Liver Nonalcoholic Steatohepatitis Research Articles

Efficacy of chemically induced human hepatic progenitor cells from diseased liver against nonalcoholic steatohepatitis model.

Numerous chemical reprogramming techniques have been reported, rendering them applicable to regenerative medicine research. The aim of our study was to evaluate the therapeutic potential of human CLiP derived from clinical specimens transplanted into a nonalcoholic steatohepatitis (NASH) mouse model of liver fibrosis. We successfully generated chemically induced liver progenitor (CLiP), which exhibited progenitor-like characteristics, through stimulation with low-molecular-weight compounds. We elucidated their cell differentiation ability and therapeutic effects. However, the therapeutic efficacy of human CLiP generated from clinical samples on liver fibrosis, such as liver cirrhosis, remains unproven. Following a 4 week period, transplanted human CLiP in the NASH model differentiated into mature hepatocytes and demonstrated suppressive effects on liver injury markers (i.e., aspartate transaminase and alanine transaminase). Although genes related to inflammation and fat deposition did not change in the human CLiP transplantation group, liver fibrosis-related factors (Acta2 and Col1A1) showed suppressive effects on gene expression following transplantation, with approximately a 60% reduction in collagen fibers. Importantly, human CLiP could be efficiently induced from hepatocytes isolated from the cirrhotic liver, underscoring the feasibility of using autologous hepatocytes to produce human CLiP. Our findings demonstrate the effectiveness of human CLiP transplantation as a viable cellular therapy for liver fibrosis, including NASH liver. These results hold promise for the development of liver antifibrosis therapy utilizing human CLiP within the field of liver regenerative medicine.

Correlative Quantitative Raman Chemical Imaging and MCR-ALS in Mouse NASH Model Reveals Direct Relationships between Diet and Resultant Liver Pathology.

Raman imaging has the capability to provide unlabeled, spatially aware analysis of chemical components, with no a priori assumptions. Several lifestyle diseases such as nonalcoholic steatohepatitis (NASH) can appear in the liver as changes in the nature, abundance, and distribution of lipids, proteins, and other biomolecules and are detectable by Raman imaging. In order to identify which of these liver-associated changes occur as a direct result of the diet and which are secondary effects, we developed correlative imaging and analysis of diet and liver samples. Oleic acid was found to be a direct contributor to NASH liver composition, whereas protein and collagen distributions were found to be affected in a manner consistent with early fibrotic transformation, as a secondary consequence of the high-fat diet.

Therapeutic Targeting of PKM2 Ameliorates NASH Fibrosis Progression in a Macrophage-Specific and Liver-Specific Manner

Nonalcoholic steatohepatitis (NASH) may soon become the leading cause of end-stage liver disease worldwide with limited treatment options. Liver fibrosis, which is driven by chronic inflammation and hepatic stellate cell (HSC) activation, critically determines morbidity and mortality in patients with NASH. Pyruvate kinase M2 (PKM2) is involved in immune activation and inflammatory liver diseases; however, its role and therapeutic potential in NASH-related fibrosis remain largely unexplored. Bioinformatics screening and analysis of human and murine NASH livers indicated that PKM2 was upregulated in nonparenchymal cells (NPCs), especially macrophages, in the livers of patients with fibrotic NASH. Macrophage-specific PKM2 knockout (PKM2FL/FLLysM-Cre) significantly ameliorated hepatic inflammation and fibrosis severity in three distinct NASH models induced by a methionine- and choline-deficient (MCD) diet, a high-fat high-cholesterol (HFHC) diet, and a western diet plus weekly carbon tetrachloride injection (WD/CCl4). Single-cell transcriptomic analysis indicated that deletion of PKM2 in macrophages reduced profibrotic Ly6Chigh macrophage infiltration. Mechanistically, PKM2-dependent glycolysis promoted NLR family pyrin domain containing 3 (NLRP3) activation in proinflammatory macrophages, which induced HSC activation and fibrogenesis. A pharmacological PKM2 agonist efficiently attenuated the profibrotic crosstalk between macrophages and HSCs in vitro and in vivo. Translationally, ablation of PKM2 in NPCs by cholesterol-conjugated heteroduplex oligonucleotides, a novel oligonucleotide drug that preferentially accumulates in the liver, dose-dependently reversed NASH-related fibrosis without causing observable hepatotoxicity. The present study highlights the pivotal role of macrophage PKM2 in advancing NASH fibrogenesis. Thus, therapeutic modulation of PKM2 in a macrophage-specific or liver-specific manner may serve as a novel strategy to combat NASH-related fibrosis.

Assessing the combined impact of fatty liver-induced TGF-β1 and LPS-activated macrophages in fibrosis through a novel 3D serial section methodology.

Non-alcoholic steatohepatitis (NASH), caused by fat buildup, can lead to liver inflammation and damage. Elucidation of the spatial distribution of fibrotic tissue in the fatty liver in NASH can be immensely useful to understand its pathogenesis. Thus, we developed a novel serial section-3D (SS3D) technique that combines high-resolution image acquisition with 3D construction software, which enabled highly detailed analysis of the mouse liver and extraction and quantification of stained tissues. Moreover, we studied the underexplored mechanism of fibrosis progression in the fatty liver in NASH by subjecting the mice to a high-fat diet (HFD), followed by lipopolysaccharide (LPS) administration. The HFD/LPS (+) group showed extensive fibrosis compared with control; additionally, the area of these fibrotic regions in the HFD/LPS (+) group was almost double that of control using our SS3D technique. LPS administration led to an increase in Tnfα and Il1β mRNA expression and the number of macrophages in the liver. On the other hand, transforming growth factor-β1 (Tgfβ1) mRNA increased in HFD group compared to that of control group without LPS-administration. In addition, COL1A1 levels increased in hepatic stellate cell (HSC)-like XL-2 cells when treated with recombinant TGF-β1, which attenuated with recombinant latency-associated protein (rLAP). This attenuation was rescued with LPS-activated macrophages. Therefore, we demonstrated that fatty liver produced "latent-form" of TGF-β1, which activated by macrophages via inflammatory cytokines such as TNFα and IL1β, resulting in activation of HSCs leading to the production of COL1A1. Moreover, we established the effectiveness of our SS3D technique in creating 3D images of fibrotic tissue, which can be used to study other diseases as well.

A 5:2 intermittent fasting regimen ameliorates NASH and fibrosis and blunts HCC development via hepatic PPARα and PCK1

The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 invivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara invivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.

Increased hepatoprotective effects of the novel farnesoid X receptor agonist INT-787 versus obeticholic acid in a mouse model of nonalcoholic steatohepatitis.

The nuclear farnesoid X receptor (FXR), a master regulator of bile acid and metabolic homeostasis, is a key target for treatment of nonalcoholic steatohepatitis (NASH). This study compared efficacy of FXR agonists obeticholic acid (OCA) and INT-787 by liver histopathology, plasma biomarkers of liver damage, and hepatic gene expression profiles in the Amylin liver NASH (AMLN) diet-induced and biopsy-confirmed Lepob/ob mouse model of NASH. Lepob/ob mice were fed the AMLN diet for 12 weeks before liver biopsy and subsequent treatment with vehicle, OCA, or INT-787 for 8 weeks. Hepatic steatosis, inflammation, and fibrosis (liver lipids, galectin-3, and collagen 1a1 [Col1a1], respectively), as well as plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, were assessed. Hepatic gene expression was assessed in Lepob/ob mice that were fed the AMLN diet for 14 weeks then treated with vehicle, OCA, or INT-787 for 2 weeks. INT-787, which is equipotent to OCA but more hydrophilic, significantly reduced liver lipids, galectin-3, and Col1a1 compared with vehicle, and to a greater extent than OCA. INT-787 significantly reduced plasma ALT and AST levels, whereas OCA did not. INT-787 modulated a substantially greater number of genes associated with FXR signaling, lipid metabolism, and stellate cell activation relative to OCA in hepatic tissue. These findings demonstrate greater efficacy of INT-787 treatment compared with OCA in improving liver histopathology, decreasing liver enzyme levels, and enhancing gene regulation, suggesting superior clinical potential of INT-787 for the treatment of NASH and other chronic liver diseases.

Targeting EFHD2 inhibits interferon-γ signaling and ameliorates non-alcoholic steatohepatitis

The precise pathomechanisms underlying the development of non-alcoholic steatohepatitis (NASH, also known as metabolic dysfunction-associated steatohepatitis [MASH]) remain incompletely understood. In this study, we investigated the potential role of EF-hand domain family member D2 (EFHD2), a novel molecule specific to immune cells, in the pathogenesis of NASH. Hepatic EFHD2 expression was characterized in patients with NASH and two diet-induced NASH mouse models. Single-cell RNA sequencing (scRNA-seq) and double-immunohistochemistry were employed to explore EFHD2 expression patterns in NASH livers. The effects of global and myeloid-specific EFHD2 deletion on NASH and NASH-related hepatocellular carcinoma were assessed. Molecular mechanisms underlying EFHD2 function were investigated, while chemical and genetic investigations were performed to assess its potential as a therapeutic target. EFHD2 expression was significantly elevated in hepatic macrophages/monocytes in both patients with NASH and mice. Deletion of EFHD2, either globally or specifically in myeloid cells, improved hepatic steatosis, reduced immune cell infiltration, inhibited lipid peroxidation-induced ferroptosis, and attenuated fibrosis in NASH. Additionally, it hindered the development of NASH-related hepatocellular carcinoma. Specifically, deletion of myeloid EFHD2 prevented the replacement of TIM4+ resident Kupffer cells by infiltrated monocytes and reversed the decreases in patrolling monocytes and CD4+/CD8+ T cell ratio in NASH. Mechanistically, our investigation revealed that EFHD2 in myeloid cells interacts with cytosolic YWHAZ (14-3-3ζ), facilitating the translocation of IFNγR2 (interferon-γ receptor-2) onto the plasma membrane. This interaction mediates interferon-γ signaling, which triggers immune and inflammatory responses in macrophages during NASH. Finally, a novel stapled α-helical peptide targeting EFHD2 was shown to be effective in protecting against NASH pathology in mice. Our study reveals a pivotal immunomodulatory and inflammatory role of EFHD2 in NASH, underscoring EFHD2 as a promising druggable target for NASH treatment. Non-alcoholic steatohepatitis (NASH) represents an advanced stage of non-alcoholic fatty liver disease (NAFLD); however, not all patients with NAFLD progress to NASH. A key challenge is identifying the factors that trigger inflammation, which propels the transition from simple fatty liver to NASH. Our research pinpointed EFHD2 as a pivotal driver of NASH, orchestrating the over-activation of interferon-γ signaling within the liver during NASH progression. A stapled peptide designed to target EFHD2 exhibited therapeutic promise in NASH mice. These findings support the potential of EFHD2 as a therapeutic target in NASH.

Abstract 2662: Deciphering the cellular and molecular determinants of immunotherapy resistance in NASH-associated hepatocellular carcinoma by single-cell analysis

Abstract Introduction: Non-alcoholic steatohepatitis (NASH) has emerged as a prominent risk and the most rapidly increasing aetiology of hepatocellular carcinoma (HCC). Despite the effectiveness of immune checkpoint blockade (ICB), like anti-programmed cell death-ligand 1 (anti-PD-L1) antibodies, in a subset of HCC patients, NASH has been shown to impair the anti-tumor immunity in ICB-treated HCC. We aim to elucidate the mechanisms responsible for ICB resistance in NASH-HCC at single-cell resolution. Methods: Orthotopic NASH-HCC mouse models were generated by orthotopically injecting liver cancer cell line into NASH liver induced by methionine-and choline-deficient diet (MCD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Multi-color flow cytometry and single-cell RNA-sequencing (scRNA-seq) were utilized to dissect the liver and tumor immune microenvironment upon anti-PD-L1 treatment. Results: Anti-PD-L1 treatment failed to alleviate tumor burden in NASH mice, indicating that orthotopic NASH-HCC mouse models were resistant to ICB therapy. Compared to the control liver, multi-color flow cytometry demonstrated that CD11b+F4/80+CD206+M2 macrophages accumulated in NASH liver and further increased in liver and tumor upon anti-PD-L1 treatment. Tumor-infiltrating M2 macrophages exhibited the most significant and robust correlation with tumor weight among all immune cell types. Moreover, PD-1 expression on tumor-infiltrating CD8+ T cells positively correlated with tumor weight and intratumoral M2 macrophages. Furthermore, scRNA-seq analysis identified a NASH-specific tumor-infiltrating monocyte subcluster, which may differentiate into an ‘M2-like’ macrophage subtype upon anti-PD-L1 treatment as demonstrated by cell trajectory analysis. Conclusion: Our study provides insights into the significance of monocyte/macrophage reprogramming in ICB resistance in NASH-HCC. Further investigation is conducted to identify the molecular mechanisms during monocyte-macrophage differentiation and explore potential targeted strategies to overcome ICB resistance in NASH-HCC. Acknowledgment: This study is supported by the General Research Fund (14120621 and 14119023), the Collaborative Research Fund (C4045-18W), the Li Ka Shing Foundation and CUHK Strategic Seed Funding for Collaborative Research Scheme. Keywords: hepatocellular carcinoma, single-cell RNA-sequencing, anti-PD-L1, macrophages. Citation Format: Lingyun Zhang, Yiling Zhang, Wenshu Tang, Zhewen Xiong, Xiaoyu Liu, Zhixian Liang, Yuk Wah Tsang, Weiqin Yang, Xiaohang Long, Alfred Sze Lok Cheng. Deciphering the cellular and molecular determinants of immunotherapy resistance in NASH-associated hepatocellular carcinoma by single-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2662.

Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/− mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

Identification of pyroptosis-related gene signature in nonalcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the major causes of liver-related morbidity and mortality globally. It ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) characterized by ballooning and hepatic inflammation. In the past few years, pyroptosis has been shown as a type of programmed cell death that triggers inflammation and plays a role in the development of NASH. However, the roles of pyroptosis-related genes (PRGs) in NASH remained unclear. In this study, we studied the expression level of pyroptosis-related genes (PRGs) in NASH and healthy controls, developed a diagnostic model of NASH based on PRGs and explored the pathological mechanisms associated with pyroptosis. We further compared immune status between NASH and healthy controls, analyzed immune status in different subtypes of NASH. We identified altogether twenty PRGs that were differentially expressed between NASH and normal liver tissues. Then, a novel diagnostic model consisting of seven PRGs including CASP3, ELANE, GZMA, CASP4, CASP9, IL6 and TP63 for NASH was constructed with an area under the ROC curve (AUC) of 0.978 (CI 0.965–0.99). Obvious variations in immune status between healthy controls and NASH cases were detected. Subsequently, the consensus clustering method based on differentially expressed PRGs was constructed to divide all NASH cases into two distinct pyroptosis subtypes with different immune and biological characteristics. Pyroptosis-related genes may play an important role in NASH and can provide new insights into the diagnosis and underlying mechanisms of NASH.

Clinical characterization of a hypersensitivity mixed bacterial and fungal dermatitis in a translational model of porcine NASH.

The development of animal models of chronic liver disease via diet modification is a promising avenue for translational research but can lead to unexpected side effects that impact model adoption. While these side effects are well characterized in rodent models of nonalcoholic steatohepatitis (NASH), limited knowledge of these effects exists for novel porcine models of NASH. To close this gap, the present study investigates the side effects of diet-based NASH induction in pigs, with a systematic analysis of the pathologic mechanisms underlying dermatitis development and evaluation of treatment approaches. Twelve pigs (10 large domestic pigs, 2 Goettingen minipigs) were fed a methionine- and choline-deficient, high-fat diet for 8 weeks to induce NASH. A retrospective review of each animal's clinical record was performed to identify the side effects of the diet. Following the identification of diet-associated dermatitis, severity was judged by using a novel gradation system that characterized the individual lesions and body regions resulting in a cumulative evaluation. In addition to this clinical assessment, the etiology of the dermatitis was investigated via histopathologic and microbiologic testing. Furthermore, the success of prophylactic and therapeutic treatment approaches was evaluated by considering dermatitis development and clinical course. All study animals demonstrated unexpected side effects of the methionine- and choline-deficient, high fat diet. In addition to marked dermatitis, study pigs showed impaired weight gain and developed steatorrhea and anemia. Based on the skin gradation system, five animals developed severe dermatitis, four animals moderate dermatitis, and three animals mild diet-associated dermatitis. Histological and microbiological evaluation of the affected skin showed signs of a hypersensitivity reaction with secondary infection by bacteria and fungi. The analysis showed that preemptive bathing extended the lesion-free duration by nearly 20 days. Furthermore, bathing in combination with a targeted antibiotic treatment represented a helpful treatment approach for diet-associated dermatitis. The provision of a methionine- and choline-deficient, high fat diet represents an effective approach for inducing NASH liver disease in pigs but predisposes study animals to multiple side effects. These side effects are universal to animals on study but can be adequately managed and do not represent a significant limitation of this model.

Exosomal miR-122, miR-128, miR-200, miR-298, and miR-342 as novel diagnostic biomarkers in NAFL/NASH: Impact of LPS/TLR-4/FoxO3 pathway.

Nonalcoholic fatty liver disease (NAFLD) is a common liver disorder affecting a quarter of the global residents. Progression of NAFL into nonalcoholic steatohepatitis (NASH) may cause cirrhosis, liver cancer, and failure. Gut microbiota imbalance causes microbial components translocation into the circulation, triggering liver inflammation and NASH-related fibrosis. MicroRNAs (miRNAs) regulate gene expression via repressing target genes. Exosomal miRNAs are diagnostic and prognostic biomarkers for NAFL and NASH liver damage. Our work investigated the role of the gut microbiota in NAFLD pathogenesis via the lipopolysaccharide/toll-like receptor 4/Forkhead box protein O3 (LPS/TLR-4/FoxO3) pathway and certain miRNAs as noninvasive biomarkers for NAFL or its development to NASH. miRNAexpression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 50 NAFL patients, 50 NASH patients, and 50 normal controls. Plasma LPS, TLR-4, adiponectin, peroxisome proliferator-activated receptor γ (PPAR-γ), and FoxO3 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). In NAFL and NASH patients, miR-122, miR-128, FoxO3, TLR-4, LPS, and PPAR-γ were upregulated while miR-200, miR-298, miR-342, and adiponectin were downregulated compared with the normalcontrol. The examined miRNAs might distinguish NAFL and NASH patients from the normal controlusing receiver operating characteristic analysis. Our study is the first to examine these miRNAs in NAFLD. Our findings imply that these are potentially promising biomarkers for noninvasive early NAFL diagnosis and NASH progression. Understanding the LPS/TLR-4/FoxO3 pathway involvement in NAFL/NASH pathogenesis may aid disease management.

Association of GCKR and MBOAT7 genetic polymorphisms with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the most important causes of chronic liver disease (CLD) in both Western and Asian populations. There is wide inter-individual variability in the occurrence of NAFLD and progression to non-alcoholic steatohepatitis (NASH) even after correcting environmental factors, and its true explanation can be provided by heritability. Two such genetic variations, the glucokinase regulator (GCKR) and membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes, in NAFLD patients were studied in the Indian population. A cross sectional analytical study was conducted in the Department of Gastroenterology at a tertiary care centre. In total 100 subjects in the age range of 18-65 years were included in the study; 50 were patients with NAFLD including fatty liver, NASH and NASH related cirrhosis, and 50 were healthy subjects (No NAFLD). The polymorphisms rs780094 and rs1260326 for GCKR and rs641738 for MBOAT7 were determined using PCR followed by the PCR-RFLP. GCKR rs780094 minor allele A was more common in NAFLD patients (p = 0.00001). Within the spectrum of NAFLD, the A allele was present frequently among cirrhotics as compared to NASH and fatty liver (p = 0.00001). Morbidly obese individuals showed significant association with the homozygous A allele (p = 0.028). These results were not seen with GCKR rs1260326 across all alleles. In MBOAT7 (rs641738) the frequency of the minor allele T for NAFLD was 84% vs. 80% in healthy subjects (p = 0.79). The association of the T allele among the spectrum of NAFLD was not statistically significant (p = 0.79). GCKR genetic variant rs780094 was found to be significantly associated with NAFLD. The MBOAT7 (rs641738) genetic variant was not found to be significantly associated with NAFLD.

Key genes involved in nonalcoholic steatohepatitis improvement after bariatric surgery.

Nonalcoholic steatohepatitis (NASH) is the advanced stage of nonalcoholic fatty liver disease (NAFLD), one of the most prevalent chronic liver diseases. The effectiveness of bariatric surgery in treating NASH and preventing or even reversing liver fibrosis has been demonstrated in numerous clinical studies, but the underlying mechanisms and crucial variables remain unknown. Using the GSE135251 dataset, we examined the gene expression levels of NASH and healthy livers. Then, the differentially expressed genes (DEGs) of patients with NASH, at baseline and one year after bariatric surgery, were identified in GSE83452. We overlapped the hub genes performed by protein-protein interaction (PPI) networks and DEGs with different expression trends in both datasets to obtain key genes. Genomic enrichment analysis (GSEA) and genomic variation analysis (GSVA) were performed to search for signaling pathways of key genes. Meanwhile, key molecules that regulate the key genes are found through the construction of the ceRNA network. NASH mice were induced by a high-fat diet (HFD) and underwent sleeve gastrectomy (SG). We then cross-linked the DEGs in clinical and animal samples using quantitative polymerase chain reaction (qPCR) and validated the key genes. Seven key genes (FASN, SCD, CD68, HMGCS1, SQLE, CXCL10, IGF1) with different expression trends in GSE135251 and GSE83452 were obtained with the top 30 hub genes selected by PPI. The expression of seven key genes in mice after SG was validated by qPCR. Combined with the qPCR results from NASH mice, the four genes FASN, SCD, HMGCS1, and CXCL10 are consistent with the biological analysis. The GSEA results showed that the 'cholesterol homeostasis' pathway was enriched in the FASN, SCD, HMGCS1, and SQLE high-expression groups. The high-expression groups of CD68 and CXCL10 were extremely enriched in inflammation-related pathways. The construction of the ceRNA network obtained microRNAs and ceRNAs that can regulate seven key genes expression. In summary, this study contributes to our understanding of the mechanisms by which bariatric surgery improves NASH, and to the development of potential biomarkers for the treatment of NASH.

The Effect of Curcuma and Garcinia Peel on Rat PPARα Gene Expression Induced by High-Fat Diet

High levels of fat in the blood are a risk factor for nonalcoholic steatohepatitis liver disease. Indonesian medicinal plants that can decrease blood fat levels are turmeric and mangosteen peel. One of the mechanisms of blood fat-lowering drugs is to increase the expression of the PPARα gene. The purpose of this study was to assess the effect of turmeric and mangosteen peel on the expression of the PPARα gene in the rat liver induced by a high-fat diet. This research was conducted at Maranatha Biomedical Research Laboratory in February–October 2021, using male Wistar rats that were divided into 5 groups (n=5): negative control groups (no treatment), positive control groups (high-fat diet), turmeric, mangosteen, and fenofibrate groups, that was given high-fat diet continued by ethanol extract of turmeric, ethanol extract of mangosteen peel, and fenofibrate. At the end of the study, the animals were terminated, and the liver was extracted for RNA extraction and semi-quantitative PCR. The results showed that there was an increase in PPARα gene expression in the turmeric group and fenofibrate group, which were significantly different from the positive control group that received a high-fat diet (p<0.05) and between the fenofibrate group compared to negative controls that received standard chow diet (p<0.05). In conclusion, turmeric and fenofibrate are suggested to increase the expression of the PPARα gene in the liver induced by a high-fat diet.

Seladelpar combined with complementary therapies improves fibrosis, inflammation, and liver injury in a mouse model of nonalcoholic steatohepatitis.

Seladelpar, a selective peroxisome proliferator-activated receptor δ (PPARδ) agonist, improves markers of hepatic injury in human liver diseases, but histological improvement of nonalcoholic steatohepatitis (NASH) and liver fibrosis has been challenging with any single agent. To discover how complementary agents could work with seladelpar to achieve optimal outcomes, this study evaluated a variety of therapeutics (alone and in combination) in a mouse model of NASH. Mice on a high-fat amylin liver NASH (AMLN) diet were treated for 12 wk with seladelpar, GLP-1-R (glucagon-like peptide-1 receptor) agonist liraglutide, apoptosis signal-regulating kinase 1 (ASK1) inhibitor selonsertib, farnesoid X receptor (FXR) agonist obeticholic acid, and with seladelpar in combination with liraglutide or selonsertib. Seladelpar treatment markedly improved plasma markers of liver function. Seladelpar alone or in combination resulted in stark reductions in liver fibrosis (hydroxyproline, new collagen synthesis rate, mRNA indices of fibrosis, and fibrosis staining) compared with vehicle and the other single agents. Robust reductions in liver steatosis were also observed. Seladelpar produced a reorganization of metabolic gene expression, particularly for those genes promoting peroxisomal and mitochondrial lipid oxidation. In summary, substantial improvements in NASH and NASH-induced fibrosis were observed with seladelpar alone and in combination with liraglutide in this model. Broad gene expression analysis suggests seladelpar should be effective in concert with diverse mechanisms of action.NEW & NOTEWORTHY NASH is a chronic, progressive, and increasingly problematic liver disease that has been resistant to treatment with individual therapeutics. In this study using a diet-induced mouse model of NASH, we found that the PPARδ agonist seladelpar reduced fibrosis and NASH pathology alone and in combinations with a GLP-1-R agonist (liraglutide) or an ASK1 inhibitor (selonsertib). Liver transcriptome analysis comparing each agent and coadministration suggests seladelpar should be effective in combination with a variety of therapeutics.

Multifaceted action of stem cell-derived extracellular vesicles for nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease associated with metabolic syndrome. Extracellular vesicles (EVs) are essential signaling mediators containing functional biomolecules. EVs are secreted from various cell types, and recent studies have shown that mesenchymal stem cell-derived EVs have therapeutic potential against immune and metabolic diseases. In this study, we investigated whether EVs from induced mesenchymal stem cells (iMSC-EVs) regulate AMPK signaling and lipid metabolism using cell-based studies and two different mouse models of NASH (methionine/choline-deficient diet-induced and ob/ob mice). Protein analysis revealed that iMSC-EVs carry cargo proteins with the potential to regulate lipid metabolism. iMSC-EVs inhibited free fatty acid release from adipose tissues by downregulating the activity of lipolytic genes in NASH. In addition, iMSC-EVs improved hepatic steatosis by modulating AMPK signaling, which plays essential role in metabolic homeostasis in the liver. Moreover, iMSC-EVs reduced CD36 expression, contributing to the blockade of free fatty acid transport to the liver of NASH mice. Finally, iMSC-EVs reduced inflammation, endoplasmic reticulum stress, and apoptosis while promoting hepatic regeneration of the NASH liver. In conclusion, iMSC-EVs can potentially serve as cell-free therapeutics for NASH owing to their multifaceted modality.

Evaluation of the efficacy of mitochondrial fission inhibitor (Mdivi-1) using non-alcoholic steatohepatitis (NASH) liver organoids.

Non-alcoholic steatohepatitis (NASH) is known to progress to cirrhosis and hepatocellular carcinoma in some patients. Although NASH is associated with abnormal mitochondrial function related to lipid metabolism, mechanisms for the development and effective treatments are still unclear. Therefore, new approaches to elucidate the pathophysiology are needed. In the previous study, we generated liver organoids from different stages of NASH model mice that could recapitulate the part of NASH pathology. In the present study, we investigated the relationship between mitochondrial function and NASH disease by comparing NASH liver organoids (NLO) and control liver organoids (CLO). Compared with CLO, mitochondrial and organoid morphology was abnormal in NLO, with increased expression of mitochondrial mitogen protein, DRP1, and mitochondria-derived reactive oxygen species (ROS) production. Treatment of NLO with a DPR1 inhibitor, Mdivi-1 resulted in the improvement of morphology and the decreased expression of fibrosis-related markers, Col1a1 and Acta2. In addition, treatment of NASH model mice with Mdivi-1 showed a decrease in fatty liver. Mdivi-1 treatment also prevented fibrosis and ROS production in the liver. These results indicate that NLO undergoes enhanced metabolism and abnormal mitochondrial morphology compared with CLO. It was also suggested that Mdivi-1 may be useful as a therapeutic agent to ameliorate NASH pathology.

Modified multi-Rayleigh model-based statistical analysis of ultrasound envelope for quantification of liver steatosis and fibrosis

PurposeQuantitative diagnosis of the degree of fibrosis progression is currently a focus of attention for fatty liver in nonalcoholic steatohepatitis (NASH). However, previous studies have focused on either lipid droplets or fibrotic tissue, and few have reported the evaluation of both in patients whose livers contain adipose and fibrous features. Our aim was to evaluate fibrosis tissue and lipid droplets in the liver.MethodsWe used an analytical method combining the multi-Rayleigh (MRA) model and a healthy liver structure filter (HLSF) as a technique for statistical analysis of the amplitude envelope to estimate fat and fibrotic volumes in clinical datasets with different degrees of fat and fibrosis progression.ResultsFat mass was estimated based on the non-MRA fraction corresponding to the signal characteristics of aggregated lipid droplets. Non-MRA fraction has a positive correlation with fat mass and is effective for detecting moderate and severe fatty livers. Progression of fibrosis was estimated using MRA parameters in combination with the HLSF. The proposed method was used to extract non-healthy areas with characteristics of fibrotic tissue. Fibrosis in early fatty liver suggested the possibility of evaluation. On the other hand, fat was identified as a factor that reduced the accuracy of estimating fibrosis progression in moderate and severe fatty livers.ConclusionThe proposed method was used to simultaneously evaluate fat mass and fibrosis progression in early fatty liver, suggesting the possibility of quantitative evaluation for discriminating between lipid droplets and fibrous tissue in the early fatty liver.

Human liver stem cell-derived extracellular vesicles modulate long non-coding RNA expression profile in an in vivo model of non-alcoholic steatohepatitis

Aim: Modifications in long non-coding RNA (lncRNA) expression are associated with inflammation and fibrosis in chronic liver diseases. It has been recently demonstrated that human liver stem cells (HLSCs) and their extracellular vesicles (EVs) can effectively reduce inflammation and fibrosis in a murine model of non-alcoholic steatohepatitis (NASH). Now it has been evaluated whether EVs can modify the expression of inflammation-related lncRNAs in NASH liver. Methods: To induce NASH, severe combined immunodeficient mice were fed with a methionine-choline-deficient diet for 4 weeks. After 2 weeks of diet, 2.5 × 109 EVs were intravenously injected twice a week. An array of 84 inflammation-related lncRNAs was performed on the RNA isolated from NASH livers, and the expression of 14 selected lncRNAs was then validated by real-time polymerase chain reaction (PCR) analysis. Expression levels of maternally expressed gene 3 (Meg3) were further evaluated in vitro, in an activated human hepatic immortalized stellate cell line (LX-2) stimulated with EVs. Results: The screening showed an altered lncRNA expression profile in the liver of NASH mice, in respect to control healthy mice. EV treatment modulated several inflammation-related lncRNAs in NASH livers. Real-time PCR validation of array results indicated that EVs restored to normal levels the expression of 10 lncRNAs altered in NASH. In particular, EV stimulation reduced Meg3 expression levels, which were increased in NASH as well as in activated LX-2. Conclusions: HLSC-EVs regulate the expression of inflammation-related lncRNAs impaired in NASH livers and in an in vitro model of liver fibrosis.