Carbon Tetrachloride-induced Hepatitis Research Articles

ANTIOXIDANT ACTIVITY AND ENERGY PROTECTIVE EFFECT OF HEDYSARUM ALPINUM L. EXTRACT IN ACUTE CCL4-INDUCED HEPATITIS

Relevance. The course of physiological processes in the norm is significantly influenced by the pro- and antioxidant, as well as the energy status of the body. In the liver, biochemical disturbances in the functioning of these systems contribute to the development or aggravate the course of patholog-ical conditions such as chronic hepatitis, cholelithiasis, chronic cholecystitis, etc. In this regard, the assessment of the influence of the studied herbal remedies that have a hepatoprotective effect on energy metabolism and the state of the antioxidant system of the body in experimental pathological processes is still relevant. The purpose of the study was to evaluate the effect of Hedysarum alpinum L. dry extract on antioxidant status and energy metabolism in white rats with acute carbon tetrachloride-induced hepatitis. Material and methods. The studies were carried out on white Wistar rats. Acute carbon tetrachloride hepatitis was induced by subcutaneous injec-tion of a 50% oily solution of CCl4 for 4 days at a dose of 1.0 ml/kg. The dry extract H. alpinum was administered per os to animals at doses of 100 and 200 mg/kg. On the 7th and 14th days of the experiment, the energy metabolism and antioxidant status of the liver were assessed. The content of ATP, lactate, pyruvate, GSH the concentration of malondialdehyde (MDA), the activity of catalase, glutathione peroxidase, glutathione reductase, super-oxide dismutase (SOD) in the liver homogenate were determined. The evaluation of the antiradical activity of the studied agent was carried out in vitro: binding of 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS˙+). Results. The dry extract H. alpinum was administered to animals at doses of 100 and 200 mg/kg, increases the activity of SOD, catalase, glutathione peroxidase, glutathione reductase and the content of GSH, reduces the content of MDA, increases the content of ATP, reduces the level of lactate; cor-rects the lactate/pyruvate ratio compared to the control. The dry extract of H. alpinum demonstrated the most pronounced antioxidant activity and en-ergy-protective effect at a dose of 200 mg/kg. The extract of H. alpinum characterized by a pronounced radical-binding activity against DPPH˙ and ABTS˙+ (IC50DPPH˙=25.1 µg/ml; IC50ABTS˙+=47.1 µg/ml), which was due to the high content in its composition of phenolic compounds (2-C-β-D-glucopyranosyl-1,3,6,7-tetraoxyxanthone, rutin, catechins, xanthones), flavonols and polysaccharides. Conclusions. The dry extract H. alpinum at doses of 100 and 200 mg/kg reduces the intensity of oxidative stress and corrects the state of energy metabolism in carbon tetrachloride-induced acute liver injury in white rats, showing a pronounced antioxidant, antiradical and energy-protective effects.

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

Inhibition of oxidative stress and NLRP3 inflammasome by Saikosaponin-d alleviates acute liver injury in carbon tetrachloride-induced hepatitis in mice.

NLRP3 inflammasome activation results in severe liver inflammation and injury. Saikosaponin-d (SSd) possesses anti-inflammatory and hepatoprotective effects. This study aimed to determine the protective effects of SSd on carbon tetrachloride (CCl4)-induced acute liver injury in mice, and whether oxidative stress and NLRP3 inflammasome activation participate in the process.The CCl4 mice model and controls were induced. The mice were treated with SSd at 1, 1.5, or 2.0 mg/kg in a total volume of 100 µl/25 g of body weight. Liver injury was assessed by histopathology. Oxidative stress was determined using mitochondrial superoxide production (MSP), malondialdehyde (MDA) content, and superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities. NLRP3, ASC, and Caspase 1 were determined by real-time PCR and western blot. IL-1β and IL-18 levels were determined by ELISA.Significantly elevated oxidative stress was induced in the liver by CCl4, as demonstrated by histopathology and increases of MDA and MSP levels and decreases of SOD, GPx, and CAT activities (all P < 0.01). SSd significantly decreased the MDA and MSP levels and increased the activities of SOD, GPx, and CAT (all P < 0.05). The mRNA expression of NLRP3, ASC, and Caspase 1, and the protein expression of Caspase 1-p10, NLRP3, ASC, IL-1β, and IL-18 were significantly increased after CCl4 induction (all P < 0.01). These changes were reversed by SSd (all P < 0.05).Suppression of the oxidative stress and NLRP3 inflammasome activation were involved in SSd-alleviated acute liver injury in CCl4-induced hepatitis.

The study of pharmacodynamics of alcoholic and aqueous extracts from Bupleurum aureum fisch

Drug-induced liver injury has approximately 10 percent of adverse effects associated with pharmacotherapy. This issue is particularly important to pediatrics. The multifactorial pathogenesis of the liver injury identifies the prospects of finding new hepatoprotectors of the plant origin with a wide spectrum of the pharmacological activity. Bupleurum aureum (hare’s ear) has long been used in folk medicine for the liver diseases treatment. This plant has a choleretic, wound-healing, and tonic effect. Aqueous and alcoholic extracts of Bupleurum aureum contain a large amount of flavonoids (quercetin, isorhamnetin, rutin, narcissine), tannins and phytosterols. The aim of this study was to investigate the potential hepatoprotective and membrane-stabilizing effects of herbal extracts of the aerial part of Bupleurum Aureum on carbon tetrachloride-induced hepatitis in vivo. The study of pharmacodynamics of alcoholic and aqueous extracts from Bupleurum aureum Fisch. was carried out under conditions of the rats’ acute experimental hepatitis caused by tetrachloromethane. It has been found that prophylactic introduction of extracts of the aerial part of Bupleurum Aureum to rats restores the bile secretion and synthetic function of the liver. It has been determined that the herbal extracts studied have hepatoprotective and membrane protective properties. High effectiveness and a wide spectrum of pharmacological properties of extracts of Bupleurum Aureum can be described by their chemical composition. The alcoholic extract of Bupleurum aureum has been found to possess the maximum efficiency. This extract is not inferior to found reference drugs “Silibor” and “Quercetine”.

Protective role of Cynanchum acutum L. extracts on carbon tetrachloride-induced hepatotoxicity in rat

AIM: Evaluation of the hepatoprotective activity of Cynanchum acutum total alcohol and successive extracts. SETTINGS AND DESIGN: Total alcohol extract and successive extracts (ether, chloroform, ethyl acetate and butanol) of C. acutum were evaluated at different doses for hepatoprotective activity. MATERIALS AND METHODS: Carbon tetrachloride-induced hepatitis method was used. The activity of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) and serum levels of total protein (TP), albumin (Alb), total bilirubin (TB), total cholesterol and triglyceride levels were determined to evaluate liver damage in rats. STATISTICAL ANALYSIS: One-way analysis of variance (ANOVA) followed by Bonferoni’s test to determine the intergroup variability by using SPSS version 14.0. RESULTS: Pre-treatment of rats with the investigated extract (50, 100, 200 mg/kg), successive extracts (10 mg/ kg) effectively protected rats against CCl4-induced hepatic damage, resulting in reduction in the elevated serum activities of liver marker enzymes (ALT, AST and ALP) in addition to elevating the lowered serum levels of TP and Alb and lowering the serum levels of TB, triglyceride and cholesterol when compared with the normal control rats, where the total extract at dose 200 mg/kg was the most effective extract. Total alcohol extract (200 mg/kg) administrated daily to rats for 15 consecutive days did not show any alterations on liver and kidney functions. CONCLUSIONS: The high flavonoidal content of the investigated extract could be responsible for the potent hepatoprotective activity where the mechanism was related to the ability of the extract to inhibit lipid peroxidation in the liver by inhibiting the free radical mediated damage.

Effects of indoleamine 2,3‐dioxygenases in carbon tetrachloride‐induced hepatitis model of rats

Indoleamine 2,3-dioxygenase (IDO) converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, to investigate the effects of IDO in carbon tetrachloride (CCl(4) )-induced hepatitis model, the levels of IDO enzymic activities in the mock group, the control group and the 1-methyl-D-tryptophan (1-MT)-treated group were confirmed by determination of l-kynurenine concentrations. Serum alanine aminotransferase levels in 1-MT-treated rats after CCl(4) injection significantly increased compared with those in mock and control groups. In CCl(4)-induced hepatitis models, tumour necrosis factor-α (TNF-α) is critical in the development of liver injury. The mRNA expression and secretion levels of TNF-α in the liver from 1-MT-treated rats were more enhanced compared with those in the mock and the control groups. Moreover, the levels of cytokine and chemokine from mock, control group and 1-MT-treated rats after treated with CCl(4) were analyzed by ELISA, and the level of interleukin-6 was found to increase in 1-MT-treated rats. It was concluded that the deficiency of IDO exacerbated liver injury in CCl(4)-induced hepatitis and its effect may be connected with TNF-α and interleukin-6.

Safety of Liver Surface Instillation of Plasmid DNA in Normal and Carbon Tetrachloride-Induced Hepatitis Mice

We previously demonstrated liver- and lobe-selective gene transfer following instillation of plasmid DNA (pDNA) onto the liver surface in mice. Safety concerns must be resolved prior to future clinical use. Thus, we investigated safety of liver surface instillation of pDNA in normal and hepatitis mice. pDNA was instilled onto the liver surface in normal and carbon tetrachloride-induced hepatitis mice. Gene expression (luciferase activity) and serum transaminase activities were measured at appropriate time points. Transfection efficiency and target site selectivity were almost the same between the instillation of pDNA using a micropipette and a catheter. Serum transaminase activities 6 h after instillation of pDNA or a vehicle treatment using a micropipette were significantly higher than the no treatment mice, whereas instillation using a catheter did not raise serum transaminase activities throughout the tested time points, suggesting the safety of instillation using a catheter. This safety was also confirmed in hepatitis mice. A dose escalation study verified that liver surface instillation using a catheter did not raise serum transaminase at high doses with saturation of gene expression not only in normal mice, but also in hepatitis mice, supporting the safety of this method. We demonstrated that liver surface instillation of pDNA using a catheter did not raise serum transaminase activities. Information in this study will be useful for future clinical use of liver surface instillation of pDNA using a catheter.

Glycyrrhetic acid-loaded microparticles: liver-specific delivery and therapeutic potential against carbon tetrachloride-induced hepatitis.

The microparticles (MPs) of an anti-hepatotoxic drug, glycyrrhetic acid (GLA), were prepared using poly(DL-lactic acid-co-glycolic acid) as a drug carrier, and their in-vitro properties, biodistribution and therapeutic effects were investigated. The MPs showed a particle diameter distribution of 1.0-1.4 microm and a drug content of approximately 10% (w/w). In the in-vitro release in a mixture of methanol and phosphate-buffered saline pH 7.4 (3:7, v/v), slow release was observed after an initial burst release of approximately 30% (w/w). After i.v. administration of MPs in normal mice, GLA was mainly distributed to the liver. After i.v. administration in normal mice, the MPs maintained a much higher liver concentration than did GLA solution, and the plasma concentration also tended to be higher for MPs than for GLA solution. As to therapeutic effect, the liver was damaged by repeated injection of carbon tetrachloride (CCl(4)) in mice every 48 h, and the drugs were administered intravenously as a single dose 3 h after the first injection of CCl(4). At 10 mg GLA eq. kg(-1), the MPs significantly suppressed the plasma level of glutamic pyruvic transaminase for at least 141 h after administration, while GLA solution did not become significantly effective within 45 h post-administration. MPs are suggested as a possible useful system to prolong the therapeutic effect of GLA.

Hepatoprotective effect of fetal tissue cytosol and its thermostable fraction in rats with carbon tetrachloride-induced hepatitis

Pretreatment of rats with cytosol of fetal tissue or its thermostable fraction prevented death of animals from CCl4 intoxication, decreased serum transaminase activities and level of TBA-reactive products, and normalized the prooxidant/antioxidant balance in the liver. The effect of cytosol was more pronounced than that of its thermostable fraction.

Hepatocyte-Targeted MR Contrast Agents: Contrast Enhanced Detection of Liver Cancer in Diffusely Damaged Liver

The performance of hepatocyte-targeted magnetic resonance (MR) contrast agents in the detection of liver tumor was tested in rats with hepatitis. Hepatocyte-targeted MR contrast agents (paramagnetic hepatobiliary complex [manganese-DPDP] and superparamagnetic iron oxide coated with arabinogalactan [SPIO-AG]) were injected into normal rats and rats with carbon tetrachloride-induced hepatitis. Before and after injection of either contrast agent, ex vivo relaxometry (0.94T) or in vivo MR imaging (1.0T) were performed. The obtained liver and tumor T1 and T2 relaxation times, liver and tumor signal-to-noise ratios (SNR), and tumor-liver contrast-to-noise ratios (CNR) of control rats and rats with hepatitis were compared. Both relaxometry and MR imaging showed that MnDPDP and SPIO-AG selectively enhanced liver tissue in controls and in rats with hepatitis to the same degree, and little tumor enhancement was seen in either group. As a result, no significant difference between control rats and rats with hepatitis was observed in the postcontrast tumor-liver CNR. For a MnDPDP-enhanced CNR with spin echo (SE) of 310/15, the results were -10.4+/-3.6 in control rats vs. -11.5+/-1.4 in rats with hepatitis; for a SPIO-AG-enhanced CNR with SE 2000/45 and 2000/90, respectively, the results were 30.7+/-9.2 and 18.7+/-4.7 in control rats vs. 31.9+/-7.1 and 17.7+/-2.4 in rats with hepatitis. These results indicate that hepatocyte-targeted contrast agents effectively enhance liver tissue and enhance liver-tumor image contrast despite hepatocellular dysfunction.

Design of polymeric prodrugs of prostaglandin E1 having galactose residue for hepatocyte targeting

Based on the relationship between in vivo disposition of macromolecules and their physicochemical and biological characteristics obtained through clearance concept-based pharmacokinetic analysis, polymeric prodrugs of prostaglandin E1(PGE1) were designed stepwise and evaluated on their targeting and therapeutic efficiencies. First poly-l-lysine (PLL) and poly-l-glutamic acid (PLGA) with an ethylenediamine (ED) spacer were modified with 2-imino-2-methoxyethyl 1-thiogalactoside to obtain galactosylated derivatives. After intravenous injection in mice, Gal-ED-PLGA was selectively taken up by the liver parenchymal cells via receptor-mediated endocytosis, while Gal-PLL accumulated in the liver as well as PLL mostly due to electrostatic interaction. Although Gal-ED-PLGA showed good targeting efficacy, its PGE1 conjugate synthesized with activated PGE1 by carbonyldiimidazole method failed to show therapeutic effects probably due to inactivation of PGE1 during conjugation and lack of release in the tissue. In order to overcome these problems, we next conjugated PGE1 to galactosylated poly-(l-glutamic acid) hydrazide (Gal-HZ-PLGA) in which PGE1 was easily coupled to Gal-HZ-PLGA via a hydrazone bond in weak acidic solution (pH 5) at room temperature. The PGE1-Gal-HZ-PLGA conjugate labeled with [111In] or [3H]PGE1 rapidly accumulated in the liver parenchymal cells. In addition, the PGE1 conjugate effectively inhibited the increase of the GPT level in plasma, while free PGE1 indicated no therapeutic efficacy even at more than ten times higher doses, in carbon tetrachloride-induced hepatitis mice. These findings suggest potentials of polymeric targeting systems of PGE1 to hepatocyte utilizing galactose recognition.

Hepatoprotective properties of Crepis rueppellii and Anisotes trisulcus: Two traditional medicinal plants of Yemen

Pharmacological investigations were carried out to evaluate the hepatoprotective effects of Crepis rueppellii and Anisotes trisulcus. Ethanolic extracts of these plants were investigated for their ability to reduce mortality of mice after ethanol intoxication and to lower the activities of plasma glutamic-pyruvic transaminase (GPT) after carbon tetrachloride-induced hepatitis in rats. Crépis and Anisotes extracts and a 50 : 50 mixture of both at 200 mg/kg presented significant hepatoprotective effects in both experimental situations. The traditional therapeutic indications of these plants have been largely confirmed.

Hepatic Δ(9) and Δ(6) desaturase activities during the recovery period following carbon tetrachloride poisoningdesaturase activities during the recovery period following carbon tetrachloride poisoning.

The liver microsomal Δ(9) and Δ(6) desaturase activities have been studied in rats with carbon tetrachloride-induced hepatitis. Immediately after poisoning, significant decreases were observed for both types of desaturase activity. However, recovery kinetics were slower for the Δ(6) desaturase than for the Δ(9) desaturase. The activities of NADH-ferricyanide and NADH-cytochrome C reductases, proteins involved in the electron transfers associated with microsomal desaturation, were also measured. There was a fall in both activities after poisoning, but this decrease was less than that of the desaturase activities.