Intestinal Atp8b1 dysfunction causes hepatic choline deficiency and steatohepatitis.

Choline is an essential nutrient, and its deficiency causes steatohepatitis. Dietary phosphatidylcholine (PC) is digested into lysoPC (LPC), glycerophosphocholine, and choline in the intestinal lumen and is the primary source of systemic choline. However, the major PC metabolites absorbed in the intestinal tract remain unidentified. ATP8B1 is a P4-ATPase phospholipid flippase expressed in the apical membrane of the epithelium. Here, we use intestinal epithelial cell (IEC)-specific Atp8b1-knockout (Atp8b1IEC-KO) mice. These mice progress to steatohepatitis by 4 weeks. Metabolomic analysis and cell-based assays show that loss of Atp8b1 in IEC causes LPC malabsorption and thereby hepatic choline deficiency. Feeding choline-supplemented diets to lactating mice achieves complete recovery from steatohepatitis in Atp8b1IEC-KO mice. Analysis of samples from pediatric patients with ATP8B1 deficiency suggests its translational potential. This study indicates that Atp8b1 regulates hepatic choline levels through intestinal LPC absorption, encouraging the evaluation of choline supplementation therapy for steatohepatitis caused by ATP8B1 dysfunction.

Icariin Supplementation Suppresses the Markers of Ferroptosis and Attenuates the Progression of Nonalcoholic Steatohepatitis in Mice Fed a Methionine Choline-Deficient Diet.

Icariin, a flavonoid abundant in the herb Epimedium, exhibits anti-ferroptotic activity. However, its impact on nonalcoholic steatohepatitis (NASH) development remains unclear. This study aimed to investigate the potential role of icariin in mitigating methionine choline-deficient (MCD) diet-induced NASH in C57BL/6J mice. The results showed that icariin treatment significantly reduced serum alanine aminotrasferase and aspartate aminotransferase activities while improving steatosis, inflammation, ballooning, and fibrosis in the liver tissues of mice fed the MCD diet. These improvements were accompanied by a substantial reduction in the hepatic iron contents and levels of malondialdehyde and 4-hydroxynonenal, as well as an increase in the activities of catalase and superoxide dismutase. Notably, icariin treatment suppressed the hepatic protein levels of ferroptosis markers such as acyl-CoA synthetase long-chain family member 4 and arachidonate 12-lipoxygenase, which were induced by the MCD diet. Furthermore, transmission electron microscopy confirmed the restoration of morphological changes in the mitochondria, a hallmark characteristic of ferroptosis, by icariin. Additionally, icariin treatment significantly increased the protein levels of Nrf2, a cystine/glutamate transporter (xCT), and glutathione peroxidase 4 (GPX4). In conclusion, our study suggests that icariin has the potential to attenuate NASH, possibly by suppressing ferroptosis via the Nrf2-xCT/GPX4 pathway.

Integration of transcriptomics and metabonomics revealed the protective effects of hemp seed oil against methionine-choline-deficient diet-induced non-alcoholic steatohepatitis in mice.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease with few therapeutic options available currently. Hemp seed oil extracted from the seeds of hemp (Cannabis sativa L.) has significant nutritional and biological properties due to the unique composition of polyunsaturated fatty acids and various antioxidant compounds. However, little is known about the beneficial effects and molecular mechanisms of hemp seed oil on NASH. Here, the hepatoprotective effects of hemp seed oil on methionine-choline-deficient (MCD) diet-induced NASH in C57BL/6 mice were explored via integration of transcriptomics and metabolomics. Hemp seed oil could improve hepatic steatosis, inflammation and fibrosis in mice with MCD diet-induced NASH. In a nuclear magnetic resonance (NMR)-based metabonomic study, the hepatic and urinary metabolic profiles of mice supplemented with hemp seed oil showed a tendency to recover to healthy controls compared to those of NASH mice. Eight potential biomarkers associated with NASH in both liver tissue and urine were restored to near normal levels by administration of hemp seed oil. The proposed pathways were mainly involved in pyrimidine metabolism, one-carbon metabolism, amino acid metabolism, glycolysis and the tricarboxylic acid (TCA) cycle. Hepatic transcriptomics based on Illumina RNA-Seq sequencing showed that hemp seed oil exerted anti-NASH activities by regulating multiple signaling pathways, e.g., downregulation of the TNF signaling pathway, the IL-17 signaling pathway, the MAPK signaling pathway and the NF-κB signaling pathway, which played a pivotal role in the pathogenesis of NASH. In particular, integration of metabonomic and transcriptomic results suggested that hemp seed oil could attenuate NASH-related liver fibrosis by inhibition of glutaminolysis. These results provided new insights into the hepatoprotective effects of hemp seed oil against MCD diet-induced NASH and hemp seed oil might have potential as an effective therapy for NASH.

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

Non-Alcoholic Steatohepatitis (NASH) is the progressive form of Non-Alcoholic Fatty Liver Disease (NAFLD). NASH is distinguished by severe hepatic fibrosis and inflammation. The plant-derived, non-psychotropic compound cannabigerol (CBG) has potential anti-inflammatory effects similar to other cannabinoids. However, the impact of CBG on NASH pathology is still unknown. This study demonstrated the therapeutic potential of CBG in reducing hepatic steatosis, fibrosis, and inflammation. METHODS: 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. RESULTS: 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)-ß1. CONCLUSIONS: 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.

Role of Choline in Ocular Diseases.

Choline is essential for maintaining the structure and function of cells in humans. Choline plays an important role in eye health and disease. It is a precursor of acetylcholine, a neurotransmitter of the parasympathetic nervous system, and it is involved in the production and secretion of tears by the lacrimal glands. It also contributes to the stability of the cells and tears on the ocular surface and is involved in retinal development and differentiation. Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline supplementation may be effective for treating these diseases.

Salvia-Nelumbinis naturalis extract protects mice against MCD diet-induced steatohepatitis via activation of colonic FXR-FGF15 pathway.

Salvia-Nelumbinis naturalis (SNN) formula is a traditional Chinese medicine prescription, and has been confirmed to be effective in treating non-alcoholic steatohepatitis (NASH), but the underlying mechanisms are still unknown. Here we showed that 4-week SNN administration alleviated methionine-choline-deficiency (MCD) diet-induced hepatic steatosis and inflammation as well as serum levels of alanine transaminase (ALT) increase in C57BL/6 mice. Fecal 16S rDNA sequencing indicated that SNN altered the structure of gut microbiota and partially reversed the gut dysbiosis. Simultaneously, we analyzed the fecal BA profile using liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQMS) -based metabolomics, and found that SNN modulated fecal BA profile, predominantly increased the microbiomes related BA species (e.g. nordeoxycholic acid) which in turn, activated farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) signaling pathway in the colon but not the ileum. The activation of intestinal FXR-FGF15 signaling was accompanied by increase of liver protein kinase B (PKB/Akt) phosphorylation, and decrease of p-65 subunit of NF-κB phosphorylation, resulting in less liver CD68 positive macrophages, and inflammatory cytokine IL-1ß and TNF-α expression. Our results established the link between SNN treatment, gut microbiota, BA profile and NASH, which might shed light into the mechanisms behind the beneficial effects of SNN on NASH, thus provide evidence for the clinical application of SNN.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Comparison of murine steatohepatitis models identifies a dietary intervention with robust fibrosis, ductular reaction, and rapid progression to cirrhosis and cancer.

Progressive fibrosis, functional liver failure, and cancer are the central liver-related outcomes of nonalcoholic steatohepatitis (NASH) but notoriously difficult to achieve in mouse models. We performed a direct, quantitative comparison of hepatic fibrosis progression in well-defined methionine- and choline-deficient (MCD) and choline-deficient, amino-acid defined (CDAA) diets with increasing fat content (10-60% by calories) in C57Bl/6J and BALB/cAnNCrl mice. In C57Bl/6J mice, MCD feeding resulted in moderate fibrosis at week 8 (up to twofold increase in total hepatic collagen content) and progressive weight loss irrespective of dietary fat. In contrast, CDAA-fed mice did not lose weight and developed progressive fibrosis starting from week 4. High dietary fat in the CDAA diet model induced the lipid metabolism genes for sterol regulatory element-binding protein and stearoyl-CoA desaturase-2 and increased ductular reaction and fibrosis in a dose-dependent manner. Longitudinal analysis of CDAA with 60% fat (HF-CDAA) feeding revealed pronounced ductular reaction and perisinusoidal bridging fibrosis, with a sevenfold increase of hepatic collagen at week 12, which showed limited spontaneous reversibility. At 24 wk, HF-CDAA mice developed signs of cirrhosis with pan-lobular "chicken wire" fibrosis, 10-fold hydroxyproline increase, regenerative nodules, portal hypertension and elevated serum bilirubin and ammonia levels; 80% of mice (8/10) developed multiple glypican-3- and/or glutamine synthetase-positive hepatocellular carcinomas (HCC). High-fat (60%) supplementation of MCD in C57Bl/6J or feeding the HF-CDAA diet fibrosis-prone BALB/cAnNCrl strain failed to result in increased fibrosis. In conclusion, HF-CDAA feeding in C57Bl/6J mice was identified as an optimal model of steatohepatitis with robust fibrosis and ductular proliferations that progress to cirrhosis and HCC within 24 wk. This robust model will aid the testing of interventions and drugs for severe NASH.NEW & NOTEWORTHY Via quantitative comparison of several dietary models, we report HF-CDAA feeding in C57Bl/6 mice as an excellent model recapitulating several key aspects of fibrotic NASH: 1) robust, poorly reversible liver fibrosis, 2) prominent ductular reaction, and 3) progression to cirrhosis, portal hypertension, and liver cancer within 24 wk. High fat dose-dependently activates SREBP2/SCD2 genes and drives liver fibrosis in e HF-CDAA model. These features qualify the model as a robust and practical tool to study mechanisms and novel treatments addressing severe human NASH.

Methionine-Choline Deprivation Impairs Adult Hippocampal Neurogenesis in C57BL/6 Mice.

Methionine and choline, which are essential nutrients for mammalian animals, are important for cell composition, as metabolic factors, and for the synthesis of other biochemical compounds for cell metabolism. Methionine and choline, which are methyl group donors, play key roles in the homocysteine cycle and neuronal development and maintenance. In this study, we investigated the effects of methionine and choline deficiency on adult hippocampal neurogenesis and neural stem cell (NSC) lineage in the adult stage. For this study, we divided C57BL/6 mice into three groups as follows: normal chow (NC)-fed, methionine choline sufficient (MCS) diet-fed, and methionine choline deficient (MCD) diet-fed mice. The mice were fed the NC, MCS, and MCD diets for 4 weeks from the age of 8 weeks. MCD diet-fed mice showed significantly decreased proliferation and differentiation of NSCs when compared with the NC diet-fed or MCS diet-fed mice. In addition, the survival of newly generated neurons was critically impaired in the MCD diet-fed mice. We confirmed a decrease in the proliferation and differentiation of NSCs after 4 weeks of MCD diet administration, compared with that in NC- and MCS diet-fed mice. MCD diet critically impaired NSCs survival and survival of neurons during the 4 weeks. The number of phosphorylated cyclic AMP response element binding (pCREB) protein immunoreactive nuclei was decreased in the MCD diet-fed mice compared with that in the NC- or MCS diet-fed group. These results suggest that suitable levels of methionine and choline are essential for the maintenance of hippocampal neurogenesis in mice and affect NSC proliferation and differentiation through phosphorylation of CREB.

Choline Supplementation in Cystic Fibrosis-The Metabolic and Clinical Impact.

BACKGROUND: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat. METHODS: 10 adult male CF patients were recruited (11/2014⁻1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84⁻91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D9]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis. RESULTS: Supplementation increased plasma choline from 4.8 (4.1⁻6.2) µmol/L to 10.5 (8.5⁻15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D9-labeled PC was decreased (12.2 [10.5⁻18.3] µmol/L vs. 17.7 [15.5⁻22.4] µmol/L, p < 0.01), indicating D9-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9⁻74.8)% to 78.3 (60.1⁻83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37⁻8.82)% to 0.84 (0.56⁻1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. CONCLUSIONS: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials.

Choline-related-inherited metabolic diseases-A mini review.

In humans, the important water soluble, vitamin-like nutrient choline, is taken up with the diet or recycled in the liver. Deficiencies of choline have only been reported in experimental situations or total parenteral nutrition. Currently, no recommended dietary allowances are published; only an adequate daily intake is defined. Choline is involved in three main physiological processes: structural integrity and lipid-derived signaling for cell membranes, cholinergic neurotransmission, and methylation. Choline is gaining increasing public attention due to studies reporting a relation of low choline levels to subclinical organ dysfunction (nonalcoholic fatty liver or muscle damage), stunting, and neural tube defects. Furthermore, positive effects on memory and a lowering of cardiovascular risks and inflammatory markers have been proposed. On the other hand, dietary choline has been associated with increased atherosclerosis in mice. This mini review will provide a summary of the biochemical pathways, in which choline is involved and their respective inborn errors of metabolism (caused by mutations in SLC5A7, CHAT, SLC44A1, CHKB, PCYT1A, CEPT1, CAD; DHODH, UMPS, FMO3, DMGDH, and GNMT). The broad phenotypic spectrum ranging from malodor, intellectual disability, to epilepsy, anemia, or congenital myasthenic syndrome is presented, highlighting the central role of choline within human metabolism.

ß-Arrestin1 alleviates acute pancreatitis via repression of NF-κBp65 activation.

BACKGROUND AND AIM: ß-Arrestins (ß-arrs) are regulators and mediators of G protein-coupled receptor signaling that are functionally involved in inflammation. Nuclear factor-κB p65 (NF-κBp65) activation has been observed early in the onset of pancreatitis. However, the effect of ß-arrs in acute pancreatitis (AP) is unclear. The aim of this study is to investigate whether ß-arrs are involved in AP through activation of NF-κBp65. METHODS: Acute pancreatitis was induced by either caerulein injection or choline-deficient supplemented with ethionine diet (CDE). ß-arr1 wild-type and ß-arr1 knockout mice were used in the experiment. The survival rate was calculated in the CDE model mice. Histological and western blot analyses were performed in the caerulein model. Inflammatory mediators were detected by real-time polymerase chain reaction in the caerulein-induced AP mice. Furthermore, AR42J and PANC-1 cell lines were used to further study the effects of ß-arr1 in caerulein-induced pancreatic cells. RESULTS: ß-Arr1 but not ß-arr2 is significantly downregulated in caerulein-induced AP in mice. Targeted deletion of ß-arr1 notably upregulated expression of the pancreatic inflammatory mediators including tumor necrosis factor α and interleukin 1ß as well as interleukin 6 and aggravated AP in caerulein-induced mice. ß-Arr1 deficiency increased mortality in mice with CDE-induced AP. Further, ß-arr1 deficiency enhanced caerulein-induced phosphorylation of NF-κBp65 both in vivo and in vitro. CONCLUSION: ß-Arr1 alleviates AP via repression of NF-κBp65 activation, and it is a potentially therapeutic target for AP.

Wuzhi capsule (Schisandra Sphenanthera extract) attenuates liver steatosis and inflammation during non-alcoholic fatty liver disease development.

OBJECTIVE: Wuzhi (WZ) capsule contains an ethanol extract of Schisandra sphenanthera. The efficacy of WZ in treating non-alcoholic fatty liver disease (NAFLD) has not yet been elucidated. The present study assessed the effects of WZ on NAFLD. MATERIAL AND METHODS: A C57BL/6 male mouse model of NAFLD was established by feeding the animals a methionine-choline-deficient (MCD) diet. Mice fed the basal diet were used as controls. Both groups were randomly administered WZ or vehicle by gavage for 5 weeks. Body weight change, liver/body weight ratio, metabolic parameters, and histological changes were assessed. Serum levels of IL-1ß, IL-6, IL-10, and TNF-α were analysed by ELISA; mRNA expression of these genes in the liver was studied by real-time PCR. Western blotting was used to analyse the protein levels of PPAR-α, PPAR-γ, MCAD, LCAD, and p65 in the liver. RESULTS: After 5 weeks of the MCD diet, the liver/body weight ratio of WZ mice was higher than that of control mice. Liver histology revealed significantly less steatosis, inflammation, and necrosis, which was confirmed by decreased intrahepatic triglycerides and serum ALT in WZ-treated mice. WZ also reduced the liver mRNA expression of IL-1ß, IL-6, and TNF-α and the serum levels of IL-1ß and IL-6. Sensitivity to steatohepatitis due to WZ administration correlated significantly with alterations in the expression of PPAR-α/γ, as well as the NF-κB signalling pathway. CONCLUSIONS: WZ plays a protective role against MCD-induced steatohepatitis. The underlying mechanism likely involves the upregulation of PPAR-α/γ and downregulation of the NF-κB signalling pathway. Based on its beneficial effects on the liver, WZ is a promising therapeutic for NAFLD patients.

Administration of methyl palmitate prevents non-alcoholic steatohepatitis (NASH) by induction of PPAR-α.

BACKGROUND AND AIMS: The lack of valid therapeutic approach that can ameliorate the manifestations of NASH is a barrier to therapeutic development. Therefore, we investigate the novel role of Methyl Palmitate (MP) in preventing NASH and the possible mechanism involved. METHODS: 50 Male C57BL/6 J mice were randomly divided into 5 groups (n = 10). The control group was fed control diet; model group was fed MCD diet; MP 1 group was fed MCD diet supplemented with MP (75 mg/kg/day); MP 2 group was fed MCD plus MP diet (150 mg/kg/day); and MP 3 group was fed MCD plus MP diet (300 mg/kg/day). Histological staining's, and commercially available kits for serum ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. RESULTS: Mice fed MCD diet developed NASH, which was markedly improved by MP in a dose-dependent manner. MP treatment improved hepatic content of TG, TC, MDA, SOD and GSH and serum levels of ALT and AST. In vivo studies showed that MP treatment activated PPARα expression, that in turns, promoted ß-oxidation protein and gene expressions, suppressed TNFα, MCP1, TGFß1 and Colla1 protein and gene expression levels, contributing to the prevention of NASH. CONCLUSIONS: Our results indicated that MP could successfully prevent NASH. This effect of MP was mediated through induction of PPARα pathway. This study provides a novel therapeutic target that plays pivotal role in the prevention of NASH.

Gypenoside UL4-Rich Gynostemma pentaphyllum Extract Exerts a Hepatoprotective Effect on Diet-Induced Nonalcoholic Fatty Liver Disease.

Nonalcoholic steatohepatitis (NASH) arises from nonalcoholic fatty liver disease (NAFLD) as a consequence of oxidative stress. Gynostemma pentaphyllum extract (GPE) is proven to be beneficial for patients suffering from NAFLD. However, the precise mechanism by which GPE confers these benefits remains largely unknown. The purpose of this study was to investigate the underlying mechanism and to determine whether supplementation with the newly discovered GPE gypenoside UL4 mitigates NASH progression. Male c57BL/6 mice were fed a normal chow diet, a methionine choline-deficient (MCD) diet, or an MCD diet supplemented with various doses of UL4-rich GPE for eight weeks. GPE supplementation suppressed oxidative stress induced by the MCD diet by increasing levels of sirtuin 6 and phase 2 anti-oxidant enzymes in mouse liver and HepG2 cells. Additionally, GPE supplementation prevented diet-induced hepatic fat accumulation, hepatocellular injury, inflammation, and fibrosis in mice fed the MCD diet. These results indicate the possible therapeutic potential of dietary supplementation of UL4-rich GPE in preventing the development of fatty liver and its progression to NASH.

Dysregulated Bile Transporters and Impaired Tight Junctions During Chronic Liver Injury in Mice.

BACKGROUND & AIMS: Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. METHODS: C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. RESULTS: Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. CONCLUSIONS: We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.

Elevation of tumour markers TGF-ß, M2-PK, OV-6 and AFP in hepatocellular carcinoma (HCC)-induced rats and their suppression by microalgae Chlorella vulgaris.

BACKGROUND: Chlorella vulgaris (ChV), a unicellular green algae has been reported to have anticancer and antioxidant effects. The aim of this study was to determine the chemopreventive effect of ChV on liver cancer induced rats by determining the level and expression of several liver tumour markers. METHODS: Male Wistar rats (200-250 g) were divided into 4 groups according to the diet given: control group (normal diet), ChV group with three different doses (50, 150 and 300 mg/kg body weight), liver cancer- induced group (choline deficient diet + 0.1% ethionine in drinking water or CDE group), and the treatment group (CDE group treated with three different doses of ChV). Rats were killed at 0, 4, 8 and 12 weeks of experiment and blood and tissue samples were taken from all groups for the determination of tumour markers expression alpha-fetoprotein (AFP), transforming growth factor-ß (TGF-ß), M2-pyruvate kinase (M2-PK) and specific antigen for oval cells (OV-6). RESULTS: Serum level of TGF-ß increased significantly (p < 0.05) in CDE rats. However, ChV at all doses managed to decrease (p < 0.05) its levels to control values. Expressions of liver tumour markers AFP, TGF-ß, M2-PK and OV-6 were significantly higher (p < 0.05) in tissues of CDE rats when compared to control showing an increased number of cancer cells during hepatocarcinogenesis. ChV at all doses reduced their expressions significantly (p < 0.05). CONCLUSIONS: Chlorella vulgaris has chemopreventive effect by downregulating the expression of tumour markers M2-PK, OV-6, AFP and TGF-ß, in HCC-induced rats.

Intestinal immune responses of Jian carp against Aeromonas hydrophila depressed by choline deficiency: Varied change patterns of mRNA levels of cytokines, tight junction proteins and related signaling molecules among three intestinal segments.

This study aimed to investigate the effects of choline deficiency on intestinal inflammation of fish after Aeromonas hydrophila infection and the potential molecular mechanisms. Juvenile Jian carp (Cyprinus carpio var. Jian) were fed two diets containing choline at 165 (deficient group) and 607 mg/kg diet respectively for 65 days. Choline deficiency decreased intestinal lysozyme activity, C3 and IgM contents, increased acid phosphatase activity, downregulated mRNA levels of antimicrobial peptides [liver-expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin and defensin], cytokines [interleukin (IL) 6a, tumor necrosis factor α (TNF-α), interferon γ2b (IFN-γ2b), IL-6b and transforming growth factor ß2 (TGF-ß2) only in proximal intestine, IL-10 in mid and distal intestine], immune-related signaling molecules [Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor kappa B (NF-κB), inhibitor of NF-κB (IκB), Janus kinase 3 (JAK3), and signal transducers and activators of transcription 5 (STAT5)], tight junction proteins (claudin 3b, claudin 3c, claudin 11 and occludin), and mitogen-activated protein kinases p38 (p38MAPK) in proximal and distal intestine of juvenile Jian carp after A. hydrophila challenge. In contrast, choline deficiency upregulated mRNA levels of antimicrobial peptides (LEAP-2A, LEAP-2B, hepcidin and defensin), cytokines (IL-6b, IFN-γ2b and TGF-ß2), immune-related signaling molecules (TLR4, MyD88, NF-κB, IκB, JAK3, STAT4 in three intestinal segments, and STAT6), claudin 11, and p38MAPK in mid intestine of fish. This study provides new finding that choline deficiency-induced immune responses against A. hydrophila infection were varied among three intestinal segments in fish.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

Imatinib accelerates progenitor cell-mediated liver regeneration in choline-deficient ethionine-supplemented diet-fed mice.

Severe chronic hepatic injury can induce complex reparative processes. Ductular reaction and the appearance of small hepatocytes are standard components of this response, which is thought to have both adverse (e.g. fibrosis, carcinogenesis) and beneficial (regeneration) consequences. This complex tissue reaction is regulated by orchestrated cytokine action. We have investigated the influence of the tyrosine kinase inhibitor imatinib on a regenerative process. Ductular reaction was induced in mice by the widely used choline-deficient ethionine-supplemented diet (CDE). Test animals were treated daily with imatinib. After 6 weeks of treatment, imatinib successfully reduced the extent of ductular reaction and fibrosis in the CDE model. Furthermore, the number of small hepatocytes increased, and these cells had high proliferative activity, were positive for hepatocyte nuclear factor 4 and expressed high levels of albumin and peroxisome proliferator-activated receptor alpha. The overall functional zonality of the hepatic parenchyma (cytochrome P450 2E1 and glucose 6 phosphatase activity; endogenous biotin content) was maintained. The expression of platelet-derived growth factor receptor beta, which is the major target of imatinib, was downregulated. The anti-fibrotic activity of imatinib has already been reported in several experimental models. Additionally, in the CDE model imatinib was able to enhance regeneration and preserve the functional arrangement of hepatic lobules. These results suggest that imatinib might promote the recovery of the liver following parenchymal injury through the inhibition of platelet-derived growth factor receptor beta.