CCl4 Treatment Research Articles

Inhibition of the mitochondrial complex-1 protects against carbon tetrachloride-induced acute liver injury

Mitochondrial dysfunction has been documented to play a crucial role in the pathogenesis of liver injury. In the present study, we investigated the role of rotenone, a mitochondrial complex-1 inhibitor, in carbon tetrachloride (CCl4) -induced acute liver injury, as well as the underlying mechanisms. Before CCl4 administration, the mice were pretreated with rotenone at a dose of 250 ppm in food for three days. Then CCl4 was administered to the mice for 16 h by intraperitoneal injection. The liver injury, mitochondrial status, oxidative stress, and inflammation were examined. Strikingly, CCl4 treatment markedly induced liver injury as shown by enhanced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and morphological lesions (HE stating), which was significantly attenuated by rotenone treatment in line with the reduced activity of mitochondrial complex-1. Meanwhile, oxidative stress markers of malondialdehyde (MDA), 4-hydroxynonenal (HNE), and dihydroethidium (DHE) and the inflammatory markers of IL-1β, MCP-1, TNF-α, TLR-4, and IL-6 were also significantly suppressed by rotenone. More importantly, the mitochondrial abnormalities shown by the reduction of SOD2, mitochondrial transcription factor A (TFAM), mitochondrial NADH dehydrogenase subunit 1 (mtND1), and Cytb were significantly restored, indicating that rotenone protected against mitochondrial damage induced by CCl4 in liver. Moreover, rotenone treatment alone did not significantly alter liver morphology and liver enzymes ALT and AST. CYP2E1, a metabolic enzyme of CCl4, was also not significantly affected by rotenone. In conclusion, rotenone protected the liver from CCl4-induced damage possibly by inhibiting the mitochondrial oxidative stress and inflammation.

An aqueous extract of Stevia rebaudiana variety Morita II prevents liver damage in a rat model of cirrhosis that mimics the human disease

Introduction and aimStevia has exhibited antioxidant, antihyperglycemic, antihypertensive and anti-inflammatory properties in several in vivo and in vitro models. The objective of this study was to investigate the ability of an aqueous extract of stevia (AES) to prevent experimental cirrhosis in rats and to explore its mechanism of action. Materials and methodsLiver cirrhosis was induced by administering carbon tetrachloride (CCl4) (400mg/kg by i.p. injection 3 times a week for 12 weeks); AES was administered (100mg/kg by gavage daily) during the CCl4 treatment. Fibrosis was evaluated with histological, biochemical and molecular approaches, and liver damage was assessed with standardized procedures. The profibrotic pathways were analyzed by western blotting, qRT-PCR and immunohistochemistry. Results and conclusionsChronic CCl4 administration increased nuclear factor kappa B (NF-κB) and proinflammatory cytokine production as well as oxidative parameters such as lipid peroxidation and 4-hydroxynonenal levels, whereas GSH and nuclear factor-E2-related factor 2 (Nrf2) levels were decreased. CCl4 induced profibrogenic mediator expression, hepatic stellate cell (HSC) activation and, consequently, extracellular matrix production. AES exhibited antioxidant, anti-inflammatory and antifibrotic properties, probably because of its capacity to induce Nrf2 expression, reduce NF-κB expression and block several profibrogenic signaling pathways, subsequently inhibiting HSC activation and preventing fibrosis induced by chronic CCl4 administration.

Protective effect of the glucagon-like peptide-1 analogue liraglutide on carbon tetrachloride-induced acute liver injury in mice

Acute liver injury seriously endangers human health. Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, has antioxidative effects in addition to being widely used in the treatment of type 2 diabetes and was reported to ameliorate liver diseases. The aim of this study was to evaluate the hepatoprotective effects of liraglutide on carbon tetrachloride (CCl4)-induced acute liver injury in mice and to investigate the mechanisms involved in this protective effect. Male BALB/c mice were pre-treated with liraglutide (200 μg/kg/day) by hypodermic injection for 3 days before a 0.1% (v/v) CCl4 (10 ml/kg, dissolved in olive oil) intraperitoneal injection, or post-treated with liraglutide once immediately after a CCl4 intraperitoneal injection. The experimental data showed that liraglutide treatment significantly decreased the serum ALT and AST levels and ameliorated the liver histopathological changes induced by CCl4. In addition, liraglutide pre-treatment dramatically increased the number of proliferating cell nuclear antigen (PCNA)-positive hepatocytes and significantly reduced hepatocyte apoptosis after CCl4 treatment. As a consequence, liraglutide pre-treatment significantly prevented CCl4-induced malondialdehyde (MDA) production and increased the activity of the antioxidant superoxide dismutase (SOD) enzyme. In addition, liraglutide pre-treatment significantly ameliorated mitochondrial respiratory functions and ultrastructural features. Furthermore, liraglutide pre-treatment enhances the activation of the NRF2/HO-1 signaling pathway. In summary, liraglutide protects against CCl4-induced acute liver injury by protecting mitochondrial functions and inhibiting oxidative stress, which may partly involve the activation of NRF2/HO-1 signaling pathway.

Metabolic mechanisms of Fuzheng-Huayu formula against liver fibrosis in rats

Ethnopharmacological relevanceFuzheng-Huayu formula (FZHY) is traditionally used to treat liver fibrosis in clinic. The study was conducted to investigate the metabolic mechanisms of FZHY against liver fibrosis in rats. Materials and methodsRats with CCl4 -induced liver fibrosis were treated with FZHY and its components, including amygdalin, cordyceps polysaccharide and gypenoside, respecitively. Liver fibrosis and function were assesed by histopathological examination, Western blot and serum biochemical detection. Metabolic profiling of liver tissue, serum and urine in each group were detected by gas chromatography-mass spectrometry (GC-MS) and transcriptomic changes were tested by gene chip. RT-qPCR was used to validate levels of different expressed genes (DEGs) with statistical significance. Metabolic network together with DEGs was constructed based on KEGG database. ResultsFZHY effectively improved liver fibrosis better than the mixture or single use of gypenoside, cordyceps sinensis mycelia and amygdalin. FZHY treatment widely modulated the metabolic profiles perturbed by liver fibrosis, involving several important metabolic pathways, including glycolysis/gluconeogenesis, glucose-alanine cycle, citrate cycle, galactose metabolism, tryptophan metabolism, urea cycle, etc. It also increased alanine and decreased glucose levels in liver tissue and decreased both of them in serum and urine, which were dysregulated by CCl4 treatment. Additionally, FZHY also upregulated expression of metabolic enzymes including Hk2, Adh1 and Gpt increased, and downregulated Gs and Acss2. ConclusionFZHY improved liver fibrosis in rats via altering the metabolic pathways and regulating gene expression of involved metabolic enzymes.

Neuron‐specific knockout of FXR attenuates the neurological complications of chronic liver disease

Hepatic encephalopathy (HE) describes the neurological deficits that result from liver impairment. Liver disease is associated with an increase of circulating bile acids that can cross the blood brain barrier and activate FXR receptors in neurons. We have previously demonstrated that aberrant bile acid signaling via activation of neuronal FXR contributes to HE pathogenesis in rodent models of acute liver failure. However, a role for FXR‐mediated bile acid signaling in HE due to chronic liver cirrhosis is undefined.MethodsNeuron‐specific FXR knockout mice were generated by crossing Floxed FXR mice (FXRfl) with SNAP‐25 cre recombinase mice. The resulting mice were designated FXRΔneu. C57Bl/6 (WT), FXRfl, and FXRΔneu were treated with carbon tetrachloride (CCl4; 1 ml/kg) by oral gavage twice per week for 12 weeks. Neurobehavioral indices and neuromuscular deficits were assessed by open field test, rotarod, grip strength test and gait analysis. After 12 weeks, tissue was collected and liver damage was assessed by serum chemistry and H&E staining. Total bile acid content was assessed in the cortex and cerebellum using colorimetric assays. The expression of ASBT, FXR, and its downstream effector SHP was assessed by qPCR and immunofluorescence. Microglia activation was assessed by Iba1 immunofluorescence. The expression of proinflammatory cytokines were assessed by qPCR and EIA. In parallel, total bile acids, ASBT and FXR expression were assessed in brain tissue from cirrhotic patients with HE, compared to cirrhotics without HE and age‐ and gender‐matched controls that had been collected and banked by the Australian Brain Bank Network.ResultsTotal bile acid content was elevated in the cortex and cerebellum in CCl4‐treated WT mice and in cirrhotic patients with HE, compared to cirrhotics without HE and non‐liver impaired controls. Furthermore, ASBT, FXR and SHP expression were increased in the frontal cortex of mice and humans with HE, but not in non‐HE control samples. WT and FXRfl mice treated with CCl4 had significant deficits observed in every neurobehavioral and neuromuscular test performed, as well as marked microglia activation and increased proinflammatory cytokine expression compared to vehicle‐treated mice. These neurological deficits and neuroinflammation were attenuated in FXRΔneu mice after CCl4 treatment, even though liver damage was comparable in all genotypes of mice used.ConclusionsTaken together, these data indicate that neuronal expression of FXR plays an important role in the development of HE. Specific targeting of FXR activation in the brain may prove fruitful in the development of novel therapeutics for the management of HE.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization, characterization and hepatoprotective effect of polysaccharides isolated from Stipa parviflora Desf. against CCl4 induced liver injury in rats using surface response methodology (RSM).

The roots of Stipa species have been used for treatment and prevention of a various number of diseases. To the best of my knowledge, little information is regarding the antioxidant and hepatoprotective activities of polysaccharides from Stipa parviflora (SPP). Hence the polysaccharides from this plant sample have been investigated here. In addition, Box-Behnken design and response surface methodology were used to optimize the hot extraction conditions. The optimal conditions were determined as: extraction time 120 min, extraction temperature 70 °C and ratio of water to raw material 30. Crude SPP was composed of mannose, ribose, glucose, galactose, pyranose and arabinose. In addition, the SPP showed a strong antioxidant capacity in vitro. In vivo experiments showed that CCl4 treatment caused significant oxidative damage in hepatic tissues, whereas these problems were inhibited by SPP treatment. Hepatoprotective potential of SPP was evaluated by estimating the levels of serum markers like ALT and AST levels. SPP treated rat liver anti-oxidant parameters (SOD, CAT and GPx) were significantly antagonized for the pro-oxidant effect of CCl4. Histopathological studies proved that the SPP extract has significant hepatoprotective activity. In conclusion, the SPP fraction isolated from S. parviflora showed significant hepatoprotective potential which could be related to active compounds present in this fraction.

Hemodynamic changes in hepatic sinusoids of hepatic steatosis mice.

BACKGROUNDFatty liver (FL) is now a worldwide disease. For decades, researchers have been kept trying to elucidate the mechanism of FL at the molecular level, but rarely involve the study of morphology and medical physics. Traditionally, it was believed that hemodynamic changes occur only when fibrosis occurs, but it has been proved that these changes already show in steatosis stage, which may help to reveal the pathogenesis and its progress. Because the pseudolobules are not formed during the steatosis stage, this phenomenon may be caused by the compression of the liver microcirculation and changes in the hemodynamics.AIMTo understand the pathogenesis of hepatic steatosis and to study the hemodynamic changes associated with hepatic steatosis.METHODSEight-week-old male C57BL/6 mice were divided into three groups randomly (control group, 2-wk group, and 4-wk group), with 16 mice per group. A hepatic steatosis model was established by subcutaneous injection of carbon tetrachloride in mice. After establishing the model, liver tissue from mice was stained with hematoxylin and eosin (HE), and oil red O stains. Blood was collected from the angular vein, and hemorheological parameters were estimated. A two-photon fluorescence microscope was used to examine the flow properties of red blood cells in the hepatic sinusoids.RESULTSOil red O staining indicated lipid accumulation in the liver after CCl4 treatment. HE staining indicated narrowing of the hepatic sinusoidal vessels. No significant difference was observed between the 2-wk and 4-wk groups of mice on morphological examination. Hemorheological tests included whole blood viscosity (mPas, γ = 10 s-1/γ = 100 s-1) (8.83 ± 2.22/4.69 ± 1.16, 7.73 ± 2.46/4.22 ± 1.32, and 8.06 ± 2.88/4.22 ± 1.50), red blood cell volume (%) (51.00 ± 4.00, 42.00 ± 5.00, and 40.00 ± 3.00), the content of plasma fibrinase (g/L) (3.80 ± 0.50, 2.90 ± 0.80, and 2.30 ± 0.70), erythrocyte deformation index (%) (44.49 ± 5.81, 48.00 ± 15.29, and 44.36 ± 15.01), erythrocyte electrophoresis rate (mm/s per V/m) (0.55 ± 0.11, 0.50 ± 0.11, and 0.60 ± 0.20), revealing pathological changes in plasma components and red blood cells of hepatic steatosis. Assessment of blood flow velocity in the hepatic sinusoids with a laser Doppler flowmeter (mL/min per 100 g) (94.43 ± 14.64, 80.00 ± 12.12, and 67.26 ± 5.92) and two-photon laser scanning microscope (μm/s) (325.68 ± 112.66, 213.53 ± 65.33, and 173.26 ± 44.02) revealed that as the modeling time increased, the blood flow velocity in the hepatic sinusoids decreased gradually, and the diameter of the hepatic sinusoids became smaller (μm) (10.28 ± 1.40, 6.84 ± 0.93, and 5.82 ± 0.79).CONCLUSIONThe inner diameter of the hepatic sinusoids decreases along with the decrease in the blood flow velocity within the sinusoids and the changes in the systemic hemorheology.

Vitronectins produced by human cirrhotic liver and CCl4-treated rats differ in their glycosylation pattern and tissue remodeling activity.

Liver cirrhosis (LC) is a disease characterized by pathological accumulation and alteration of extracellular matrix (ECM) proteins; the interaction between two such proteins, collagen and vitronectin (VN), is considered to be the key to controlling ECM remodeling in liver cirrhosis. If it is possible to control the modification of oligosaccharides on VN, it may be possible to retard progression of liver cirrhosis. In this study, we examined the relationship between changes in VN glycosylation and activity related to the remodeling of hepatic tissue in human LC and a rat model of LC generated using carbon tetrachloride (CCl4). Plasma concentrations of VN in human LC declined to approximately two‐thirds that in normal plasma, but the ratio of active VN, which has collagen‐binding activities, increased 2.8 times in LC plasma. In contrast, purified LC‐VN exhibited similar binding activities toward type I, IV, and V collagens to those of normal VN. Lectin reactivities and carbohydrate analyses of LC‐VN revealed that branching, fucosylation, and sialylation of N‐glycans were higher than those of normal VN. On the other hand, the plasma level of rat CCl4‐VN increased and the ratio of active molecules to collagen in plasma decreased. Increased fucosylation of LC‐VN was not detected in carbohydrates of CCl4‐VN. The changes in rat VN due to CCl4 treatment did not correspond to the changes in plasma levels of human VN caused by LC, the ratio of active molecules, or carbohydrate composition, thereby indicating that CCl4‐treated rats are not an appropriate model for studying VNs in human LC. Glycosidase treatment of VNs supported the hypothesis that the collagen‐binding activity of VN is modulated by alterations of glycosylation during LC, which may contribute to (a) the matrix incorporation of VN and (b) tissue fibrosis.

Hepatoprotective effects of ginseng saponins in a mouse model of carbon tetrachloride-induced liver injury

Purpose: To investigate the effects of ginsenosides Rg1 and Rb1 on carbon tetrachloride (CCl4)-induced liver injury in mice.Methods: Thirty-two mice were randomly divided into four groups. In control group, mice were administered sodium carboxymethylcellulose (CMC-Na) by intraperitoneal injection for seven days. In CCl4 treatment group, mice were treated as control group for the first seven days and then intraperitoneally injected with CCl4 in olive oil on day 8. In Rb1- and Rg1-treatment group, the mice were intraperitoneally injected with Rb1 or Rg1 (each 30 mg/kg, dissolved in 0.5 % CMC-Na), respectively for seven days, followed by intraperitoneal injection with CCl4 in olive oil on day 8. Histological damage was examined by haematoxylin and eosin (H&E) staining. Serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) were assessed enzymatically. Tissue IL-6 and IL-8 levels were measured by ELISA. Gene and protein levels of transforming growth factor (TGF)-β, Smad2, and Smad3 were analyzed by real-time PCR (RT-PCR) and western blotting, respectively.Results: CCl4 treatment caused histological damage in mouse liver, and increased the levels of ALT, AST (five-fold), IL-6 (three-fold), and IL-8 (five-fold), and elevated expressions of TGF-β, Smad2, and Smad3. Ginsenosides Rg1 or Rb1 pre-treatment attenuated liver injury by decreasing the serum levels of ALT (from 700 to 200 UI/L) AST (from 550 pg/mL to 250 pg/ml), IL-6 (from 1,100 to 750 pg/mL), and IL-8 (from 600 to 200 pg/mL), and inhibiting the expressions of TGF-β, Smad2, and Smad3.Conclusion: Ginsenosides (Rg1 and Rb1) attenuate CCl4-induced liver injury and inflammation by regulating TGF-β/Smad signalling pathway.Keywords: Ginseng saponin, Ginsenosides, Rg1, Rb1, Hepatoprotective effect, TGF-β/Smad signalling pathway

Interleukin-1 Receptor Antagonist Modulates Liver Inflammation and Fibrosis in Mice in a Model-Dependent Manner.

Background: Interleukin-1 (IL-1)β and IL-1 receptor antagonist (IL-1Ra) have been proposed as important mediators during chronic liver diseases. We aimed to determine whether the modulation of IL-1β signaling with IL-1Ra impacts on liver fibrosis. Methods: We assessed the effects of IL-1β on human hepatic stellate cells (HSC) and in mouse models of liver fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4). Results: Human HSCs treated with IL-1β had increased IL-1β, IL-1Ra, and MMP-9 expressions in vitro. HSCs treated with IL-1β had reduced α-smooth muscle actin expression. These effects were all prevented by IL-1Ra treatment. In the BDL model, liver fibrosis and Kuppfer cell numbers were increased in IL-1Ra KO mice compared to wild type mice and wild type mice treated with IL-1Ra. In contrast, after CCl-4 treatment, fibrosis, HSC and Kupffer cell numbers were decreased in IL-1Ra KO mice compared to the other groups. IL-1Ra treatment provided a modest protective effect in the BDL model and was pro-fibrotic in the CCl-4 model. Conclusions: We demonstrated bivalent effects of IL-1Ra during liver fibrosis in mice. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Altogether these data suggest that blocking IL-1-mediated inflammation may be beneficial only in selective liver fibrotic disease.

MFGE8 protects against CCl4 -induced liver injury by reducing apoptosis and promoting proliferation of hepatocytes.

Milk fat globule-EGF factor 8 (MFGE8) has been reported to play various roles in acute injury and inflammation response. However, the role of MFGE8 in liver injury is poorly investigated. The present research was designed to clarify the expression and function of MFGE8 in carbon tetrachloride (CCl4 )-induced liver injury. Using serum cytokine arrays, we selected a promising cytokine MFGE8 as the candidate in the process of hepatitis-fibrosis-hepatocellular carcinoma (HCC) progression, based on the elevated expression in both hepatic fibrosis and HCC models. We validated the increased expression of MFGE8 in liver tissues and serum samples of acute and chronic CCl4 -induced mice. Immunohistochemistry staining of mouse liver tissues indicated that elevated MFGE8 expression was mainly derived from the injured hepatocytes. In addition, MFGE8 expression in the supernatant of primary hepatocytes was accumulated with prolongation of culture time, and CCl4 treatment further increased the expression of MFGE8. Moreover, a strong correlation between serum MFGE8 expression and liver transaminase activities suggested that MFGE8 may be a novel candidate in liver injury. Intriguingly, mice pretreated with MFGE8 were protected from CCl4 -induced liver injury through antiapoptosis role in the early stage and proproliferation role in the late stage. MFGE8 reduced apoptosis by inhibiting the activation of IRE1α/ASK1/JNK pathway and promoted proliferation by phosphorylation of ERK and AKT. Moreover, serum MFGE8 expression was increased in hepatitis patients while decreased in liver cirrhosis patients. All the results suggest MFGE8 as a novel marker and promising therapeutic agent of liver injury.

Macrophage-derived HMGB1 is dispensable for tissue fibrogenesis.

Alarmins and damage‐associated molecular patterns (DAMPs) are powerful inflammatory mediators, capable of initiating and maintaining sterile inflammation during acute or chronic tissue injury. Recent evidence suggests that alarmins/DAMPs may also trigger tissue regeneration and repair, suggesting a potential contribution to tissue fibrogenesis. High mobility group B1 (HMGB1), a bona fide alarmin/DAMP, may be released passively by necrotic cells or actively secreted by innate immune cells. Macrophages can release large amounts of HMGB1 and play a key role in wound healing and regeneration processes. Here, we hypothesized that macrophages may be a key source of HMGB1 and thereby contribute to wound healing and fibrogenesis. Surprisingly, cell‐specific deletion approaches, demonstrated that macrophage‐derived HMGB1 is not involved in tissue fibrogenesis in multiple organs with different underlying pathologies. Compared to control HMGB1Flox mice, mice with macrophage‐specific HMGB1 deletion (HMGB1ΔMac) do not display any modification of fibrogenesis in the liver after CCL4 or thioacetamide treatment and bile duct ligation; in the kidney following unilateral ureter obstruction; and in the heart after transverse aortic constriction. Of note, even under thermoneutral housing, known to exacerbate inflammation and fibrosis features, HMGB1ΔMac mice do not show impairment of fibrogenesis. In conclusion, our study clearly establishes that macrophage‐derived HMGB1 does not contribute to tissue repair and fibrogenesis.

Hepatoprotective Effects of Camel Milk and Urine on Carbon Tetrachloride (CCL4) induced Liver Damage

Carbon tetrachloride (CCL4) is a highly toxic chemical agent and is the most commonly used drug to experimentally induce liver damage. Aim of the study the aim is to investigate the possible protective role of both camel milk and urine on CCL4 induced liver damage. Method 20 rates were used in the study and they were classified into 4 different groups. Group I control, Group II CCL4 group, Group III (milk+CCL4), Group IV (urine+ CCL4). Biochemical parameters in addition to liver enzymes were estimated to evaluated the liver damage. Results the CCl4 treatment markedly affected the liver-specific enzyme activities. A significant (P < 0.05) increase in serum AST (135± 13.6 IU/L), ALT (157± 24.3 IU/L), and ALP (1209 ± 2.59 IU/L) activities was observed in the CCl4-treated rats compared with those of the control rats (104 ± 6.9 IU/L, 101±14.6 IU/L, 146.8±11.2 IU/L) respectively. Concluded from this study that Camel urine has protective effect against CCL4 induced liver damage more than Camel milk.

SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

Prolonged parenchymal cell death leads to activation of fibrogenic cells and extracellular matrix accumulation and eventually liver fibrosis. Autophagy, a major catabolic process of intracellular degradation and recycling, participates in hepatic fibrosis. However, the precise role of autophagy in the pathogenesis of hepatic fibrosis is controversial. The present study aims to investigate the key role of small VCP/p97 interacting protein (SVIP) against CCl4-induced hepatic fibrosis via activating autophagy. Autophagy could be activated by SVIP in HepG2 cells, but starvation cannot increase SVIP expression in vitro and in vivo. Moreover, SVIP expression, in agreement with autophagic activity and the volume of lipid droplets, first increases and then decreases during the progression of liver fibrosis with CCl4 treatment in vivo and in vivo. Further, overexpression of SVIP can protect HepG2 cells from the toxicity of CCl4, which could be enhanced by starvation. Finally, starvation keeps SVIP and autophagy at such high levels in the rat livers that markedly delays the progress of hepatic fibrosis. Probably, the protective effect of SVIP is associated with stabilizing nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) and transcription factor EB (TFEB). The current study provides insight into the biological role of SVIP and autophagy in regulating hepatic fibrosis, targeting SVIP might be a novel therapeutic strategy in the future.

Solanum nigrum confers protection against CCl4-induced experimental hepatotoxicity by increasing hepatic protein synthesis and regulation of energy metabolism

BackgroundHepatotoxicity induced by carbon tetrachloride (CCl4) is used as an experimental model to screen phytochemicals with liver protecting activity. Solanum nigrum (SN) is a shrub which is widely distributed throughout India. Traditionally the plant has been used for curing various ailments related to gastroenterology and hepatology. The present study was performed to evaluate the hepatoprotective property of S. nigrum extract standardized for its flavonoid content against CCl4-induced hepatotoxicity.MethodsThe liver protecting property of SN was evaluated by means of various biochemical parameters and histopathologically. S. nigrum was administered to Swiss strain female albino mice with either 100/ 200/ 300 mg/kg body weight/day for 30 days along with CCl4 which is a well established model to induce hepatotoxicity.ResultsAdministration of CCl4 for 30 days caused a significant increase in liver marker enzymes and a decrease in hepatic DNA, RNA and protein levels which was effectively mitigated by treatment with the plant extract in a dose dependent manner. Similarly co-treatment of the extract along with the hepatotoxin improved hepatic energy status by increasing the activities of succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase). Histopathological findings indicated severe vacuolization and necrotic changes after CCl4 treatment which was mitigated by the co-administration of SN extract in a dose-dependent manner.ConclusionsCarbon tetracholoride is known to exert hepatotoxicty by forming adducts with tissue macromolecules through covalent interactions. S. nigrum extract was found to effectively mitigate CCl4-induced changes in hepatic macromolecular content and energy status of liver tissue. The present study has identified the standardized S. nigrum as an effective hepatoprotective agent probably due to its role in improving the protein and energy levels in the hepatic tissue.

Protective effects of phytochemicals of Capparis spinosa seeds with cisplatin and CCl4 toxicity in mice

The phenolic compound content and the beneficial effects of Capparis spinosa seed extracts (CSSE) against the toxicity induced using cisplatin and CCl4 treatment were studied. The amounts of total phenolics, flavonoids and condensed tannins were 83 mg gallic acid equivalents, ~92 mg rutin equivalents, and 1 mg tannic acid equivalents/g d.w., respectively. Results showed that CSSE pretreatment restored the higher content of serum creatinine, urea and uric acid, and increased activities of aspartate aminotransferase, and alanine aminotransferase induced by cisplatin and CCl4 treatment. Furthermore, CSSE pretreatment decreased the protein carbonyls and malondialdehyde, and increased the activities of superoxide dismutase, catalase, and glutathione peroxidase. Histopathological studies confirmed that CSSE decreased the degree of tissues fibrosis. The results obtained confirmed the potential nutritional and medicinal benefits of C. spinosa and highlighted the potential use of the seeds as a source of bioantioxidants for the food and pharmaceutical industries.

Histopathological and Molecular Signatures of a Mouse Model of Acute-on-Chronic Alcoholic Liver Injury Demonstrate Concordance With Human Alcoholic Hepatitis.

Human alcoholic hepatitis (AH) carries a high mortality rate. AH is an acute-on-chronic form of liver injury characterized by hepatic steatosis, ballooned hepatocytes, neutrophil infiltration, and pericellular fibrosis. We aimed to study the pathogenesis of AH in an animal model which combines chronic hepatic fibrosis with intragastric alcohol administration. Adult male C57BL6/J mice were treated with CCl4 (0.2 ml/kg, 2×weekly by intraperitoneal injections for 6 weeks) to induce chronic liver fibrosis. Then, ethyl alcohol (up to 25 g/kg/day for 3 weeks) was administered continuously to mice via a gastric feeding tube, with or without one-half dose of CCl4. Liver and serum markers and liver transcriptome were evaluated to characterize acute-on-chronic-alcoholic liver disease in our model. CCl4 or alcohol treatment alone induced liver fibrosis or steatohepatitis, respectively, findings that were consistent with expected pathology. Combined treatment resulted in a marked exacerbation of liver injury, as evident by the development of inflammation, steatosis, and pericellular fibrosis, pathological features of human AH. E. coli and Candida were also detected in livers of mice cotreated with CCl4 and alcohol, indicating pathogen translocation from gut to liver, similar to human AH. Importantly, liver transcriptomic changes specific to combined treatment group demonstrated close concordance with pathways perturbed in patients with severe AH. Overall, mice treated with CCl4 and alcohol displayed key molecular and pathological characteristics of human AH-pericellular fibrosis, increased hepatic bacterial load, and dysregulation of the same molecular pathways. This model may be useful for developing therapeutics for AH.

Inositol-Requiring Enzyme 1 Alpha Endoribonuclease Specific Inhibitor STF-083010 Alleviates Carbon Tetrachloride Induced Liver Injury and Liver Fibrosis in Mice.

Accumulating data demonstrated that hepatic endoplasmic reticulum (ER) stress was involved in the pathogenesis of liver fibrosis. Long-term chronic hepatocyte death contributed to liver fibrosis initiation and progression. Previous researches reported that ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α) was first activated in the process of liver fibrosis. STF-083010 was an IRE1α RNase specific inhibitor. This study aimed to explore the effects of STF-083010 on carbon tetrachloride (CCl4)-induced liver injury and subsequent liver fibrosis. Mice were intraperitoneally (i.p.) injected with CCl4 (0.15 ml/kg) for 8 weeks. In STF-083010+CCl4 group, mice were injected with STF-083010 (30 mg/kg, i.p.), twice a week, beginning from the 6th week after CCl4 injection. CCl4 treatment markedly enhanced the levels of serum ALT, TBIL, DBIL and TBA, and STF-083010 had obviously extenuated CCl4-induced exaltation of ALT, DBIL, and TBA levels. CCl4-induced hepatic hydroxyproline and collagen I, major indicators of liver fibrosis, were alleviated by STF-083010. Additionally, CCl4-induced α-smooth muscle actin, a marker for hepatic stellate cells activation, was obviously attenuated in STF-083010-treated mice. Moreover, CCl4-induced upregulation of inflammatory cytokines was suppressed by STF-083010. Mechanistic exploration found that hepatic miR-122 was downregulated in CCl4-treated mice. Hepatic MCP1, CTGF, P4HA1, Col1α1, and Mmp9, target genes of miR-122, were upregulated in CCl4-treated mice. Interestingly, STF-083010 reversed CCl4-induced hepatic miR-122 downregulation. Correspondingly, STF-083010 inhibited CCl4-induced upregulation of miR-122 target genes. This study provides partial evidence that STF-083010 alleviated CCl4-induced liver injury and thus protected against liver fibrosis associated with hepatic miR-122.

Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis

This is the first study to investigate the hepatoprotective effect of CQ on acute liver injury caused by carbon tetrachloride (CCl4) in a murine model and the underlying molecular mechanisms. Ninety-six mice were randomly divided into the control (n = 8), CQ (n = 8), CCl4 (n = 40), and CCl4 + CQ (n = 40) treatment groups. In the CCl4 group, mice were intraperitoneally (i.p) injected with 0.3% CCl4 (10 mL/kg, dissolved in olive oil); in the CCl4 + CQ group, mice were i.p injected with CQ at 50 mg/kg at 2, 24, and 48 h before CCl4 administration. The mice in the control and CQ groups were administered with an equal vehicle or CQ (50 mg/kg). Mice were killed at 2, 6, 12, 24, 48 h post CCl4 treatment and their livers were harvested for analysis. The results showed that CQ pre-treatment markedly inhibited CCl4-induced acute liver injury, which was evidenced by decreased serum transaminase, aspartate transaminase and lower histological scores of liver injury. CQ pretreatment downregulated the CCl4-induced hepatic tissue expression of high-mobility group box 1 (HMGB1) and the levels of serum HMGB1 as well as IL-6 and TNF-α. Furthermore, CQ pre-treatment inhibited autophagy, downregulated NF-kB expression, upregulated p53 expression, increased the ratio of Bax/Bcl-2, and increased the activation of caspase-3 in hepatic tissue. This is the first study to demonstrate that CQ ameliorates CCl4-induced acute liver injury via the inhibition of HMGB1-mediated inflammatory responses and the stimulation of pro-apoptotic pathways to modulate the apoptotic and inflammatory responses associated with progress of liver damage.

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways.

The aims of the present study were to investigate the capacity of stevia leaves to prevent experimental cirrhosis induced by chronic administration of carbon tetrachloride (CCl4 ) in rats and to explore the action mechanism involved. Liver cirrhosis was established by CCl4 treatment (400mg/kg i.p. three times a week for 12weeks); stevia powder was administered (100mg/kg by gavage daily) during the CCl4 treatment. Serum markers of liver damage and hydroxyproline were evaluated and histopathological analyses were carried out. The profibrotic pathways were analyzed by western blot and immunohistochemistry. We found for the first time that stevia cotreatment prevented the elevation of serum markers of necrosis and cholestasis and the occurrence of liver fibrosis. It is worth noting that stevia downregulated several profibrogenic pathways, including the reduction of hepatic myofibroblasts and decreased matrix metalloproteinase (MMP)2 and MMP13 expression, thereby blocking the liberation of transforming growth factor-β from the extracellular matrix. Notably, stevia reduced the phosphorylation of pSmad3L, the most profibrogenic and mitogenic Smad, by inhibiting the activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. Interestingly, Smad7, an important antifibrotic molecule, was upregulated by stevia treatment in cirrhotic rats. These multitarget mechanisms led to the prevention of experimental cirrhosis. Because stevia possesses a reasonable safety profile, our results indicate that it could be useful in the clinical setting to treat chronic liver diseases.