Methionine-choline Deficient Diet-induced Nonalcoholic Steatohepatitis Research Articles

Rifaximin alleviates MCD diet-induced NASH in mice by restoring the gut microbiota and intestinal barrier

AimsDue to the increasing global incidence rate of nonalcoholic steatohepatitis (NASH) combined with the lack of effective treatment methods for this disease, there is an urgent need to find new treatment strategies. The aim of this study was to investigate the efficacy of rifaximin in preventing and treating NASH and the related mechanism. Materials and methodsA NASH model was constructed by feeding male C57BL/6 mice a methionine-choline-deficient (MCD) diet for 4 weeks. Rifaximin was administered for 1 week before MCD diet feeding or during the last week of MCD diet feeding to investigate its preventive or therapeutic effects. Liver pathology, hepatic enzyme levels and metabolic indices were measured to evaluate the effects of rifaximin on NASH. Intestinal barrier integrity was measured via the Ussing chamber system and western blotting. 16S rDNA sequencing was conducted to investigate the fecal microbiota composition. Western blotting was performed to evaluate peroxisome proliferator activated receptor (PPAR)α and PPARγ protein levels. Key findingsRifaximin effectively alleviated MCD diet-induced NASH. The microbiota composition in MCD diet-fed mice was significantly altered, and intestinal barrier integrity was disrupted. Dysbiosis and intestinal barrier dysfunction were reversed by rifaximin. In addition, rifaximin modulated PPARα and PPARγ expression in the liver. SignificanceRifaximin effectively alleviated MCD diet-induced NASH by restoring the gut microbiota and reversing intestinal barrier dysfunction, suggesting that rifaximin treatment is a new approach for preventing and treating NASH.

Chrysin and its nanoliposome ameliorated non-alcoholic steatohepatitis via inhibiting TLR4 signalling pathway.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease histologically characterized by liver steatosis, hepatocellular injury, inflammation and fibrosis, resulting in cirrhosis and hepatocellular carcinoma, but effective measures and obvious pathogenesis for NASH remain elusive. Chrysin (CH) has been reported to have anti-inflammatory effects but shows lower bioavailability. In this study, a chrysin nanoliposome (CH-NL) was first prepared and characterized. Then, we used the methionine-choline-deficient (MCD) diet to induce a mouse model of NASH. Finally, the effects of CH and CH-NL on NASH were evaluated in the liver of NASH mice. The results showed that CH or CH-NL significantly reduced the accumulation of lipids in hepatocytes, alleviated liver injury, decreased the generation of radical oxygen species, and attenuated the accumulation of collagen fibre in the liver of NASH mice. In addition, CH and its nano-liposomes markedly inhibited the production of inflammatory cytokines and inflammatory cell infiltration in the liver of NASH mice. Further studies found that CH-NL and CH-NL downregulated the MCD diet-induced activation of Toll-like receptor 4 (TLR4) signalling pathway in the liver of mice. CH and its nanoliposome alleviated MCD diet-induced NASH in mice, which might be through inhibiting TLR4 signalling pathway.

Fluid Mechanics Approach to Perfusion Quantification: Vasculature Computational Fluid Dynamics Simulation, Quantitative Transport Mapping (QTM) Analysis of Dynamics Contrast Enhanced MRI, and Application in Nonalcoholic Fatty Liver Disease Classification.

We quantify liver perfusion using quantitative transport mapping (QTM) method that is free of arterial input function (AIF). QTM method is validated in a vasculature computational fluid dynamics (CFD) simulation and is applied for processing dynamic contrast enhanced (DCE) MRI images in differentiating liver with nonalcoholic fatty liver disease (NAFLD) from healthy controls using pathology reference in a preclinical rabbit model. QTM method was validated on a liver perfusion simulation based on fluid dynamics using a rat liver vasculature model and the mass transport equation. In the NAFLD grading task, DCE MRI images of 7 adult rabbits with methionine choline-deficient diet-induced nonalcoholic steatohepatitis (NASH), 8 adult rabbits with simple steatosis (SS) were acquired and processed using QTM method and dual-input two compartment Kety's method respectively. Statistical analysis was performed on six perfusion parameters: velocity magnitude | u | derived from QTM, liver arterial blood flow LBFa, liver venous blood flow LBFv, permeability Ktrans, blood volume Vp and extravascular space volume Ve averaged in liver ROI. In the simulation, QTM method successfully reconstructed blood flow, reduced error by 48% compared to Kety's method. In the preclinical study, only QTM |u| showed significant difference between high grade NAFLD group and low grade NAFLD group. QTM postprocesses DCE-MRI automatically through deconvolution in space and time to solve the inverse problem of the transport equation. Comparing with Kety's method, QTM method showed higher accuracy and better differentiation in NAFLD classification task. We propose to apply QTM method in liver DCE MRI perfusion quantification.

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.

CREBH alleviates mitochondrial oxidative stress through SIRT3 mediating deacetylation of MnSOD and suppression of Nlrp3 inflammasome in NASH.

Lipotoxicity and unresolved oxidative stress are key drivers of metabolic inflammation in nonalcoholic steatohepatitis (NASH). cAMP-response element binding protein H(CREBH) is a liver-specific transcription factor and regulates the glucose and lipid metabolism of NASH. However, its role in mitochondrial oxidative stress and its association with sirtuin 3 (SIRT3), a master regulator of deacetylation for mitochondrial proteins, remains elusive. In this study, AML-12cells were treated with palmitic acid to imitate the pathological changes of NASH in vitro and 8-week-old male C57BL/6J mice were fed with a high-fat (HF) diet or a methionine-choline-deficient (MCD) diet to build the widely accepted in vivo model of NASH. We found that lipid overload induced mitochondrial oxidative stress and stimulated the expression of CREBH and SIRT3. CREBH overexpression alleviated the mitochondrial oxidative stress. Moreover, CREBH promoted SIRT3 expression, which regulated the deacetylation of manganese superoxide dismutase (MnSOD) and inhibited NOD-Like Receptor Pyrin Domain Containing 3 (Nlrp3) inflammasome activation whereas suppression of SIRT3 damaged the protecting ability of CREBH in mitochondrial oxidative stress. CREBH knockout mice were highly susceptible to HF and MCD diet-induced NASH with more severe oxidative stress. Collectively, our results firstly provided the support that CREBH could serve as a protective factor in the progression of NASH by regulating the acetylation of MnSOD and the activation of Nlrp3 inflammasome through SIRT3. These results suggest that CREBH might be a valuable therapeutic candidate for NASH.

The PKM2 activator TEPP-46 attenuates MCD feeding-induced nonalcoholic steatohepatitis by inhibiting the activation of Kupffer cells.

The present study aimed to investigate the effect and molecular mechanism of the PKM2 small molecule agonist TEPP-46 on the development of methionine choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis (NASH) in mice. In this study, C57BL/6 mice were fed an MCD diet for 15 days to establish a NASH model. The protein expression levels of pyruvate kinase M2 (PKM2), PKM1, hypoxia-inducible factor-1α (HIF-1α) and NLRP3 in liver Kupffer cells (KCs) were measured by Western blotting. Immunofluorescence analysis was used to analyze the nuclear translocation of PKM2 in KCs, and the levels of IL-1β and TNF-α in mouse serum and the cell polarization indexes were determined. The MCD diet-fed mice were injected with 30 mg/kg of TEPP-46 intraperitoneally every 5 days. After 15 days, the liver tissue and peripheral blood were collected for analysis. We found the NASH model was successfully established after the mice were fed an MCD diet for 15 days. MCD feeding promoted the expression of the PKM2 monomer/dimer and inhibited the expression of the PKM2 tetramer in KCs. Immunofluorescence analysis further confirmed that MCD feeding inhibited the nuclear translocation of PKM2. Besides, MCD feeding promoted the expression of HIF-1α and NLRP3 in KCs, promoted M1 KCs polarization and inhibited M2 KCs polarization. Intraperitoneal injection 30 mg/kg of TEPP-46 significantly inhibited the development of MCD diet-induced NASH, alleviated the pathological changes in the liver, improved liver function, promoted the expression of the PKM2 tetramer in KCs, and inhibited the expression of HIF-1α and NLRP3. This study demonstrated that TEPP-46, a small molecule agonist of PKM2, may inhibit the nuclear translocation of PKM2 and the activation of KCs by promoting the expression of PKM2 tetramers in KCs, thus inhibiting the development of MCD diet-induced NASH in mice.

Esculin protects against methionine choline-deficient diet-induced non-alcoholic steatohepatitis by regulating the Sirt1/NF- κ B 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.

Citrus reticulata Blanco peel extract ameliorates hepatic steatosis, oxidative stress and inflammation in HF and MCD diet-induced NASH C57BL/6 J mice

Excessive lipid deposition, oxidative stress and inflammation in liver tissues are regarded as crucial inducers of nonalcoholic steatohepatitis (NASH), which is the most frequent chronic liver disease and closely related to obesity and insulin resistance. In this work, the preventive and therapeutic effects of Citrus reticulata Blanco (Jizigan) peel extract (JZE) on NASH induced by high fat (HF) diet and methionine choline-deficient (MCD) diet in C57BL/6 mice were investigated. We found that daily supplementation of JZE with an HF diet effectively ameliorated glucose tolerance and insulin resistance. In addition, the key indexes of lipid profiles, oxidative stress, hepatic steatosis and inflammatory factors were also ameliorated in both NASH mouse models. Furthermore, JZE treatment activated nuclear factor erythroid-2-related factor 2 (Nrf2) in the livers of diet- induced NASH mice. Our study suggests that JZE might alleviate NASH via the activation of Nrf2 signaling and that citrus Jizigan could be used as a dietary therapy for NASH and related metabolic syndrome.

Sweroside Prevents Non-Alcoholic Steatohepatitis by Suppressing Activation of the NLRP3 Inflammasome.

Non-alcoholic steatohepatitis (NASH), a type of non-alcoholic fatty liver disease, is characterized as steatosis and inflammation in the liver. NLRP3 inflammasome activation is associated with NASH pathology. We hypothesized that suppressing the NLRP3 inflammasome could be effective in preventing NASH. We searched substances that could inhibit the activation of the NLRP3 inflammasome and identified sweroside as an NLRP3 inhibitor. We investigated whether sweroside can be applied to prevent the pathological symptoms associated with NASH in a methionine–choline-deficient (MCD) diet-induced NASH mouse model. The activation of the NLRP3 inflammasome was determined by detecting the production of caspase-1 and IL-1β from pro-caspase-1 and pro-IL-1β in primary mouse macrophages and mouse liver. In a NASH model, mice were fed an MCD diet for two weeks with daily intraperitoneal injections of sweroside. Sweroside effectively inhibited NLRP3 inflammasome activation in primary macrophages as shown by a decrease in IL-1β and caspase-1 production. In a MCD diet-induced NASH mouse model, intraperitoneal injection of sweroside significantly reduced serum aspartate transaminase and alanine transaminase levels, hepatic immune cell infiltration, hepatic triglyceride accumulation, and liver fibrosis. The improvement of NASH symptoms by sweroside was accompanied with its inhibitory effects on the hepatic NLRP3 inflammasome as hepatic IL-1β and caspase-1 were decreased. Furthermore, sweroside blocked de novo synthesis of mitochondrial DNA in the liver, contributing to suppression of the NLRP3 inflammasome. These results suggest that targeting the NLRP3 inflammasome with sweroside could be beneficially employed to improve NASH symptoms.

Interleukin-22 Ameliorates Neutrophil-Driven Nonalcoholic Steatohepatitis Through Multiple Targets.

Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C-X-C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high-fat diet (HFD)-fed mice. The aim of this study was to test whether overexpression of CXCL1 itself in the liver can induce NASH in HFD-fed mice and to test the therapeutic potential of IL-22 in this new NASH model. Overexpression of Cxcl1 in the liver alone promotes steatosis-to-NASH progression in HFD-fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as apoptosis signal-regulating kinase 1 and p38 mitogen-activated protein kinase) activation. Myeloid cell-specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1-induced NASH and stress kinase activation in HFD-fed mice. Treatment with interleukin (IL)-22, a cytokine with multiple targets, ameliorated CXCL1/HFD-induced NASH or methionine-choline deficient diet-induced NASH in mice. Mechanistically, IL-22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL-22 treatment attenuated the inflammatory functions of hepatocyte-derived, mitochondrial DNA-enriched extracellular vesicles, thereby suppressing liver inflammation in NASH. Hepatic overexpression of CXCL1 is sufficient to drive steatosis-to-NASH progression in HFD-fed mice through neutrophil-derived reactive oxygen species and activation of stress kinases, which can be reversed by IL-22 treatment via the induction of metallothionein.

Allantoin improves methionine-choline deficient diet-induced nonalcoholic steatohepatitis in mice through involvement in endoplasmic reticulum stress and hepatocytes apoptosis-related genes expressions.

Objective(s):Non-alcoholic steatohepatitis (NASH) is defined by steatosis and inflammation in the hepatocytes, which can progress to cirrhosis and possibly hepatocellular carcinoma. However, current treatments are not entirely effective. Allantoin is one of the principal compounds in many plants and an imidazoline I receptor agonist as well. Allantoin has positive effects on glucose metabolism and inflammation. In this study, the effects of allantoin on the NASH induced animals and the pathways involved have been evaluated. Materials and Methods:C57/BL6 male mice received saline and allantoin as the control groups. In the next group, NASH was induced by the methionine-choline-deficient diet (MCD) for eight weeks. In the NASH+allantoin group, allantoin was injected four weeks in the mice feeding on an MCD diet. Histopathological evaluations, serum analysis, ELISA assay, and real-time RT-PCR were performed. Results:Allantoin administration decreased serum alanine aminotransferase (ALT), cholesterol, low-density lipoprotein (LDL), hepatic lipid accumulation, and liver tumor necrosis factor (TNFα) level. Also, treatment with allantoin down-regulated the gene expression of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (AFT6), TNFα, sterol regulatory element binding proteins 1c (SREBP1c), fatty acid synthase (FAS), Bax/Bcl2 ratio, caspase3, and P53. On the other hand, peroxisome proliferator-activated receptor alpha (PPARα), apolipoprotein B (Apo B), and acetyl-coenzyme acetyltransferase 1 (ACAT1) gene expression increased after allantoin injection. Conclusion:This study indicated that allantoin could improve animal induced NASH by changes in the expression of endoplasmic reticulum stress-related genes and apoptotic pathways.

Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in β-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor α. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of β-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Macrophage-Specific Hypoxia-Inducible Factor-1α Contributes to Impaired Autophagic Flux in Nonalcoholic Steatohepatitis.

Inflammatory cell activation drives diverse cellular programming during hepatic diseases. Hypoxia-inducible factors (HIFs) have recently been identified as important regulators of immunity and inflammation. In nonalcoholic steatohepatitis (NASH), HIF-1α is upregulated in hepatocytes, where it induces steatosis; however, the role of HIF-1α in macrophages under metabolic stress has not been explored. In this study, we found increased HIF-1α levels in hepatic macrophages in methionine-choline-deficient (MCD) diet-fed mice and in macrophages of patients with NASH compared with controls. The HIF-1α increase was concomitant with elevated levels of autophagy markers BNIP3, Beclin-1, LC3-II, and p62 in both mouse and human macrophages. LysMCre HIFdPA fl/fl mice, which have HIF-1α levels stabilized in macrophages, showed higher steatosis and liver inflammation compared with HIFdPA fl/fl mice on MCD diet. In vitro and ex vivo experiments reveal that saturated fatty acid, palmitic acid (PA), both induces HIF-1α and impairs autophagic flux in macrophages. Using small interfering RNA-mediated knock-down and overexpression of HIF-1α in macrophages, we demonstrated that PA impairs autophagy via HIF-1α. We found that HIF-1α mediates NF-κB activation and MCP-1 production and that HIF-1α-mediated impairment of macrophage autophagy increases IL-1β production, contributing to MCD diet-induced NASH. Conclusion: Palmitic acid impairs autophagy via HIF-1α activation in macrophages. HIF-1α and impaired autophagy are present in NASH in vivo in mouse macrophages and in human blood monocytes. We identified that HIF-1α activation and decreased autophagic flux stimulate inflammation in macrophages through upregulation of NF-κB activation. These results suggest that macrophage activation in NASH involves a complex interplay between HIF-1α and autophagy as these pathways promote proinflammatory overactivation in MCD diet-induced NASH.

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.

Cangju Qinggan Jiangzhi Decoction Reduces the Development of NonAlcoholic Steatohepatitis and Activation of Kupffer Cells

Background/Aims: Nonalcoholic steatohepatitis (NASH) is defined as lipid accumulation with hepatic injury, inflammation and early to moderate fibrosis. Kupffer cells play a crucial role in promoting hepatic inflammation, which further facilitates the development of NASH. Here we investigated the effects of Cangju Qinggan Jiangzhi decoction (CQJD) on high fat diet (HFD) and methionine-choline deficient (MCD) induced mouse NASH pathogenesis. Methods: Mouse NASH models were developed by HFD and MCD diet. The treated mice were divided into three groups: the control group (n = 10), the low-dose CQJD treatment group (n = 10) and the high-dose CQJD treatment group (n = 10). The hepatic injury, inflammation, and apoptotic molecules were evaluated by H&E staining, immunohistochemistry and real-time PCR. Kupffer cells were isolated from control mice and CQJD-treated mice after stimulation by lipopolysaccharide (LPS) and/or palmitic acid. The level of the inflammatory cytokines TNFα, IL1β, and CCL2 was measured by ELISA. Results: The HFD-fed mice displayed significant metabolic, inflammatory, and oxidative stress-related alterations due to hepatic lipid accumulation. CQJD treatment largely normalized the hepatic injury, lowered the ALT/AST level, and reduced the severity of liver inflammation, as revealed by the decreased inflammatory cytokines levels. In vitro, CQJD blocked the activation of LPS- or palmitic acid-primed Kupffer cells in a dose-dependent manner. In the MCD diet-induced NASH mice, similar therapeutic effects of CQJD were also observed. Conclusion: CQJD ameliorates mouse nonalcoholic steatohepatitis. The reduction in liver injury and inflammation induced by CQJD is associated with reduced activation of Kupffer cells. Our results suggest that CQJD is a promising therapeutic strategy in clinical steatohepatitis.

G-Aminobutyric acid promotes methionine-choline deficient diet-induced nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is one of the most common liver diseases and a major cause of liver fibrosis worldwide. Γ-Aminobutyric acid (GABA) is one of the most abundant inhibitory neurotransmitters in the central nervous system. Recently, it has been reported that GABAergic signaling pathways are found in various non-neuronal tissues including the immune system and play a functional role. In the present study, we investigated whether administration of GABA has effects on NASH through its immunomodulatory effects. To test this hypothesis, C57BL/6 mice were fed a methionine–choline-deficient (MCD) diet for 8 weeks. After four weeks into MCD feeding, mice were provided with plain water (control) or water containing 2 mg/mL of GABA for the subsequent 4 weeks. Using this MCD diet-induced NASH model, we found that mice receiving GABA showed more severe steatohepatitis and liver fibrosis than control mice. This increased liver damage was confirmed by higher levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) compared to the control group. In accordance with increased liver steatohepatitis, NASH-related and inflammatory gene expression (collagen α1, tissue inhibitor of metalloproteinase-1, TNF-α) in the liver was markedly increased in GABA-treated mice. Furthermore, GABA directly enhanced production of inflammatory cytokines including IL-6 and TNF-α in LPS activated RAW macrophage cells and increased TIB–73 hepatocyte death. Such effects were abolished when GABA was treated with bicuculline, a competitive antagonist of GABA receptors. These results suggest that oral administration of GABA may be involved in changes of the liver immune milieu and conferred detrimental effects on NASH progression.

Involvement of resistin-like molecule β in the development of methionine-choline deficient diet-induced non-alcoholic steatohepatitis in mice

Resistin-like molecule β (RELMβ) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMβ to non-alcoholic steatohepatitis (NASH) development. First, RELMβ knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMβ and that RELMβ expression levels in the colon and the numbers of RELMβ-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMβ-KO mice to distinguish between the contributions of RELMβ in these two organs. These experiments revealed the requirement of RELMβ in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMβ-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMβ in the gut and Kupffer cells to NASH development, raising the possibility of RELMβ being a novel therapeutic target for NASH.

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.

Lactobacillus caseistrain Shirota protects against nonalcoholic steatohepatitis development in a rodent model

Gut microbiota alterations are associated with various disorders. In this study, gut microbiota changes were investigated in a methionine-choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis (NASH) rodent model, and the effects of administering Lactobacillus casei strain Shirota (LcS) on the development of NASH were also investigated. Mice were divided into three groups, given the normal chow diet (NCD), MCD diet, or the MCD diet plus daily oral administration of LcS for 6 wk. Gut microbiota analyses for the three groups revealed that lactic acid bacteria such as Bifidobacterium and Lactobacillus in feces were markedly reduced by the MCD diet. Interestingly, oral administration of LcS to MCD diet-fed mice increased not only the L. casei subgroup but also other lactic acid bacteria. Subsequently, NASH development was evaluated based on hepatic histochemical findings, serum parameters, and various mRNA and/or protein expression levels. LcS intervention markedly suppressed MCD-diet-induced NASH development, with reduced serum lipopolysaccharide concentrations, suppression of inflammation and fibrosis in the liver, and reduced colon inflammation. Therefore, reduced populations of lactic acid bacteria in the colon may be involved in the pathogenesis of MCD diet-induced NASH, suggesting normalization of gut microbiota to be effective for treating NASH.

NALP3 inflammasome activation is MYD88-, but not TLR4-dependent and involves both parenchymal and bone marrow derived cells in non-alcoholic steatohepatitis (NASH)

Background and Aims: Endogenous danger signals from damaged hepatocytes in NASH might up-regulate and activate the inflammasome, that is a multiprotein complex that leads to IL-1ß secretion. We reported NALP3 inflammasome up-regulation activation in NASH. Here, we aimed to investigate the role of toll-like receptor 4 (TLR4) and the common TLR adaptor, MyD88 in the activation of NALP3 inflammasome. Furthermore, we aimed to investigate which cell types are involved in the inflammasome activation in the liver in NASH. Methods: Wild type (WT), TLR4- and MyD88-deficient (KO) mice, and WT mice transplanted with MyD88 KO bone marrow (WT/MyD88KO-BM) or MyD88KO mice with WT bone marrow (MyD88KO/WT-BM) received methionine-choline-deficient (MCD) or -supplemented (MCS) diet. Results: MCD diet-induced NASH resulted in up-regulation of TLR4, MyD88 and NALP3 inflammasome (NALP3, ASC, pro-caspase-1) mRNA, and mature IL-1β (17kDa) protein and caspase-1 activation in the liver compared to MCS diet feeding in WT mice. Total body deficiency in TLR4 although attenuated the MCD diet-induced liver injury and steatosis, but did not prevent the either the NALP3 up-regulation or the IL-1β production. MyD88 is a common adaptor molecule of most of the TLRs, including TLR4, but also TLR9. Total body deficiency in MyD88 prevented up-regulation of NALP3 mRNA and mature IL-1β production and significantly attenuated liver damage and steatosis in NASH. Selective MyD88-deficiency in bone marrow-derived or parenchymal cells prevented NALP3 mRNA up-regulation and attenuated IL-1β production in both chimeras (WT/MyD88KO-BM and MyD88KO/WT-BM)). Interestingly, WT/MyD88KO-BM mice were protected from MCD diet induced steatosis and liver damage, but liver injury was comparable between MCD diet-fed WT and MyD88KO/WT-BM mice suggesting that BM-derived cells contribute to NASH in a MyD88-dependent manner. Conclusions: We demonstrate for the first time that NALP3 inflammasome activation requires MyD88-dependent signaling in the MCD diet-induced NASH, but the deficiency of TLR4 is not enough to prevent the infammasome priming and activation. Finally, we show that both bone marrow-derived and parenchymal cells contribute to IL-1β production and the absence of MyD88 in bone marrow-derived cells attenuates steatohepatitis.