Methionine-choline Deficient Group Research Articles

Effects of Ganjianglingzhu Decoction on Lean Non-Alcoholic Fatty Liver Disease in Mice Based on Untargeted Metabolomics.

Non-alcoholic fatty liver disease (NAFLD) is usually associated with obesity. However, it is crucial to recognize that NAFLD can also occur in lean individuals, which is frequently overlooked. Without an approved pharmacological therapy for lean NAFLD, we aimed to investigate whether the Ganjianglingzhu (GJLZ) decoction, a representative traditional Chinese medicine (TCM), protects against lean NAFLD and explore the potential mechanism underlying these protective effects. The mouse model of lean NAFLD was established with a methionine-choline-deficient (MCD) diet in male C57BL/6 mice to be compared with the control group fed the methionine-choline-sufficient (MCS) diet. After four weeks, physiological saline, a low dose of GJLZ decoction (GL), or a high dose of GJLZ decoction (GH) was administered daily by gavage to the MCD group; the MCS group was given physiological saline by gavage. Untargeted metabolomics techniques were used to explore further the potential mechanism of the effects of GJLZ on lean NAFLD. Different doses of GJLZ decoction were able to ameliorate steatosis, inflammation, fibrosis, and oxidative stress in the liver; GL performed a better effect on lean NAFLD. In addition, 78 candidate differential metabolites were screened and identified. Combined with metabolite pathway enrichment analysis, GL was capable of regulating the glucose and lipid metabolite pathway in lean NAFLD and regulating the glycerophospholipid metabolism by altering the levels of sn-3-O-(geranylgeranyl)glycerol 1-phosphate and lysoPC(P-18:0/0:0). GJLZ may protect against the development of lean NAFLD by regulating glucose and lipid metabolism, inhibiting the levels of sn-3-O-(geranylgeranyl)glycerol 1-phosphate and lysoPC(P-18:0/0:0) in glycerophospholipid metabolism.

Theabrownin ameliorates liver inflammation, oxidative stress, and fibrosis in MCD diet-fed C57BL/6J mice.

Nonalcoholic steatohepatitis (NASH), also known as metabolic steatohepatitis, is a clinical syndrome with pathological changes like alcoholic hepatitis but without a history of excessive alcohol consumption. NASH is closely related to metabolic disorders such as obesity, insulin resistance, type 2 diabetes mellitus, and hyperlipidemia. Its main characteristics are hepatocyte steatosis with hepatocyte injury and inflammation. In severe cases, it can develop into liver cirrhosis. At present, there is no special treatment for NASH. Theabrownin (TB) is the main pigment substance in fermented tea. Theabrownin has beneficial effects on lipid metabolism and intestinal flora. However, the effect of theabrownin on NASH has not been studied. This study was aimed at exploring the effects of theabrownin from Fuzhuan brick tea on NASH. 8-week-old mice were randomly assigned to three groups and fed with chow diet (CD), methionine and choline sufficient (MCS) diet (MCS Ctrl), which is a Methionine/choline deficient (MCD) control diet, and MCD diet. After 5 weeks of feeding, the MCD group mice were randomly divided into two groups and were gavaged with double distilled water (MCD Ctrl) or theabrownin (MCD TB) (200mg/kg body weight, dissolved in double distilled water) every day for another 4 weeks respectively, while continuing MCD diet feeding. We found that theabrownin treatment could not improve liver mass loss and steatosis. However, theabrownin ameliorated liver injury and decreased liver inflammatory response. Theabrownin also alleviated liver oxidative stress and fibrosis. Furthermore, our results showed that theabrownin increased hepatic level of fibroblast growth factor 21 (FGF21) and reduced the phosphorylation of mitogen-activated protein kinase p38 in MCD diet-fed mice.

Low-Dose Administration of Cannabigerol Attenuates Inflammation and Fibrosis Associated with Methionine/Choline Deficient Diet-Induced NASH Model via Modulation of Cannabinoid Receptor

8-week-old C57BL/6 male mice were fed with methionine/choline deficient (MCD) diet or control (CTR) diets for five weeks. At the beginning of week 4, mice were divided into three sub-groups and injected with either a vehicle, a low or high dose of CBG for two weeks. Overall health of the mice, Hepatic steatosis, fibrosis, and inflammation were evaluated. Increased liver-to-body weight ratio was observed in mice fed with MCD diet, while a low dose of CBG treatment rescued the liver-to-body weight ratio. Hepatic ballooning and leukocyte infiltration were decreased in MCD mice with a low dose of CBG treatment, whereas the CBG treatment did not change the hepatic steatosis. The high dose CBG administration increased inflammation and fibrosis. Similarly, the expression of cannabinoid receptor (CB)1 and CB2 showed decreased expression with the low CBG dose but not with the high CBG dose intervention in the MCD group and were co-localized with mast cells. Additionally, the decreased mast cells were accompanied by decreased expression of transforming growth factor (TGF)-β1. Collectively, the low dose of CBG alleviated hepatic fibrosis and inflammation in MCD-induced NASH, however, the high dose of CBG treatment showed enhanced liver damage when compared to MCD only group. These results will provide pre-clinical data to guide future intervention studies in humans addressing the potential uses of CBG for inflammatory liver pathologies, as well as open the door for further investigation into systemic inflammatory pathologies.

Morphological Changes in Major Salivary Glands in Mice Treated With a Choline and Methionine Deficient Diet.

It has been shown that the methionine-choline deficient (MCD) diet induces hepatocarcinogenesis, but not in extrahepatic organs, such as the testis, and pancreas, although may increase chemical-induced carcinogenesis in the colon, mammary gland, esophagus, and pancreas. Accumulating evidence suggests that salivary glands are very susceptible to stress conditions, such as radiation, hyperglycemia, and exposure to xenobiotics in vivo. This study aimed to analyze the histological changes on the major salivary glands (parotid, submandibular, and sublingual) after MCD diet administration. Male Swiss mice were submitted to ad libitum access to the control (AIN-76) or MCD diet for 28 days. The rebound group received the MCD diet for 24 days and the control diet for 10 days. Using the AxioImager A2 microscope, the hematoxylin-eosin (HE) stained specimens (4 mm) were evaluated for tissue degeneration, nuclear hyperchromatism and atrophy. In the parotid gland from the MCD group, tissue degeneration, pyknosis, apoptosis and atrophy were observed, which remained in the rebound group, associated with hyperchromatism. In the submandibular gland from both MCD and rebound groups, severe tissue disorganization was associated with cell pleomorphism, hyperchromatic cells, apoptosis, increased eosinophilia, and inflammatory infiltrate. Finally, in the sublingual gland, there were no histological alterations in the experimental groups compared to the control. MCD can induce pre-neoplastic changes in the mouse parotid and submandibular glands, which are not reversed by a change in the diet.

Ethyl pyruvate supplemented in drinking water ameliorates experimental nonalcoholic steatohepatitis

Inflammation and oxidative stress play a significant role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Ethyl pyruvate (EP) is a novel anti-inflammatory agent and a potent reactive oxygen species (ROS) scavenger. Therefore, EP supplemented in drinking water may alleviate experimental NASH in this study (even though 0.3% of EP cannot attenuate the simple non-aggressive fatty liver). The methionine-choline-deficient (MCD) diet was given to the C57BL/6 male mice for 3 weeks to induce NASH. The NASH animals were randomized into 3 treatment groups: animals in the MCD alone group were treated with normal drinking water alone; animals in the delayed EP group were given 3% (v/v) of EP supplemented in normal drinking water, the treatment started 10 days after MCD diet feeding; animals in the early EP therapy group were treated the same as the delayed EP group except that EP treatment started the same day when MCD diet was given; the control mice were fed with normal chow and treated with normal drinking water (n = 10 for each group). Compared to MCD group with normal drinking water, early EP treatment significantly decreased serum ALT and improved NASH histopathology; delayed EP therapy only attenuated NASH in 50% (5/10) of the animals. The beneficial effects were associated with decreased hepatic TNF-a and IL-6 mRNA expression on early 5 days, inhibited NF-kB activation, reduced liver tissue malondialdehyde levels, and decreased intestinal bacterial translocation (BT). In conclusion: EP supplemented in drinking water attenuates experimental NASH.

Withdrawal Notice: Therapeutic Effect of Prdx1 on NAFLD Mice May be Related to the Activation of Nrf-2/HO-1 Pathway

It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneously submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submitting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript the authors agree that the copyright of their article is transferred to the publishers if and when the article is accepted for publication.

Epigallocatechin Gallate Protects Mice against Methionine-Choline-Deficient-Diet-Induced Nonalcoholic Steatohepatitis by Improving Gut Microbiota To Attenuate Hepatic Injury and Regulate Metabolism.

Epigallocatechin gallate (EGCG) has been regarded as a protective bioactive polyphenol in green tea against nonalcoholic steatohepatitis (NASH), but the mechanism remains poorly deciphered. Herein, we assessed the role and mechanism of EGCG on gut microbiota and the metabolism in NASH development. Forty-eight male C57BL/6J mice were fed with either a methionine–choline-sufficient diet or a methionine–choline-deficient (MCD) diet with or without EGCG administration for 4 weeks. Liver injury, inflammation, lipid accumulation, and iron overload were examined. 16S ribosomal RNA sequencing was used to detect the fecal microbiome. In our research, we observed that EGCG notably improved MCD-diet-derived gut microbiota dysbiosis, as proved by a distinctively clustered separation from that of the MCD group and by the decrease of the Oxalobacter, Oscillibacter, Coprococcus_1, and Desulfovibrio genera and enrichment of norank_f__Bacteroidales_S24_7_group, Alloprevotella, and Bacteroides. Spearman-correlation heatmap analysis indicated that Bacteroides and Alloprevotella induced by EGCG were strongly negatively correlated with lipid accumulation. Functional enzymes of the gut microbiome were predicted by PICRUSt based on the operation classification unit. The results revealed that 1468 enzymes were involved in various metabolic pathways, and 371 enzymes showed distinct changes between untreated and EGCG-treated mice. Long-chain-fatty-acid-CoA ligase ACSBG played a distinct role in fatty acid metabolism and ferroptosis and was significantly negatively correlated with Bacteroides. Altogether, the salutary effect of EGCG on NASH might be via shifting gut flora and certain enzymes from genera. Our study thus takes a step toward NASH prevention and therapy.

Expression of Notch family is altered in non‑alcoholic fatty liver disease

The aim of the present study was to explore the dynamic relationship between Notch and non-alcoholic fatty liver disease (NAFLD), both in vitro and in vivo. The LX2, Huh7 and MIHA hepatic cell lines were used to establish a cell steatosis model induced by palmitic acid (PA) at different concentrations (0.1, 0.25 and 0.5 mM). Cell proliferation and migration were assessed using a 5-bromo-2′-deoxyuridine kit and a wound healing assay. The dosage of 0.25 mM PA for 36–48 h treatment was chosen for subsequent experiments. Steatotic cells were identified by Oil Red O staining. Feeding mice a methionine-choline-deficient (MCD) diet is known induce a model of NAFLD, compared with a methionine-choline-sufficient (MCS) diet. Therefore, Notch family mRNA expression was evaluated in the liver of MCD-fed mice at varying time points (days 5, 10, 21 and 70) using reverse transcription-quantitative PCR. Notch expression levels were also assessed in cell lines at 12, 24, 36 and 48 h after PA treatment. Notch signaling molecules changed in the PA or MCD model over time. In vitro, the mRNA levels of Notch1, −2 and −4 increased in all cell lines after 12-h PA treatment. At 24 h, these genes were upregulated only in LX2 cells, while showing a ‘down-up’ pattern in MIHA cells (i.e. these genes were downregulated at 24 h but upregulated at 36 h). However, expression of Notch1, −2, −3 and −4 mRNA rose significantly in the early stage (day 10) of NAFLD. At week 3, the levels of Notch1 and −2 were higher in the MCD group than in the MCS group, while the reverse was observed for Notch3 and −4. Expression of these four genes increased again in the late stage (day 70) of NAFLD. Therefore, these results indicated that Notch family members Notch1-4 were involved in the development of NAFLD and played an important role in steatosis in this model.

Heme Oxygenase-1 Suppresses Wnt Signaling Pathway in Nonalcoholic Steatohepatitis-Related Liver Fibrosis.

Methods Mice were fed with a methionine-choline-deficient (MCD) diet for 8 weeks to induce steatohepatitis-related liver fibrosis and were treated with HO-1 inducer Hemin and inhibitor ZnPP. Mouse sera were collected for the biochemical analysis, and livers were obtained for further histological observation and gene expression analysis. HSC-T6 cells were cultured for the in vitro study and were administrated with Hemin and si-HO-1 to induce or inhibit the expression of HO-1. qPCR and Western blot were used to assess the mRNA and protein levels of genes. Results MCD-fed mice developed marked macrovesicular steatosis, focal necrosis, and inflammatory infiltration and pericellular fibrosis in liver sections. Administration of Hemin could significantly ameliorate the severity of steatosis, inflammation, and fibrosis and also could decrease the serum ALT and AST. We demonstrated that HO-1 induction was able to downregulate the key regulator of the canonical Wnt pathway Wnt1 and the noncanonical Wnt pathway Wnt5a. The downstream factors of the Wnt pathway β-catenin and NFAT5 were inhibited by Hemin, but GSK-3β was upregulated compared to the MCD group, which were consistent with the in vitro study. Hemin markedly inhibited the TGF-β1/Smad signaling pathway in both in vivo and in vitro studies. Conclusion Our study demonstrated that HO-1 inhibited the activation of canonical and noncanonical Wnt signaling pathways in NASH-related liver fibrosis. Thus, these results may suggest a new therapeutic strategy for NASH-related liver fibrosis.

Butyrate Protects Mice Against Methionine-Choline-Deficient Diet-Induced Non-alcoholic Steatohepatitis by Improving Gut Barrier Function, Attenuating Inflammation and Reducing Endotoxin Levels.

Butyrate exerts protective effects against non-alcoholic steatohepatitis (NASH), but the underlying mechanisms are unclear. We aimed to investigate the role of butyrate-induced gut microbiota and metabolism in NASH development. Sixty-five C57BL/6J mice were divided into four groups (n = 15–17 per group) and were fed either a methionine–choline-sufficient (MCS) diet or methionine–choline-deficient (MCD) diet with or without sodium butyrate (SoB; 0.6 g/kg body weight) supplementation for 6 weeks. Liver injury, systematic inflammation, and gut barrier function were determined. Fecal microbiome and metabolome were analyzed using 16S rRNA deep sequencing and gas chromatography-mass spectrometry (GC-MS). The results showed that butyrate alleviated the MCD diet-induced microbiome dysbiosis, as evidenced by a significantly clustered configuration separate from that of the MCD group and by the depletion of Bilophila and Rikenellaceae and enrichment of promising probiotic genera Akkermansia, Roseburia, Coprococcus, Coprobacillus, Delftia, Sutterella, and Coriobacteriaceae genera. The fecal metabolomic profile was also substantially improved by butyrate; several butyrate-responsive metabolites involved in lipid metabolism and other pathways, such as stearic acid, behenic acid, oleic acid, linoleic acid, squalene, and arachidonic acid, were identified. Correlation analysis of the interaction matrix indicated that the modified gut microbiota and fecal metabolites induced by butyrate were strongly correlated with the alleviation of hepatic injury, fibrosis progression, inflammation, and lipid metabolism and intestinal barrier dysfunction. In conclusion, our results demonstrated that butyrate exerts protective effects against NASH development, and these effects may be driven by the protective gut microbiome and metabolome induced by butyrate. This study thus provides new insights into NASH prevention.

The Effects of Betaine on the Nuclear Fractal Dimension, Chromatin Texture, and Proliferative Activity in Hepatocytes in Mouse Model of Nonalcoholic Fatty Liver Disease.

The effects of betaine on hepatocytes chromatin architecture changes were examined by using fractal and gray-level co-occurrence matrix (GLCM) analysis in methionine/choline-deficient (MCD) diet-induced, nonalcoholic fatty liver disease (NAFLD). Male C57BL/6 mice were divided into groups: (1) Control: standard diet; (2) BET: standard diet and betaine supplementation through drinking water (solution 1.5%); (3) MCD group: MCD diet for 6 weeks; (4) MCD+BET: fed with MCD diet + betaine for 6 weeks. Liver tissue was collected for histopathology, immunohistochemistry, and determination of fractal dimension and GLCM parameters. MCD diet induced diffuse micro- and macrovesicular steatosis accompanied with increased Ki67-positive hepatocyte nuclei. Steatosis and Ki67 immunopositivity were less prominent in the MCD+BET group compared with the MCD group. Angular second moment (ASM) and inverse difference moment (IDM) (textural homogeneity markers) were significantly increased in the MCD+BET group versus the MCD group (p<0.001), even though no difference between the MCD and the control group was evident. Heterogeneity parameters, contrast, and correlation were significantly increased in the MCD group versus the control (p<0.001). On the other hand, betaine treatment significantly reduced correlation, contrast, and entropy compared with the MCD group (p<0.001). Betaine attenuated MCD diet-induced NAFLD by reducing fat accumulation and inhibiting hepatocyte proliferation. Betaine supplementation increased nuclear homogeneity and chromatin complexity with reduction of entropy, contrast, and correlation.

S100A4 Gene is Crucial for Methionine-Choline-Deficient Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice.

PurposeTo explore the influence of S100 calcium binding protein A4 (S100A4) knockout (KO) on methionine-choline-deficient (MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice.Materials and MethodsS100A4 KO mice (n=20) and their wild-type (WT) counterparts (n=20) were randomly divided into KO/MCD, Ko/methionine-choline-sufficient (MCS), WT/MCD, and WT/MCS groups. After 8 weeks of feeding, blood lipid and liver function-related indexes were measured. HE, Oil Red O, and Masson stainings were used to observe the changes of liver histopathology. Additionally, expressions of S100A4 and proinflammatory and profibrogenic cytokines were detected by qRT-PCR and Western blot, while hepatocyte apoptosis was revealed by TUNEL staining.ResultsSerum levels of aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol in mice were increased after 8-week MCD feeding, and hepatocytes performed varying balloon-like changes with increased inflammatory cell infiltration and collagen fibers; however, these effects were improved in mice of KO/MCD group. Meanwhile, total NAFLD activity scores and fibrosis were lower compared to WT+MCD group. Compared to WT/MCS group, S100A4 expression in liver tissue of WT/MCD group was enhanced. The expression of proinflammatory (TNF-α, IL-1β, IL-6) and profibrogenic cytokines (TGF-β1, COL1A1, α-SMA) in MCD-induced NAFLD mice were increased, as well as apoptotic index (AI). For MCD group, the expressions of proinflammatory and profibrogenic cytokines and AI in KO mice were lower than those of WT mice.ConclusionS100A4 was detected to be upregulated in NAFLD, while S100A4 KO alleviated liver fibrosis and inflammation, in addition to inhibiting hepatocyte apoptosis.

Liver Graft Susceptibility during Static Cold Storage and Dynamic Machine Perfusion: DCD versus Fatty Livers.

We compared static preservation (cold storage, CS, 4 °C) with dynamic preservation (machine perfusion, MP, 20 °C) followed by reperfusion using marginal livers: a model of donation after cardiac death (DCD) livers and two models of fatty livers, the methionine-choline deficient (MCD) diet model, and obese Zucker (fa/fa) rats. CS injury in DCD livers was reversed by an oxygenated washout (OW): hepatic damage, bile flow, and the ATP/ADP ratio in the OW + CS group was comparable with the ratio obtained with MP. Using fatty livers, CS preservation induced a marked release in hepatic and biliary enzymes in obese Zucker rats when compared with the MCD group. The same trend occurred for bile flow. No difference was found when comparing MP in MCD and obese Zucker rats. Fatty acid analysis demonstrated that the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was, respectively, 1.5 and 0.71 in obese Zucker and MCD rats. While preservation damage in DCD livers is associated with the ATP/ADP recovered with OW, injury in fatty livers is linked to fatty acid constituents: livers from obese. Zucker rats, with greater content in saturated FA, might be more prone to CS injury. On the contrary, MCD livers with elevated PUFA content might be less susceptible to hypothermia.

Kombucha tea prevents obese mice from developing hepatic steatosis and liver damage.

Nonalcoholic fatty liver disease (NAFLD) is associated with the increased accumulation of hepatocellular lipids. Although Kombucha tea (KT) has emerged as a substance protecting the liver from damage, the effects of KT in NAFLD remain unclear. Hence, we investigated whether KT influenced hepatic steatosis. Db/db mice were fed either control or methionine/choline-deficient (MCD) diets for 4 weeks. The MCD diet group was treated with KT or water for 3 weeks. KT treatment alleviated macrovesicular steatosis compared to the MCD-fed group. The levels of triglyceride, ALT, and AST also decreased in the KT+MCD-treated db/db mice. RNA expression in the MCD+KT group showed reduced triglyceride synthesis and uptake of fatty acids. Immunostaining and western blot assays for active caspase-3 demonstrated a lower level of apoptosis in the MCD+KT than in the MCD group. These results demonstrate that KT attenuated lipid accumulation and protected the liver from damage, promoting liver restoration in mice.

Innovative targets involved in the progression of NASH: Asymmetric-dymethylarginine (ADMA) and its cationic transporters

Background: The methionine choline-deficient (MCD) diet results in liver injury similar to humannonalcoholic steatohepatitis (NASH). Recent data demonstrated that after methionine administration an increase in plasma asymmetric-dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, and a decrease in hepatic dimethylarginine-dimethylaminohydrolase (DDAH-1), enzyme involved in ADMA metabolism, occur. Aim: Using an experimental model of NASH induced by MCD diet, this study investigated whether changes in serum levels of ADMA and tissue content of DDAH-1 occur [Kusku-Kiraz Z., Genc S., Bekpinar S., et al. Circulating levels of apelin, glucagon-like peptide and visfatin in hypercholesterolemic–hyperhomocysteinemic guinea-pigs: their relation with NO metabolism. Molecular and Cellular Biochemistry 2015;400:69–75;Magne J., Huneau J.-F., Borderie D., Mathe V., Bos C., Mariotti F. Plasma asymmetric and symmetric dimethylarginine in a rat model of endothelial dysfunction induced by acute hyperhomocysteinemia. Amino Acids 2015, http://dx.doi.org/10.1007/s00726-015-1959-4]. Materials and methods: Male Wistar rats underwent to NASH induced by 8 weeks of feeding with MCD diet. Serum and hepatic biopsies at 2, 4 and 8 weeks were used: serum enzymes (AST, ALT) and ADMA were evaluated. Hepatic biopsies were used for the analysis of mRNA expression of DDAH-1 and ADMA transporters (CAT-1, CAT-2A and CAT-2B). Tissue total lipids, lipid peroxides (TBARS), glutathione and DDAH activity were also quantified. Results: As expected, an increase in serum AST and ALT and in hepatic total lipids were detected in MCD animals. A time-dependent decrease in serum ADMA and increase in mRNA expression of DDAH-1 were found in MCD group. Higher mRNA expression of CAT-1, lower mRNA expression of CAT-2A and no changes in CAT-2B were found after 8 weeks using MCD diet. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high TBARS levels and low glutathione content. Conclusions: A decrease in serum ADMA levels and an increase in DDAH-1mRNA found inMCD rats were associated with a reduction in DDAH activity due to the marked oxidative stress observed. Changes in ADMA levels and its transporters represent innovative factors involved in the onset and the progression of hepatic alteration correlated with MCD-diet-induced NASH. Funding: Supported by Fondazione Cariplo, grant n◦ 2011-0439.

Mosapride citrate improves nonalcoholic steatohepatitis with increased fecal lactic acid bacteria and plasma glucagon-like peptide-1 level in a rodent model.

Several lines of evidence have suggested a role of gut microbiota in the etiology of nonalcoholic steatohepatitis (NASH). NASH subjects reportedly showed a prolonged orocecal transit time coexistent with small intestinal bacterial overgrowth. We considered the possibility that enhanced gastrointestinal motility would influence gut microbiota and thus investigated the effects of the gastroprokinetic agent mosapride citrate (MC) on gut microbiota and the development of NASH using a methionine-choline deficient (MCD) diet-fed rodent model. Mice were divided into three groups, given the normal chow diet (NCD), the MCD diet, or the MCD diet containing 10 mg·kg(-1)·day(-1) of MC (MCD plus MC) for 6 wk. NASH development was evaluated based on hepatic histochemical findings, serum parameters and various mRNA and/or protein expression levels. MC treatment suppressed MCD diet-induced NASH development, with reduced serum lipopolysaccharide and increased plasma glucagon-like peptide-1 (GLP-1) concentrations. Calculation of the relative abundance of each strain based on gut microbiota analyses indicated lactic acid bacteria specifically, such as Bifidobacterium and Lactobacillus, in feces to be decreased in the MCD, compared with the NCD group. Interestingly, the reduction in lactic acid bacteria in the MCD diet group was reversed in the MCD plus MC group. In addition, colon inflammation observed in the MCD diet group was reduced in the MCD plus MC group. Therefore, MC showed a protective effect against MCD diet-induced NASH development in our rodent model, with possible involvements of increased fecal lactic acid bacteria, protection against colon inflammation and elevated plasma GLP-1.

Yo Jyo Hen Shi Ko (YHK) Modulates the Expression of Proteins Involved in de novo Lipogenesis and Lipid Exportation in Experimental Nonalcoholic Steatohepatitis (NASH)

Previous study by our group showed the protective effect of Yo Jyo Hen Shi Ko (YHK) a natural compound in experimental nonalcoholic steatohepatitis (NASH). The aim of this study was to evaluate whether YHK modulates lipid metabolism.NASH was induced in male ob/ob mice by methionine/choline-deficient (MCD) diet for 4 weeks. YHK-treated animals (YHK) received YHK solution orally (20 mg/kg/day) by gavage while MCD (n=6) group received only vehicle. The control animals (CTRL; n=6) received standard diet. Liver fragments were collected for mRNA and protein isolation. The analysis of gene expression and protein was performed by RT-qPCR and western blot, respectively.A significant decrease in srebp1c mRNA and protein expression and fasn mRNA expression was observed in MCD+YHK group. A significant increase in MTP protein expression was observed in the MCD+YHK vs MCD group while a decreased expression was observed in the MCD vs CTRL group. The expression of the scd1 in the MCD group was diminished. The Perilipin protein expression was augmented in the MCD group in comparison with MCD+YHK and CTRL groups.YHK modulated genes involved in the synthesis and exportation of hepatic lipids, probably limiting hepatocyte lipid accumulation, reducing lipogenesis and upregulating lipid exportation suggesting that the YHK can be a promising drug for treat non-alcoholic fatty liver disease (NAFLD).

Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture

Non-alcoholic fatty liver disease can progress to steatohepatitis and fibrosis, and is also associated with impaired liver regeneration. The pathophysiology remains elusive. We recently showed that severe steatosis is associated with an increase in portal pressure, suggesting liver flow impairment. The objective of this study is to directly assess total intrahepatic resistance and its potential functional and structural determinants in an in situ perfusion model. Male Wistar rats fed a control (n=30) or a methionine–choline-deficient (MCD) diet (n=30) for 4 weeks were compared. Liver tissue and serum analysis, in vivo haemodynamic measurements, in situ perfusion experiments and vascular corrosion casts were performed. The MCD group showed severe steatosis without inflammation or fibrosis on histology. Serum levels and liver tissue gene expression of interleukin (IL)-6, tumour necrosis factor-α, IL-1β and interferon-γ, liver tissue myeloperoxidase activity and liver immunohistochemistry with anti-CD68 and anti-α smooth muscle actin were comparable between groups, excluding significant inflammation. Flow-pressure curves were significantly different between groups for all flows (slope values: 0.1636±0.0605 mm Hg/ml/min in controls vs 0.7270±0.0408 mm Hg/ml/min in MCD-fed rats, P<0.001), indicating an increased intrahepatic resistance, which was haemodynamically significant (portocaval pressure gradient 2.2±1.1 vs 8.2±1.3 mm Hg in controls vs MCD, P<0.001). Dose-response curves to acetylcholine were significantly reduced in MCD-fed rats (P<0.001) as was the responsiveness to methoxamine (P<0.001). Vascular corrosion casts showed a replacement of the regular sinusoidal anatomy by a disorganized pattern with multiple interconnections and vascular extensions. Liver phosphorylated endothelial NO synthase (eNOS)/eNOS and serum nitrite/nitrate were not increased in severe steatosis, whereas liver thromboxane synthase expression, liver endothelin-1 (ET-1) expression and serum andothelin-1 concentration were significantly increased. Severe steatosis induces a haemodynamically significant increase in intrahepatic resistance, which precedes inflammation and fibrogenesis. Both functional (endothelial dysfunction and increased thromboxane and ET-1 synthesis) and structural factors are involved. This phenomenon might significantly contribute to steatosis-related disease.

Peroxisome proliferator activated receptor gamma activation and overexpression prevent hepatocellular apoptosis of nutritional fibrotic steatohepatitis in mice

To elucidate the effect of targeted gene modulation of peroxisome proliferator activated receptor gamma (PPARg) on hepatocellular apoptosis in nutritional fibrotic steatohepatitis in mice. C57BL/6J mice were fed with high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPARg (Ad-PPARg), adenovirus-beta-galactosidase (Ad-LacZ), Ad-PPARg plus PPARg agonist rosiglitazone, or PPARg antagonist 2-chloro-5-nitro- benzanilide (GW9662), respectively. H and E stain was performed for observation of hepatocellular apoptosis, hepatic steatosis, inflammation and fibrosis in the liver sections. The expression levels of mRNA and protein of PPARg and apoptosis related genes, Fas, Fas Ligand (FasL), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine-containing aspartate-specific proteases-3 (caspase-3) were detected by real-time RT-PCR and Western blot assay, respectively. Mice fed with MCD diet for 8 weeks showed severe hepatic injury including steatosis, hepatocellular apoptosis, inflammatory infiltration and fibrosis, concomitancy with enhanced expression of pro-apoptosis genes, Fas, FasL, Bax and caspase-3 and increased expression of anti-apoptosis gene Bcl-2, by comparing with the control group. The mRNA expression levels of these genes were 3.59+/-0.35 vs 1.11+/-0.37, 4.37+/-1.03 vs 1.09+/-0.33, 4.27+/-0.48 vs 1.03+/-0.10, 4.93+/-0.67 vs 1.12+/-0.24 and 3.95+/-0.34 vs 1.20+/-0.19, and LSD-t values were 2.49, 3.28, 3.25, 3.80 and 2.75, as compared with the control group, P is less than 0.01; the protein expression levels were 1.96+/-0.07 vs 0.45+/-0.07, 0.53+/-0.07 vs 0.22+/-0.02, 1.32+/-0.06 vs 0.59+/-0.03, 1.51+/-0.23 vs 0.36+/-0.09 and 0.57+/-0.01 vs 0.29+/-0.01, and LSD-t values were 1.51, 0.31, 0.73, 1.14 and 0.28, P is less than 0.01. Administration of PPARg agonist rosiglitazone and/or Ad-PPARg significantly ameliorated hepatic steatosis, hepatocellular apoptosis, necro inflammation and fibrosis. These effects were associated with repressed expression of pro-apoptosis genes and up-regulated expression of anti-apoptosis gene. After rosiglitazone treatment, the mRNA expression levels were 3.78+/-0.58, 3.66+/-0.83, 3.04+/-0.37, 2.54+/-0.62 and 4.42+/-0.42, and LSD-t values were 0.18, 0.71, 1.23, 2.39 and 0.46, as compared with MCD group, the P values were 0.627, 0.241, less than 0.01, less than 0.01 and 0.278, the protein expression levels were 1.06+/-0.03, 0.30+/-0.01, 0.70+/-0.05, 1.19+/-0.30 and 0.90+/-0.01, and LSD-t values were 0.90, 0.23, 0.62, 0.31 and 0.34, the P values were less than 0.01, less than 0.01, less than 0.01, 0.122, less than 0.01. After Ad-PPARg treatment, the mRNA expression levels were 2.31+/-0.16, 2.71+/-0.23, 2.52+/-0.27, 1.79+/-0.32 and 5.97+/-0.72, and LSD-t values were 1.28, 1.66, 1.75, 3.13 and 2.02, as compared with MCD group, P is less than 0.05; the protein expression levels were 1.73+/-0.07, 0.43+/-0.04, 1.01+/-0.08, 1.31+/-0.10 and 1.56+/-0.04, and LSD-t values were 0.23, 0.10, 0.30, 0.20 and 0.99, with P values equal 0.009, 0.01, less than 0.01, 0.322 and less than 0.01. This study provided evidences for the protective role of activation and overexpression of PPARg in ameliorating hepatocellular apoptosis in mice with hepatic fibrosing steatohepatitis.

Prevention and Reversion of Nonalcoholic Steatohepatitis in OB/OB Mice by S-Nitroso-N-Acetylcysteine Treatment

Objective: To evaluate the role oral administration of S-nitroso-N-acetylcysteine (SNAC), a NO donor drug, in the prevention and reversion of NASH in two different animal models.Methods: NASH was induced in male ob/ob mice by methionine-choline deficient (MCD) and high-fat (H) diets. Two animal groups received or not SNAC orally for four weeks since the beginning of the treatment. Two other groups were submitted to MCD and H diets for 60 days receiving SNAC only from the 31st to the 60th day.Results: SNAC administration inhibited the development of NASH in all groups, leading to a marked decrease in macro and microvacuolar steatosis and in hepatic lipid peroxidation in the MCD group. SNAC treatment reversed the development of NASH in animals treated for 60 days with MCD or H diets, which received SNAC only from the 31st to the 60th day.Conclusions: Oral administration of SNAC markedly inhibited and reversed NASH induced by MCD and H diets in ob/ob mice.