Cultured Mouse Hepatocytes Research Articles

Chemical constituents from Sambucus williamsii Hance fruits and hepatoprotective effects in mouse hepatocytes

Chemical investigation of Chinese folk medicine Sambucus williamsii Hance has resulted in the isolation and characterisation of seventeen compounds from the n-BuOH extract of its fruits, including two new phenylethanoid glycosides and fifteen known compounds. Structures of new compounds were elucidated primarily on the basis of their extensive spectroscopic data including 2D NMR. In addition, the n-BuOH extract from the fruits of S. williamsii was found to show a protective effect on D-galactosamine (D-GalN)-induced cytotoxicity in primary cultured mouse hepatocytes. So the hepatoprotective effects of principal constituents from it were tested by MTT assays. The results showed that Compounds 13, 16 and 17 displayed hepatoprotective effects.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Abstract 540: Activation of Srebp1c Processing is Required in Flx-Induced Hepatic Lipid Accumulation

Fluoxetine (FLX), a typical drug belonging to the category of selective serotonin reuptake inhibitors (SSRI), is the most widely prescribed psychoactive drug in the treatment of depression and other mood disorders. It has been demonstrated that the administration of FLX increases the possibility of weight gain and dyslipidemia. We find previously that dysregulation of lipogenic and lipolytic genes is critical in FLX-promoted hepatic lipid accumulation. Therefore, a chronic mild stress depression model and cultured primary mouse hepatocytes were used to investigate the effects and underlying mechanisms of FLX on the promoted hepatic lipid accumulation. The evidence have shown that FLX increases the concentrations of triglyceride (TG) and total cholesterol (TC) in the liver tissues of depressive mice, while only increases TG in the liver tissues of normal mice. FLX induces lipid accumulation in both normal and depressive mice by upregulating the lipogeneic genes Acetyl-CoA carboxylase 1 (ACC1) and Fatty acid synthase (FAS) expression and downregulating the lipolytic genes carboxylesterase 1 (CES1) and CES2. Using primary cultured mouse hepatocytes, it is shown that FLX promotes the expression of transcription factor SREBP1c as well as its proteolytic cleavage and nuclear translocation. FLX significantly suppresses SREBP1c proteolytic cleavage in hepatocytes after the incubation lasting as short as 3 hours, which is a more prompt way than the elevated expression of SREBP1c. Further experiments show that the specific inhibitors of proteases S1P and S2P attenuate FLX-induced SREBP1c activation and hepatic lipid accumulation. As SCAP (SREBP cleavage-activating protein) escorts SREBPs from the endoplasmic reticulum to the Golgi complex where proteases cleave SREBPs and therefore is required for SREBP activation, we find that FLX promotes the protein level of SCAP in a concentration- and time-dependent manner. In conclusion, FLX directly acts on the hepatocytes by facilitating the expression and proteolytic activation of SREBP1c to promote hepatic lipid accumulation. The findings not only provide new insight into the understanding of the mechanisms for SSRI-mediated dyslipidemia effects, but also suggest a novel therapeutic target to interfere.

Quantitative Determination of Stilbenoids and Dihydroisocoumarins in Shorea roxburghii and Evaluation of Their Hepatoprotective Activity.

A simultaneous quantitative analytical method for 13 stilbenoids including (−)-hopeaphenol (1), (+)-isohopeaphenol (2), hemsleyanol D (3), (−)-ampelopsin H (4), vaticanols A (5), E (6), and G (7), (+)-α-viniferin (8), pauciflorol A (9), hopeafuran (10), (−)-balanocarpol (11), (−)-ampelopsin A (12), and trans-resveratrol 10-C-β-d-glucopyranoside (13), and two dihydroisocoumarins, phayomphenols A1 (14) and A2 (15) in the extract of Shorea roxburghii (dipterocarpaceae) was developed. According to the established protocol, distributions of these 15 polyphenols (1–15) in the bark and wood parts of S. roxburghii and a related plant Cotylelobium melanoxylon were evaluated. In addition, the principal polyphenols (1, 2, 8, 13–15) exhibited hepatoprotective effects against d-galactosamine (d-galN)/lipopolysaccharide (LPS)-induced liver injury in mice at a dose of 100 or 200 mg/kg, p.o. To characterize the mechanisms of action, the isolates were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes; (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages; and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of these polyphenols (1, 2, and 8) were suggested to be dependent on the inhibition of LPS-induced macrophage activation and reduction of sensitivity of hepatocytes to TNF-α. However, none of the isolates reduced the cytotoxicity caused by d-GalN.

붕어와 마우스의 간세포 배양에서 열 스트레스에 의해 유도되는 heat shock factor1 (HSF1)의 비교

Heat shock proteins (HSPs) are induced in response to various physiological or environmental stressors. However, the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). Fish models provide an ideal system for examining the biochemical and molecular mechanisms of adaptation to various temperatures and water environments. In this study, we examined the pattern differentials of heat shock factor 1 (HSF1) and expression of heat shock protein 70 (HSP70) in response to thermal stress in goldfish and mouse hepatocyte cultures by immune-blot analysis. Goldfish HSF1 (gfHSF1) changed from a monomer to a trimer at 33°C and showed slightly at 37°C, whereas mouse HSF1 (mHSF1) did so at 42°C. This experiment showed similar results to a previous study, indicating that gfHSF1 and mHSF1 play different temperature in the stress response. We also examined the activation conditions of the purified recombinant proteins in human HSF1 (hmHSF1) and gfHSF1 using CD spectroscopy and immune-blot analysis. The purified recombinant HSF1s were treated from 25°C to 42°C. Structural changes were observed in hmHSF1 and gfHSF1 according to the heat-treatment conditions. These results revealed that both mammal HSF1 (human and mouse HSF1) and fish HSF1 exhibited temperature-dependent changes; however, their optimal activation temperatures differed.

Acetaminophen toxicity in rat and mouse hepatocytes in vitro

Context: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. Objectives: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). Results: APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury. Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.

Protective effect of Cistanchis A on ethanol-induced damage in primary cultured mouse hepatocytes.

Cistanoside A (C. A) was one of phenylethanol glycosides isolated from Cistanche deserticola, a tonic in traditional Chinese medicine. In our previous research, we demonstrated that Cistanoside A (C. A) possess the protective activities on CCl4 induced hepatotoxicity in mice, such as increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and improving respiratory chain function in mitochondria. Meanwhile, our previous research also demonstrated C.A possess protective activities on alcohol induced hepatotoxicity in mice, shown in ameliorate the hepatic function indices, lightening steatosis and inflammatory infiltration, increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and alleviating energy metabolism in mitochondria. The aim of this research was to evaluate the effects of Cistanoside A (C. A) on ethanol-induced damage in primary cultured mouse hepatocytes, and probe into the mechanism related. Using fluorescent staining, flow cytometer, immunohistochemistry analysis, and Western blotting, we demonstrated that C.A could enhance the survival rate of the primary cultured hepatocytes, alleviate apoptosis and necrosis, the mechanism was involved with enhance the expression of apoptosis inhibition factor bcl-2, and inhibition the expression of immediate early genes c-fos.

Hepatoprotective Limonoids from Andiroba (Carapa guianensis).

Three gedunin-type limonoids, gedunin (1), 6α-acetoxygedunin (2), and 7-deacetoxy-7-oxogedunin (3), which were isolated from the seed and flower oils of andiroba (Carapa guianensis Aublet, Meliaceae), exhibited hepatoprotective effects at doses of 25 mg/kg, p.o. against d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. To characterize the mechanisms of action of 1–3 and clarify the structural requirements for their hepatoprotective effects, 17 related limonoids (1–17) isolated from the seed and/or flower oils of C. guianensis were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of 1–3 are likely to involve the inhibition of LPS-induced macrophage activation and reduced sensitivity of hepatocytes to TNF-α; however, these compounds did not decrease the cytotoxicity caused by d-GalN. In addition, the structural requirements of limonoids (1–17) for inhibition of LPS-induced NO production in mouse peritoneal macrophages and TNF-α-induced cytotoxicity in L929 cells were evaluated.

Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4′-trimethoxy-2′-hydroxy-chalcone (CH1) and 3′-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas.

Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes.

Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism.

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

Analysis of aquaporin expression in liver with a focus on hepatocytes.

A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

A new mechanism of lipotoxicity: Calcium channel blockers as a treatment for nonalcoholic steatohepatitis?

A new mechanism of lipotoxicity: Calcium channel blockers as a treatment for nonalcoholic steatohepatitis?

Salidroside ameliorates insulin resistance through activation of a mitochondria-associated AMPK/PI3K/Akt/GSK3β pathway.

Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms. The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg(-1) ·day(-1)) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro. Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3β (GSK3β) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio. Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3β pathway.

Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I

Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD+ ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30µM EFV and submaximal effects at 50µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD+ ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical.

Biologically Active Peptides Isolated from Dill Anethum graveolens L

Peptide mixtures with molecular weights of 1000-2000 Da and in vivo membrano-trophic activity against mouse hepatocyte culture at very low concentrations were isolated from dill Anethum graveolens L. leaves. It has been found that plant peptides in aqueous solution formed larger nanosized particles of approximately 90 nm with a secondary structure mainly composed of β-structures and random coil structures. We demonstrated that peptides isolated from A. graveolens in vitro at an ultra-low dosage affected the size of the area of pigmented cells of amphibian liver, which are analogous to Kupffer cells of the mammalian liver, using roller organotypic newt liver culture models.

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes.

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury.

Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours. MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored.