Protective effect of Echinochrome against intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats / Efeito protetor de Echinochrome contra colestase intra-hepática induzida por isotiocianato de alfa-naftilo em ratos

Abstract The present study was designed to evaluate the protective effects of echinochrome (Ech) on intrahepatic cholestasis in rats induced by a single (i.p.) injection of alpha-naphthylisothiocyanate (ANIT) (75 mg/kg body weight). The rats were pre-treated orally for 48hr (one dose / 24hr) with Ech (1, 5 and 10 mg/kg body weight) or ursodeoxycholic acid (UDCA) 80 mg/kg body weight drug then, injected with ANIT. ANIT markedly increased serum activities of alanine amino transaminase (ALT), aspartate amino transaminase (AST) and alkaline phosphatase (ALP), which was accompanied by a massive inflammation of epithelial cells on bile duct at 24h after ANIT injection. ANIT also increased the levels of total protein (TP), total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), however decrease albumin content (ALB). In addition ANIT increased hepatic MDA and NO level and decreased GSH level and GST activity. The Ech exerted hepatoprotective and anticholestatic effects as assessed by a significant decrease in the activities of serum AST, ALT and ALP, and the levels of TP, TB, DB and IB as well as liver MDA level and NO level. In conclusion, Ech was found to possess hepatoprotective effect against intrahepatic cholestasis induced by hepatotoxin such as ANIT.

Resumo O presente estudo destinou-se a avaliar os efeitos protetores do Ech na colestase intra-hepática em ratos induzidos por uma única injeção (i.p.) de alfa-naftilisotiocianato (ANIT) (75 mg / kg de peso corporal). Os ratos foram pré-tratados oralmente durante 48 horas (uma dose / 24 horas) com cada (1, 5 e 10 mg / kg de peso corporal) e ácido ursodeoxicólico (UDCA) 80 mg / kg de peso corporal, em seguida, injetado com ANIT. ANIT atividades de soro marcadamente aumentadas de alanina amino transaminasa (ALT), aspartato de amino transaminases (AST) e fosfatase alcalina (ALP), que foi acompanhada por uma inflamação maciça de células epiteliais no ducto biliar às 24h após a injeção de ANIT. A ANIT também aumentou os níveis de proteína total (TP), bilirrubina total (TB), bilirrubina direta (DB), bilirrubina indireta (IB), no entanto, diminuem o teor de albumina (ALB). Além disso, a ANIT aumentou o nível de MDA hepático e NO e diminuiu o nível de GSH e a atividade de GST. O Ech exerceu efeitos hepatoprotectores e anticolestáticos como avaliado por uma diminuição significativa nas atividades de AST sérica, ALT e ALP e os níveis de TP, TB, DB e IB, bem como o nível de MDA no fígado e o nível de NO. Ech foi encontrado para possuir efeito protetor no fígado contra a colestase intra-hepática induzida por hepatotoxina, como a ANIT.

Changes of pharmacokinetics of 6,7-dimethoxycoumarin in a rat model of alpha-naphthylisothiocyanate-induced experimental hepatic injury after Yinchenhao Decoction () treatment / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the changes of pharmacokinetics of 6,7-dimethoxycoumarin in a rat model of alpha-naphthylisothiocyanate (ANIT)-induced experimental hepatic injury after oral administration of Yinchenhao Decoction (, YCHD) using an ultra pressure liquid chromatography (UPLC) method.</p><p><b>METHODS</b>Rats were divided into a normal group and a model group, after modeled by 4% ANIT (75 mg/kg) for 48 h, they were orally administrated with YCHD extract at the dose of 0.324 g/kg, and then blood was collected from their orbital sinus after different intervals. Changes in liver function were monitored by the levels of liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] and bilirubins [total bilirubin (TBIL), direct bilirubin (DBIL)], the concentration of 6,7-dimethoxycoumarin in plasma were measured by UPLC, and the pharmaceutical parameters were calculated with DAS2.1.1 software.</p><p><b>RESULTS</b>The concentration-time curve of both normal and modeled rats after oral administration of YCHD was obtained. Their time to maximum plasma concentration (t(max)) were both 0.25 h, the maximum concentration (C(max)) were 4.533 μg/mL and 6.885 μg/mL, and their area under concentration-time curve (AUC)(0→24h) were 16.272 and 32.981, respectively. There was a 51.88% and 100.46% increase in C(max) and AUC(0-t) (P<0.05), but there showed a 45.52% and 92.93% reduction in clearance of drug and volum of distribution (P<0.05), respectively.</p><p><b>CONCLUSIONS</b>Hepatic injury could significantly influence the pharmacokinetics of 6,7-dimethoxycoumarin after oral administration of YCHD, the absorption and distribution process was accelerated in liver injured rats, but the metabolism and elimination process was slowed. And this may lead to a significant accumulation of 6,7-dimethoxycoumarin in the body.</p>

Evaluation study on urine metabolomics in yinhuang rat model induced by triplet factors of rhubarb, ethanol, and α-nephthylisothiolyanate / 中国结合医学杂志

<p><b>OBJECTIVE</b>To ascertain the biomarkers capable to characterize the animal composite model of Chinese medicine (CM) yinhuang syndrome induced by triplet factors of rhubarb, ethanol, and α-nephthylisothiolyanate (abbreviated as R, E, and A below) through metabolomic study and to evaluate the established model by means of studying the sources of markers based on the changes of metabolites produced from various combinations of the three modeling drugs.</p><p><b>METHODS</b>Eighty Wistar rats allocated equally in eight groups (A-H) were treated with saline, R+E+A, R, E, A, R+E, R+A, and E+A, respectively. Rats' 12 h urine in the 14 successive experimental days were collected separately using metabolic cages and analyzed by ultra performance liquid chromatograph/time of flight mass spectrometer (UPLC/TOF-MS) to create the metabolic contour graph of urine in different groups for identifying the differences between them. The similarities and differences of metabolic network among various groups were represented from microcosmic viewpoint by pattern recognition method (principal component analysis).</p><p><b>RESULTS</b>Controlled by group A, the landing points in principal component map of various groups were apparently assorted, especially obvious on the 14th day; 19 biomarkers, which capable to represent the genesis and development process of the yinhuang syndrome in the triplet factors-induced rat model, were identified.</p><p><b>CONCLUSION</b>Metabolomic method is successfully used in evaluating the animal model of CM syndrome. Furthermore, according to the holistic view and substance changes in vivo, the influences of disease on organism were comprehensively analyzed, and the pathogenic mechanism of CM yinhuang syndrome was explored at the level of metabolomics in vivo as well.</p>

Effect of emodin on P-gp expression in intrahepatic cholestatic rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of emodin on acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT) in rats.</p><p><b>METHOD</b>Acute cholestatic model in rats was induced by ANIT. Normal control group, emodin group without ANIT treatment, model group and emodin group with ANIT treatment were set up. Liver function and pathological changes of hepatic tissue were examined. Real-time fluorescent quantitative RT-PCR was used to detect the mRNA levels of the hepatic transport protein genes mdr1a (multidrug resistance protein 1a), mdr1b (multidrug resistance protein 1b) mdr2 (multidrug resistance protein 2), The expression of P-gp were determined by Western blotting analysis.</p><p><b>RESULT</b>Compared to the model group, Emodin treatment resulted in significant reductions in serum total bilirubin (TBiL), direct bilirubin (DBiL), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA) (P < 0.01 or P < 0.05). By examining the liver pathology, it was found that hepatic cellular change and necrosis, inflammatory cell infiltration and bile duct proliferation were notably alleviated in emodin model with ANIT treatment. Analysis of gene expression in livers from emodin-treated cholestatic rats revealed that mdr1a, mdr1b and mdr2 could be up-regulated (P < 0.01 or P < 0.05), expression of P-gp was increased in accordance with its mRNA (P < 0.05).</p><p><b>CONCLUSION</b>Emodin has a protective effect on hepatocytes and a restoring activity on cholestatic hepatitis. Mechanism of its action may be related to induce expression of the bile-metabolism-related transporter P-gp in the liver to prevent bile acids and other toxic compounds overaccumulation in hepatocytes and hepatic toxicity.</p>

Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride / 中国结合医学杂志

<p><b>OBJECTIVE</b>To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine.</p><p><b>METHODS</b>The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition.</p><p><b>RESULTS</b>The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05).</p><p><b>CONCLUSION</b>YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).</p>

Effect and mechanism of emodin on cholestatic hepatitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the therapeutic effect and mechanism of emodin on cholestatic hepatitis.</p><p><b>METHODS</b>Rats were divided into 5 groups: 1 group was untreated, the other 4 groups were treated with alpha-naphthylisothiocyanate (ANIT), ANIT and emodin, ANIT and ursodeoxycholic acid, or ANIT and dexamethasone, respectively. At 24 h, 48 h and 72 h after the treatment, NF-kappa B, early growth response factor-1 (Egr-1), cytokine-induced neutrophil chemoattractant 1 (CINC-1), macrophage inflammatory protein 2 (MIP-2), intercellular adhesion molecule 1 (ICAM-1),tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) were assayed by immunohistochemistry, real-time PCR , western-blot and ELISA. The level of malondialdehyde (MDA), superoxide Dismutase(SOD) and myeloperoxidase (MPO) were assayed by thiobarbituric acid method, xanthine oxidase method and colorimetric method, respectively.</p><p><b>RESULTS</b>(1) Compared to the controls, emodin had a notable effect on total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT) at all time points (all P less than 0.05). Compared to ursodeoxycholic acid, emodin had a notable effect on TB and DB at 24 h after the treatments, however, after 48 h, emodin had a notable effect only on TB (all P less than 0.05). Compared to Dexamethasone, emodin had a notable effect on TB at 48 h time point, and it had a notable effect on ALT at all time points (all P less than 0.05). (2) The nuclei NF-kappa B p65 staining was significantly increased at 24 h and 48 h after ANIT treatment (all P less than 0.05), and emodin treatment could block the increase (all P less than 0.05). (3) Egr-1 mRNA level was not affected by emodin treatment (P more than 0.05); levels of CINC-1, MIP-2 mRNA and ICAM-1 protein were significantly decreased after emodin treatment (all P less than 0.05). (4) The levels of TNF alpha and IL-6 were decreased after emodin treatment(all P less than 0.05). (5) The levels of MDA at all time points and MPO at 24 h, 48 h time points were notably down-regulated by emodin treatment, while the level of SOD was markedly elevated at all time points after emodin treatment (all P less than 0.05).</p><p><b>CONCLUSIONS</b>Emodin treatment can reduce the levels of TB, DB and ALT in ANIT induced-cholestatic hepatitis. The effect may be due to inhibition of NF-kappa B signal pathway.</p>

Role of early growth response factor-1 signal pathway in acute intrahepatic cholestatic hepatic injury in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the role of early growth response factor-1 (Egr-1) signal pathway in acute intrahepatic cholestatic liver injury in rats.</p><p><b>METHODS</b>A single dose (50 mg/kg) of alpha-naphthylisothiocyanate (ANIT) was administered by gavage to each experimental rat to induce intrahepatic cholestasis. Liver tissue and serum specimens were collected from rats at 24, 48 and 72 h after the intoxication. The values of Egr-1, cytokine induced neutrophil chemoattractant 1(CINC-1), macrophage inflammatory protein-2 (MIP-2) mRNA, the protein expression of inducible nitricoxide synthase (iNOS) and the values of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) were assayed by real-time PCR, immunohistochemistry, and ELISA, respectively. The levels of MDA, SOD, NO and MPO were assayed by thiobarbituric acid method, xanthine oxidase method, nitric acid deoxidizing assay, and colorimetric method, respectively.</p><p><b>RESULTS</b>In the model group at 24, 48, 72 h after intoxication, the values of CINC-1 and MIP-2 mRNA were higher than those of the controls (P < 0.05). In the model group at 24, 48 h after intoxication, the value of Egr-1 mRNA was higher than that of the controls (P < 0.05), but there was no significant difference at 72 h (P < 0.05). Of the model group, the absorbance value of iNOS was lower than that of the controls at 24, 48 and 72 h (P < 0.05). Of the model group at 24, 48, 72 h after intoxication, the values of TNF alpha, IL-6, MPO and MDA were higher than those of the controls (P < 0.05), but the values of NO and SOD were lower than those of the controls (P < 0.05).</p><p><b>CONCLUSIONS</b>Egr-1 signal pathway is involved in acute intrahepatic cholestatic liver injury induced by ANIT. After Egr-1 was activated, CINC-1 and MIP-2 are activated consequently and attract neutrophils into the liver. TNF alpha and IL-6 are activated at the same time, so neutrophils are activated and the resulting lipid peroxidation and MDA increased, injuring the liver. iNOS and NO may play a protective role in acute intrahepatic cholestatic liver injury induced by ANIT.</p>

Induction of tumour necrosis factor alpha in experimental animals treated with hepatotoxicants.

Administrations of hepatotoxicants namely carbon tetrachloride (CCl4:0.4 ml in 1.2 ml of liquid paraffin) and ANIT (1 ml of 1.5% solution in liquid paraffin) in Charles foster rats (force fed) and D-galactosamine (8 mg in water per swiss albino mouse, ip) induce the release of TNF-alpha in case of CCl4 and D-galactosamine. High TNF-alpha level was observed up to 48 hr in CCl4 and up to 24 hr in D-galactosamine treated animals. Elevated levels of biochemical like ALP and SGPT are also recorded. TNF-alpha level can be measured of tissue damage and prognosis in case of hepatitis.