Centrilobular Areas Research Articles

Nutri-Protection and Mediterranean Diet: Bitter Apricot Kernel and Amygdalin Treatment Effects on a Battery of Oxidative Stress and Apoptosis Biomarkers

The role of nutrition against oxidative stress is increasingly debated in the literature. Much of the traditional food in Mediterranean diet are claimed to possess antioxidant properties but are in need of greater empirical support. This study was designed to test the putative protective effect of 3% and 5% bitter apricot kernel containing food (frequently consumed in Mediterranean diet) treated with amygdalin on apoptosis and oxidative stress in a preclinical model: carbon tetrachloride (CCl4)-induced hepatic damage in Sprage Dawley rats. The animals (n = 64) were divided into eight groups as follows: (i) control, (ii) CCl4; (iii) Amygdalin, (iv) Amygdalin and CCl4, (v) Bitter Apricot Kernel (3%), (vi) Bitter apricot kernel (5%), (vii) CCl4 +Bitter apricot kernel (3%), (viii) CCl4 and bitter apricot kernel (5%). Chronic liver injury was induced by intraperitoneally administering carbon tetrachloride (CCl4) (1 mg/kg body weight for 3 d at the end of 28 days) to rats. The area of liver injury was found significantly decreased with 5% bitter apricot kernel feeding. Serum AST, ALT, TOS activities and hepatic Bcl 2 and NFƙB levels were elevated following CCl4 administration. However, their activities were markedly reduced by supplementation with the bitter apricot kernel (P < 0.05). Serum TAS and hepatic Bax, Caspase 3, Nrf2 levels were decreased by CCl4 admistration. However, they increased in the bitter apricot kernel group versus CCl4 groups. Histopathological examination revealed massive necrosis in the centrilobular area and degenerative changes caused by CCl4 were ameliorated by dietary supplementation with bitter apricot kernel concentrates. These results suggest that bitter apricot kernel concentrates have hepatoprotective effects and may improve the symptoms of liver injuries.

Hepatoprotective effects ofRubus coreanusmiquel concentrates on liver injuries induced by carbon tetrachloride in rats

As well-being foods pursuing healthy life are becoming popular, interest in Rubus coreanus Miquel (RCM) fruit, a type of Korean blackberry, is increasing due to its medicinal actions including protecting the liver, brightening the eyes, and alleviating diabetes. This study was carried out to evaluate the hepatoprotective effects of RCM concentrates on liver injuries induced by carbon tetrachloride (CCl4) in rats. RCM, produced in June ~ July 2008 at Chunbook, Gochang (South Korea), was finely mashed. The seeds were removed and the juices were condensed. Thirty-two Sprague-Dawley rats were divided into four groups according to treatment: normal (eight rats), CCl4, 1% RCM, and 2% RCM. Experimental diets were provided to the experimental animals for 4 weeks. We measure total cholesterol, high density lipoprotein-cholesterol (HDL-C), aspartate amino transferase (AST), alanine amino transferase (ALT), and alkaline phosphatase (ALP) levels. Part of the livers was isolated for histopathological evaluation, and analyzed for lipid peroxide (TBARS), superoxide dismutase (SOD) and liver proteins. The activities of serum AST, ALT, and ALP were elevated following CCl4 administration. Levels of hepatic TBARS were also significantly increased in the CCl4 groups. However, hepatic TBARS levels and the activities of serum enzymes were markedly reduced by supplementation with the RCM concentrates (P < 0.05). Hepatic SOD activity increased in the RCM concentrates group versus CCl4 groups. Histopathological examination revealed massive necrosis in the centrilobular area and degenerative changes caused by CCl4 were ameliorated by dietary supplementation with RCM concentrates. These results suggest that RCM concentrates have hepatoprotective effects and may improve the symptoms of liver injuries.

Steatohepatitis Secondary to Long-Term Glucocorticoid Treatment for Congenital Adrenal Hyperplasia: A Potential Diagnostic Pitfall

A 24-year-old woman with congenital adrenal hyperplasia (CAH) was referred for evaluation of elevated liver enzyme activities over the preceding 6 months. The patient was diagnosed with CAH at the age 12 when she presented with irregular menses and hirsutism. Since then, she had been on dexamethasone to maintain a normal menstrual cycle and prevent hirsutism and acne. She had no history of chronic liver disease and drank alcohol socially. An extensive workup for other treatable causes of liver disease was unrevealing. Therefore, a liver biopsy was performed, which revealed extensive ballooned degenerative hepatocytes containing Mallory-Denk hyalines. The ballooned hepatocytes were located predominantly in centrilobular areas and without any accompanying steatosis. Even though the histopathologic features are most compatible with alcoholic and/or nonalcoholic steatohepatitis, it was not supported by the patient's medical history and clinical presentation. The patient had a normal body mass index and only occasional alcohol use. Based on the biopsy finding and clinical presentation, we postulated that the abnormal liver enzyme and pathological features seen on the liver biopsy were secondary to CAH and long-term use of glucocorticoid. A few studies have shown that patients with CAH often develop metabolic abnormalities and insulin resistance, particularly women treated with glucocorticoid for several years. To our knowledge, this is the first report describing steatohepatitis secondary to CAH and prolonged glucocorticoid treatment. It is important to be aware that steatohepatitis can develop in these patients due to long-term glucocorticoid use and potentially lead to progressive liver damage. Furthermore, in patients with CAH who develop abnormal liver enzyme activities a liver biopsy is warranted to assess for steatohepatitis and any associated fibrosis. If indeed fibrosis is already present, a consultation with the endocrinologist should be undertaken in an effort to lower the dose of the glucocorticoids as much as possible while still controlling the symptoms of the disease.

Codistribution of Collagen Type IV and Laminin in Liver Fibrosis of Elderly Cadavers: Immunohistochemical Marker of Perisinusoidal Basement Membrane Formation

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane. Formation of perisinusoidal basement membranes beneath the endothelium is an integral feature of capillarization of sinusoids that is a significant pathology found in advanced fibrosis. Liver fibrosis is prevalent in elderly cadavers; however, basement membrane formation in these liver samples has yet to be studied. Collagen type IV and laminin are major basement membrane proteins and their codistribution around sinusoids provides an immunohistochemical marker of basement membrane formation. Here, we examined the intralobular sites of perisinusoidal basement membrane formation in elderly cadaveric livers having various stages of fibrosis. Collagen IV and laminin codistributed in basement membranes of portal and septal ductular and vascular structures, providing a positive control. In the parenchyma, collagen IV immunostaining of sinusoids was panlobular in all stages of fibrosis, and the stain was continuous along the sinusoids. In contrast, laminin was not detected in livers, showing minimal fibrotic change. It was rarely seen in perisinusoidal/pericellular fibrosis, but frequently in septa formation, bridging fibrosis, and cirrhosis. The laminin stain was patchy, occurring principally in sinusoids of periportal and periseptal areas, less commonly in mid-lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin codistributed with collagen IV in these sinusoidal locations, thus marking the sites of perisinusoidal basement membrane formation in aged fibrotic livers. This development is presumably related to aging of the liver and exacerbated by liver injury caused by advanced liver fibrosis, possibly resulting in sinusoidal capillarization.

Co‐distribution of Collagen Type IV and Laminin Around Sinusoids in Liver Fibrosis of Elderly Cadavers: Immunohistochemical Marker of Perisinusoidal Basement Membrane Formation

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane (BM). Production of sinusoidal BM along with loss of fenestrae characterizes capillarization of sinusoids that is a significant pathology accompanying advanced fibrotic changes. Liver fibrosis is prevalent in elderly cadavers; however, BM formation in these liver samples has yet to be studied. Collagen type IV (col IV) and laminin are major BM proteins and their co‐distribution around sinusoids provides an immunohistochemical marker of BM formation. Here we examined sites of sinusoidal BM formation in elderly cadaveric livers with advanced septal fibrosis. We found co‐distribution of col IV and laminin in BM of bile ducts and blood vessels in portal tracts, providing a positive control. However, the thickened walls of fibrotic central veins were positive for col IV but not for laminin. Within the lobules, Col IV immunostaining of the sinusoidal lining appeared continuous and was panlobular. In contrast, laminin staining was patchy, occurring commonly in sinusoids close to fibrotic portal tracts and septa, less commonly in mid‐lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin co‐distributed with col IV in these sinusoidal locations, thus marking the sites of perisinusoidal BM formation in the elderly fibrotic liver, possibly in association with the capillarization of sinusoids.

Tumor necrosis factor‐α promotes bile ductular transdifferentiation of mature rat hepatocytes in vitro

We previously showed that mature hepatocytes could transdifferentiate into bile ductular cells when placed in a collagen-rich microenvironment. To explore the mechanism of transdifferentiation, we examined whether inflammatory cytokines affected the phenotype of hepatocytes in a three-dimensional culture system. Spheroidal aggregates of rat hepatocytes were embedded within a type I collagen gel matrix and cultured in the presence of various cytokines. In the control, hepatocytes gradually lost expression of albumin, tyrosine aminotransferase, and hepatocyte nuclear factor (HNF)-4α, while aberrantly expressed bile ductular markers, including cytokeratin 19 (CK 19) and spermatogenic immunoglobulin superfamily (SgIGSF). Among the cytokines examined, tumor necrosis factor (TNF)-α inhibited expression of albumin and HNF-4α, both at mRNA and protein levels. After culturing for 2 weeks with TNF-α, hepatocytic spheroids were transformed into extensively branching tubular structures composed of CK 19- and SgIGSF-positive small cuboidal cells. These cells responded to secretin with an increase in secretion and expressed functional bile duct markers. TNF-α also induced the phosphorylation of Jun N-terminal kinase (JNK) and c-Jun, and the morphogenesis was inhibited by SP600125, a specific JNK inhibitor. Furthermore, in chronic rat liver injury induced by CCl(4) , ductular reaction in the centrilobular area demonstrated strong nuclear staining of phosphorylated c-Jun. Our results demonstrate that TNF-α promotes the ductular transdifferentiation of hepatocytes and suggest a role of TNF-α in the pathogenesis of ductular reaction.

Hard Metal Lung Disease Successfully Treated with Inhaled Corticosteroids

We herein report a case of hard metal lung disease that was successfully treated with inhaled corticosteroids. A 46-year-old man was admitted to our hospital due to coughing and an abnormal shadow on a chest radiograph. He had worked as a hard metal tool sharpener for five years. Chest computed tomography scans showed centrilobular micronodules and areas of ground-glass opacity in the bilateral lung fields. Video-associated lung biopsy specimens revealed bronchocentric interstitial pneumonia and cellular bronchiolitis. A high-energy dispersion X-ray microanalysis detected tungsten. The patient was diagnosed with hard metal lung disease. Inhaled corticosteroid therapy (800 μg of ciclesonide hydrofluoroalkane daily) resolved the patient's symptoms, elevated KL-6 level, abnormal areas of chest opacity and obstructive, restrictive and diffusion impairments.

Change in the gene expression of the &lt;i&gt;N&lt;/i&gt;-methyl-D-aspartate receptor 2C subunit by dietary β-naphthoflavone, indole-3-carbinol, or acetaminophen in the rat liver

We have previously demonstrated super-induced expression of the Grin2c gene encoding the N-methyl-D-aspartate receptor 2C subunit during the process of liver enlargement induced by phenobarbital, clofibrate, piperonyl butoxide, or lead nitrate. In the present study, hepatic Grin2c gene expression levels were assessed by real-time RT-PCR in male F344 rats fed for 3 days, 4 weeks, and 13 weeks a diet containing either β-naphthoflavone (BNF) (5,000 ppm), indole-3-carbinol (I3C) (2,000 ppm), or acetaminophen (AA) (12,500 ppm until the first 14 days; 10,000 ppm from 15 days on), each of which is capable of inducing hepatocellular hypertrophy. Especially, either the 4-week or the 13-week treatment with each chemical, except for BNF, resulted in a drastic increase in the expression level of the Grin2c gene. DNA microarray analysis using RNAs of 13-week-treated rats showed that in the I3C- and AA-treated rats, the fold-increase rates of the Grin2c gene ranked second and first, respectively, among the genes analyzed. Histopathological analyses indicated that the slight hepatocellular hypertrophy in the periportal area and the hepatocellular necrosis in a portion of the centrilobular area developed in the BNF-treated and AA-treated rats, respectively. In addition, relative liver weight was significantly higher in the rats treated with BNF and I3C than in the control rats. The present findings suggest the possibility that the induction of Grin2c gene expression is not necessarily dependent on only the development of liver enlargement, although the significance of this induction remains unclear.

Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions

Ionized calcium binding adaptor molecule 1 (Iba1) is associated with membrane ruffling and motility of cells. Galectin-3 (Gal-3) is a β-galactoside binding animal lectin, and regulates fibrogenesis probably through transforming growth factor-β1. To evaluate macrophage properties, expressions of Iba1 and Gal-3 were investigated, in relation to macrophages expressing CD68 (ED1; reflecting increased phagocytosis) and CD163 (ED2; implying proinflammatory factor productions) in centrilobular lesions induced in rat livers with thioacetamide (TAA; 300 mg/kg body weight, once intraperitoneally). In agreement with expression patterns of CD68(+) and CD163(+) macrophages, cells reacting to Iba1 and Gal-3 were increased in numbers on post-injection (PI) days 1-5, peaking on day 2; thereafter, the positive cells gradually decreased to control levels until PI days 7 and 10. The increased expressions of Iba1 and Gal-3 were confirmed at mRNA levels by the RT-PCR. Double immunofluorescence staining on PI days 2 and 3 demonstrated Iba1 expression in 15-46% of CD68(+) and CD163(+) macrophages, and Gal-3 expression in 65-82% of CD68(+) and CD163(+) macrophages; Gal-3 expression was observed in 84-93% of Iba1(+) cells. Interestingly, Gal-3 was also expressed in a small number of α-smooth muscle actin-positive myofibroblasts in fibrotic lesions developed in injured centrilobular areas. These findings indicate that macrophages with various functions can participate in development of liver lesions and resultant fibrosis. Besides CD68 and CD163, Iba1 and Gal-3 immunohistochemistry for macrophages would be useful to analyze the pathogenesis behind developing hepatotoxicity.

Mouse Liver Protein Sulfhydryl Depletion after Acetaminophen Exposure

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

Patterns of Histological Changes following Hepatic Electrolytic Ablation in an Ex-Vivo Perfused Model

Electrolytic ablation (EA) destroys the liver by releasing toxic radicles and producing modifications in the local pH without increasing the tissue temperature. We assessed the histological changes produced by EA using an ex-vivo perfused model. Five porcine livers were harvested, preserved in ice and reperfused for six hours in an extracorporeal circuit using autologous normothermic blood. One hour after reperfusion EA was performed and liver biopsies collected at the end of the experiments. The main necrotic zone consisted of coagulative necrosis, sinusoidal dilatation and haemorrhage with an unusual morphological pattern. The coagulative necrosis and haemorrhage affected mainly the peripheral area of the lobule with relative sparing of the area surrounding the centrilobular vein. Contrasting with this sinusoidal dilatation appeared to be more prominent in the centrilobular area. EA produces patterns of tissue destruction that have not been observed with the more commonly used thermal techniques. Further studies should obtain more information about the influence of adjacent biliary and vascular structures so that appropriate clinical trials can be designed.

Integrated Histopathological and Urinary Metabonomic Investigation of the Pathogenesis of Microcystin-LR Toxicosis

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Hepatoprotective effects of Rubus coreanus Miquel concentrates on liver injuries induced by carbon tetrachloride in rats

As well‐being foods pursuing healthy life are getting popular, and interests for Rubus coreanus Miquel (RCM) fruit, kind of Korean blackberry, are heightening as its medical actions, which are protecting liver, brightening eyes, and in curing diabetes, are known. This study was carried out to evaluate hepatoprotective effects of RCM concentrates on liver injuries induced by carbon tetrachloride (CCl4) in rats.RCM, produced in June~July 2008 at Chunbook, Gochang, mashed fine. The seeds of RCM were removed and the juices were condensed. 32 Sprague‐Dawley rats were divided into normal group, CCl4 group (8 rats), 1% RCM group, and 2% RCM group. Experimental diets were provided to experimental animals for four weeks. We measure blood total cholesterol, HDL‐cholesterol, Glutamic oxaloacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), and Alkaline phosphatase (ALP). Part of the livers was separated for hitopathological observation, and measured lipid peroxide (TBARS) and liver protein.The activities of serum GOT, GPT and ALP were elevated by CCl4 administration and levels of hepatic TBARS were also significantly increased in CCl4 groups. However, the hepatic TBARS levels and activities of serum enzymes were markedly reduced by supplementation of the RCM concentrates (p&lt;0.05). In histopathological observation, massive necrosis in centrilobular area and degenerative changes by CCl4 were recovered by dietary supplementation of RCM concentrates.These findings indicate that RCM concentrates have hepatoprotective effects and may improve symptoms of liver injuries.

Complement Activation in Acetaminophen-Induced Liver Injury in Mice

Overdose with acetaminophen (APAP) results in acute liver failure in humans and experimental animals. Complement comprises more than 30 proteins that can participate in tissue injury and/or repair, but the role of complement activation in APAP-induced hepatotoxicity has not been evaluated. Treatment of male, C57BL6J mice with APAP (200-400 mg/kg) resulted in liver injury as evidenced by increased activity of alanine aminotransferase (ALT) in plasma and hepatocellular necrosis. Plasma concentration of the complement component C3 was significantly reduced 6 h after treatment with APAP, indicating complement activation, and C3b (detected by immunostaining) accumulated in the centrilobular areas of liver lobules. Pretreatment with cobra venom factor (CVF; 15 U/mouse) to deplete complement components abolished APAP-mediated C3b accumulation, and this was accompanied by reductions in plasma ALT activity, hepatocellular necrosis, hepatic neutrophil accumulation, and expression of inflammatory genes (interleukin-6, interleukin-10, and plasminogen activation inhibitor-1) at 24 h after APAP treatment. Loss of hepatocellular GSH was similar in APAP-treated mice pretreated with either saline or CVF, suggesting that CVF pretreatment did not affect APAP bioactivation. Mice with a genetic deficiency in C3 had reduced ALT activity 6 and 12 h after APAP administration compared with wild-type animals. These results reveal a key role for complement activation in hepatic inflammation and progression of injury during the pathogenesis of APAP-induced hepatotoxicity.

Hepatic angiosarcoma mimicking sinusoidal obstruction syndrome/venoocclusive disease: a pathologic-radiologic correlation

We present a case of a 63-year-old man with liver dysfunction and biopsy findings of venoocclusive disease (VOD) who, at autopsy, was discovered to have multifocal hepatic angiosarcoma. After double lung transplantation, he initially presented with signs of liver failure and portal hypertension resulting in recurrent high-volume ascites. Clinically, VOD was considered, and tacrolimus was discontinued, due to its known association with VOD. This, however, did not result in clinical improvement, and computed tomography eventually revealed the development of multiple low-attenuating hepatic lesions over the course of several months. Biopsies of the masses and background liver demonstrated changes most consistent with VOD, characterized by sinusoidal congestion affecting the centrilobular areas with associated hepatocyte atrophy and dropout. A reticulin stain highlighted deposition of reticulin fibers within the sinusoids and central veins. Scattered sinusoidal atypical cells were identified; however, a definitive diagnosis of malignancy was not possible. He eventually passed away because of complications of liver disease. At autopsy, there were multiple firm, red-brown masses identified throughout both hepatic lobes. Upon histologic review, the masses were shown to be angiosarcoma. Away from the tumor, the liver also demonstrated features of VOD. It is likely that the histologic appearance of VOD in the background liver probably represents secondary changes due to injury to the hepatic sinusoids by the primary malignancy. We conclude that it is necessary to consider the possibility of unsampled vascular malignancy when hepatic masses are identified on imaging and histology is consistent with VOD.

Preliminary morphological and biochemical changes in rat liver following postnatal exposure to sodium arsenite

The effects of sodium arsenite exposure on the hepatic maturation period of cellular and functional reorganization in developing rat livers were evaluated. Animals received intraperitoneal injections of sodium arsenite (1.5 mg/kg body weight) or distilled water on days 9 to 28 after birth. On day 29, the animals were sacrificed either by cervical dislocation or by perfusion fixation. The perfusion fixed liver tissue was processed for paraffin embedding, sectioning and hematoxylin and eosin staining. The fresh liver tissue was processed for cryo-sectioning followed by Sudan Black B staining and for biochemical estimation of reduced glutathione. Microscopic observation revealed comparable preserved hepatic lobular patterns and distributions of uninucleate and binucleate hepatocytes in the control and the experimental groups. The mean nuclear area and diameter of the hepatocytes was increased in the experimental group. Lipid droplet distribution pattern in Sudan Black B stained sections revealed higher staining intensity towards the centrilobular area in both groups. Semiquantitative estimation of staining intensity showed lower mean gray values in zone 3 than in zones 2 and 1 (suggestive of the setting in of the adult pattern) in both groups. The reduced glutathione levels in the liver tissue and the altered nuclear size of the hepatocytes in the experimental group suggested the impairment of morphological and biochemical processes induced by arsenic exposure during the postnatal period.

Cannabis sativa exacerbates hepatic injury caused by acetaminophen or carbon tetrachloride in rats

The effect of Cannabis sativa extract on acute liver injury caused by acetaminophen or carbon tetrachloride (CCl4) was studied in rats. Cannabis sativa was given at doses of 5 or 10 mg/kg (expressed as Δ9-tetrahydrocannabinol) once daily intraperitoneally (i.p.) for 2 days and simultaneously with acetaminophen or CCl4. Rats were killed 24 h after acetaminophen or CCl4 administration. Reduced glutathione (GSH), lipid peroxidation (malondialdehyde; MDA) and nitric oxide (nitrite/nitrate) concentrations were measured in the liver. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined in serum. Hepatic injury was also determined via histological examination of liver sections. The administration of only cannabis for 2 days had no significant effect on serum liver enzymes or on the hepatic levels of GSH, MDA or nitric oxide. However, in rats intoxicated with acetaminophen, Cannabis sativa at 5 or 10 mg/kg resulted in a significant increase in serum GOT by 17.6% and 19.5%, respectively, compared with the acetaminophen control group. In the CCl4-induced acute liver injury, the levels of AST, ALT and ALP in serum were significantly elevated by Cannabis sativa extract in a dose-dependent manner by 23.7–29.1%, 14.4–21.3% and 17.6–22.1%, respectively. In both models of hepatic injury, Cannabis sativa resulted in a significant increase in the level of liver MDA and nitric oxide and a significant decrease in GSH compared with the corresponding acetaminophen or CCl4 control group. These changes were dose dependent. Histological examination showed an increase in centrilobular necrotic areas in acetaminophen or CCl4-treated rats administered with Cannabis sativa. Histochemical investigation revealed a decrease in intracellular protein contents caused by CCl4 or acetaminophen, and these were further decreased by Cannabis sativa. It is concluded that short-term administration of Cannabis sativa enhances acute hepatic damage caused by CCl4 or acetaminophen in rats.

Dangfei Liganning Capsules ( ) Attenuate the Susceptibility of Rat Nonalcoholic Fatty Liver to Carbon Tetrachloride Toxicity

To test whether nonalcoholic hepatic steatosis sensitizes carbon tetrachloride (CCl4)-induced liver injury, and to assess the therapeutic effect of Chinese medicine extracts of Dangfei Liganning capsules and their potential underlying mechanisms. Male Wistar rats were fed a high-fat diet to induce nonalcoholic fatty liver disease (NAFLD) or a normal diet (N). Eight weeks later, a nonlethal dose of CCl4 was applied intraperitoneally. From the start, HF-CCl4 rats were administered daily Dangyao extracts (D), Dangfei Liganning capsules (DF), or Diammonium Glycyrrhizinate (G) intragastrically. Rats were sacrificed 48 h after CCl4 administration. In addition to serum biochemistry, liver histopathology was observed using hematoxylin-eosin (HE) and oil red O staining, and hepatic levels of triglyceride (TG), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), caspase-3 activation and cytochrome P450 (CYP2E1) expression were assessed. There was almost no response to the nonlethal dose of CCl4 in the N control group. However, the HF group demonstrated massive steatosis, and elevated levels of serum ALT and AST, liver MDA, CYP2E1, and caspase-3 activation, whereas the levels of GSH and SOD were significantly decreased. All indexes assessed were dramatically worse in the HF-CCl4 group compared to the HF group, in addition to the more severe steatosis, hepatocyte ballooning, and inflammatory infiltration apparent in the centrilobular area. The medicines we tested affected the pathological changes in HF-CCl4 rats to differing degrees: DF and G led to improvements in all of the above examined indexes, including an obvious improvement in histopathology, and DF improved serum ALT and MDA levels more markedly than G, whereas D extracts produced only mild liver injury attenuation. Liver with NAFLD is more sensitive to hepatotoxicity; furthermore, the disrupted balance of oxidative stress and anti-oxidant defense contributes to the underlying mechanisms. Dangfei Liganning capsules potentially decrease this toxic susceptibility and alleviate liver injury in non-alcoholic fatty liver.

Immunohistochemical quantification of the Tumor Necrosis Factor (TNF) receptor II expression in the hepatic tissue after systemic administration of the DNA-SWCNT.

The Tumor Necrosis Factor is a key mediator in hepatic inflammatory response during acute exposure to xenobiotics. The cellular effects of TNF are mediated via two cell surface receptors, TNF receptor 1 and TNF receptor 2. The purpose of this study was to examine the expression and to identify the cellular localization of TNFR2 in hepatic tissue by immunohistochemistry after systemic administration of the SWCNT. In the same time the cellular infiltration and the weight of the liver was correlated with the TNF alpha receptor 2 expression. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or SWCNT-DNA (1.5ml, 2.925 mg/kg) for 48 h. The hepatic response associated with SWCNT systemic administration was characterized by increased expression of the TNF receptor II especially in the hepatocytes from the centrilobular and midzonal areas of hepatic lobule. This induced elevation of the TNF receptor II is not followed by hepatic necrosis, inflammatory infiltration or significant changes in liver weight.

Expression of the megalin C-terminal fragment by macrophages during liver fibrogenesis in mice

The low-density-lipoprotein receptor megalin (LRP2, gp330) is strongly expressed in the kidney, where it is responsible for the resorption of metabolites from primary urine. One of the main ligands is the complex of retinol and retinol binding protein. Megalin has been hypothesized to be part of the retinol storage system in liver. Considering the role of hepatic stellate cells in retinol storage and fibrogenesis we investigated mouse strains that developed different degrees of fibrosis after challenge with CCl 4. Immunoblotting revealed the invariable expression of the megalin C-terminal fragment independent of liver damage in all strains. However, only a specific cell population in centrilobular areas of fibrotic livers from DBA/2J mice, which were most susceptible for CCl 4-induced fibrogenesis in our study, was stained using megalin-specific antibodies. Double immunostaining indicated that a subset of hepatic macrophages might represent the megalin-expressing cells in fibrotic liver. Fluorescence activated cell sorting based isolation of hepatic macrophages and megalin specific expression analysis demonstrated the transcription of the whole megalin gene in liver macrophages. We argue that megalin might exhibit a proinflammatory effect by the uptake of retinoids in recruited monocytes, which thereby differentiate to liver macrophages and potentiate fibrogenesis by the release of proinflammatory mediators. Otherwise, megalin might be activated in macrophages during advanced fibrogenesis and act as a negative regulator of proinflammatory genes.