Diabetes mellitus triggers oxidative stress in the liver of alloxan-treated rats: A mechanism for diabetic chronic liver disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection

PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.

Absence of chemopreventive influence of propolis on the rat liver altered foci development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of toxicity after one-months treatment with Bauhinia forficata decoction in streptozotocin-induced diabetic rats

Background: Previous experiments have shown that a decoction of Bauhinia forficata leaves reduces the changes in carbohydrate and protein metabolism that occur in rats with streptozotocin-induced diabetes. In the present investigation, the serum activities of enzymes known to be reliable toxicity markers were monitored in normal and streptozotocin-diabetic rats to discover whether the use of B. forficata decoction has toxic effects on liver, muscle or pancreas tissue or on renal microcirculation. Methods: An experimental group of normal and streptozotocin-diabetic rats received an aqueous decoction of fresh B. forficata leaves (150 g/L) by mouth for 33 days while a control group of normal and diabetic rats received water for the same length of time. The serum activity of the toxicity markers lactate dehydrogenase, creatine kinase, amylase, angiotensin-converting enzyme and bilirubin were assayed before receiving B. forficata decoction and on day 19 and 33 of treatment. Results: The toxicity markers in normal and diabetic rats were not altered by the diabetes itself nor by treatment with decoction. Whether or not they received B. forficata decoction the normal rats showed a significant increase in serum amylase activity during the experimental period while there was a tendency for the diabetic rats, both treated and untreated with decoction, to have lower serum amylase activities than the normal rats. Conclusions: Administration of an aqueous decoction of B. forficata is a potential treatment for diabetes and does not produce toxic effects measurable with the enzyme markers used in our study. © 2004 Pepato et al; licensee BioMed Central Ltd.

Water-soluble extract of Coleus barbatus modulates weight gain, energy utilization and lipid metabolism in secondary biliary cirrhosis: an experimental study in young rats.

PURPOSE: To test if a water extract of Coleus barbatus (WEB) has any effect on weight gain, food energy utilization and lipid metabolism in young rats with obstructive cholestasis. METHODS: Forty 21 day old (P21) Wistar rats, in groups of 10, were submitted to one of the following treatments: a sham operation with daily water or WEB administration, double ligature and resection of the bile duct with daily water or WEB administration. At P49 they were submitted for euthanasia when the following were determined: ingested feed (IF), energy utilization (EU) and weight gain (WG) from P29 to P49, together with total serum cholesterol (TC) and triacylglycerol (TG) concentrations, liver wet weight (LWW) and fat content (LFC). Two Way ANOVA and the S.N.K. test for paired comparisons were employed to study the effects of cholestasis and those of WEB and their interactions (p < or = 0.05). RESULTS: Cholestasis, independently of WEB, and WEB, independently of cholestasis both reduced IF, EU, and WG but there was no significant interaction between the two factors. Cholestasis, independently of WEB, increased LWW, LFC, the TC and TG The WEB, independently of cholestasis, reduced these values, and there was a significant interaction between the two factors; such that these effects were more accentuated in animals with cholestasis. CONCLUSION: The WEB reduced IF, WG, and EU, both in the presence and absence of cholestasis in the same proportion. It also partially inhibited the increase in LWW, LFC, TC and TG caused by cholestasis.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

Critical role of interleukin 5 and eosinophils in concanavalin A-induced hepatitis in mice.

BACKGROUND & AIMS: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model. METHODS: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils. RESULTS: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not. CONCLUSIONS: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.

CD4+ T cells play an important role in acute experimental pancreatitis in mice

BACKGROUND & AIMS: Few data are available on the potential role of T lymphocytes in experimental acute pancreatitis. The aim of this study was to characterize their role in the inflammatory cascade of acute pancreatitis. METHODS: To type this issue, acute pancreatitis was induced by repeated injections of cerulein in nude mice and in vivo CD4(+) or CD8(+) T cell-depleted mice. The role of T lymphocyte-costimulatory pathways was evaluated using anti-CD40 ligand or anti-B7-1 and -B7-2 monoclonal blocking antibodies. The role of Fas-Fas ligand was explored using Fas ligand-targeted mutant (generalized lymphoproliferative disease) mice. Severity of acute pancreatitis was assessed by serum hydrolase levels and histology. Intrapancreatic interleukin 12, interferon gamma, Fas ligand, and CD40 ligand messenger RNA were detected by reverse-transcription polymerase chain reaction. Intrapancreatic T lymphocytes were identified by immunohistochemistry. RESULTS: In control mice, T cells, most of them CD4(+) T cells, are present in the pancreas and are recruited during acute pancreatitis. In nude mice, histological lesions and serum hydrolase levels are significantly decreased. T-lymphocyte transfer into nude mice partially restores the severity of acute pancreatitis and intrapancreatic interferon gamma, interleukin 12, and Fas ligand gene transcription. The severity of pancreatitis is also reduced by in vivo CD4(+) (but not CD8(+)) T-cell depletion and in Fas ligand-targeted mutant mice. Blocking CD40-CD40 ligand or B7-CD28 costimulatory pathways has no effect on the severity of pancreatitis. CONCLUSIONS: T lymphocytes, particularly CD4(+) T cells, play a pivotal role in the development of tissue injury during acute experimental pancreatitis in mice.

Induction of alloxan/streptozotocin diabetes in dogs: a revised experimental technique

The diabetic dog represents an excellent model for use in many aspects of diabetic research. The present paper describes, in detail, a reproducible experimental protocol for the successful induction of chemical diabetes in beagles using a combination of the 2 pancreatic beta-cell cytoxic agents alloxan and streptozotocin.

An experimental study of quinine blindness

An experimental model of quinine induced blindness is presented. Electrophysiological, angiographical and morphological examinations were made. The occurrence of blindness and any recovery from blindness was dependent upon the dose of quinine taken. As no evidence of acute retinal ischaemia was found it is concluded that quinine is retinotoxic.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection.

Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection. We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance. In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages. Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.

Investigation on key molecular targets responsible for antidiabetic properties of Symplocos cochinchinensis (Lour.) S. Moore

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in insulin secretion, insulin action or both. Currently there are 387 million people with diabetes worldwide and is expected to affect 592 million people by 2035. Insulin resistance in peripheral tissues and pancreatic beta cell dysfunction are the major challenges in the pathophysiology of diabetes. Diabetic secondary complications (like liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from persistent hyperglycemia and dyslipidemia can be disabling or even life threatening. Current medications are effective for control and management of hyperglycemia but undesirable effects, inefficiency against secondary complications and high cost are still serious issues in the present prognosis of this disorder. Hence the search for more effective and safer therapeutic agents of natural origin has been found to be highly demanding and attract attention in the present drug discovery research. The data available from Ayurveda on various medicinal plants for treatment of diabetes can efficiently yield potential new lead as antidiabetic agents. For wider acceptability and popularity of herbal remedies available in Ayurveda scientific validation by the elucidation of mechanism of action is very much essential. Modern biological techniques are available now to elucidate the biochemical basis of the effectiveness of these medicinal plants. Keeping this idea the research programme under this thesis has been planned to evaluate the molecular mechanism responsible for the antidiabetic property of Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown Ayurvedic antidiabetic preparation. A general introduction of diabetes, its pathophysiology, secondary complications and current treatment options, innovative solutions based on phytomedicine etc has been described in Chapter 1. The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract. Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in vitro methods against multiple targets such as control of postprandial hyperglycemia, insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos cochinchinensis (SCE) against hyperglycemia associated secondary complications in streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE 500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22. Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21, DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c, decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and renal oxidative stress markers compared to NC. DC rats also exhibited increased level of plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations of biochemical parameters in a dose dependent manner including histopathological alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and function markers compared to DC group. Considerable amount of liver and muscle glycogen was replenished by SCE treatment in diabetic animals. Although metformin showed better effect, the activity of SCE was very much comparable with this drug. xxii The possible molecular mechanism behind the protective property of S. cochinchinensis against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE 250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance, HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues. Overall results suggest that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant activities.

Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats

Primary sensory afferent neurons modulate the hyperdynamic circulation in Cirrhotic rats with portal hypertension.The stomach of cirrhotic rats is prone to damage induced by ethanol, a phenomenon associated with reduced gastric hyperemic response to acid-back diffusion. The aim of this study was to examine the impact of ablation of capsaicin-sensitive neurons and the tachykinin NK(1) receptor antagonist A5330 on the susceptibility of the portal hypertensive gastric mucosa, to ethanol-induced injury and its effects on gastric cyclooxygenase (COX) and nitric oxide synthase (NOS) mRNA expression. Capsaicin was administered to neonatal, male, Wistar rats and the animals were allowed to grow. Cirrhosis was then induced by bile duct ligation in adult rats while controls had sham operation. Ethanol-induced gastric damage was assessed using ex vivo gastric chamber experiments. Gastric blood flow was measured as well as COX/NOS mRNA expression. Topical application of ethanol produced significant gastric damage in cirrhotic rats compared to controls, which was reversed in capsaicin- and A5330-treated animals. Mean arterial and portal pressure was normalized in capsaicin-treated cirrhotic rats. Capsaicin and A5330 administration restored gastric blood flow responses to topical application of ethanol followed by acid in cirrhotic rats. Differential COX and NOS mRNA expression was noted in bile duct ligated rats relative to controls. Capsaicin treatment significantly modified gastric eNOS/iNOS/COX-2 mRNA expression in cirrhotic rats. Capsaicin-sensitive neurons modulate the susceptibility of the portal hypertensive gastric mucosa to injury induced by ethanol via tachykinin NK(1) receptors and signalling of prostaglandin and NO production/release. (c) 2008 Elsevier B.V. All rights reserved.