Time-Restricted Feeding Ameliorates Methionine-Choline Deficient Diet-Induced Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) is an inflammatory form of non-alcoholic fatty liver disease (NAFLD), closely associated with disease progression, cirrhosis, liver failure, and hepatocellular carcinoma. Time-restricted feeding (TRF) has been shown to decrease body weight and adiposity and improve metabolic outcomes; however, the effect of TRF on NASH has not yet been fully understood. We had previously reported that inositol polyphosphate multikinase (IPMK) mediates hepatic insulin signaling. Importantly, we have found that TRF increases hepatic IPMK levels. Therefore, we investigated whether there is a causal link between TRF and IPMK in a mouse model of NASH, i.e., methionine- and choline-deficient diet (MCDD)-induced steatohepatitis. Here, we show that TRF alleviated markers of NASH, i.e., reduced hepatic steatosis, liver triglycerides (TG), serum alanine transaminase (ALT) and aspartate aminotransferase (AST), inflammation, and fibrosis in MCDD mice. Interestingly, MCDD led to a significant reduction in IPMK levels, and the deletion of hepatic IPMK exacerbates the NASH phenotype induced by MCDD, accompanied by increased gene expression of pro-inflammatory chemokines. Conversely, TRF restored IPMK levels and significantly reduced gene expression of proinflammatory cytokines and chemokines. Our results demonstrate that TRF attenuates MCDD-induced NASH via IPMK-mediated changes in hepatic steatosis and inflammation.

Apical papilla stem cell-derived exosomes regulate lipid metabolism and alleviate inflammation in the MCD-induced mouse NASH model.

Stem cells from the apical papilla(SCAPs) exhibit remarkable tissue repair capabilities, demonstrate anti-inflammatory and pro-angiogenic effects, positioning them as promising assets in the realm of regenerative medicine. Recently, the focus has shifted towards exosomes derived from stem cells, perceived as safer alternatives while retaining comparable physiological functions. This study delves into the therapeutic implications of exosomes derived from SCAPs in the methionine-choline-deficient (MCD) diet-induced mice non-alcoholic steatohepatitis (NASH) model. We extracted exosomes from SCAPs. During the last two weeks of the MCD diet, mice were intravenously administered SCAPs-derived exosomes at two distinct concentrations (50 µg/mouse and 100 µg/mouse) biweekly. Thorough examinations of physiological and biochemical indicators were performed to meticulously evaluate the impact of exosomes derived from SCAPs on the advancement of NASH in mice induced by MCD diet. This findings revealed significant reductions in body weight loss and liver damage induced by the MCD diet following exosomes treatment. Moreover, hepatic fat accumulation was notably alleviated. Mechanistically, the treatment with exosomes led to an upregulation of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) levels in the liver, enhancing hepatic fatty acid oxidation and transporter gene expression while inhibiting genes associated with fatty acid synthesis. Additionally, exosomes treatment increased the transcription levels of key liver mitochondrial marker proteins and the essential mitochondrial biogenesis factor. Furthermore, the levels of serum inflammatory factors and hepatic tissue inflammatory factor mRNA expression were significantly reduced, likely due to the anti-inflammatory phenotype induced by exosomes in macrophages. The above conclusion suggests that SCAPs-exosomes can improve NASH.

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.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 µmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 µmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Nonalcoholic steatohepatitis-associated hepatocarcinogenesis in mice fed a modified choline-deficient, methionine-lowered, L-amino acid-defined diet and the role of signal changes.

Nonalcoholic steatohepatitis (NASH) can progress to cirrhosis and even hepatocellular carcinoma (HCC). The incidence of NASH-associated HCC is increasing, posing a serious public health threat. Unfortunately, the underlying pathological mechanisms, including the possible differences between neoplastic and non-neoplastic lesions, remain largely unknown. Previously, we reported a dietary mouse NASH model with a choline-deficient, methionine-lowered, L-amino-acid-defined, high-fat diet containing shortening without trans fatty acids (CDAA-HF-T[-]), which rapidly induces fibrosis and proliferative lesions in the liver. This study aimed to develop a mouse CDAA-HF-T(-) model capable of assessing NASH-associated hepatocarcinogenesis and identifying key signaling factors involved in its underlying mechanisms. Multiple large masses, histopathologically hepatocellular adenomas and carcinomas, and hemangiosarcomas were detected in the liver samples of mice fed CDAA-HF-T(-) for 52 or 63 weeks, along with highly advanced fibrosis and numerous foamy, phagocytic macrophages in the adjacent nontumoral area. Multiple metastatic nodules were found in the lungs of one of the animals, and lymphoid clusters were found in all CDAA-HF-T(-) group mice. In the Ingenuity Pathways Analysis of RNA expression data, the CDAA-HF-T(-) feeding revealed common signal changes in nontumoral and tumoral liver tissues, including increased IL-8 and RhoGTPases signaling and decreased lipid metabolism. Meanwhile, macrophage inflammatory protein 2 (MIP-2) expression levels were upregulated in nontumoral liver tissue from the end of Week 13 of CDAA-HF-T(-) feeding to the end of Week 63. On the other hand, MIP-2 was expressed on macrophages in non-tumor areas and hepatocytes in tumor areas. Therefore, the CDAA-HF-T(-) mouse model is useful for assessing NASH and NASH-associated hepatocarcinogenesis, and IL-8 signaling plays important roles in NASH-associated carcinogenesis and cirrhosis, but it may also play different roles in nontumoral liver tissue and tumorigenesis.

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.

Inhibition of lysophosphatidic acid receptor 6 upregulated by the choline-deficient l-amino acid-defined diet prevents hepatocarcinogenesis in mice.

Hepatocellular carcinoma (HCC) is one of the most worrying tumors worldwide today, and its epidemiology is on the rise. Traditional pharmacological approaches have shown unfavorable results and exhibited many side effects. Hence, there is a need for new efficacious molecules with fewer side effects and improvements on traditional approaches. We previously showed that lysophosphatidic acid (LPA) supports hepatocarcinogenesis, and its effects are mainly mediated by LPA receptor 6 (LPAR6). We also reported that 9-xanthylacetic acid (XAA) acts as an antagonist of LPAR6 to inhibit the growth of HCC. Here, we report that LPAR6 is involved in the choline-deficient l-amino acid-defined (CDAA) diet-induced hepatocarcinogenesis in mice. Our data demonstrate that CDAA diet-induced metabolic imbalance stimulates LPAR6 expression in mice and that XAA counteracts diet-induced effects on hepatic lipid accumulation, fibrosis, inflammation, and HCC development. These conclusions are corroborated by results on LPAR6 gain and loss-of-function in HCC cells.

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.

Osteoprotegerin deficiency aggravates methionine-choline-deficient diet-induced nonalcoholic steatohepatitis in mice.

Clinical studies have shown that osteoprotegerin (OPG) is reduced in patients with nonalcoholic steatohepatitis (NASH), but the underlying mechanisms are unclear. The current study focuses on the role of OPG in the NASH pathogenesis. OPG knockout mice and wild-type control mice fed a methionine choline-deficient diet (MCD) for 4 weeks resulted in an animal model of NASH. Measurement of triglycerides (TG) in serum and liver to assess steatosis. Hematoxylin eosin (HE), Sirius Red and Masson staining were used to assess the liver damage. Transcriptome sequencing analysis, qPCR and western blot were to analyze changes in lipid metabolism and inflammation-related indicators in the liver. In vivo knockout of OPG resulted in a reduction of TG levels in the liver and a significant increase in serum ALT and AST. The expression of inflammatory factors and fibrosis genes was significantly upregulated in the livers of OPG knockout mice. Transcriptome sequencing analysis showed that OPG knockout significantly enhanced MCD diet-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Mechanistically, OPG may inhibit MAPK signaling pathway activity by upregulating the expression of dual specificity phosphatase 14 (DUSP14), thereby reducing inflammatory injury. OPG could regulate the activity of the MAPK signaling pathway via DUSP14, thus regulating the expression of some inflammatory factors in NASH, it may be a promising target for the treatment of NASH.

Exploring the Therapeutic Effects of Black Ginseng on Non-Alcoholic Fatty Liver Disease by Using Network Pharmacology and Molecular Docking.

This study aimed to investigate the therapeutic effect of black ginseng (BG) on non-alcoholic fatty liver disease (NAFLD) using network pharmacology combined with the molecular docking strategy. The saponin composition of BG was analyzed by liquid chromatography-mass spectrometry (LC/MS) instrument. Then the network pharmacology was applied to explore the potential targets and related mechanisms of BG in the treatment of NAFLD. After screening out key targets, molecular docking was used to predict the binding modes between ginsenoside and target. Finally, a methionine and choline deficiency (MCD) diet-induced NAFLD mice model was established to further confirm the therapeutic effect of BG on NAFLD. Twenty-four ginsenosides were annotated based on the MS and tandem MS information. Ten proteins were screened out as key targets closely related to BG treatment of NAFLD. The molecular docking showed that most of the ginsenosides had good binding affinities with AKT1. The validation experiment revealed that BG administration could reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the MCD diet-induced histological changes in liver tissue. Moreover, BG could upregulate the phosphorylation level of AKT in the liver of NAFLD mice, thereby exerting the therapeutic effect on NAFLD. Further studies on the active ginsenosides as well as their synergistic action on NAFLD will be required to reveal the underlying mechanisms in-depth. This study demonstrates that network pharmacological prediction in conjunction with molecular docking is a viable technique for screening the active chemicals and related targets of BG, which can be applied to other herbal medicines.

Melatonin Attenuates Inflammation, Oxidative Stress, and DNA Damage in Mice with Nonalcoholic Steatohepatitis Induced by a Methionine- and Choline-Deficient Diet.

Nonalcoholic steatohepatitis (NASH) is a disease with a high incidence worldwide, but its diagnosis and treatment are poorly managed. In this study, NASH pathophysiology and DNA damage biomarkers were investigated in mice with NASH treated and untreated with melatonin (MLT). C57BL/6 mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks to develop NASH. Melatonin was administered at 20 mg/kg during the last 2 weeks. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured, and hepatic tissue was dissected for histological analysis, evaluation of lipoperoxidation, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as nuclear factor-erythroid 2 (Nrf2), tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and transforming growth factor beta (TGF-ß) expression by immunohistochemistry. DNA damage was evaluated using Comet assay, while a micronucleus test in bone marrow was performed to assess the genomic instability associated with the disease. Melatonin decreased AST and ALT, liver inflammatory processes, balloonization, and fibrosis in mice with NASH, decreasing TNF-α, iNOS, and TGF-ß, as well as oxidative stress, shown by reducing lipoperoxidation and intensifying Nrf2 expression. The SOD and GPx activities were increased, while CAT was decreased by treatment with MLT. Although the micronucleus frequency was not increased in mice with NASH, a protective effect on DNA was observed with MLT treatment in blood and liver tissues using Comet assay. As conclusions, MLT slows down the progression of NASH, reducing hepatic oxidative stress and inflammatory processes, inhibiting DNA damage via anti-inflammatory and antioxidant actions.

Chlorogenic acid alleviated liver fibrosis in methionine and choline deficient diet-induced nonalcoholic steatohepatitis in mice and its mechanism.

Nonalcoholic steatohepatitis, one of the most common chronic liver diseases, is a progressive form of nonalcoholic fatty liver disease accompanied by the development of liver fibrosis. Chlorogenic acid (CGA) is a natural polyphenolic compound. This study aims to observe the CGA-provided alleviation on liver fibrosis in methionine and choline deficient (MCD) diet-induced nonalcoholic steatohepatitis in mice and to elucidate its engaged mechanism. CGA attenuated hepatocellular injury, decreased the elevated hepatic lipids accumulation and attenuated liver fibrosis by reducing hepatic collagen deposition in mice fed with MCD diet. CGA abrogated the activation of hepatic stellate cells (HSCs) and promoted mitochondrial biogenesis both in vivo and in vitro. Moreover, the CGA-provided inhibition on HSCs activation in vitro was obviously disappeared after the application of peroxisome proliferator-activated receptor gamma, coactivator 1alpha (PGC1α) siRNA. CGA reduced the enhanced hepatic extracellular matrix (ECM) expression and the elevated serum high-mobility group box 1 (HMGB1) content in mice fed with MCD diet. CGA decreased the HMGB1-induced ECM production in both human liver sinusoidal endothelial cells and human umbilical vein endothelial cells. CGA also weakly promoted mitochondrial biogenesis in both liver sinusoidal endothelial cells and human umbilical vein endothelial cells incubated with HMGB1. Hence, CGA ameliorated hepatic fibrosis in mice fed with MCD diet through inhibiting HSCs activation via promoting mitochondrial biogenesis and reducing the HMGB1-initiated ECM production in hepatic vascular endothelial cells.

Choline deficiency-related multi-omics characteristics are susceptible factors for chemotherapy-induced thrombocytopenia.

The XELOX chemotherapy protocol that includes capecitabine and oxaliplatin is the routine treatment for colorectal cancer (CRC), but it can cause chemotherapy-related adverse events such as thrombocytopenia (TCP). To identify predictive biomarkers and clarify the mechanism of TCP susceptibility, we conducted integrative analysis using normal colorectal tissue (CRT), plasma, and urine samples collected before CRC patients received adjuvant XELOX chemotherapy. RNA-sequencing and DNA methylation arrays were performed on CRT samples, while liquid chromatography-mass spectrometry was performed on CRT, plasma, and urine samples. Differentially expressed features (DEFs) from each uni-omics analysis were then subjected to integrative analysis using Multi-Omics Factor Analysis (MOFA). Choline-deficiency in plasma and CRT was found as the most critical TCP-related feature. Based on bioinformatic analysis and literature research, we further concluded that choline-deficiency was the possible reason for most of the other TCP-related multi-omics DEFs, including metabolites representing reduced sphingolipid de novo synthesis and elevated solute carrier-mediated transmembrane transportation in CRT and plasma, DNA hypermethylation and elevated expression of genes involved in neuronal system genes. In terms of thrombocytopoiesis, these TCP-related DEFs may cause atypical maintenance and differentiation of megakaryocyte, resulting a suppressed ability of thrombocytopoiesis, making patients more susceptible to chemotherapy-induced TCP. At last, prediction models were developed and validated with reasonably good discrimination. The area under curves (AUCs) of training sets were all > 0.9, while validation sets had AUCs between 0.778 and 0.926. In conclusion, our results produced reliable marker systems for predicting TCP and promising target for developing precision treatment to prevent TCP.

Formyl peptide receptor 2 determines sex-specific differences in the progression of nonalcoholic fatty liver disease and steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is an important health concern worldwide and progresses into nonalcoholic steatohepatitis (NASH). Although prevalence and severity of NAFLD/NASH are higher in men than premenopausal women, it remains unclear how sex affects NAFLD/NASH pathophysiology. Formyl peptide receptor 2 (FPR2) modulates inflammatory responses in several organs; however, its role in the liver is unknown. Here we show that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH. NASH-like liver injury was induced in both sexes during choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) feeding, but compared with females, male mice had more severe hepatic damage. Fpr2 was more highly expressed in hepatocytes and healthy livers from females than males, and FPR2 deletion exacerbated liver damage in CDAHFD-fed female mice. Estradiol induced Fpr2 expression, which protected hepatocytes and the liver from damage. In conclusion, our results demonstrate that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH, suggesting a novel therapeutic target for NAFLD/NASH.

Preventive effects of black soybean polyphenols on non-alcoholic fatty liver disease in three different mouse models.

Non-alcoholic fatty liver disease (NAFLD) and its advanced stage, non-alcoholic steatohepatitis (NASH), are a major health issue throughout the world. Certain food components such as polyphenols are expected to possess preventive effects on NAFLD and NASH. In this study, the preventive effects of black soybean polyphenols were examined by using three NAFLD/NASH animal models. In a choline-deficient and L-amino acid-defined high-fat diet-induced NASH model, the intake of black soybean polyphenols decreased oxidative stress, but failed in attenuating liver injury and decreasing the expression of alpha-smooth muscle actin (α-SMA). In a Western diet with sucrose and fructose containing sweetened water-induced NAFLD model, black soybean polyphenols suppressed hepatic lipid accumulation, oxidative stress, aminotransferase activities in the plasma, inflammatory cytokine expression, and α-SMA expression accompanied by modulation of lipid metabolism. In a combination of Western diet and carbon tetrachloride model, black soybean polyphenols also suppressed hepatic lipid accumulation, oxidative stress, aminotransferase activities in the plasma, and α-SMA expression. In conclusion, black soybean is an attractive food for the prevention of NAFLD and NASH due to its strong antioxidant activity.

Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Esculin protects against methionine choline-deficient diet-induced non-alcoholic steatohepatitis by regulating the Sirt1/NF-κB p65 pathway.

CONTEXT: Esculin, an active coumarin compound, has been demonstrated to exert anti-inflammatory effects. However, its potential role in non-alcoholic steatohepatitis (NASH) remains unclear. OBJECTIVE: This study explored the hepatoprotective effect and the molecular mechanism of esculin in methionine choline-deficient (MCD) diet-induced NASH. MATERIALS AND METHODS: Fifty C57BL/6J mice were divided into five groups: control, model, low dosage esculin (oral, 20 mg/kg), high dosage esculin (oral, 40 mg/kg), and silybin (oral, 105 mg/kg). All animals were fed a MCD diet, except those in the control group (control diet), for 6 weeks. RESULTS: Esculin (20 and 40 mg/kg) inhibited MCD diet-induced hepatic lipid content (triglyceride: 16.95 ± 0.67 and 14.85 ± 0.78 vs. 21.21 ± 1.13 mg/g; total cholesterol: 5.10 ± 0.34 and 4.08 ± 0.47 vs. 7.31 ± 0.58 mg/g), fibrosis, and inflammation (ALT: 379.61 ± 40.30 and 312.72 ± 21.45 vs. 559.51 ± 37.01 U/L; AST: 428.22 ± 34.29 and 328.23 ± 23.21 vs. 579.36 ± 31.93 U/L). In vitro, esculin reduced tumour necrosis factor-α, interleukin-6, fibronectin, and collagen 4A1 levels, but had no effect on lipid levels in HepG2 cells induced by free fatty acid. Esculin increased Sirt1 expression levels and decreased NF-κB acetylation levels in vivo and in vitro. Interfering with Sirt1 expression attenuated the beneficial effect of esculin on inflammatory and fibrotic factor production in HepG2 cells. CONCLUSIONS: These findings demonstrate that esculin ameliorates MCD diet-induced NASH by regulating the Sirt1/ac-NF-κB signalling pathway. Esculin could thus be employed as a therapy for NASH.

Resolution of severe hepatosteatosis in a cystic fibrosis patient with multifactorial choline deficiency: A case report.

In cystic fibrosis (CF), 85% to 90% of patients develop exocrine pancreatic insufficiency. Despite enzyme substitution, low pancreatic phospholipase A2 (sPLaseA2-IB) activity causes fecal loss of bile phosphatidylcholine and choline deficiency. We report on a female patient who has CF and progressive hepatosteatosis from 4.5 y onward. At 22.3 y, the liver comprised 27% fat (2385 mL volume) and transaminases were strongly increased. Plasma choline was 1.9 µmol/L (normal: 8-12 mol/L). Supplementation with 3 ×  1g/d choline chloride decreased liver fat and volume (3 mo: 8.2%; 1912 mL) and normalized transaminases. Plasma choline increased to only 5.6 µmol/L upon supplementation, with high trimethylamine oxide levels (12-35 µmol/L; normal: 3 ± 1 mol/L) proving intestinal microbial choline degradation. The patient was homozygous for rs12325817, a frequent single-nucleotide polymorphism in the PEMT gene, associated with severe hepatosteatosis in response to choline deficiency. Resolution of steatosis required 2 y (4.5% fat). Discontinuation/resumption of choline supplementation resulted in rapid relapse/resolution of steatosis, increased transaminases, and abdominal pain.

Hepatocyte-specific deletion of Nlrp6 in mice exacerbates the development of non-alcoholic steatohepatitis.

OBJECTIVE: Previous studies have established that deficiency in Nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain containing 6 (Nlrp6) changes the configuration of the gut microbiota, which leads to hepatic steatosis. Here, we aimed to determine the hepatic function of Nlrp6 in lipid metabolism and inflammation and its role in the development of non-alcoholic steatohepatitis (NASH). METHODS: Nlrp6Loxp/Loxp and hepatocyte-specific Nlrp6-knockout mice were fed a high-fat diet (HFD) or methionine-choline deficient (MCD) diet to induce fatty liver or steatohepatitis, respectively. Primary hepatocytes were isolated to further explore the underlying mechanisms in vitro. In addition, we used adenovirus to overexpress Nlrp6 in ob/ob mice to demonstrate its role in NASH. RESULTS: Hepatic Nlrp6 expression was downregulated in NASH patients and in obese mice. Hepatocyte-specific Nlrp6 deficiency promoted HFD- or MCD diet-induced lipid accumulation and inflammation, whereas Nlrp6 overexpression in ob/ob mice had beneficial effects. In vitro studies demonstrated that knockdown of Nlrp6 aggravated hepatic steatosis and inflammation in hepatocytes, but its overexpression markedly attenuated these abnormalities. Moreover, both in vitro and in vivo study demonstrated that Nlrp6 inhibited Cd36-mediated lipid uptake. Nlrp6 deficiency-enhanced fatty acid uptake was blocked by a Cd36 inhibitor in hepatocytes. Nlrp6 ablation increased the expression of proinflammatory cytokines, likely as a result of increased NF-κB phosphorylation and activation. Mechanistically, Nlrp6 promoted the degradation of transforming growth factor-ß-activated kinase 1 (TAK1)-binding protein 2/3 (TAB2/3) via a lysosomal-dependent pathway, which suppressed NF-κB activation. CONCLUSIONS: Nlrp6 may play a key role in the pathological process of NASH by inhibiting Cd36 and NF-κB pathways. It may be a potential therapeutic target for NASH.

Deletion of TRPC3 or TRPC6 Fails to Attenuate the Formation of Inflammation and Fibrosis in Non-alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice.