Liver Tissue Injury Research Articles

Protective Effect of Melatonin on Carbon Tetrachloride - induced Hepatic Fibrogenesis in Rats

Background: Liver cirrhosis is a critical stage of chronic liver diseases that canproduce liver failure, portal hypertension and hepatic carcinoma. Sustained oxidativestress plays a key role in cell damage and fibrosis induced during liver cirrhosis. Aimof the work: The aim of the present study was to examine the potential protectiveeffect of exogenous melatonin co-treatment on liver tissue injury and oxidative stressprocesses during induction of early phase of liver fibrosis by carbon tetrachloride(CCl4) injection in rats. Methods: Hepatic fibrogenesis model was induced in thisstudy by subcutaneous injection of rats by carbon tetrachloride (CCl4). Eighteenadult, female albino rats were randomly divided into 3 groups (n = 6): control group(group I), carbon tetrachloride treated group (group II) and CCl4 + melatonin cotreatedgroup (group III). Rats in CCl4 treated group were injected subcutaneouslywith sterile CCl4 (2 ml/kg of body weight) in a ratio of 1:1 with olive oil twice a weekfor 8 weeks. Rats of group III (melatonin co-treated group) were injected with CCl4 inthe same manner as in group II and received intraperitoneal melatonin injection in adose of 20 mg/kg twice a week for 8 weeks, starting from the beginning of CCl4injection. Rats in normal control group were injected subcutaneously with olive oil atthe same dose and frequency as those in CCl4 treated group. At the end of theexperiment, rats were sacrificed, blood samples were collected for biochemical assay.Liver from each animal was removed for histopathological examination.Measurement of oxidative stress markers in serum was done by chemical estimationof serum levels of free radicals: lipid peroxides (LPO) and nitric oxide (NO).Antioxidant enzymes were estimated by chemical measurement of glutathioneperoxidase (GSH-Px) and superoxide dismutase (SOD) in the serum. Liver injury wasassessed by evaluation of serum levels of liver enzymes (alanine aminotransferase(ALT), and aspartate aminotransferase (AST)). Determination of development ofearly phase of hepatic fibrogenesis was done by chemical measurement of serum levelof hyaluronic acid (HA) using enzyme immunoassay (ELISA), and byhistopathological examinations of hepatic tissues to detect early fibrotic changes aswell as other histological damage of hepatic tissue caused by CCl4 injection with orwithout melatonin administration. Results: Results of the present study showed thatCCl4 treatment to rats of group II caused highly significant increase in serum levelsof oxidative stress markers (lipid peroxides and nitric oxide), decrease in serum levelsof antioxidant markers (glutathione peroxidase and superoxide dismutase), increaseof serum levels of hepatic enzymes (ALT and AST) as well as increased serum level ofhyaluronic acid (HA) 8 weeks after CCl4 injections when compared with controlgroup. Melatonin co-treatment to animals of group III caused significant reduction in serum levels of lipid peroxides (LPS) and nitric oxide (NO), significant increase inplasma levels of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD),significant reduction in serum levels of liver enzymes (ALT and AST) as well assignificant decrease in serum level of hyaluronic acid (HA) 8 weeks after CCl4injections when compared with group II. Histopathological study of liver tissue ofanimals of CCl4 treated group showed various manifestation of hepatic cell damageand early phase of fibrogenesis as necrosis, degeneration, collagen deposition andfew fibrous threads extending into the hepatic lobules. Histopathological study ofhepatic tissue of melatonin co-treated group showed that melatonin caused markedamelioration of histological manifestations of hepatic cell degeneration and absenceof any sign of fibrogenesis with nearly normalization of the histological appearanceof the hepatic tissue. Compared with CCl4 treated group (group II), histologicalappearance of hepatic tissue of rats in melatonin co-treated group (group III) showedsignificant improvement. Conclusion: Results of this study suggest that melatonin hasa substantial hepatoprotective effect in a rat hepatic fibrosis model induced by an 8-weeks’ CCl4 regimen. The protective effect of melatonin may be due to both its directradical scavenging properties and indirect effect as a regulator of antioxidantsystems. Therefore, the study proposes that melatonin may be a valuable drug forinhibition of unwanted fibrosis in patients exposed to different hepatotoxic agents.

Caffeine does not enhance radiosensitivity of normal liver tissue in vivo

The aim of this study was to determine whether caffeine enhanced radiosensitivity of normal liver tissue in a rat radiation-induced liver disease model. Buffalo rat McA-RH7777 hepatocellular cancer cells and BRL3A normal liver cells were irradiated, and cell cycle distribution and apoptosis rates were analyzed. A rat model of radiation-induced liver disease was established, rats were randomized into four groups: control; caffeine alone; irradiation (IR) alone; and caffeine plus IR (Caff + IR) group. Apoptosis rates in normal rat liver tissue after IR were evaluated by TUNEL staining and caspase-3 Western blot. Transaminase activity was measured and histopathological examination was done after IR. Caffeine abrogated IR-induced G2 phase arrest (Caff + IR vs. IR: 40.9 ± 4.0 vs. 60.7 ± 5.5%, at 12 h after IR) and increased apoptosis rates (Caff + IR vs. IR: 56.1 ± 6.8 vs. 35.5 ± 4.0%, at 72 h after IR) in McA-RH7777 cells, but did not affect IR-induced G2 phase arrest and apoptosis rates at any time point after IR in BRL3A cells. Caffeine did not enhance apoptosis, transaminase activity, or histopathological injury of normal rat liver tissue at any time points after IR. This study suggests that caffeine might not enhance radiosensitivity of normal liver tissue in vivo. In an earlier study, we reported that caffeine enhanced radiosensitivity of human hepatocellular cancer in a nude mice model. Together, these results offer feasibility of clinical application.

Oral administration of tributyrin increases concentration of butyrate in the portal vein and prevents lipopolysaccharide-induced liver injury in rats

Short-chain fatty acids, especially butyrate, have various biological activities including inhibition of tumor necrosis factor (TNF)-α secretion, via attenuation of nuclear factor-κB (NF-κB) activation. Here, we evaluated the protective effect of oral administration of tributyrin, a prodrug of butyrate, on lipopolysaccharide (LPS)-induced liver injury in rats. Rats were divided into four groups: normal control, tributyrin, LPS, and tributyrin/LPS (treated with tributyrin 1h before LPS). Plasma levels of butyrate and TNF-α, expression of TNF-α, NF-κB, Toll-like receptor (TLR) 2, and TLR4 mRNA in liver, blood biochemical tests, and histopathological analysis of liver were performed. Oral tributyrin increased plasma butyrate level in the portal vein to 2.4mM at 1h and 0.7mM at 2.5h. Tributyrin attenuated NF-κB activation and liver tissue injury associated with LPS injection. The increases in TNF-α level, and hepatic TLR2 mRNA expression were lower in the tributyrin/LPS group. We believe that this study provides the first evidence that orally administered tributyrin increases butyrate level in the hepato-portal system and attenuates liver injury and subsequent inflammatory responses. Oral tributyrin increased plasma butyrate in the portal vein and attenuated liver injury in endotoxemic rats.

Filtration Leukocytapheresis Therapy Ameliorates Lipopolysaccharide-Induced Systemic Inflammation in a Rat Model

Systemic inflammation, which is associated with various conditions such as sepsis, pneumonia, and trauma, can lead to multiple organ dysfunction syndrome. Systemic inflammation can be life-threatening and is often associated with conditions seen in the intensive care unit. Leukocytes exert a proinflammatory effect and damage various tissues during systemic inflammation. The purpose of this study was to determine whether leukocytapheresis therapy can prevent lipopolysaccharide (LPS)-induced systemic inflammation in a rat model. Male Wistar rats weighing 250 to 300 g were used for all experiments. Rats received an LPS injection, followed 6 h later by filtration leukocytapheresis or mock treatment for 30 min under sevoflurane anesthesia. Systemic inflammation was induced in rats by intravenous LPS injection (7.5 mg/kg) followed by filtration leukocytapheresis. Following blood filtration, we evaluated lung and liver histology, serum cytokine levels, and survival rate of rats for each treatment group. Histologic examination revealed markedly reduced inflammatory injury in lung and liver tissue harvested from rats 24 h after leukocytapheresis therapy compared with mock treatment. LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion was also inhibited by leukocytapheresis therapy. Moreover, survival was significantly increased in rats treated with high-efficiency leukocytapheresis compared to mock-treated rats (P<0.05). Taken as a whole, our findings indicate that filtration leukocytapheresis therapy protects against LPS-induced systemic inflammation. Therefore, leukocytapheresis shows potential as a new therapy for various systemic inflammatory diseases.

Antioxidant, free radical scavenging, anti-inflammatory and hepatoprotective potential of the extract from Parathelypteris nipponica (Franch. et Sav.) Ching

Aim of the study The present study was conducted to evaluate the antioxidant, free radical scavenging, hepatoprotective and anti-inflammatory potential of Parathelypteris nipponica (Franch. et Sav.) Ching. Methods and results Antioxidant activity of the methanolic extract of Parathelypteris nipponica (Franch. et Sav.) Ching (TMPN) was studied using in vitro and in vivo models, total flavonoids content in TMPN was found to be 262 ± 5.6 mg/g (w/w). The TMPN exhibited strong antioxidant activity with EC 50 values in reductive ability (0.18 ± 0.02 mg/ml) and ferric thiocyanate (FTC) assay (0.10 ± 0.01 mg/ml), strong free radical scavenging activity as evidenced by the low EC 50 values in DPPH (1,1-diphenyl-2-picrylhydrazyl) (2.00 ± 0.02 mg/ml), superoxide anion (0.60 ± 0.05 mg/ml), OH radicals (0.26 ± 0.03 mg/ml), and hydrogen peroxide (0.45 ± 0.03 mg/ml) methods. Acute toxicity study revealed that the LD 50 value of the extract was more than the dose 2000 mg/kg bodyweight of mice. Hepatoprotective activity of TMPN was determined by the carbon tetrachloride (CCl 4)-induced oxidative tissue injury in rat liver, the extract showed significant hepatoprotective activity that was evident by enzymatic examination and histopathological study. In assessing anti-inflammatory activity the carrageenan-induced rat paw oedema test was used, the extract reduced carrageenan-induced rat paw oedema in dose-dependent manner, achieving high degree of anti-inflammatory activity. Conclusion This study provides a scientific basis for the ethnomedical claims that Parathelypteris nipponica (Franch. et Sav.) Ching is effective against inflammation and liver injury.

Liver Hypoxia and Tissue Injury Are Specific to Sickle Cell Mice in An Experimental Model of Sickle Cell Vaso-Occlusion.

Abstract 2569Poster Board II-546Sickle cell vaso-occlusion causes tissue ischemia, injury, and severe pain in patients with sickle cell disease (SCD). Investigations of the pathophysiology of vaso-occlusion have largely focused on the mechanisms for microcirculatory obstruction, rather than the tissue damage. The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vaso-occlusive pain. Animal models can help to study mechanisms of how vaso-occlusive microcirculatory abnormalities cause tissue damage. Sickle cell mice originally developed by Pastzy (“Berkeley sickle mice”) expressing exclusively human sickle hemoglobin have severe hemolytic anemia and physiologic fragility, and are well-suited for examining an acute vaso-occlusive challenge. HYPOTHESIS:Moderate hypoxic exposure will cause liver hypoxia and tissue injury only in homozygote sickle mice, as signs of vaso-occlusive injury. METHODS:We examined vaso-occlusion by challenging male sickle mice with moderate normobaric hypoxia (10% oxygen) for up to 2 hours then restoring them to normoxia and assaying plasma alanine aminotransferase (ALT) as a quantitative measure of hepatic injury. Pimonidazole (HypoxyProbe 60mg/kg, NPI, Inc) was injected intraperitoneally before hypoxia, in order to provide irreversible labeling of cells with pO2 less than 10 torr. Controls included sickle mice without hypoxia, and non-sickle mice with the same hypoxic exposure. Mice were euthanized for blood and tissue harvest. Liver, kidney, and lungs were fixed in formalin, and processed for immunohistochemical staining (HP-100 kit, NPI). A veterinary pathologist unaware of the treatment group scored histologic sections under light microscopy for the extent of staining on a scale of 1 to 4, and intensity of staining on a scale of 1 to 3. RESULTS:Sickle cell mice have significantly greater extent of Hypoxyprobe labeling of liver than wild-type mice with the same hypoxia-reoxygenation challenge (2.6 + 0.5 vs 1.3 + 0.5, p=0.027 by t-test). At normoxic baseline, liver HypoxyProbe staining from sickle mice and wild-type controls had the same extent (1 vs 1) and intensity of liver staining. Kidney had a higher baseline of Hypoxyprobe staining in all normoxic mice, and this did not increase with hypoxia in sickle mice. Lungs had very little Hypoxyprobe staining at baseline, and a few bronchial cell stained under hypoxia in sickle mice, but the change was not statistically significant. ALT was mildly elevated in sickle mice at normoxic baseline, and rose 3-fold to a peak by 18 hours after hypoxia, then declined back to baseline by 48 – 72 hours. CONCLUSION:The extent of HypoxyProbe staining shows that sickle mice suffer significantly greater liver tissue hypoxia with hypoxic exposure than controls with the same mild hypoxia. Sickle mice in normoxia have no greater extent of HypoxyProbe staining than control mice. These results are consistent with hypoxia-triggered vaso-occlusion as the cause of ALT elevation in the sickle mice. The two outcome measures after hypoxic challenge of sickle mice, HypoxyProbe staining for tissue hypoxia and plasma ALT for tissue injury, provide a bioassay system for pre-clinical screening of antisickling therapy. Disclosures:No relevant conflicts of interest to declare.

Amelioratory effect of vitamin E on organophosphorus insecticide diazinon-induced oxidative stress in mice liver

Considering that the involvement of reactive oxygen species (ROS) has been implicated in the toxicity of organophosphate insecticides (OPIs), the aim of this study was to investigate the ameliorative properties of vitamin E (vitE) against the subchronic effect of diazinon (DZN) on oxidative damage markers such as lipid peroxidation (LPO) and the antioxidant defense system (ADS) in the liver of male MFI albino mice. The groups were intraperitoneally (i.p) administered with either vehicle or vitE (100 mg/kg body weight) or ¼ LD 50 of DZN (16.25 mg/kg b.w.) or ½ LD 50 of DZN; 32.5 mg/kg b.w) or ¼ LD 50–DZN + vitE or ½ LD 50 + vitE every consecutive day for 14 days. Hepatic damage markers analysis revealed that alanine transferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were significantly decreased in both DZN doses. Also, the significantly increased levels of biomarkers of oxidative stress as LPO and protein carbonyl (PC) and the decreased antioxidant defenses like reduced glutathione (GSH), and free radical scavenger enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Rx) were noted in DZN-treated groups as compared to control group. Distinctly lower levels of GSH and increased levels of LPO, along with alterations in endogenous antioxidant enzymes were evident in hepatic toxicity of DZN which is dose-dependent. Hepatic specific marker enzymes were restored to normalcy in mice supplemented with vitE following treatment with DZN which otherwise was decreased in the DZN-treated mice. The results show that co-treatment of vitE with DZN prevents or diminishes the oxidative stress of DZN-treated mice and may act as a putative protective agent against DZN-induced liver tissue injury.

Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity

Phalloidin causes severe liver damage characterized by marked cholestasis, which is due in part to irreversible polymerization of actin filaments. Liver uptake of this toxin through the transporter OATP1B1 is inhibited by the bile acid derivative BALU-1, which does not inhibit the sodium-dependent bile acid transporter NTCP. The aim of the present study was to investigate whether BALU-1 prevents liver uptake of phalloidin without impairing endogenous bile acid handling and hence may have protective effects against the hepatotoxicity induced by this toxin. In anaesthetized rats, i.v. administration of BALU-1 increased bile flow more than taurocholic acid (TCA). Phalloidin administration decreased basal (− 60%) and TCA-stimulated bile flow (− 55%) without impairing bile acid output. Phalloidin-induced cholestasis was accompanied by liver necrosis, nephrotoxicity and haematuria. In BALU-1-treated animals, phalloidin-induced cholestasis was partially prevented. Moreover haematuria was not observed, which was consistent with histological evidences of BALU-1-prevented injury of liver and kidney tissue. HPLC-MS/MS analysis revealed that BALU-1 was secreted in bile mainly in non-conjugated form, although a small proportion (< 5%) of tauro-BALU-1 was detected. BALU-1 did not inhibit the biliary secretion of endogenous bile acids. When highly choleretic bile acids, – ursodeoxycholic (UDCA) and dehydrocholic acid (DHCA) – were administered, they were found less efficient than BALU-1 in preventing phalloidin-induced cholestasis. Biliary phalloidin elimination was low but it was increased by BALU-1 > TCA > DHCA > UDCA. In conclusion, BALU-1 is able to protect against phalloidin-induced hepatotoxicity, probably due to an inhibition of the liver uptake and an enhanced biliary secretion of this toxin.

Impaired liver regeneration following partial hepatectomy using the Pringle maneuver: Protective effect of mesna

We investigated the role of the prophylactic administration of the antioxidant 2-mercaptoethane sulfonate (mesna) on the hepatocyte-regenerating capacity following partial hepatectomy (PH) with concurrent Pringle maneuver. Wistar rats were subjected to PH (70% hepatectomy), 30 min Pringle maneuver, PH plus Pringle with or without mesna pretreatment (400 mg/kg, per os, 3 h before Pringle), or sham operation. At 24 h, 48 h, 72 h, and 1 week after operation, relative liver weight, hepatocyte mitotic activity (mitotic index), the histopathological score and serum aspartate aminotransferase, and alanine aminotransferase concentrations were assessed. At 1 h after operation, oxidative stress markers (glutathione to glutathione disulfide ratio, malondialdehyde concentration, and superoxide dismutase activity) and nuclear factor-kappaB (NF-kappaB) activity were assessed. Hepatectomy stimulated the regenerating process and induced mild oxidative stress and the activation of NF-kappaB in hepatocytes, while causing tissue injury in the remnant liver. When PH was performed under Pringle maneuver, hepatocyte mitotic activity was substantially suppressed, although Pringle alone initiated a delayed regenerating response. Furthermore, Pringle maneuver deteriorated oxidative stress markers, markedly increased NF-kappaB activity, and aggravated tissue injury, as compared to hepatectomy alone. Mesna pretreatment prevented the Pringle-induced antimitotic effect and the induction of oxidative stress, inhibited the activation of NF-kappaB, while attenuating liver injury after PH under Pringle. The excessive activation of NF-kappaB is related to the suppression of hepatocyte-regenerating activity following PH with concurrent liver ischemia. Mesna pretreatment protects the liver against the Pringle-induced antimitotic effect after PH via the prevention of oxidative stress and the inhibition of NF-kappaB activation.

Thoracic epidural anesthesia reverses sepsis-induced hepatic hyperperfusion and reduces leukocyte adhesion in septic rats

IntroductionLiver dysfunction is a common feature of severe sepsis and is associated with a poor outcome. Both liver perfusion and hepatic inflammatory response in sepsis might be affected by sympathetic nerve activity. However, the effects of thoracic epidural anesthesia (TEA), which is associated with regional sympathetic block, on septic liver injury are unknown. Therefore, we investigated hepatic microcirculation and inflammatory response during TEA in septic rats.MethodsForty-five male Sprague-Dawley-rats were instrumented with thoracic epidural catheters and randomized to receive a sham procedure (Sham), cecal ligation and puncture (CLP) without epidural anesthesia (Sepsis) and CLP with epidural infusion of 15 ul/h bupivacaine 0.5% (Sepsis + TEA). All animals received 2 ml/100 g/h NaCl 0.9%. In 24 (n = 8 in each group) rats, sinusoidal diameter, loss of sinusoidal perfusion and sinusoidal blood flow as well as temporary and permanent leukocyte adhesion to sinusoidal and venolar endothelium were recorded by intravital microscopy after 24 hours. In 21 (n = 7 in each group) separate rats, cardiac output was measured by thermodilution. Blood pressure, heart rate, serum transaminase activity, serum TNF-alpha concentration and histologic signs of tissue injury were recorded.ResultsWhereas cardiac output remained constant in all groups, sinusoidal blood flow increased in the Sepsis group and was normalized in rats subjected to sepsis and TEA. Sepsis-induced sinusoidal vasoconstriction was not ameliorated by TEA. In the Sepsis + TEA group, the increase in temporary venolar leukocyte adherence was blunted. In contrast to this, sinusoidal leukocyte adherence was not ameliorated in the Sepsis + TEA group. Sepsis-related release of TNF-alpha and liver tissue injury were not affected by Sepsis + TEA.ConclusionsThis study demonstrates that TEA reverses sepsis-induced alterations in hepatic perfusion and ameliorates hepatic leukocyte recruitment in sepsis.

Taurine supplementation prevents ethanol-induced decrease in serum adiponectin and reduces hepatic steatosis in rats #

Chronic ethanol feeding decreases expression of adiponectin by adipocytes and circulating adiponectin. Adiponectin treatment during chronic ethanol feeding prevents liver injury in mice. Chronic ethanol feeding also increases oxidative and endoplasmic reticulum (ER) stress in adipose tissue. Here we tested the hypothesis that supplemental taurine, an amino acid that functions as a chemical chaperone/osmolyte and enhances cellular antioxidant activity, would prevent ethanol-induced decreases in adiponectin expression and attenuate liver injury. Serum adiponectin concentrations decreased as early as 4 to 7 days after feeding rats a 36% ethanol diet. This rapid decrease was associated with increased oxidative, but not ER, stress in subcutaneous adipose tissue. Taurine prevented ethanol-induced oxidative stress and increased inflammatory cytokine expression in adipose tissue. Ethanol feeding also rapidly decreased expression of transcription factors regulating adiponectin expression (CCAAT/enhancer binding protein alpha; peroxisome proliferator-activated receptor alpha/gamma) in subcutaneous adipose tissue. Taurine prevented the ethanol-induced decrease in CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor alpha, normalizing adiponectin messenger (m)RNA and serum adiponectin concentrations. In the liver, taurine prevented ethanol-induced oxidative stress and attenuated tumor necrosis factor alpha expression and steatosis, at least in part, by increasing expression of genes involved in fatty acid oxidation. In subcutaneous adipose tissue, taurine decreased ethanol-induced oxidative stress and cytokine expression, as well as normalized expression of adiponectin mRNA. Taurine prevented ethanol-induced decreases in serum adiponectin; normalized adiponectin was associated with a reduction in hepatic oxidative stress, tumor necrosis factor alpha expression, and steatosis. Taken together, these data demonstrate that taurine has important protective effects against ethanol-induced tissue injury in both adipose and liver tissue.

Alpinia katsumadai hayata prevents mouse sepsis induced by cecal ligation and puncture through promoting bacterial clearance and downregulating systemic inflammation

Sepsis continues to be a challenge in clinic. Therapeutic strategies focus on local host defenses and the inhibition of overwhelming inflammation response. The present study aimed to investigate the protective effects and the underlying mechanisms of the ethanol extract of Alpinia katsumadai Hayata seeds (EAKH) on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. It was shown that oral administration of EAKH at 1 h before and 2 h after CLP significantly elevated the survival rate and the mean arterial pressure of mice. Histological examination and serum ALT/AST assessment demonstrated that EAKH protected the animals from lung and liver tissue injury and dysfunction. Although EAKH was devoid of direct bacteriostatic or bacteriocidal activities, it facilitated peritoneal bacteria clearance and increased leukocyte migration into peritoneal cavity of septic mice. Furthermore, EAKH remarkably decreased serum pro-inflammatory cytokine (TNF-alpha, IL-1beta and NO) levels in septic mice. These findings demonstrated that EAKH has preventive effects on mouse sepsis induced by CLP, which may be attributed to elevating local defense via promoting leukocyte migration to infection focus and attenuating systemic inflammation.

Fatal Autoimmune Hepatitis Induced by Concurrent Loss of Naturally Arising Regulatory T Cells and PD-1-Mediated Signaling

Because of the lack of animal models developing spontaneous autoimmune hepatitis (AIH), the molecular mechanisms involved in the development of AIH are still unclear. This study aims to examine the regulatory roles of naturally arising CD4(+)CD25(+) regulatory T (Treg) cells and programmed cell death 1 (PD-1)-mediated signaling in the development of AIH. To induce a concurrent loss of Treg cells and PD-1-mediated signaling, neonatal thymectomy (NTx), which severely reduces the number of Treg cells, was performed on PD-1(-/-) mice. After the NTx, we performed histologic examination, assessed autoantibody production and infiltrating cells in the liver, and conducted adoptive transfer experiments. In contrast to NTx mice and PD-1(-/-) mice, NTx-PD-1(-/-) mice produced antinuclear antibodies and developed fatal hepatitis characterized by a CD4(+) and CD8(+) T-cell infiltration invading the parenchyma with massive lobular necrosis. Induction of AIH in NTx-PD-1(-/-) mice was suppressed by transfer of Treg cells, even derived from PD-1(-/-) mice. Transfer of total but not CD4(+) T-cell-depleted splenocytes from NTx-PD-1(-/-) mice into RAG2(-/-) mice induced the development of severe hepatitis. In contrast, the transfer of CD8(+) T-cell-depleted splenocytes triggered only mononuclear infiltrates without massive necrosis of the parenchyma. NTx-PD-1(-/-) mice are the first mouse model of spontaneous fatal AIH. The concurrent loss of Treg cells and PD-1-mediated signaling can induce the development of fatal AIH. Autoreactive CD4(+) T cells are essential for induction of AIH, whereas CD8(+) T cells play an important role in progression to fatal hepatic damage.

A study on antioxidant, free radical scavenging, anti-inflammatory and hepatoprotective actions of Aegiceras corniculatum (stem) extracts

Aim of the study The present study was conducted to evaluate the antioxidant, anti-inflammatory and hepatoprotective potential of Aegiceras corniculatum Linn. Blanco (Aegicerataceae). Methods and results The n-hexane, ethyl acetate and methanol extracts, derived from Aegiceras corniculatum stems, scavenged superoxide anions (O 2 −) and hydroxyl radicals ( OH) in nitro blue tetrazolium reduction and deoxyribose degradation assays, respectively. All the extracts inhibited the process of lipid peroxidation at its initiation step. Additionally, in rat liver microsomes n-hexane and ethyl acetate extracts also caused termination of radical chain reaction supporting their scavenging action towards lipid peroxy radicals (LOO ). Moreover, increased production of O 2 − in human neutrophils, stimulated by phorbol-12-myristate-13-acetate (PMA) and/or opsonized zymosan were also suppressed (IC 50 ∼3–20 μg/mL). Thereby, revealing the ability of plant extracts to antagonize the oxidative stress via interference with NADPH oxidase metabolic pathway. These in vitro results coincide with the reduction in the glucose oxidase-induced paw edema in mice in the presence of ethyl acetate and methanol extracts (10, 50, and 100 mg/kg, i.p.). Plant extracts (250, 500 and 1000 mg/kg, p.o.) also significantly protected the carbon tetrachloride (CCl4)-induced oxidative tissue injury in rat liver. This was reflected by a ∼60% decline in the levels of serum aminotransferase enzymes. Conclusion Aegiceras corniculatum extracts found to possess pronounced antioxidant effect that may be at least in part related to its anti-inflammatory and hepatoprotective activities. This study provides a scientific basis for the ethnomedical claims that Aegiceras corniculatum is effective against inflammation and liver injury.

Relaxin as a Protective Substance in Preservation Solutions for Organ Transplantation, as Shown in an Isolated Perfused Rat Liver Model

Reperfusion injury, a well-known problem in organ transplantation, results from multiple pathologic mechanisms, including platelet/mast cell activation and peroxidation of cell membrane lipids. Relaxin was originally described as an insulin-like hormone produced in the ovaries during pregnancy. It causes vessel dilation and inhibition of platelet and mast cell activation. The present study investigated the protective effect of relaxin against reperfusion injury in liver tissue. We used a model of isolated perfused rat liver to simulate liver transplantation. Organ preservation was performed identical to human transplantation in 20 male Wistar rats. During preservation we applied 64 ng/mL relaxin. In contrast controls (n = 10) had no relaxin treatment. To quantify cell damage, we measured malonyldialdehyde (MDA; end product of lipid peroxidation) and myeloperoxidase activity (MPO; marker for accumulation of neutrophil granulocytes) in the perfusates. The livers were examined immunohistochemically for the same parameters. Relaxin as an additional substance in preservation solutions decreased perfusate MPO and MDA levels by up to 30%, as shown by immunohistochemistry. Our preliminary data suggested that relaxin is a promising agent to reduce hepatocyte damage caused by ischemia-reperfusion injury. Quantitative analysis of MDA and MPO levels in the perfusate is the subject of an ongoing study.

Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids: Involvement of Regulatory T Cells

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Delta(9)-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.

The Protective Effect of CAPE on Hepatic Ischemia/Reperfusion Injury in Rats

The transcription factor nuclear factor-kappa B (NF-kappaB) exerts a pivotal role in the pathogenesis of hepatic ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent and specific NF-kappaB inhibitor, presents protective effects on I/R injury in some tissues. This study aimed to evaluate the effect of CAPE on hepatic I/R injury in rats. Wistar rats were submitted to a sham operation, 60 min ischemia, or 60 min ischemia plus saline or CAPE treatment followed by 6 h reperfusion. Liver tissue injury was evaluated by alanine aminotransferase, aspartate aminotransferase, and tissue glutathione measurement, and histological damage score. Apoptotic hepatocytes were determined by the transferase-mediated dUTP-biotin nick-end labeling assay. Hepatic neutrophil accumulation was assessed by the naphthol method. Lipid peroxidation and NF-kappaB activation were evaluated by 4-hydroxynonenal and NF-kappaB p65 immunohistochemistry, respectively. Animals submitted to ischemia showed a marked increase of alanine aminotransferase and aspartate aminotransferase after reperfusion, but with lower levels in CAPE group. Tissue glutathione content declined gradually during ischemia to reperfusion and was partially recovered with CAPE treatment. The histological damage score, apoptosis index, and neutrophil infiltration, as well as 4-hydroxynonenal and NF-kappaB p65 nuclear labeling, were higher in the liver of animals submitted to I/R compared to the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. CAPE was able to protect the liver against normothermic I/R injury in rats. This effect may be associated with the inhibition of the NF-kappaB signaling pathway and decrease of the acute inflammatory response following I/R in the liver.

Alleviation of ischemia/reperfusion injury in ob/ob mice by inhibiting UCP-2 expression in fatty liver

To investigate the protective effect of target suppression of uncoupling protein-2 (UCP-2) on ischemia/reperfusion (I/R) injury in fatty liver in ob/ob mice. Plasmids suppressing UCP-2 expression were constructed, and transfected into fatty liver cells cultured in vitro and the ob/ob mouse I/R injury model. Serum tumor necrosis factor (TNF)- alpha levels, UCP-2 mRNA expression, alanine aminotransferase (ALT) levels in ob/ob mice were tested, and the pathological changes in fatty liver were observed in experimental and control groups. In ob/ob mouse I/R models, serum TNF-alpha levels were significantly higher than in normal controls. After the plasmids were transfected into the cultured cells and animal models, expression of UCP-2 mRNA was significantly reduced as compared with that in the control group (2(1.56+/-0.15) vs 2(-0.45+/-0.15), P<0.05). In ob/ob mouse models, in which expression of UCP-2 was suppressed, serum ALT levels were significantly lower than those of other groups, and pathological analysis revealed that injury of liver tissues was significantly alleviated. The target suppression of UCP-2 expression in fatty liver can alleviate the I/R injury in the ob/ob mice.

The Protective Effect of Aprotinin and α-Tocopherol on Ischemia-Reperfusion Injury of the Rat Liver

Background Liver injury caused by ischemia-reperfusion (I/R) processes is a complication of hepatic resection surgery and transplantation, particularly using grafts from marginal donors. Despite improvements in organ preservation and advances in surgical techniques, I/R injury remains a significant clinical problem. In this study, we investigated whether aprotinin provided protection against the adverse effects of I/R injury in liver tissue. Methods Forty rats were randomized into four groups ( n = 10): group I: (control group) I/R + no medication; group II: sham-operated group + no medication or I/R; group III: I/R + aprotinin; group IV: I/R + α-tocopherol. Malondialdehyde (MDA) was measured in the liver tissue and superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as lactate dehydrogenase (LDH) in rat serum. Results Administration of aprotinin and α-tocopherol before I/R resulted in significant reductions of MDA levels compared to the I/R alone group (group I; P = .01 and P < .01, respectively). Administration of aprotinin or α-tocopherol prior to I/R resulted in significant increases in SOD and CAT levels compared with the I/R group ( P < .05 each). Compared to the I/R group, significant decreases in plasma AST, ALT, and LDH levels were observed both in the aprotinin and in the α-tocopherol group ( P < .05). Histological evaluation revealed the injury grade to be relatively lower among groups III and IV compared to group I. Discussion In conclusion, rat hepatic structures in aprotinin and α-tocopherol administered groups were well protected. Therefore, aprotinin may provide protection against the adverse effects of I/R injury in liver transplantation.

IL-19 IS INVOLVED IN THE PATHOGENESIS OF ENDOTOXIC SHOCK

IL-19, a proinflammatory cytokine, belongs to the IL-10 family. IL-19 is induced in systemic inflammatory response syndrome, but its pathophysiological function in sepsis is unclear. Our aim was to determine the roles of IL-19 in endotoxin-induced tissue damage in vivo and in vitro. We examined serum levels of IL-19 in sepsis patients and healthy volunteers, determined the in vitro effects of IL-19 on lung epithelial cells, liver cells, and neutrophils, and analyzed the tissue expression of IL-19 and its receptors in murine endotoxic shock. Electroporation-mediated gene transfer of mouse IL-19-soluble receptor plasmid DNA was used to determine the effects of IL-19 depletion in preventing endotoxic shock-induced tissue damage in mice. We found that serum levels of IL-19 were higher in patients than in healthy volunteers (n = 28, P = 0.001). IL-19 induced apoptosis in lung epithelial cells and reactive oxygen species production in liver cells in vitro. IL-19 also promoted neutrophil chemotaxis, reduced neutrophil apoptosis, and induced the production of proinflammatory cytokines and chemokines (IL-1[beta], IL-6, IL-8, CCL5, and CXCL9) in lung epithelial cells. In LPS-challenged mice, transcripts of IL-19 and its receptors were up-regulated in heart, lung, liver, and kidney tissue. Neutrophil infiltration in lung and liver tissue, and serum levels of alanine transaminase and aspartate transaminase, were lower in mice electroporated with IL-19-soluble receptor plasmid DNA before LPS treatment compared with control mice. These results suggest that up-regulated IL-19 may be involved in lung and liver tissue injury in murine endotoxic shock.