide dismutase (SOD), and other antioxidative systems, leading to an increase in lipid peroxidation. We analyzed the effects of protein deficiency on liver Cu, Fe, Zn, Mn, and Se in carbon tetrachloride (CCl₄)-induced liver cirrhosis, the relation of protein undernutrition and these trace elements with the activity of some hepatic antioxidative enzymatic mechanisms, and the relation of all of them with morphological and biochemical changes in 40 male adult Sprague-Dawley rats divided in four groups. Liver cirrhosis was induced by intraperitoneal injection of CCl₄ to 10 rats fed a 2% protein diet and another 10 fed a 18% protein control diet; two further groups included rats without cirrhosis fed the 2% protein and the 18% protein diets. The study period lasted 6 wk. GPX, SOD, and lipid peroxidation products as well as Zn, Cu, Mn, Se, and Fe were determined in liver samples. We found that liver GPX and Se were reduced in the cirrhotic animals, especially in the low-protein-fed ones, protein deficiency, but not cirrhosis, exerting the main effects. A close correlation was found between liver GPX and serum albumin and weight loss and an inverse one among GPX and hepatocyte ballooning, liver fibrosis, and fat, histomorphometrically determined. These results suggest a pathogenetic role of decreased GPX in the progression of liver disease, which may become enhanced by concomitant protein undernutrition. In addition to iron, the levels of which were increased in the malnourished rats, no differences were found regarding the other trace elements, SOD activity, and lipid peroxidation products.     

Effect of alcoholic cirrhosis on ethane and pentane levels in breath

Lipid peroxidation can be monitored by measuring one or several highly volatile alkanes in exhaled air. The concentrations of ethane and pentane were determined in breath samples from patients with alcoholic and non-alcoholic cirrhosis as well as from healthy subjects. The greatest increase of exhaled pentane was found in 17 patients with alcoholic cirrhosis (2.85 +/- 2.37 pmol/ml) in comparison with 10 patients with non-alcoholic cirrhosis (0.71 +/- 0.33 pmol/ml) and 10 control subjects (0.59 +/- 0.41 pmol/ml). On the contrary, no significant difference was detected as far as exhaled ethane is concerned. These data suggest that: a) gas-chromatographic determination of exhaled pentane may play a significant role in detecting alcohol-induced liver disease; b) hepatic injury may be mediated by lipid peroxidation in these patients.     

基于16S rRNA基因和细菌基因组间重复序列的DNA指纹图谱技术对肝硬化大鼠肝移植后肠道菌群多样性的研究

目的应用PCR-DGGE和rep-PCR技术对肝硬化大鼠肝移植后肠道菌群多样性进行研究,探讨肠道菌群多样性的变化,并比较这两种方法在菌群分析中的作用。方法首先建立CCL4诱导肝硬化大鼠的肝移植模型,收取对照组、肝硬化成模时、肝移植后7 d和肝移植后30 d的大鼠粪便,提取细菌基因组DNA,采用PCR-DGGE和rep-PCR[BOX-PCR,ERIC-PCR,ERIC2-PCR,(GTG)5-PCR,REP-PCR]进行DNA指纹图谱分析。结果PCR-DGGE可明确将正常大鼠、肝硬化大鼠、肝移植大鼠分为3个簇,并显示出肝硬化、肝移植大鼠肠道菌群多样性明显增多。rep-PCR技术也可将各组分开,其中ERIC-PCR、ERIC2-PCR及REP-PCR三者扩增条带各组差异有显著性,鉴别效果更好。结论应用基于16S rRNA基因和细菌基因组间重复序列的指纹图谱技术对肝硬化大鼠肝移植后肠道微生态的研究具有重要价值,可对临床肝硬化、肝移植患者肠道微生态制剂的使用起到指导作用。     

The degree of hepatic regeneration after partial hepatectomy in rats with peritonitis and the role of lipid peroxidation

Bile accumulation in the peritoneal cavity after partial hepatectomy reduces hepatic regeneration. In 70% of hepatectomized rats with bile peritonitis, hepatic DNA synthesis showed a delayed initiation and diminished peak level. Because intraperitoneal bile significantly accelerated lipid peroxidation and decreased energy metabolism in the liver remnant, all hepatectomized rats with bile peritonitis died within 7 days. Subcutaneous administration of exogenous combined antioxidants SOD and catalase dramatically reduced lipid peroxidation and improved the survival rate. Although the slightly elevated serum endotoxin level in rats with peritonitis may play a role in the inhibition of hepatic regeneration, the result suggest that intraperitoneal accumulation of bile components may also directly accelerate lipid peroxidation in the liver remnant, inhibiting the hepatic regeneration.     

Estimation of collagen content of liver specimens. Variation among animals and among hepatic lobes in cirrhotic rats

We undertook a study to evaluate the correlation between morphometric evaluation and colorimetric determination of hepatic collagen content, and to analyze the variation among animals as well as among lobes of the same liver in hepatic collagen content after CCl4-induced micronodular cirrhosis. The results revealed a significant correlation (r = 0.9458; p less than 0.001) between the morphometric and colorimetric methods of collagen evaluation of liver specimens; both methods also significantly distinguished data obtained from controls and from cirrhotic rats (p less than 0.0005). After induction of micronodular cirrhosis by chronic CCl4 administration, a highly significant variation in hepatic collagen content was observed among animals (p less than 0.0001). By contrast, no significant difference in collagen content was observed (p less than 0.05) among hepatic lobes of a given animal. These results indicate that in this animal model of liver cirrhosis, interpretation of biochemical data would benefit by being related to the severity of the hepatic collagen infiltration of each animal. Our data also show that representative values for total hepatic collagen infiltration can be obtained from a single liver specimen; we suggest, however, that the specimen be taken from a major lobe of the liver and that a sufficiently large number of animals be used to avoid occasional sampling errors.     

IGF1 gene transfer into skeletal muscle using recombinant adeno-associated virus in a rat model of liver cirrhosis

Systemic administration of recombinant IGF1 at low levels has been shown to improve hepatic function, nutritional status and testicular atrophy in rats with CCl4-induced cirrhosis. We have developed a recombinant adeno-associated (rAAV) viral vector containing the cDNA for rat IGF1 and confirmed the expression of IGF1 after intramuscular injection of this vector in a rat model of liver cirrhosis. Although weight of injected muscles was significantly increased in rats with mild cirrhosis, this was not the case in rats with advanced, de-compensated cirrhosis. Furthermore, we found no significant amelioration of liver damage in treated rats at any stage of liver cirrhosis. Our results suggest that IGF1 gene transfer into muscle results in a local effect, at least at the vector dose employed here.     

Age-related changes in antioxidant enzyme activities in the small intestine and liver from Wistar rats.

The present study was designed to determine age-related changes in intestinal and hepatic antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and glutathione-S-transferase (GST), and lipid peroxidation in male Wistar rats (n = 8) aged 2 wk, 2.5 mon, 5 mon, 10 mon, and 23 mon. In the small intestine, cytosolic SOD, GSH-PX activities and lipid peroxidation were not affected by age, but intestinal GST activity was noticeably enhanced as age increased. In particular, intestinal GST activity in 23 mon old rats was 3 times as strong as that in 2 wk old rats. In the liver, the activity of hepatic cytosolic SOD was not affected by age, whereas GSH-PX and GST activities in rats aged 10 mon and 23 mon were much stronger than those in rats aged 2 wk, 2.5 mon, and 5 mon. The increased lipid peroxidation in 2.5 mon and 5 mon old rats was observed when compared with that of other groups. It is therefore concluded from the results presented here that age greatly increases GST activity in the small intestinal mucosae and increasing GSH-PX, GST activities and lipid peroxidation in the liver from male Wistar rats.     

Hepatic production of apolar aldehydes in rats with carbon tetrachloride-induced cirrhosis

The aim of this study was to identify apolar aldehydes in liver homogenates from rats with CCl₄-induced cirrhosis and, as a corollary, the antioxidant effect of zinc administration. The study was performed in five control rats and in ten cirrhotic rats which were further sub-divided into two groups to receive either a standard diet or one supplemented with zinc. The percentage of hepatic fibrosis, plasma malondialdehyde concentration and alanine aminotransferase activity were measured as well as the following aldehydes: hexanal, octanal, decanal, 2-hexenal, 2-octenal, 2-nonenal, 2,4-heptadienal and 2,4-decadienal. Of the 10 cirrhotic rats, 4 had elevated concentrations of the highly toxic 2,4-dialkenals which coincided with a higher percentage of fibrosis and plasma alanine aminotransferase activity. These aldehydes were not observed in the control group. Zinc administration was associated with a reduction of the hepatic malondialdehyde concentration and an amelioration on the degree of hepatic injury. In conclusion, this study demonstrates the presence of the highly toxic 2,4-dialkenals in hepatic tissue of rats whith CCl₄-induced cirrhosis. Results obtained would suggest that these particular aldehydes may be related to the severity of the hepatic injury.     

Dysregulation of hepatic iron with aging: implications for heat stress-induced oxidative liver injury

Environmental heat stress is associated with an age-related increase in hepatic oxidative damage and an exaggerated state of oxidative stress. The purpose of this investigation was to evaluate the regulation of hepatic iron after heat stress. A secondary aim was to determine a potential role for iron in heat stress-induced liver injury. Hyperthermia-induced alterations in hepatic iron were evaluated in young (6 mo) and old (24 mo) Fischer 344 rats by exposing them to a two-heat stress protocol. Livers were harvested at several time points after the second heating and assayed for labile and nonheme iron. In the control condition, there was no difference in labile iron between age groups. Both labile iron and storage iron were not altered by hyperthermia in young rats, but both were increased immediately after heating in old rats. To evaluate a role for iron in liver injury, hepatic iron content was manipulated in young and old rats, and then both groups were exposed to heat stress. Iron administration to young rats significantly increased hepatic iron content and ferritin but did not affect markers of lipid peroxidation under control conditions or after heat stress. In old rats, iron chelation with deferoxamine prevented the increase in nonheme iron, labile iron, ferritin, and lipid peroxidation after heat stress. These results suggest that iron may play a role in hepatic injury after hyperthermia. Thus, dysregulation of iron may contribute to the gradual decline in cellular and physiological function that occurs with aging.     

Lipid peroxidation induced by cyclophosphamide

Intraperitoneal administration of a single dose of cyclophosphamide (CP) to rats was found to produce hepatic glutathione depletion and to enhance NADPH-mediated lipid peroxidation in the 15,000 x g supernatant fraction of the liver. These effects were associated with CP in a dose- and a time-dependent manner. The data suggest that the glutathione depletion is, at least in part, responsible for the enhancement in lipid peroxidation induced by CP.     

Antioxidant enzyme systems in rat liver and skeletal muscle. Influences of selenium deficiency, chronic training, and acute exercise

The influences of selenium deficiency (Se-D), chronic training, and an acute bout of exercise on hepatic and skeletal muscle antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), as well as glutathione S-transferase (GST) and tissue lipid peroxidation, were investigated in post-weaning male Sprague-Dawley rats. Se-D per se depleted GPX in both liver and skeletal muscle but had no effect on SOD or catalase activity. One hour of treadmill running (20 m/min, 0% grade and 27 m/min, 15% grade for untrained and trained rats, respectively) significantly elevated hepatic catalase and cytosolic SOD activity; more prominent activations were found in the Se-D or untrained rats, whereas skeletal muscle antioxidant enzymes were little affected. Ten weeks of training (1 h/day, 5 days/week at 27 m/min, 15% grade) increased hepatic mitochondrial SOD by 23% (P less than 0.05) in Se-D rats. Both hepatic mitochondrial and cytosolic GPX were decreased by training whereas GPX was increased twofold in skeletal muscle mitochondria. Se-independent GPX was elevated by training only in the skeletal muscle mitochondria of Se-D rats. Lipid peroxidation (malondialdehyde formation) was increased by an acute bout of exercise in hepatic mitochondria of the untrained rats and in skeletal muscle mitochondria of the Se-D rats. These data indicate that antioxidant enzymes in liver and skeletal muscle are capable of adapting to selenium deficiency and exercise to minimize oxidative injury caused by free radicals.     

The content of reduced glutathione in rat liver after poisoning with white phosphorus

The content of hepatic GSH was evaluated in rats after poisoning with white phosphorus. In addition, liver damage following the administration of the hepatotoxin was assessed by determining hepatic triglyceride accumulation. Experiments in parallel were carried out in an attempt to evaluate the enhanced susceptibility of hepatic tissue to peroxidative decomposition of unsaturated lipids 'in vitro', as measured by the production of TBA-reacting substances. Our data indicate that only in the early stage of intoxication is it possible to detect a slight decrease of GSH content in the liver, while during the subsequent stages the concentration of GSH was unaffected. At 6 hours of intoxication the level of hepatic triglycerides was significantly increased. Pretreatment with GSH was followed by an amelioration of fatty infiltration, but the content of hepatic GSH was unchanged. The production of TBA-reacting products was found enhanced only at 6 hours of intoxication. These results are discussed in relation to the role of lipid peroxidation in liver injury by white phosphorus.     

Differential lipid peroxidative response of rat liver and lung tissues to glutathione depletion induced in vivo by diethyl maleate: effect of the antioxidant flavonoid (+)-cyanidanol-3

The administration of a single dose of diethyl maleate (DEM) to fed rats elicited a drastic decrease in the content of reduced glutathione (GSH) both in liver and lung tissues after 6 h of treatment. Cellular GSH depletion induced by DEM was accompanied by a marked increase in pulmonary lipid peroxidation which was completely abolished by (+)-cyanidanol-3, without changes in the liver. Superoxide dismutase (SOD) activity remained unchanged in both tissues in this situation. Hepatic and pulmonary GSH depletion induced by a second dose of DEM given 24 h later produced a further increase in lung lipid peroxidation and a diminution of pulmonary SOD activity. In this condition, hepatic lipid peroxidation and SOD activity were not altered. These results indicate that lung and liver tissues exhibit a different lipid peroxidative response to chemically-induced GSH depletion.     

肝硬化大鼠细菌易位以及甜菜碱的干预作用

【目的】研究肝硬化大鼠的细菌易位情况,并探讨甜菜碱对其的干预作用。【方法】随机将48只健康雄性SD(Sprague dawley,SD)大鼠分为四组:正常对照组(N),甜菜碱干预的正常对照组(NB),肝硬化模型组(M),甜菜碱干预的肝硬化模型组(MB)。采用复合致病因素法诱导大鼠肝硬化,NB组和MB组使用1 000 mg/(kg w·d)的甜菜碱水溶液灌胃,N组和M组使用等体积饮用水灌胃;HE染色观察肝脏与小肠损伤情况,并检测各组动物脏器指数与细菌易位情况。【结果】M组大鼠体重增长缓慢(与N组比较,4周和6周时间点均P=0,P0.01);与M组相比,MB组大鼠体重增长较快,至6周时间点体重差异显著(P=0.023,P0.05)。与N组相比,M组动物4周时间点肝脏指数显著升高(P=0,P0.01);6周时间点肝脏(P=0,P0.01)、脾脏指数(P=0.038,P0.05)均显著升高,肾脏指数显著降低(P=0.019,P0.05);与M组相比,6周时间点MB组动物肝脏指数(P=0.038,P0.05)明显降低,肾脏指数(P=0.011,P0.05)明显升高。M组动物肝、肠组织发生明显病理学改变;MB组动物病理学改变减轻。M组发生细菌易位的大鼠数量升高,4周时主要易位于MLN,6周时主要易位于MLN和肾脏;MB组发生细菌易位的大鼠数量有所减少,其中6周时易位至MLN的大鼠数量明显减少(P=0.046,P0.05)。【结论】复合致病因素诱导的肝硬化大鼠,发生细菌易位的器官主要是MLN和肾脏,易位的细菌可通过淋巴管道转位,并随肝硬化病程进展趋于严重。甜菜碱除了其转甲基的保护作用以外,很可能还通过对肠道的保护作用,在一定程度上阻止了肝硬化动物细菌易位的发生,从而发挥了对肝脏的保护作用     

Thalidomide decreases intrahepatic resistance in cirrhotic rats

Increased intrahepatic resistance (IHR) within cirrhotic liver is caused by increased endotoxemia, cytokines tumor necrosis factor-α (TNF-α), vasoconstrictor thromboxane A₂ (TXA₂), and disrupted microvasculatures. We evaluated the effects of thalidomide-related inhibition of TNF-α upon the hepatic microcirculation of cirrhosis in rats. Portal venous pressure (PVP), hepatic TNF-α, expression of thromboxane synthase (TXS), and leukocyte common antigen (LCA) were measured in bile-duct-ligated (BDL) rats receiving 1 month of thalidomide (BDL-thalido rats). Portal perfusion pressure (PPP), IHR, and hepatic TXA₂ production were measured in the isolated liver perfusion system. Intravital microscopy was used to examine hepatic microvascular disruptions. In BDL-thalido rats, PVP, PPP, IHR, hepatic TXA₂ and TNF-α, hydroxyproline content, expression of TXS and LCA, and LPS-induced leukocyte recruitment were significantly decreased. Conversely, hepatic microvascular density and perfused sinusoids were significantly increased. Thalidomide decreased PVP and IHR by reducing hepatic TXA₂ and improving hepatic microvascular disruptions in rats with biliary cirrhosis.     

Tamarix gallica ameliorates thioacetamide-induced hepatic oxidative stress and hyperproliferative response in Wistar rats

Tamarix gallica, a hepatic stimulant and tonic, was examined for its ability to inhibit thioacetamide (TAA)-induced hepatic oxidative stress, toxicity and early tumor promotion response in male Wistar rats. TAA (6.6 mmol/kg body wt. i.p) enhanced lipid peroxidation, hydrogen peroxide content, glutathione S-transferase and xanthine oxidase with reduction in the activities of hepatic antioxidant enzymes viz., glutathione peroxidase, superoxide dismutase and caused depletion in the level of hepatic glutathione content. A marked increase in liver damage markers was also observed. TAA treatment also enhanced tumor promotion markers, ornithine decarboxylase (ODC) activity and [3H] thymidine incorporation into hepatic DNA. Pretreatment of rats orally with Tamarix gallica extract (25 and 50 mg/kg body weight) prevented TAA-promoted oxidative stress and toxicity. Prophylaxis with Tamarix gallica significantly reduced the susceptibility of the hepatic microsomal membrane for iron-ascorbate induced lipid peroxidation, H2O2 content, glutathione S-transferase and xanthine oxidase activities. There was also reversal of the elevated levels of liver marker parameters and tumor promotion markers. Our data suggests that Tamarix gallica is a potent chemopreventive agent and may suppress TAA-mediated hepatic oxidative stress, toxicity, and tumor promotion response in rats.     

Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.     

Protective effects of dietary cyanidin 3-O-beta-D-glucoside on liver ischemia-reperfusion injury in rats.

We recently reported that feeding cyanidin 3-O-beta-d-glucoside (C3G), a typical anthocyanin pigment, lowered the serum thiobarbituric acid-reactive substance (TBARS) concentration and increased the oxidation resistance of the serum to lipid peroxidation in rats. These results suggest that C3G acts as a potent antioxidant in vivo when acute oxidative stress is encountered. In the present study, we evaluated whether feeding C3G suppresses oxidative injury to the liver caused by hepatic ischemia-reperfusion (I/R), which was used as a model for oxidative stress. Rats were fed a diet containing C3G (2 g/kg diet) for 14 days and then subjected to hepatic I/R. I/R treatment elevated the liver TBARS concentration and the serum activities of marker enzymes (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase) for liver injury and lowered the liver reduced glutathione concentration. Feeding C3G significantly suppressed these changes caused by hepatic I/R. Although the liver ascorbic acid concentration was also lowered by hepatic I/R, feeding C3G restored this concentration more quickly compared to the control rats. These results indicate that orally administered C3G suppresses I/R-induced oxidative damage and suggest that C3G functions as a potent antioxidant in vivo under oxidative stress.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.     

Secretion and elimination of insulin by the in vitro perfused pancreas and liver of rats with thioacetamide-induced liver cirrhosis

Pancreatic secretion and hepatic removal of insulin have been measured in thioacetamide (TAA)-induced compensated rat liver cirrhosis in perfusion experiments. Peripheral plasma concentrations of glucose and insulin were slightly decreased in TAA-treated rats. Pancreatic secretion and hepatic removal of insulin remained unchanged by the TAA-treatment. Thus, even in morphologically and biochemically proven experimental liver cirrhosis, insulin secretion and removal may not be disturbed.