Choline-deficient L-amino Acid-defined Research Articles

Association of Differentially Altered Liver Fibrosis with Deposition of TGFBi in Stabilin-Deficient Mice.

Liver sinusoidal endothelial cells (LSECs) control clearance of Transforming growth factor, beta-induced, 68kDa (TGFBi) and Periostin (POSTN) through scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2). Stabilin inhibition can ameliorate atherosclerosis in mouse models, while Stabilin-double-knockout leads to glomerulofibrosis. Fibrotic organ damage may pose a limiting factor in future anti-Stabilin therapies. While Stab1-deficient (Stab1-/-) mice were shown to exhibit higher liver fibrosis levels upon challenges, fibrosis susceptibility has not been studied in Stab2-deficient (Stab2-/-) mice. Wildtype (WT), Stab1-/- and Stab2-/- mice were fed experimental diets, and local ligand abundance, hepatic fibrosis, and ligand plasma levels were measured. Hepatic fibrosis was increased in both Stab1-/- and Stab2-/- at baseline. A pro-fibrotic short Methionine-Choline-deficient (MCD) diet induced slightly increased liver fibrosis in Stab1-/- and Stab2-/- mice. A Choline-deficient L-amino acid-defined (CDAA) diet induced liver fibrosis of similar distribution and extent in all genotypes (WT, Stab1-/- and Stab2-/-). A hepatic abundance of Stabilin ligand TGFBi correlated very highly with liver fibrosis levels. In contrast, plasma levels of TGFBi were increased only in Stab2-/- mice after the CDAA diet but not the MCD diet, indicating the differential effects of these diets. Here we show that a single Stabilin deficiency of either Stab1 or Stab2 induces mildly increased collagen depositions under homeostatic conditions. Upon experimental dietary challenge, the local abundance of Stabilin ligand TGFBi was differentially altered in Stabilin-deficient mice, indicating differentially affected LSEC scavenger functions. Since anti-Stabilin-directed therapies are in clinical evaluation for the treatment of diseases, these findings bear relevance to treatment with novel anti-Stabilin agents.

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.

Changes in Lipidomics, Metabolomics, and the Gut Microbiota in CDAA-Induced NAFLD Mice after Polyene Phosphatidylcholine Treatment.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in most parts of the world. Although there is no first-line drug approved for the treatment of NAFLD, polyene phosphatidylcholine (PPC) is used by clinicians to treat NAFLD patients. This study aimed to evaluate the efficacy of PPC on a mice model of NAFLD, and to study the PPC's mechanism of action. The mice were fed a choline-deficient, L-amino acid-defined (CDAA) diet to induce NAFLD and were subsequently treated with PPC. The treatment effects were evaluated by the liver index, histopathological examination, and routine blood chemistry analyses. Lipidomics and metabolomics analyses of 54 samples were carried out using ultraperformance liquid chromatography (UPLC) coupled to a mass spectrometer to select for changes in metabolites associated with CDAA diet-induced NAFLD and the effects of PPC treatment. The intestinal flora of mice were extracted for gene sequencing to find differences before and after the induction of NAFLD and PPC treatment. PPC significantly improved the CDAA diet-induced NAFLD condition in mice. A total of 19 metabolites including 5 polar metabolites and 14 lipids showed marked changes. In addition, significant differences in the abundance of Lactobacillus were associated with NAFLD. We inferred that the protective therapeutic effect of PPC on the liver was related to the supplement of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin (PC, LPC, and SM, resectively) and acylcarnitine metabolism. This study developed a methodology for exploring the pathogenesis of NAFLD and can be extended to other therapeutic agents for treating NAFLD.

Choline-deficient Diet-induced NAFLD Animal Model Recaptures Core Human Pathophysiology With Similar Gene Co-expression Networks.

Nonalcoholic fatty liver disease (NAFLD) is a wide spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recently, the prevalence of NAFLD has dramatically increased, and treatment is urgently needed. Animal models are often used to understand the molecular mechanisms of disease development and progression, but their relevance to human diseases has not been fully understood. This study aimed to establish the usefulness of the animal model for preclinical research, we evaluated its relevance to human disease by gene expression analysis. We performed weighted gene co-expression network analysis of liver tissues from a choline-deficient L-amino acid-defined (CDAA) diet-induced NAFLD animal model. In addition, module preservation analysis was conducted to evaluate similarity across species. Several modules were identified to be associated with disease severity, and their gene co-expression network was found to be preserved in the human NAFLD datasets. Of note, module brown (immune cell clusters involved in inflammatory responses) was positively associated with disease severity, and its gene co-expression network was highly preserved in the human datasets. Tyrobp, Laptm5 and Lgals3 were identified as hub genes in the brown module, and their increased expression was confirmed in the human datasets. CDAA diet-induced NAFLD animal model recaptured key aspects of human pathophysiology (especially immune cell functions) and is thought to be a powerful tool for understanding the molecular mechanisms of NAFLD development and progression.

Diethyldithiocarbamate inhibits the activation of hepatic stellate cells via PPARα/FABP1 in mice with non-alcoholic steatohepatitis

Activation of hepatic stellate cells (HSCs) is the main course of liver fibrosis which is positively correlated with adverse clinical outcomes in non-alcoholic steatohepatitis (NASH). Diethyldithiocarbamate (DDC) attenuates NASH related liver fibrosis in mice, but its underlying mechanisms remains unclear. In this study, the data showed that DDC inhibited the activation of HSCs in high fat choline-deficient, L-amino acid-defined (CDAA) diet induced NASH. Double Immunofluorescence analysis showed that the baseline expression of peroxisome proliferator-activated receptor α (PPARα) is high in HSCs in normal mouse liver and notably decreases in the NASH liver, indicating that PPARα might be associated with the activation of HSCs. While, DDC upregulated PPARα in HSCs in the NASH liver. Mixture of free fatty acid was used to induce steatosis of hepatocytes. Human HSCs (LX-2 cells) were activated after co-cultured with steatotic hepatocytes, and DDC inhibited the activation of LX-2 cells. Meanwhile, DDC upregulated PPARα and FABP1, and promoted the accumulation of LDs in LX-2 cells. PPARα small interfering RNA blocked these effect of DDC. These findings suggest that PPARα is associated with the activation of HSCs in the context of NASH. DDC improves NASH related fibrosis through inhibiting the activation of HSCs via PPARα/FABP1.

Rifaximin and lubiprostone mitigate liver fibrosis development by repairing gut barrier function in diet-induced rat steatohepatitis.

Although gut-derived lipopolysaccharide (LPS) affects the progression of non-alcoholic steatohepatitis (NASH) pathogenesis, few studies have focused on this relationship to develop treatments for NASH. To explore the effects of combination with rifaximin and lubiprostone on NASH liver fibrosis through the modulation of gut barrier function. To induce steatohepatitis, F344 rats were fed a choline-deficient l-amino acid-defined (CDAA) diet for 12 weeks and received oral administration of rifaximin and/or lubiprostone. Histological, molecular, and fecal microbial analyses were performed. Barrier function in Caco-2 cells were assessed by in vitro assays. Combination rifaximin/lubiprostone treatment significantly suppressed macrophage expansion, proinflammatory responses, and liver fibrosis in CDAA-fed rats by blocking hepatic translocation of LPS and activation of toll-like receptor 4 signaling. Rifaximin and lubiprostone improved intestinal permeability via restoring tight junction proteins (TJPs) with the intestinal activation of pregnane X receptor and chloride channel-2, respectively. Moreover, this combination increased the abundance of Bacteroides, Lactobacillus, and Faecalibacterium as well as decreased that of Veillonella resulting in an increase of fecal short-chain fatty acids and a decrease of intestinal sialidase activity. Both agents also directly suppressed the LPS-induced barrier dysfunction and depletion of TJPs in Caco-2 cells. The combination of rifaximin and lubiprostone may provide a novel strategy for treating NASH-related fibrosis.

Mitochondrial fission in hepatocytes as a potential therapeutic target for nonalcoholic steatohepatitis.

The mitochondria are highly plastic and dynamic organelles; mitochondrial dysfunction has been reported to play causative roles in diabetes, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). However, the relationship between mitochondrial fission and NAFLD pathogenesis remains unknown. We aimed to investigate whether alterations in mitochondrial fission could play a role in the progression of NAFLD. Mice were fed a standard diet or choline-deficient, L-amino acid-defined (CDAA) diet with vehicle or mitochondrial division inhibitor-1. Substantial enhancement of mitochondrial fission in hepatocytes was triggered by 4weeks of feeding and was associated with changes reflecting the early stage of human nonalcoholic steatohepatitis (NASH), steatotic change with liver inflammation, and hepatocyte ballooning. Excessive mitochondrial fission inhibition in hepatocytes and lipid metabolism dysregulation in adipose tissue attenuated liver inflammation and fibrogenesis but not steatosis and the systemic pathological changes in the early and chronic fibrotic NASH stages (4- and 12-week CDAA feeding). These beneficial changes due to the suppression of mitochondrial fission against the liver and systemic injuries were associated with decreased autophagic responses and endoplasmic reticulum stress in hepatocytes. Injuries to other liver cells, such as endothelial cells, Kupffer cells, and hepatic stellate cells, were also attenuated by the inhibition of mitochondrial fission in hepatocytes. Taken together, these findings suggest that excessive mitochondrial fission in hepatocytes could play a causative role in NAFLD progression by liver inflammation and fibrogenesis through altered cell cross-talk. This study provides a potential therapeutic target for NAFLD.

Rifaximin enhances the L‑carnitine‑mediated preventive effects on skeletal muscle atrophy in cirrhotic rats by modulating the gut‑liver‑muscle axis.

The gut-liver-muscle axis is associated with the development of sarcopenia in liver cirrhosis. The present study aimed to illustrate the combined effects of rifaximin and L-carnitine on skeletal muscle atrophy in cirrhotic rats with steatohepatitis. For this purpose, a total of 344 Fischer rats were fed a choline-deficient L-amino acid-defined (CDAA) diet with the daily oral administration of rifaximin (100 mg/kg) and/or L-carnitine (200 mg/kg), and measurements of psoas muscle mass index and forelimb grip strength were performed. After feeding for 12 weeks, blood samples, and liver, ileum and gastrocnemius muscle tissues were harvested. The effects of L-carnitine on rat myocytes were assessed using in vitro assays. Treatment with rifaximin attenuated hyperammonemia and liver fibrosis in the CDAA-fed rats. Moreover, it improved intestinal permeability with the restoration of tight junction proteins and suppressed the lipopolysaccharide (LPS)-mediated hepatic macrophage activation and pro-inflammatory response. In addition, rifaximin prevented skeletal muscle mass atrophy and weakness by decreasing intramuscular myostatin and pro-inflammatory cytokine levels. Moreover, rifaximin synergistically enhanced the L-carnitine-mediated improvement of skeletal muscle wasting by promoting the production of insulin-like growth factor-1 and mitochondrial biogenesis, resulting in the inhibition of the ubiquitin-proteasome system (UPS). The in vitro assays revealed that L-carnitine directly attenuated the impairment of mitochondrial biogenesis, thereby inhibiting the UPS in rat myocytes that were stimulated with LPS or tumor necrosis factor-α. On the whole, the present study demonstrates that the combination of rifaximin with L-carnitine may provide a clinical benefit for liver cirrhosis-related sarcopenia.

Angiotensin Receptor Blockers Potentiate the Protective Effect of Branched-Chain Amino Acids on Skeletal Muscle Atrophy in Cirrhotic Rats.

This study investigated the combined effect of the angiotensin II (AT-II) receptor blocker losartan and branched-chain amino acids (BCAAs) on skeletal muscle atrophy in rats with cirrhosis and steatohepatitis. Fischer 344 rats are fed a choline-deficient l-amino acid-defined (CDAA) diet for 12 weeks and treated with oral losartan (30mg kg-1 day-1 ) and/or BCAAs (Aminoleban EN, 2500mg kg-1 day-1 ). Treatment with losartan and BCAAs attenuated hepatic inflammation and fibrosis and improved skeletal muscle atrophy and strength in CDAA-fed rats. Both agents reduced intramuscular myostatin and pro-inflammatory cytokine levels, resulting in inhibition of the ubiquitin-proteasome system (UPS) through interference with the SMAD and nuclear factor-kappa B pathways, respectively. Losartan also augmented the BCAA-mediated increase of skeletal muscle mass by promoting insulin growth factor-I production and mitochondrial biogenesis. Moreover, losartan decreased the intramuscular expression of transcription factor EB (TFEB), a transcriptional inducer of E3 ubiquitin ligase regulated by AT-II. In vitro assays illustrated that losartan promoted mitochondrial biogenesis and reduced TFEB expression in AT-II-stimulated rat myocytes, thereby potentiating the inhibitory effects of BCAAs on the UPS and caspase-3 cleavage. These results indicate that this regimen could serve as a novel treatment for patients with sarcopenia and liver cirrhosis.

Hepatic stellate cells-specific LOXL1 deficiency abrogates hepatic inflammation, fibrosis, and corrects lipid metabolic abnormalities in non-obese NASH mice.

Lysyl oxidase-like-1 (LOXL1), a vital cross-linking enzyme in extracellular matrix (ECM) maintenance, promotes fibrosis via enhancement of ECM stability. However, the potential role of LOXL1 in the pathogenesis of nonalcoholic steatohepatitis (NASH) has not been previously studied. We generated Loxl1fl/fl mice to selectively delete LOXL1 in hepatic stellate cells (HSCs) (Loxl1fl/flGfapcre; Loxl1fl/fl as littermate controls) and then examined liver pathology and metabolic profiles in Loxl1fl/flGfapcre fed with either a choline-deficient L-amino acid-defined (CDAA) diet or an isocaloric control diet for 16 weeks. Thereafter, the findings from the animal model were confirmed in 23 patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD). LOXL1 was significantly increased in CDAA induced non-obese NASH compared with the control diet, and LOXL1 deficient in HSCs ameliorated CDAA-induced inflammation and fibrosis, with reduced expression of pro-inflammation and pro-fibrogenic genes in the HSCs-specific LOXL1 knockout mice model. Interestingly, LOXL1 deficient in HSCs could attenuate hepatic steatosis and reverse the metabolic disorder by restoring adipose tissue function without altering the effect of hepatic lipogenesis gene expression in non-obese NASH model. More importantly, analyses of serum LOXL1 and leptin levels from NAFLD patients revealed that LOXL1 was positively correlated with histological fibrosis progression, whereas it was inversely correlated with leptin levels, especially in non-obese NAFLD patients. LOXL1 may contribute to fibrosis progression in non-obese NAFLD, and HSCs-specific knockout of LOXL1 attenuated liver steatosis, inflammation, fibrosis, , and improved lipid metabolic abnormalities. Hence, LOXL1 inhibition may serve as a new therapeutic strategy for NASH.

Hepatic Stellate Cells-Specific Loxl1 Deficiency Abrogates Hepatic Inflammation, Fibrosis, and Corrects Lipid Metabolic Abnormalities in Non-Obese NASH Mice

Background & Aims: Lysyl oxidase-like-1 (LOXL1), a vital crosslinking enzyme in extracellular matrix (ECM) protein maintenance, is well established in fibrosis via mediating ECM stabilization. However, the potential role of LOXL1 in the pathogenesis of nonalcoholic steatohepatitis (NASH) has not been previously studied. Methods: We generated Loxl1 fl/fl mice to selectively delete Loxl1 in hepatic stellate cells (HSCs) (Loxl1fl/flGfapcre; Loxl1fl/fl as littermate controls) and then examined liver pathology and metabolic context in Loxl1fl/flGfapcre fed a choline-deficient L-amino acid-defined (CDAA) diet or an isocaloric control diet for 16 weeks. We confirmed study findings in 23 patients with biopsy-proven NAFLD. Results: LOXL1 was significantly increased in CDAA induced non-obese NASH compared with control diet. Here, utilizing a HSCs-specific deletion of Loxl1 model, we found that Loxl1 deficient in HSCs ameliorated CDAA-induced inflammation and fibrosis, with reduced expression of pro-inflammation and pro-fibrogenic genes. Interestingly, CDAA-fed Loxl1 deficient mice was associated with improved body weight and attenuated hepatic steatosis and to an up-regulation of leptin in adipose tissue and in serum, without changes in hepatic lipogenesis gene expression, compared with CDAA-fed control mice. Most importantly, analyses of serum LOXL1 and leptin levels from NAFLD patients revealed that LOXL1 was positively correlated with histological fibrosis progression, whereas was inversely correlated with leptin levels, especially in non-obese NAFLD patients. Conclusion: In a mouse model of CDAA-induced non-obese NASH, selective deletion of Loxl1 from HSCs attenuated steatohepatitis, hepatic fibrosis and improved lipid metabolic abnormalities. Hence, LOXL1 inhibition may serve as a new therapeutic strategy for NASH. Funding Statement: This study was supported by the National Natural Science Foundation of China (81670539 and 81500456). Declaration of Interests: The authors disclose no conflicts. Ethics Approval Statement: (Human) The Ethics Committee of Beijing Friendship Hospital approved study (No.: 2018-P2-228-02). (Animal) This study was approved by the Institutional Animal Care and Usage Committee of the Beijing Friendship Hospital, Capital Medical University, Beijing, China.

Endothelial GATA4 controls liver fibrosis and regeneration by preventing a pathogenic switch in angiocrine signaling

Angiocrine signaling by liver sinusoidal endothelial cells (LSECs) regulates hepatic functions such as growth, metabolic maturation, and regeneration. Recently, we identified GATA4 as the master regulator of LSEC specification during development. Herein, we studied the role of endothelial GATA4 in the adult liver and in hepatic pathogenesis. We generated adult Clec4g-icretg/0xGata4fl/fl (Gata4LSEC-KO) mice with LSEC-specific depletion of Gata4. Livers were analyzed by histology, electron microscopy, immunohistochemistry/immunofluorescence, in situ hybridization, and LSECs were isolated for gene expression profiling, ChIP- and ATAC-sequencing. Partial hepatectomy was performed to assess regeneration. We used choline-deficient, l-amino acid-defined (CDAA) diet and chronic carbon tetrachloride exposure to model liver fibrosis. Human single cell RNA-seq data sets were analyzed for endothelial alterations in healthy and cirrhotic livers. Genetic Gata4 deficiency in LSECs of adult mice caused perisinusoidal liver fibrosis, hepatopathy and impaired liver regeneration. Sinusoidal capillarization and LSEC-to-continuous endothelial transdifferentiation were accompanied by a profibrotic angiocrine switch involving de novo endothelial expression of hepatic stellate cell-activating cytokine PDGFB. Increased chromatin accessibility and amplification by activated MYC mediated angiocrine Pdgfb expression. As observed in Gata4LSEC-KO livers, CDAA diet-induced perisinusoidal liver fibrosis was associated with GATA4 repression, MYC activation and a profibrotic angiocrine switch in LSECs. Comparison of CDAA-fed Gata4LSEC-KO and control mice demonstrated that endothelial GATA4 indeed protects against dietary-induced perisinusoidal liver fibrosis. In human cirrhotic livers, GATA4-positive LSECs and endothelial GATA4 target genes were reduced, while non-LSEC endothelial cells and MYC target genes including PDGFB were enriched. Endothelial GATA4 protects against perisinusoidal liver fibrosis by repressing MYC activation and profibrotic angiocrine signaling at the chromatin level. Therapies targeting the GATA4/MYC/PDGFB/PDGFRβ axis offer a promising strategy for prevention and treatment of liver fibrosis. The liver vasculature is supposed to play a major role in the development of liver fibrosis and cirrhosis, which can lead to liver failure and liver cancer. Herein, we discovered that structural and transcriptional changes induced by genetic deletion of the transcription factor GATA4 in the hepatic endothelium were sufficient to cause liver fibrosis. Activation of the transcription factor MYC and de novo expression of the "angiocrine" growth factor PDGFB were identified as downstream drivers of fibrosis and as potential therapeutic targets for this potentially fatal disease.

Effective Combination Therapy of Angiotensin-II Receptor Blocker and Rifaximin for Hepatic Fibrosis in Rat Model of Nonalcoholic Steatohepatitis.

The progression of nonalcoholic steatohepatitis (NASH) is complicated. The multiple parallel-hits theory is advocated, which includes adipocytokines, insulin resistance, endotoxins, and oxidative stress. Pathways involving the gut–liver axis also mediate the progression of NASH. Angiotensin-II receptor blockers (ARB) suppress hepatic fibrosis via the activation of hepatic stellate cells (HSCs). Rifaximin, a nonabsorbable antibacterial agent, is used for the treatment of hepatic encephalopathy and has been recently reported to improve intestinal permeability. We examined the inhibitory effects on and mechanism of hepatic fibrogenesis by combining ARB and rifaximin administration. Fischer 344 rats were fed a choline-deficient/l-amino acid-defined (CDAA) diet for 8 weeks to generate the NASH model. The therapeutic effect of combining an ARB and rifaximin was evaluated along with hepatic fibrogenesis, the lipopolysaccharide–Toll-like receptor 4 (TLR4) regulatory cascade, and intestinal barrier function. ARBs had a potent inhibitory effect on hepatic fibrogenesis by suppressing HSC activation and hepatic expression of transforming growth factor-β and TLR4. Rifaximin reduced intestinal permeability by rescuing zonula occludens-1 (ZO-1) disruption induced by the CDAA diet and reduced portal endotoxin. Rifaximin directly affect to ZO-1 expression on intestinal epithelial cells. The combination of an ARB and rifaximin showed a stronger inhibitory effect compared to that conferred by a single agent. ARBs improve hepatic fibrosis by inhibiting HSCs, whereas rifaximin improves hepatic fibrosis by improving intestinal permeability through improving intestinal tight junction proteins (ZO-1). Therefore, the combination of ARBs and rifaximin may be a promising therapy for NASH fibrosis.

Attenuation of the Hepatoprotective Effects of Ileal Apical Sodium Dependent Bile Acid Transporter (ASBT) Inhibition in Choline-Deficient L-Amino Acid-Defined (CDAA) Diet-Fed Mice.

Non-alcoholic fatty liver disease (NAFLD) is a major growing worldwide health problem. We previously reported that interruption of the enterohepatic circulation of bile acids using a non-absorbable apical sodium-dependent bile acid transporter inhibitor (ASBTi; SC-435) reduced the development of NAFLD in high fat diet fed mice. However, the ability of ASBTi treatment to impact the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and fibrosis in a diet-induced mouse model remains untested. In the current study, we assessed whether ASBTi treatment is hepatoprotective in the choline-deficient, L-amino acid-defined (CDAA) diet model of NASH-induced fibrosis.Methods: Male C57Bl/6 mice were fed with: (A) choline-sufficient L-amino acid-defined diet (CSAA) (31 kcal% fat), (B) CSAA diet plus ASBTi (SC-435; 60 ppm), (C) CDAA diet, or (D) CDAA diet plus ASBTi. Body weight and food intake were monitored. After 22 weeks on diet, liver histology, cholesterol and triglyceride levels, and gene expression were measured. Fecal bile acid and fat excretion were measured, and intestinal fat absorption was determined using the sucrose polybehenate method.Results: ASBTi treatment reduced bodyweight gain in mice fed either the CSAA or CDAA diet, and prevented the increase in liver to body weight ratio observed in CDAA-fed mice. ASBTi significantly reduced hepatic total cholesterol levels in both CSAA and CDAA-fed mice. ASBTi-associated significant reductions in hepatic triglyceride levels and histological scoring for NAFLD activity were observed in CSAA but not CDAA-fed mice. These changes correlated with measurements of intestinal fat absorption, which was significantly reduced in ASBTi-treated mice fed the CSAA (85 vs. 94%, P < 0.001) but not CDAA diet (93 vs. 93%). As scored by Ishak staging of Sirius red stained liver sections, no hepatic fibrosis was evident in the CSAA diet mice. The CDAA diet-fed mice developed hepatic fibrosis, which was increased by the ASBTi.Conclusions: ASBT inhibition reduced intestinal fat absorption, bodyweight gain and hepatic steatosis in CSAA diet-fed mice. The effects of the ASBTi on steatosis and fat absorption were attenuated in the context of dietary choline-deficiency. Inhibition of intestinal absorption of fatty acids may be involved in the therapeutic effects of ASBTi treatment.

Investigating fibrosis and inflammation in an ex vivo NASH murine model.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, characterized by excess fat accumulation (steatosis). Nonalcoholic steatohepatitis (NASH) develops in 15-20% of NAFLD patients and frequently progresses to liver fibrosis and cirrhosis. We aimed to develop an ex vivo model of inflammation and fibrosis in steatotic murine precision-cut liver slices (PCLS). NASH was induced in C57Bl/6 mice on an amylin and choline-deficient l-amino acid-defined (CDAA) diet. PCLS were prepared from steatohepatitic (sPCLS) and control (cPCLS) livers and cultured for 48 h with LPS, TGFβ1, or elafibranor. Additionally, C57Bl/6 mice were placed on CDAA diet for 12 wk to receive elafibranor or vehicle from weeks 7 to 12. Effects were assessed by transcriptome analysis and procollagen Iα1 protein production. The diets induced features of human NASH. Upon culture, all PCLS showed an increased gene expression of fibrosis- and inflammation-related markers but decreased lipid metabolism markers. LPS and TGFβ1 affected sPCLS more pronouncedly than cPCLS. TGFβ1 increased procollagen Iα1 solely in cPCLS. Elafibranor ameliorated fibrosis and inflammation in vivo but not ex vivo, where it only increased the expression of genes modulated by PPARα. sPCLS culture induced inflammation-, fibrosis-, and lipid metabolism-related transcripts, explained by spontaneous activation. sPCLS remained responsive to proinflammatory and profibrotic stimuli on gene expression. We consider that PCLS represent a useful tool to reproducibly study NASH progression. sPCLS can be used to evaluate potential treatments for NASH, as demonstrated in our elafibranor study, and serves as a model to bridge results from rodent studies to the human system.NEW & NOTEWORTHY This study showed that nonalcoholic steatohepatitis can be studied ex vivo in precision-cut liver slices obtained from murine diet-induced fatty livers. Liver slices develop a spontaneous inflammatory and fibrogenic response during culture that can be augmented with specific modulators. Additionally, the model can be used to test the efficacy of pharmaceutical compounds (as shown in this investigation with elafibranor) and could be a tool for preclinical assessment of potential therapies.

Trans-portal hepatic infusion of cultured bone marrow-derived mesenchymal stem cells in a steatohepatitis murine model.

The incidence of nonalcoholic steatohepatitis-related liver cirrhosis is increasing. We used a steatohepatitis murine model fed a choline-deficient, l-amino acid-defined (CDAA) diet with a single injection of carbon tetrachloride (CCl4) to evaluate the efficacy of trans-portal hepatic infusion of bone marrow-derived mesenchymal stem cells (BMSCs) for liver fibrosis, liver steatosis, and oxidative stress. Mice were fed a CDAA diet and injected with a single intraperitoneal dose of CCl4 (0.5 ml/kg) after 4 weeks of CDAA diet. After 12 weeks of CDAA diet, 1 × 106 luciferase-positive syngeneic BMSCs (Luc-BMSCs) were infused into the animal spleen. An in vivo imaging system was used to confirm Luc-BMSC accumulation in the liver via the portal vein, and at 4 weeks after infusion, we compared liver fibrosis, liver steatosis, and oxidative stress. After the BMSC-infusion, serum albumin and serum total bilirubin were significantly improved. Liver fibrosis assessed by Sirius red staining, α-smooth muscle actin protein, and collagen 1A1 mRNA expression was significantly suppressed. Furthermore, liver steatosis area was significantly lower, the 8-hydroxy-2'-deoxyguanosine-positive cells were significantly fewer, and superoxide dismutase 2 protein expression of the liver was significantly increased. In conclusion, our data confirmed the efficacy of trans-portal hepatic infusion of BMSCs in a steatohepatitis murine model.

Combined effect of a farnesoid X receptor agonist and dipeptidyl peptidase-4 inhibitor on hepatic fibrosis.

Non-alcoholic steatohepatitis (NASH) has a broad clinicopathological spectrum (inflammation to severe fibrosis). The farnesoid X receptor agonist obeticholic acid (OCA) ameliorates the histological features of NASH; satisfactory antifibrotic effects have not yet been reported. Here, we investigated the combined effects of OCA + a dipeptidyl peptidase-4 inhibitor (sitagliptin) on hepatic fibrogenesis in a rat model of NASH. Fifty Fischer 344 rats were fed a choline-deficient L-amino-acid-defined (CDAA) diet for 12weeks. The in vitro and in vivo effects of OCA + sitagliptin were assessed along with hepatic fibrogenesis, lipopolysaccharide-Toll-like receptor 4 (TLR4) regulatory cascade and intestinal barrier function. Direct inhibitory effects of OCA + sitagliptin on activated hepatic stellate cells (Ac-HSCs) were assessed in vitro. Treatment with OCA + sitagliptin potentially inhibited hepatic fibrogenesis along with Ac-HSC proliferation and hepatic transforming growth factor (TGF)-β1, α1(I)-procollagen, and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression and hydroxyproline levels. Obeticholic acid inhibited hepatic TLR4 expression and increased hepatic matrix metalloproteinase-2 expression. Obeticholic acid decreased intestinal permeability by ameliorating CDAA diet-induced zonula occludens-1 disruption, whereas sitagliptin directly inhibited Ac-HSC proliferation. The in vitro suppressive effects of OCA + sitagliptin on TGF-β1 and α1(I)-procollagen mRNA expression and p38 phosphorylation in Ac-HSCs were almost consistent. Sitagliptin directly inhibited the regulation of Ac-HSC. Treatment with OCA + sitagliptin synergistically affected hepatic fibrogenesis by counteracting endotoxemia induced by intestinal barrier dysfunction and suppressing Ac-HSC proliferation. Thus, OCA + sitagliptin could be a promising therapeutic strategy for NASH.

Exogenous Administration of Low-Dose Lipopolysaccharide Potentiates Liver Fibrosis in a Choline-Deficient l-Amino-Acid-Defined Diet-Induced Murine Steatohepatitis Model

Various rodent models have been proposed for basic research; however, the pathogenesis of human nonalcoholic steatohepatitis (NASH) is difficult to closely mimic. Lipopolysaccharide (LPS) has been reported to play a pivotal role in fibrosis development during NASH progression via activation of toll-like receptor 4 (TLR4) signaling. This study aimed to clarify the impact of low-dose LPS challenge on NASH pathological progression and to establish a novel murine NASH model. C57BL/6J mice were fed a choline-deficient l-amino-acid-defined (CDAA) diet to induce NASH, and low-dose LPS (0.5 mg/kg) was intraperitoneally injected thrice a week. CDAA-fed mice showed hepatic CD14 overexpression, and low-dose LPS challenge enhanced TLR4/NF-κB signaling activation in the liver of CDAA-fed mice. LPS challenge potentiated CDAA-diet-mediated insulin resistance, hepatic steatosis with upregulated lipogenic genes, and F4/80-positive macrophage infiltration with increased proinflammatory cytokines. It is noteworthy that LPS administration extensively boosted pericellular fibrosis with the activation of hepatic stellate cells in CDAA-fed mice. Exogenous LPS administration exacerbated pericellular fibrosis in CDAA-mediated steatohepatitis in mice. These findings suggest a key role for LPS/TLR4 signaling in NASH progression, and the authors therefore propose this as a suitable model to mimic human NASH.

Dietary iron restriction leads to a reduction in hepatic fibrosis in a rat model of non-alcoholic steatohepatitis.

ABSTRACTIron overload in the liver causes oxidative stress and inflammation, which result in organ dysfunction, making it a risk factor for non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma. We aimed to evaluate the effect of dietary iron restriction on disease progression in rats fed a choline-deficient L-amino acid-defined (CDAA) diet. Male F344 rats were fed a choline-sufficient amino acid-defined (control) diet, a CDAA diet or an iron-restricted CDAA diet for 4, 8 and 12 weeks. At each time point, hepatic iron levels, oxidative stress, inflammation and fibrosis were evaluated by immunohistochemistry. The iron-restricted CDAA diet significantly decreased serum iron levels for 12 weeks compared with the CDAA diet. Histological analysis confirmed that feeding with the CDAA diet induced hepatic iron overload and that this was associated with oxidative stress (number of 8-hydroxydeoxyguanosine-positive cells), inflammation (CD68 positive area) and fibrosis (Sirius Red positive area). Iron restriction with the CDAA diet significantly led to a reduction in the hepatic iron levels, oxidative stress, inflammation and fibrosis. Therefore, dietary iron restriction could be a useful therapeutic approach for NASH patients with hepatic iron overload.

Combining probiotics and an angiotensin-II type 1 receptor blocker has beneficial effects on hepatic fibrogenesis in a rat model of non-alcoholic steatohepatitis.

Intestinal endotoxin is important for the progression of non-alcoholic steatohepatitis (NASH). Circulating endotoxin levels are elevated in most animal models of diet-induced non-alcoholic fatty liver disease (NAFLD) and NASH. Furthermore, plasma endotoxin levels are significantly higher in NAFLD patients, which is associated with small intestinal bacterial overgrowth and increased intestinal permeability. By improving the gut microbiota environment and restoring gut-barrier functions, probiotics are effective for NASH treatment in animal models. It is also widely known that hepatic fibrosis and suppression of activated hepatic stellate cells (Ac-HSCs) can be attenuated using an angiotensin-II type 1 receptor blocker (ARB). We thus evaluated the effect of combination probiotics and ARB treatment on liver fibrosis using a rat model of NASH. Fisher 344 rats were fed a choline-deficient/L-amino acid-defined (CDAA) diet for 8weeks to generate the NASH model. Animals were divided into ARB, probiotics, and ARB plus probiotics groups. Therapeutic efficacy was assessed by evaluating liver fibrosis, the lipopolysaccharide Toll-like receptor (TLR)4 regulatory cascade, and intestinal barrier function. Both probiotics and ARB inhibited liver fibrosis, with concomitant HSC activation and suppression of liver-specific transforming growth factor-β and TLR4 expression. Probiotics reduced intestinal permeability by rescuing zonula occludens-1 disruption induced by the CDAA diet. Angiotensin-II type 1 receptor blocker was found to directly suppress Ac-HSCs. Probiotics and ARB are effective in suppressing liver fibrosis through different mechanisms. Currently both drugs are in clinical use; therefore, the combination of probiotics and ARB is a promising new therapy for NASH.