The novel role of β-aescin in attenuating CCl4-induced hepatotoxicity in rats

Context: β-Aescin has anti-inflammatory, anti-oxidant and antiedematous properties.

Objective: The present study investigated the hepatoprotective effect and underlying mechanisms of β-aescin in CCl₄-induced liver damage.

Materials and methods: Thirty-five Wistar rats were divided into six groups: normal control, CCl₄ control, silymarin (50?mg/kg, p.o) and β-aescin (0.9, 1.8 and 3.6?mg/kg, i.p.) treatment for 14 d. CCl₄ (1?mL/kg, i.p. for 3 d) was administered to produce hepatic damage. Ponderal changes and liver marker enzymes were estimated. Hepatic oxidative and nitrosative stress was estimated by levels of thiobarbituric acid reactive substances (TBARS), glutathione (GSH) and nitrite/nitrate. Serum TGF-β1 and TNF-α were estimated by ELISA technique. Hepatic collagen and histopathological studies were carried out.

Results: β-Aescin (3.6?mg/kg) markedly decreased CCl₄-induced increased levels of ALT, AST, ALP (71.77 versus 206.7, 71.39 versus 171.82, 121.20 versus 259?IU/L, respectively), total bilirubin (0.41 versus 1.35?mg/dL), TBARS (2.0 versus 8.83?nmol MDA/mg protein), nitrite/nitrate (352.50 versus 745.15?μg/mL) and increased CCl₄-induced decreased GSH levels (0.095 versus 0.048?μmol/mg protein). β-Aescin (3.6?mg/kg) induced focal regenerative changes in liver and markedly decreased TBARS (2.0 versus 8.83?nmol MDA/mg protein), nitrite/nitrate (352.50 versus 745.15?μg/mL), TGF-β1 (92.28 versus 152.1?pg/mL), collagen content (110.75 versus 301.74?μmol/100?mg tissue) and TNF-α (92.82 versus 170.56?pg/mL) when compared with CCl₄ control.

Discussion and conclusion: The findings suggest that β-aescin has a protective effect on CCl₄-induced liver injury, exhibited via its anti-inflammatory, antioxidative, antinitrosative and antifibrotic properties inducing repair regeneration of liver. Hence, it can be used as a promising hepatoprotective agent.     

Exposure–Response modeling of anti-depressant treatments: the confounding role of placebo effect

Emerging evidence indicates that the selection of patients and the selection of recruitment centers in placebo-controlled, randomized clinical trials (RCTs) in depression have a substantial impact on the probability of observing a treatment effect of a novel medication. The objective of this work was to evaluate the role of placebo in characterizing the exposure–response relationship and proposes a new methodology based on band-pass filtering for assessing the relationship between drug exposure, level of clinical response conditioned to the level of placebo response, and the potency of the antidepressant drug evaluated. Clinical trial simulation (CTS) was used to demonstrate that the conventional analyses of data generated in multi-centre RCTs including centres exhibiting heterogeneous placebo response can lead to contradictory and inappropriate assessment of the exposure–response relationship. To address this issue, a novel modelling approach has been developed for establishing the exposure–response relationship by using a pharmacodynamic model accounting for the placebo effect. The proposed model demonstrated that the expected drug related treatment effect in a placebo-controlled RCT can be predicted as a function of the dose, the drug potency and the level of placebo response when data from informative recruitment centres are considered. With this novel approach, a more accurate estimate of the dose/exposure response can be derived and more informed go/no-go decisions can be made in developing drugs for psychiatric disorders.     

Application of proteomic and bioinformatic techniques for studying the hepatoprotective effect of dioscin against CCl₄-induced liver damage in mice

In this study, the significant hepatoprotective effect of dioscin against CCl?-induced acute liver damage in mice was first discovered, and the effect produced by dioscin at the dose of 100 mg/kg was equal to the action produced by silymarin at the dose of 200 mg/kg. Then, 1-dimension gel electrophoresis was used to separate the liver proteins, and five differentially expressed bands were selected. After in-gel digestion, 71 proteins were identified by nano-RP-HPLC-ESI-MS/MS/MS. Further network analysis suggested that the identified proteins formed a connected protein interaction subnetwork. Ten functional categories were selected to demonstrate the distribution of the proteins by Gene Ontology (GO) enrichment analysis. Six of the proteins, heat shock protein 5 (HSPA5), annexin 6 (ANXA6), isovaleryl-CoA dehydrogenase (IVD), ribosomal protein S6 (RPS6), cytoglobin (Cygb), and nucleoside diphosphate kinase A (NDPK-A), were validated by Western blotting assay. They might be involved in the hepatoprotective effect of dioscin, and their investigation could be useful, together with the determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels, as well as the liver histopathologic study, for the elucidation of the action mechanisms of dioscin against CCl?-induced liver injury. Our work shows that the validated proteins should be considered as biomarkers for the investigation of acute liver injury, and its results should contribute to the therapy of liver damage by dioscin in the future.     

Protective effect of L-carnitine and coenzyme Q10 on CCl₄-induced liver injury in rats

This study provides an information about the mechanisms of liver injury induced by CCl(4), and determines the influence of administration of L-carnitine or/and CoQ10 as prophylactic agents against CCl(4) deteriorative effect. The study was carried out on 80 adult male albino rats divided into eight groups, 10 animals each, as follows: four normal groups (control, treated with L-carnitine, treated with CoQ10, and treated with a combination of Lcarnitine and CoQ10) and four liver injury groups treated with CCl(4) (control, treated with L-carnitine, treated with CoQ10, and treated with a combination of L-carnitine and CoQ10). Liver injury was induced by s.c. injection of a single dose of CCl(4) (1 ml/kg). L-carnitine (50 mg/kg/day) was given i.p. for four successive days 24 hours before CCl(4) injection, and CoQ10 (200 mg/kg) was given as a single i.p. dose 24 hours before CCl(4) injection. Animals were sacrificed 24 hours after CCl(4) injection, blood samples were withdrawn and liver tissue samples were homogenized. The levels of the following parameters were determined: hepatic reduced glutathione, serum ALT and AST, hepatic lipid peroxides, hepatic vitamin C, hepatic and serum total protein, serum albumin, serum sialic acid, serum nitrite, and serum and hepatic total LDH activities and LDH isoenzymes. The obtained data revealed that CCl(4) injection produced a significant decrease in reduced glutathione content, vitamin C, total protein and albumin levels. However, there was a significant increase in serum ALT and AST activities, lipid peroxides, sialic acid, nitric oxide, serum and hepatic total LDH activities. On the other hand, groups treated with L-carnitine or/and CoQ10 prior to CCl(4) injection showed an improvement in most parameters when compared with cirrhotic control group. It has been concluded that L-carnitine and coenzyme Q10 have a pronounced prophylactic effect against liver damage induced by halogenated alkanes such as carbon tetrachloride.     

An evaluation of the protective role of α-tocopherol on free radical induced hepatotoxicity and nephrotoxicity due to chromium in rats

Aim:

To avert the health problems induced by many environmental pollutants, available antioxidants have been evaluated. The present study was aimed to investigate whether α-tocopherol could protect the hexavalent chromium (Cr VI)-induced peroxidation in the liver and kidney and to explore the underlying mechanism of the same.

Materials and Methods:

A total of 24 Wistar adult female rats were equally divided into four groups. Group 1 served as control while Groups 2 and 3 were administered K₂Cr₂O₇(10 mg/kg b.wt. s.c. single dose). In addition to (Cr VI), Group 3 also received α-tocopherol (125 mg/kg, daily) by oral gavage for 14 days. Group 4 was maintained as α-tocopherol control (dose as above). At the end of 14 days, blood samples were drawn for hematology. Subsequently, all the rats were sacrificed to collect liver and kidney samples for assay of tissue peroxidation markers, antioxidant markers and functional markers and histopathology.

Results:

Administration of chromium (Cr VI) in Group 2 significantly (P < 0.05) reduced the antioxidant markers such as superoxide dismutase and reduced glutathione along with significant (P < 0.05) increase in peroxidation markers such as malondialdehyde and protein carbonyls in the liver and kidney as compared with other groups. The functional markers in serum such as total protein was decreased significantly (P < 0.05), whereas other functional markers viz. alanine transaminase, blood urea nitrogen and creatinine were increased significantly (P < 0.05) in Group 2 as compared with the other groups. Significant (P < 0.05) decrease in hemoglobin, packed cell volume, total erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin and total leukocyte count were observed in Cr VI treated Group 2 rats. Prominent pathological changes were observed in the liver and kidney of Group 2. Co-treatment with α-tocopherol in Group 3 rats significantly (P < 0.05) reversed the Cr VI induced changes. The parameters in the study in Group 4 did not differ as compared with Group 1.

Conclusions:

α–tocopherol exhibited protective effect against Cr VI-induced damage to the liver and kidney by inhibition of lipid peroxidation owing its antioxidant activity.KEY WORDS: α-tocopherol, chromium (Cr VI), kidney, lipid peroxidation, liver     

Alleviation of CCl4-induced cirrhosis in rats by tetramethylpyrazine is associated with downregulation of leptin and TGF-β1 pathway

This study was conducted to investigate the effect of tetramethylpyrazine (TMP) on CCl₄-induced fibrosis in rats and the possible roles of leptin, TGF-β1, Smad3, and Smad7 in this process. Liver fibrosis in rats was induced by the subcutaneous injection of 60% CCl₄ (0.3?mL /100?g body weight, biweekly ) for 12 weeks. Rats in TMP prevention and treatment groups were given TMP (10?mg /100?g body weight, daily) by gavage from days 1 and 31 after the start of CCl₄ injection, respectively. The mRNA expression of leptin, OB-Rb, TGF-β1, and TGF-βRII in the liver were detected by RT-PCR, whereas Smad3 and Smad7 protein were determined by Western blot. The results showed that hepatic cirrhosis was obviously alleviated in both TMP prevention and treatment groups. The mRNA expression of leptin, OB-Rb, TGF-β1 and -βRII, and Smad3 protein were higher in the cirrhotic models. In TMP prevention and treatment groups, these markers of expression were higher, compared with that of the normal control, but were lower when compared with that of the cirrhotic model group. Smad7 protein expression was lower in the cirrhotic model group than in the normal control. Smad7 expression in TMP prevention and treatment groups was higher, compared with that in the cirrhotic model group. Liver collagen in the TMP prevention group was the lowest among all CCl₄ injection groups. In conclusion, TMP can prevent and alleviate the development of liver fibrosis in rats. The possible mechanism could involve the downregulation of leptin, Ob-Rb, TGF-β1, TGF-βRII, and Samd3, and upregulation of Smad7.     

“A” esterases and their role in regulating the toxicity of organophosphates

Esterases which can hydrolyse organophosphates without being inhibited by them are termed A esterases. Using paraoxon and pirimiphos-methyl oxon as substrates, high A esterase activity is found in the liver and plasma or serum of a range of mammalian species. In a study of serum A esterases of sheep and humans, over 80% of the activity separated into the high density lipoprotein (HDL) fraction following ultracentrifugation. When HDL fractions from sheep serum were run on Sepharose gel columns, most of the paraoxonase activity separated as a single peak of estimated molecular weight 360000, which corresponds to that of HDL₂ of humans.During the course of purification of A esterases by three different column procedures, contrasting esterase elution profiles were obtained with organophosphate and pyrethroid substrates. This was strong evidence for the existence of multiple forms of HDL A esterases.Levels of A esterase activity in plasma and liver of birds were much lower than those of mammals. This appears to be the main reason why birds are much more susceptible than mammals to organophosphates such as pirimiphos-methyl and diazinon which form active oxons that are good substrates for mammalian A esterases.No A esterase was detected in strains of rust red flour beetle (Tribolium castaneum) which were resistant to organophosphates. Similar observations have been made with strains of other insects resistant to organophosphates, raising the question to what extent esterases of this type are present in insects.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Sulfasalazine prevents the increase in TGF-β, COX-2, nuclear NFκB translocation and fibrosis in CCl4-induced liver cirrhosis in the rat

It has been demonstrated that this sulfasalazine (SF) inhibits the nuclear factor κB (NFκB) pathway, which regulates important genes during inflammation and immune answer. The aim of this work was to evaluate the effects of SF on carbon tetrachloride (CCl(4))-induced liver fibrosis. We formed the following experimental groups of rats: controls, damage induced by chronic CCl(4) (0.4?g/kg, intraperitoneally, three times a week for 8 weeks) administration and CCl(4)?+?SF (100?mg/kg/day, postoperatively for 8 weeks) administration. We determined the activities of alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), cyclooxygenase (COX)-1 and COX-2, lipid peroxidation, glutathione levels, collagen content, expression of transforming growth factor-β (TGF-β) and nuclear translocation of NFκB. SF was capable to inhibit the ALT and γ-GTP elevated levels induced with the CCl(4) administration. SF had antioxidant properties, prevented the lipid peroxidation and the imbalance of reduced and oxidized glutathione produced by CCl(4). Importantly, SF blocked the accumulation of collagen in the liver, the expression of TGF-β, the nuclear translocation of NFκB and the activity of COX-2, all induced with the administration of CCl(4) in the rat. These results show that SF has strong antifibrotic properties because of its antioxidant properties and its ability to prevent nuclear translocation of NFκB and consequently the expression of TGF-β and the activity of COX-2.     

Response of nicotine self-administration in the rat to manipulations of mu-opioid and γ-aminobutyric acid receptors in the ventral tegmental area

Rationale: The mesolimbic dopamine system has been implicated in the reinforcing effects of nicotine, a drug which appears to act at least in part through the ventral tegmental area (VTA). Other neuronal elements in the VTA are important in drug reward. In particular, mu opioid receptors in the VTA have been shown to influence cocaine reinforcement. Objective: The aim of this study was to test whether the mu opioid receptors in the VTA also regulate the intake of nicotine. Methods: This research was carried out with animals trained to self-administer nicotine or cocaine, or to respond for food. Mu receptors were targeted with the selective agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and γ-aminobutyric acid (GABA) receptors with the selective agonists baclofen and muscimol; each of these compounds was delivered by microinfusion into the VTA. Results: The mu-selective agonist DAMGO, tested over a dose range of 0.005–0.05 μg, had an effect at the highest dose only, where it produced a reduction in self-administration maintained by doses of either 10 μg/kg or 30 μg/kg per infusion of nicotine. Intra-VTA microinfusions of DAMGO did not reinstate extinguished responding previously established for nicotine, nor did they have prominent effects on operant behavior maintained by food. In contrast to the overall limited effects of DAMGO on nicotine self-administration, the GABA agonists muscimol and baclofen each reduced nicotine self-administration substantially when delivered into the VTA, whereas they were less effective against cocaine self-administration. Conclusions: The lesser effect of DAMGO microinfusions in the VTA on nicotine than cocaine self-administration is associated with the opposite efficacy of GABA agonists. These findings suggest that nicotine and cocaine differentially activate circuitry in which mu receptors are situated, especially GABAergic elements. Received: 20 October 1998 / Final version: 24 November 1999     

Is there a trigger role of peroxynitrite in the anti-arrhythmic effect of ischaemic preconditioning and peroxynitrite infusion?

This study has examined whether peroxynitrite (PN), generated during the preconditioning (PC) procedure or administered by brief intracoronary infusions, plays a trigger role in the anti-arrhythmic effects of preconditioning and peroxynitrite in anaesthetized dogs. To achieve this we infused the peroxynitrite scavenger uric acid (UA; 0.2 mg/kg/min, i.v.) over a 30 min period, just prior to a 25 min occlusion of the left anterior descending coronary artery, in preconditioned (UA+PC, n=8), peroxynitrite-treated (UA+PN, n=8) and in control (UAC; n=9) dogs. The effects were compared to those obtained from groups (PC, n=10; PN, n=10; C1, n=14) without uric acid administration. Severities of ischaemia (ST-segment elevation, inhomogeneity of electrical activation) and ventricular arrhythmias (VPBs, VT, VF), plasma nitrate/nitrite levels, as well as myocardial superoxide and nitrotyrosine productions were determined. Both preconditioning and the infusion of peroxynitrite increased nitrotyrosine formation which was abolished by the simultaneous administration of urate. Despite this, the protective effects of preconditioning (i.e. reductions in arrhythmias, superoxide and nitrotyrosine productions, as well as the increase in nitric oxide availability), occurring during the prolonged period of occlusion and reperfusion were still present. In contrast, urate completely abolished the protection resulted from peroxynitrite administration. This effect is most probably due to the fact that urate has already scavenged peroxynitrite during the infusion. Interestingly, urate itself, given prior to ischaemia and reperfusion, was also protective. We conclude that peroxynitrite in nanomolar concentrations can induce an anti-arrhythmic effect but peroxynitrite, generated during the preconditioning stimulus, is not necessary for the preconditioning-induced anti-arrhythmic protection.     

Characterization of midazolam metabolism in locusts: the role of a CYP3A4-like enzyme in the formation of 1′-OH and 4-OH midazolam

1.?The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5.

2.?Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (K_m values) were in the same range as reported in humans (in locusts: 7–23 and 33–85?µM for the formation of the 1′-OH and 4-OH metabolites, respectively).

3.?The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor.

4.?Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by ¹H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring.

5.?Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time.

6.?In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.     

Hepatoprotection of 1β-hydroxyeuscaphic acid – the major constituent from Rubus aleaefolius against CCl4-induced injury in hepatocytes cells

Abstract

Context: Rubus aleaefolius Poir. (Rosaceae) is used as a folk medicine to treat various types of hepatitis with significant effects in Fujian Province of China. The ethyl acetate fraction of R. aleaefolius root ethanol extract proved effective after our testing in vivo animal experiments.

Objective: The protective effects of a major constituent, 1β-hydroxyeuscaphic acid isolated from R. aleaefolius was first investigated against carbon tetrachloride (CCl₄)-induced injury in liver cells from hepatocytes cell line (BRL-3A).

Materials and methods: Treatment of BRL-3A with CCl₄ led to generation of free radicals detected after a 2?h incubation and produced cell injury demonstrated by increased leakage of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) in the media. Exposure to CCl₄ caused apoptosis to cells but did not induce lipid peroxidation. Following treatment with 1β-hydroxyeuscaphic acid at doses ranging from 1 to 100?µg/mL for 24?h, cellular morphology, cell growth function (MTT assay), ALT, AST, malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed and evaluated under control and exposed conditions.

Results: The IC₅₀ of 1β-hydroxyeuscaphic acid was 15?μg/mL. Exposure of injured BRL-3A at 20?μg/mL changed abnormal size, cellular shrinkage, and enhanced regulation. ALT, AST, MDA enzyme levels were reduced and SOD activity was increased.

Discussion and conclusion: Treatment with 1β-hydroxyeuscaphic acid has significant hepatoprotective activity by lowering the leakage of intracellular enzymes, reducing the oxidation of proteins and decreasing the incidence of apoptosis. These results provide a basis for confirming the traditional uses of R. aleaefolius in treating hepatic diseases.