Nonalcoholic Steatohepatitis In Mice Research Articles

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.

Integrative analysis identifies oxidative stress biomarkers in non-alcoholic fatty liver disease via machine learning and weighted gene co-expression network analysis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease globally, with the potential to progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma. Given the absence of effective treatments to halt its progression, novel molecular approaches to the NAFLD diagnosis and treatment are of paramount importance. Firstly, we downloaded oxidative stress-related genes from the GeneCards database and retrieved NAFLD-related datasets from the GEO database. Using the Limma R package and WGCNA, we identified differentially expressed genes closely associated with NAFLD. In our study, we identified 31 intersection genes by analyzing the intersection among oxidative stress-related genes, NAFLD-related genes, and genes closely associated with NAFLD as identified through Weighted Gene Co-expression Network Analysis (WGCNA). In a study of 31 intersection genes between NAFLD and Oxidative Stress (OS), we identified three hub genes using three machine learning algorithms: Least Absolute Shrinkage and Selection Operator (LASSO) regression, Support Vector Machine - Recursive Feature Elimination (SVM-RFE), and RandomForest. Subsequently, a nomogram was utilized to predict the incidence of NAFLD. The CIBERSORT algorithm was employed for immune infiltration analysis, single sample Gene Set Enrichment Analysis (ssGSEA) for functional enrichment analysis, and Protein-Protein Interaction (PPI) networks to explore the relationships between the three hub genes and other intersecting genes of NAFLD and OS. The distribution of these three hub genes across six cell clusters was determined using single-cell RNA sequencing. Finally, utilizing relevant data from the Attie Lab Diabetes Database, and liver tissues from NASH mouse model, Western Blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) assays were conducted, this further validated the significant roles of CDKN1B and TFAM in NAFLD. In the course of this research, we identified 31 genes with a strong association with oxidative stress in NAFLD. Subsequent machine learning analysis and external validation pinpointed two genes: CDKN1B and TFAM, as demonstrating the closest correlation to oxidative stress in NAFLD. This investigation found two hub genes that hold potential as novel targets for the diagnosis and treatment of NAFLD, thereby offering innovative perspectives for its clinical management.

A combined nanotherapeutic approach targeting farnesoid X receptor, ferroptosis, and fibrosis for nonalcoholic steatohepatitis treatment

Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist with favorable effects on fatty and glucose metabolism, has been considered the leading candidate drug for nonalcoholic steatohepatitis (NASH) treatment. However, its limited effectiveness in resolving liver fibrosis and lipotoxicity-induced cell death remains a major drawback. Ferroptosis, a newly recognized form of cell death characterized by uncontrolled lipid peroxidation, is involved in the progression of NASH. Nitric oxide (NO) is a versatile biological molecule that can degrade extracellular matrix. In this study, we developed a PEGylated thiolated hollow mesoporous silica nanoparticles (MSN) loaded with OCA, as well as a ferroptosis inhibitor liproxsatin-1 and a NO donor S-nitrosothiol (ONL@MSN). Biochemical analyses, histology, multiplexed flow cytometry, bulk-tissue RNA sequencing, and fecal 16S ribosomal RNA sequencing were utilized to evaluate the effects of the combined nanoparticle (ONL@MSN) in a mouse NASH model. Compared with the OCA-loaded nanoparticles (O@MSN), ONL@MSN not only protected against hepatic steatosis but also greatly ameliorated fibrosis and ferroptosis. ONL@MSN also displayed enhanced therapeutic actions on the maintenance of intrahepatic macrophages/monocytes homeostasis, inhibition of immune response/lipid peroxidation, and correction of microbiota dysbiosis. These findings present a promising synergistic nanotherapeutic strategy for the treatment of NASH by simultaneously targeting FXR, ferroptosis, and fibrosis.

Tyrosol attenuates NASH features by reprogramming the hepatic immune milieu

Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease, and no drugs have been approved for its therapy. Among plant-derived molecules, phenolic compounds of extra virgin olive oil like tyrosol (Tyr) had demonstrated multiple beneficial actions for liver health, including the modulation of inflammation in fibrosis. This study aims at assessing the protective effect and mechanism of Tyr in invitro and in vivo models of NASH, with a focus on the hepatic immune microenvironment and extrahepatic manifestations. The effect of Tyr was evaluated in cellular models of NASH, obtained by co-culturing palmitic and oleic acid-treated HepG2 cells with THP1-derived M1 macrophages and LX2 cells, and in a mouse model of NASH induced by a high fructose-high fat diet combined to CCl4 treatment. In vitro Tyr reduced fatty acid (FA) accumulation in HepG2 cells and displayed a beneficial effect on LX2 activation and macrophage differentiation. In vivo, beside reducing steatosis and fibrosis in NASH animals, Tyr prevented inflammation, as demonstrated by the reduction of hepatic inflammatory foci, and immune cells like CD86+ macrophages (p < 0.05), CD4+ (p < 0.05) and T helper effector CD4+ FoxP3- CD62L-lymphocytes (p < 0.05). Also, the prooxidant enzyme NOX1 and the mRNA expression of TGF-β1 and IL6 (p < 0.05) were reduced by Tyr. Notably, in Tyr-treated animals, a significant increase of CD4+ FoxP3+ Treg cells (p < 0.05) was observed, involved in regenerative pathways. Moreover, Tyr attenuated the fatigue and anxious behavior observed in NASH mice. In conclusion, Tyr effectively reduced NASH-related steatosis, fibrosis, oxidative stress, and inflammation, displaying a beneficial effect on the hepatic immune infiltrate, indicating its possible development as a therapeutic agent for NASH due to its multifaceted mechanism.

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

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

Multispecies transcriptomics identifies SIKE as a MAPK repressor that prevents NASH progression.

Nonalcoholic fatty liver (NAFL) remains relatively benign, but high-risk to end-stage liver diseases become highly prevalent when it progresses into nonalcoholic steatohepatitis (NASH). Our current understanding of the development of NAFL to NASH remains insufficient. In this study, we revealed MAP kinase (MAPK) activation as the most notable molecular signature associated with NASH progression across multiple species. Furthermore, we identified suppressor of IKKε (SIKE) as a conserved and potent negative controller of MAPK activation. Hepatocyte-specific overexpression of Sike prevented NASH progression in diet- and toxin-induced mouse NASH models. Mechanistically, SIKE directly interacted with TGF-β-activated kinase 1 (TAK1) and TAK1-binding protein 2 (TAB2) to interrupt their binding and subsequent TAK1-MAPK signaling activation. We found that indobufen markedly up-regulated SIKE expression and effectively improved NASH features in mice and macaques. These findings identify SIKE as a MAPK suppressor that prevents NASH progression and provide proof-of-concept evidence for targeting the SIKE-TAK1 axis as a potential NASH therapy.

Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis in mice

Sonic hedgehog (SHH) and heat shock protein 90β (HSP90β) have been implicated in nonalcoholic steatohepatitis (NASH) but their molecular mechanisms of action remain elusive. We find that HSP90β is a key SHH downstream molecule for promoting NASH process. In hepatocytes, SHH reduces HSP90β ubiquitylation through deubiquitylase USP31, thus preventing HSP90β degradation and promoting hepatic lipid synthesis. HSP90β significantly increases in NASH mouse model, leading to secretion of exosomes enriched with miR-28-5p. miR-28-5p directly targetes and decreases Rap1b levels, which in turn promotes NF-κB transcriptional activity in macrophages and stimulates the expression of inflammatory factors. Genetic deletion, pharmacological inhibition of the SHH-HSP90β axis, or delivery of miR-28-5p to macrophages in the male mice liver, impairs NASH symptomatic development. Importantly, there is a markedly higher abundance of miR-28-5p in NASH patient sera. Taken together, the SHH-HSP90β-miR-28-5p axis offers promising therapeutic targets against NASH, and serum miR-28-5p may serve as a NASH diagnostic biomarker.

Dietary intervention reverses molecular markers of hepatocellular senescence in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing. Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence. Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH.

Gypenoside XIII regulates lipid metabolism in HepG2 hepatocytes and ameliorates nonalcoholic steatohepatitis in mice.

Gypenoside XIII is isolated from Gynostemma pentaphyllum (Thunb.) Makino. In mice, G. pentaphyllum extract and gypenoside LXXV have been shown to improve non-alcoholic steatohepatitis (NASH). This study investigated whether gypenoside XIII can regulate lipid accumulation in fatty liver cells or attenuate NASH in mice. We used HepG2 hepatocytes to establish a fatty liver cell model using 0.5 mM oleic acid. Fatty liver cells were treated with different concentrations of gypenoside XIII to evaluate the molecular mechanisms of lipid metabolism. In addition, a methionine/choline-deficient diet induced NASH in C57BL/6 mice, which were given 10 mg/kg gypenoside XIII by intraperitoneal injection. In fatty liver cells, gypenoside XIII effectively suppressed lipid accumulation and lipid peroxidation. Furthermore, gypenoside XIII significantly increased SIRT1 and AMPK phosphorylation to decrease acetyl-CoA carboxylase phosphorylation, reducing fatty acid synthesis activity. Gypenoside XIII also decreased lipogenesis by suppressing sterol regulatory element-binding protein 1c and fatty acid synthase production. Gypenoside XIII also increased lipolysis and fatty acid β-oxidation by promoting adipose triglyceride lipase and carnitine palmitoyltransferase 1, respectively. In an animal model of NASH, gypenoside XIII effectively decreased the lipid vacuole size and number and reduced liver fibrosis and inflammation. These findings suggest that gypenoside XIII can regulate lipid metabolism in fatty liver cells and improve liver fibrosis in NASH mice. Therefore, gypenoside XIII has potential as a novel agent for the treatment of NASH.

Inhibition of visceral adipose tissue-derived pathogenic signals by activation of adenosine A2AR improves hepatic and cardiac dysfunction of NASH mice.

A2AR-disrupted mice is characterized by severe systemic and visceral adipose tissue (VAT) inflammation. Increasing adenosine cyclase (AC), cAMP, and protein kinase A (PKA) formation through A2AR activation suppress systemic/VAT inflammation in obese mice. This study explores the effects of 4 wk A2AR agonist PSB0777 treatment on the VAT-driven pathogenic signals in hepatic and cardiac dysfunction of nonalcoholic steatohepatitis (NASH) obese mice. Among NASH mice with cardiac dysfunction, simultaneous decrease in the A2AR, AC, cAMP, and PKA levels were observed in VAT, liver, and heart. PSB0777 treatment significantly restores AC, cAMP, PKA, and hormone-sensitive lipase (HSL) levels, decreased SREBP-1/FASN, MCP-1, and CD68 levels, reduces infiltrated CD11b+ F4/80+ cells and adipogenesis in VAT of NASH + PSB0777 mice. The changes in VAT were accompanied by the suppression of hepatic and cardiac lipogenic/inflammatory/injury/apoptotic/fibrotic markers, the normalization of cardiac contractile [sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2)] marker, and cardiac dysfunction. The in vitro approach revealed that conditioned media (CM) of VAT of NASH mice (CMnash) trigger palmitic acid (PA)-like lipotoxic (lipogenic/inflammatory/apoptotic/fibrotic) effects in AML-12 and H9c2 cell systems. Significantly, A2AR agonist pretreatment-related normalization of A2AR-AC-cAMP-PKA levels was associated with the attenuation of CMnash-related upregulation of lipotoxic markers and the normalization of lipolytic (AML-12 cells) or contractile (H9C2 cells) marker/contraction. The in vivo and in vitro experiments revealed that A2AR agonists are potential agent to inhibit the effects of VAT inflammation-driven pathogenic signals on the hepatic and cardiac lipogenesis, inflammation, injury, apoptosis, fibrosis, hypocontractility, and subsequently improve hepatic and cardiac dysfunction in NASH mice.NEW & NOTEWORTHY Protective role of adenosine A2AR receptor (A2AR) and AC-cAMP-PKA signaling against nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) possibly via its actions on adipocytes is well known in the past decade. Thus, this study evaluates pharmacological activities of A2AR agonist PSB0777, which has already demonstrated to treat NASH. In this study, the inhibition of visceral adipose tissue-derived pathogenic signals by activation of adenosine A2AR with A2AR agonist PSB0777 improves the hepatic and cardiac dysfunction of high-fat diet (HFD)-induced NASH mice.

Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Gut Microbiota Plays Essential Roles in Soyasaponin's Preventive Bioactivities against Steatohepatitis in the Methionine and Choline Deficient (MCD) Diet-Induced Non-Alcoholic Steatohepatitis (NASH) Mice.

Gut microbiota (GM) is involved in nonalcoholic steatohepatitis (NASH) development. Phytochemicals soyasaponins can prevent NASH possibly by modulating GM. This study aims to investigate the preventive bioactivities of soyasaponin monomers (SS-A1 and SS-Bb) against NASH and explores the mechanisms by targeting GM. Male C57BL/6 mice are fed with methionine and choline deficient (MCD) diet containing SS-A1 , SS-Bb, or not for 16 weeks. Antibiotics-treated pseudo germ-free (PGF) mice are fed with MCD diet containing SS-A1 , SS-Bb, or not for 8 weeks. GM is determined by 16S rRNA amplicon sequencing. Bile acids (BAs) are measured by UPLC-MS/MS. In NASH mice, SS-A1 and SS-Bb alleviate steatohepatitis and fibrosis, reduce ALT, AST, and LPS in serum, decrease TNF-α, IL-6, α-SMA, triglycerides, and cholesterol in liver. SS-A1 and SS-Bb decrease Firmicutes, Erysipelotrichaceae, unidentified-Clostridiales, Eggerthellaceae, Atopobiaceae, Aerococcus, Jeotgalicoccus, Gemella, Rikenella, increase Proteobacteria, Verrucomicrobia, Akkermansiaceae, Romboutsia, and Roseburia. SS-A1 and SS-Bb alter BAs composition in liver, serum, and feces, activate farnesoid X receptor (FXR) in liver and ileum, increase occludin and ZO-1 in intestine. However, GM clearance abrogates the preventive bioactivities of SS-A1 and SS-Bb against NASH. GM plays essential roles in soyasaponin's preventive bioactivities against steatohepatitis in MCD diet-induced NASH mice.

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.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1g/L fructose and 18.9g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2μL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

The AMPK-mTOR Pathway Is Inhibited by Chaihu Shugan Powder, Which Relieves Nonalcoholic Steatohepatitis by Suppressing Autophagic Ferroptosis.

Nonalcoholic steatohepatitis (NASH) is the advanced stage of nonalcoholic fatty liver disease (NAFLD), which is distinguished by the accumulation of fat in the liver, damage to liver cells, and inflammation. Chaihu Shugan powder (CSP), a renowned traditional Chinese medicine (TCM) blend extensively utilized in China to address liver disease, has demonstrated its efficacy in reducing lipid buildup and effectively combating inflammation. Hence, the primary objective of this research is to examine the impacts and possible mechanisms of CSP on NASH through assessments of liver histopathology, lipidomic analysis, and gene expression. To induce a mouse model of NASH, we employed a diet which deficient in methionine and choline, known as methionine-choline deficient (MCD) diet. Initially, we examined the impact of administering CSP to NASH mice by assessing the levels of serum and liver indicators. We found that CSP was able to reduce lipid buildup and inflammation in mice. In addition, a total of 1009 genes exhibited enrichment in both the autophagy and ferroptosis pathways. The liver protein levels of Adenosine monophosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR)-mediated autophagy and ferroptosis markers, such as p-AMPKα/AMPKα, p-mTOR/mTOR, Beclin-1, microtubule associated protein 1 light chain 3 gamma (LC3), p62 (sequestosome 1 [SQSTM1/p62]), Kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (Nrf-2), ferritin heavy chain 1 (FTH1), and glutathione peroxidase 4 (GPX4), were restored by CSP. Furthermore, our findings indicated that the suppression of autophagy had a repressive impact on the occurrence of ferroptosis in the mouse model, indicating that autophagy activation likely plays a role in mediating ferroptosis in NASH.

A study of the anti-fibrotic effect of ghrelin on mice with nonalcoholic steatohepatitis

Objective: To study the anti-fibrotic effect of ghrelin on high-fat diet-induced non-alcoholic steatohepatitis (NASH) in mice. Methods: 24 male C57BL/6 mice were randomly divided into a normal diet group, a normal diet + ghrelin group, a high-fat diet group, and the high-fat diet + ghrelin group. The HFD and HFD+ghrelin groups were fed high-fat diet for 16 weeks to induce non-alcoholic steatohepatitis. Among them, the NCD+ghrelin group and HFD+ghrelin group were continuously given ghrelin intervention (11nmol·kg(-1)·d(-1)) for 2 weeks after feeding for 14 weeks. 16 mice were euthanized on weekends. The plasma levels of alanine aminotransferase (ALT) and hyaluronic acid (HA) were measured in mice. The content of hydroxyproline (Hyp) was determined in liver tissue. RT-qPCR was used to detect the mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, and IV in liver tissue. A Western blot was used to detect the expression level of the α-smooth muscle actin (α-SMA) protein in liver tissue. HE staining was used to observe the morphological changes in liver tissue. VG staining was used to observe the fibrotic condition in liver tissue. Results: Compared with the NCD group, plasma ALT (266.80±146.80)U/L, HA (219.00±39.47) ng/ml levels, Hyp content (0.35±0.05)μg/mg prot (P < 0.05), mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD group were significantly increased (P < 0.05), with congestion in the hepatic central lobular veins, hepatocytes swelling, and deposition of a large amount of collagen fibers in liver tissue. Compared with the HFD group, plasma ALT (57.17±20.88)U/L, HA (75.68±8.40)μg/mg levels, Hyp content (0.19±0.07)μg/mg prot, mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD+ghrelin mice group was significantly reduced (P < 0.05), with only mild sinusoidal congestion in the liver tissue but significant improvement and reduction in liver injury and collagen fiber deposition. Conclusion: Ghrelin has a significant improvement effect on liver fibrosis in NASH mice.

Ginsenoside Rg5 inhibits lipid accumulation and hepatocyte apoptosis via the Notch1 signaling pathway in NASH mice

BackgroundNon-alcoholic steatohepatitis (NASH) is a prevalent chronic liver disease that lacks an FDA-approved treatment medicine. Despite the known antitumor and hypoglycemic properties of Ginsenoside Rg5, its effects and underlying mechanisms in the context of NASH remain largely unexplored. PurposeThis study aims to investigate the effect of Rg5 on NASH mice induced by a high-fat diet and CCl4. Study designIn vivo experiments, a mouse NASH model was established by a HFHC diet plus intraperitoneal injection of low-dose CCl4. In vitro experiments, a cellular steatosis model was established using free fatty acids (FFA) induced HepG2 cells. In addition, a fibrogenesis model was established using HSC-LX2 cells. MethodsThe effects of Ginsenoside Rg5 on lipid accumulation and oxidative damage were analyzed by ELISA kit, H&E staining, Oil Red O staining, flow cytometry and Western blot. The effects of Ginsenoside Rg5 on liver fibrosis were analyzed by Masson staining, Sirus Red staining, immunohistochemistry and Western blot. The effect of Ginsenoside Rg5 on Notch1 signaling pathway in liver was studied by protein Oil Red staining, protein immunoblotting and immunofluorescence. ResultsIn terms of lipid accumulation, Rg5 has the ability to regulate key proteins related to lipogenesis, thereby inhibiting hepatic lipid accumulation and oxidative stress. Additionally, Rg5 can reduce the occurrence of hepatocyte apoptosis by regulating the p53 protein. Moreover, after Rg5 intervention, the presence of fibrotic proteins (α-SMA, Collagen 1, TGF-β) in the liver is significantly suppressed, thus inhibiting liver fibrosis. Lastly, Rg5 leads to a decrease in the expression levels of Notch1 and its ligand Jagged-1 in the liver. ConclusionIn summary, the regulatory effects of Rg5 on the Notch1 signaling pathway play a crucial role in modulating hepatic lipid metabolism and preventing hepatocyte apoptosis, thereby impeding the progression of NASH. These findings highlight the potential of Rg5 as a promising natural product for interventions targeting NASH.

Rotundic acid improves nonalcoholic steatohepatitis in mice by regulating glycolysis and the TLR4/AP1 signaling pathway

BackgroundSteatosis and inflammation are the hallmarks of nonalcoholic steatohepatitis (NASH). Rotundic acid (RA) is among the key triterpenes of Ilicis Rotundae Cortex and has exhibited multipronged effects in terms of lowering the lipid content and alleviating inflammation. The study objective is to systematically evaluate the potential mechanisms through which RA affects the development and progression of NASH.MethodsTranscriptomic and proteomic analyses of primary hepatocytes isolated from the control, high-fat diet-induced NASH, and RA treatment groups were performed through Gene Ontology analysis and pathway enrichment. Hub genes were identified through network analysis. Integrative analysis revealed key RA-regulated pathways, which were verified by gene and protein expression studies and cell assays.ResultsHub genes were identified and enriched in the Toll-like receptor 4 (TLR4)/activator protein-1 (AP1) signaling pathway and glycolysis pathway. RA reversed glycolysis and attenuated the TLR4/AP1 pathway, thereby reducing lipid accumulation and inflammation. Additionally, lactate release in L-02 cells increased with NaAsO2-treated and significantly decreased with RA treatment, thus revealing that RA had a major impact on glycolysis.ConclusionsRA is effective in lowering the lipid content and reducing inflammation in mice with NASH by ameliorating glycolysis and TLR4/AP1 pathways, which contributes to the existing knowledge and potentially sheds light on the development of therapeutic interventions for patients with NASH.

Pharmacological and genetic increases in liver NADPH levels ameliorate NASH progression in female mice

Non-alcoholic steatohepatitis (NASH) is one of the fastest growing liver diseases worldwide, and oxidative stress is one of NASH main key drivers. Nicotinamide adenine dinucleotide phosphate (NADPH) is the ultimate donor of reductive power to a number of antioxidant defences. Here, we explored the potential of increasing NADPH levels to prevent NASH progression. We used nicotinamide riboside (NR) supplementation or a G6PD-tg mouse line harbouring an additional copy of the human G6PD gene. In a NASH mouse model induced by feeding mice a methionine-choline deficient (MCD) diet for three weeks, both tools increased the hepatic levels of NADPH and ameliorated the NASH phenotype induced by the MCD intervention, but only in female mice. Boosting NADPH levels in females increased the liver expression of the antioxidant genes Gsta3, Sod1 and Txnrd1 in NR-treated mice, or of Gsr for G6PD-tg mice. Both strategies significantly reduced hepatic lipid peroxidation. NR-treated female mice showed a reduction of steatosis accompanied by a drop of the hepatic triglyceride levels, that was not observed in G6PD-tg mice. NR-treated mice tended to reduce their lobular inflammation, showed a reduction of the NK cell population and diminished transcription of the damage marker Lcn2. G6PD-tg female mice exhibited a reduction of their lobular inflammation and hepatocyte ballooning induced by the MCD diet, that was related to a reduction of the monocyte-derived macrophage population and the Tnfa, Ccl2 and Lcn2 gene expression. As conclusion, boosting hepatic NADPH levels attenuated the oxidative lipid damage and the exhausted antioxidant gene expression specifically in female mice in two different models of NASH, preventing the progression of the inflammatory process and hepatic injury.

Discovery of 4-aminophenylacetamide derivatives as intestine-specific farnesoid X receptor antagonists for the potential treatment of nonalcoholic steatohepatitis

Farnesoid X receptor (FXR) plays a key role in bile acid homeostasis, inflammation, fibrosis, lipid and glucose metabolism and is emerging as a promising therapeutic target for nonalcoholic steatohepatitis (NASH). Emerging evidence suggested that intestine-specific FXR antagonists exhibited remarkable metabolic improvements and slowed NASH progression. In this study, we discovered several potent FXR antagonists using a multistage ligand- and structure-based virtual screening approach. Notably, compound V023-9340, which possesses a 4-aminophenylacetamide scaffold, emerged as the most potent FXR antagonist with an IC50 value of 4.27 μM. In vivo, V023-9340 demonstrated selective accumulation in the intestine, substantially ameliorating high-fat diet (HFD)-induced NASH in mice by mitigating hepatic steatosis and inflammation. Mechanistic studies revealed that V023-9340 strongly inhibited intestinal FXR while concurrently feedback-activated hepatic FXR. Further structure-activity relationship optimization employing V023-9340 has resulted in the synthesis of a more efficacious compound V02-8 with an IC50 value of 0.89 μM, which exhibited a 4.8-fold increase in FXR antagonistic activity compared to V023-9340. In summary, 4-aminophenylacetamide derivative V023-9340 represented a novel intestine-specific FXR antagonist and showed improved effects against HFD-induced NASH in mice, which may serve as a promising lead in discovering potential therapeutic drugs for NASH treatment.