Nonalcoholic Steatohepatitis In Mice Research Articles

PAMK Ameliorates Non-Alcoholic Steatohepatitis and Associated Anxiety/Depression-like Behaviors Through Restoring Gut Microbiota and Metabolites in Mice

Objectives: Long-term Western diet-induced non-alcoholic steatohepatitis (NASH) can lead to liver cirrhosis and NASH-associated hepatocellular carcinoma, which are end-stage liver diseases. Meanwhile, NASH is associated with mental burden and worsens as the disease progresses. Atractylodes Macrocephala Koidz (AMK) is one of the main ingredients of Shenling Baizhu San, and the effect of Polysaccharide from AMK ameliorates (PAMK), as an important medicinal ingredient of AMK, on NASH and associated anxiety/depression-like behaviors is still unclear. Methods: This study investigated the protective effect of PAMK on NASH and associated anxiety/depression-like behaviors through a Western diet-induced NASH mice model. Results: showed that PAMK decreased the concentrations of liver TC, TG, and serum AST and ALT, improving glucose tolerance, and reducing liver steatosis and fibrosis. Moreover, the expression of liver IL-6, IL-1β, TNF-α, IL-18 and MCP-1 could be reduced by PAMK significantly. Additionally, PAMK decreased anxiety/depression-like behaviors and expression of IL-6, IL-1β, TNF-α, and MCP-1 in the hippocampus. 16S rRNA gene sequencing revealed that PAMK diminished the Firmicutes/Bacteroidetes ratio and abundance of Faecalibaculum_rodentium, and increased the abundance of Muribaculaceae. This might be related to gene abundance of Pentose, the glucuronate interconversions pathway and carbohydrate enzymes (GH1, GH4). Serum metabolomics suggested that PC (18:5e/2:0), PC (16:2e/2:0), Lysopc 20:4, PC (16:0/2:0), and LPC 19:0 upregulated significantly after PAMK intervention, together with the enrichment of carbon metabolism and Citrate cycle pathways specially. Conclusions: PAMK as a potential prebiotic ameliorated NASH and associated anxiety/depression-like behaviors in mice, probably by regulating Faecalibaculum_rodentium, carbohydrate enzymes and lipid metabolites.

Exosomes derived from apical papilla stem cells improve NASH by regulating fatty acid metabolism and reducing inflammation

BackgroundApical papilla stem cells (SCAPs) exhibit significant potential for tissue repair, characterized by their anti-inflammatory and pro-angiogenic properties. Exosomes derived from stem cells have emerged as safer alternatives that retain comparable physiological functions. This study explores the therapeutic potential of exosomes sourced from SCAPs in the treatment of non-alcoholic steatohepatitis (NASH).MethodsA NASH mouse model was established through the administration of a high-fat diet (HFD), and SCAPs were subsequently isolated for experimental purposes. A cell model of NASH was established in vitro by treating hepatocellular carcinoma cells with oleic acid (OA) and palmitic acid (PA). Exosomes were isolated via differential centrifugation. The mice were treated with exosomes injected into the tail vein, and the hepatocytes were incubated with exosomes in vitro. After the experiment, physiological and biochemical markers were analyzed to assess the effects of exosomes derived from SCAPs on the progression of NASH in both NASH mouse models and NASH cell models.ResultsAfter exosomes treatment, the weight gain and liver damage induced by HFD were significantly reduced. Additionally, hepatic fat accumulation was markedly alleviated. Mechanistically, exosomes treatment promoted the expression of genes involved in hepatic fatty acid oxidation and transport, while simultaneously suppressing genes associated with fatty acid synthesis. Furthermore, the levels of serum inflammatory cytokines and the mRNA expression of inflammatory markers in liver tissue were significantly decreased. In vitro cell experiments produced similar results.

Discovery of ZG-2305, an Orally Bioavailable Factor Inhibiting HIF Inhibitor for the Treatment of Obesity and Fatty Liver Disease.

Genetic loss of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH) enhances both glycolysis and aerobic metabolism. FIH is thus a potential target for adiposity control and improving hepatic steatosis. We describe development of a series of novel, potent, and selective FIH inhibitors that occupy both the FIH catalytic site and a recently defined tyrosine conformational-flip pocket. ZG-2305, with a Ki of 79.6 nM for FIH, manifests 38-fold selectivity over the hypoxia-inducible factor (HIF) prolyl hydroxylase PHD2. Oral administration of ZG-2305 in the western-diet induced obesity mouse model results in improved lipid accumulation and recovery from abnormal body weight/hepatic steatosis. Amelioration of nonalcoholic steatohepatitis (NASH) related pathological phenotypes in the HF-CDAA-diet induced NASH mouse model was observed. Preliminary preclinical studies indicate ZG-2305 has good pharmacokinetic properties and an acceptable safety profile. The results imply ZG-2305 is a promising candidate for treatment of obesity and fatty liver disease.

Atractylodes lancea Rhizome Polysaccharide Alleviates MCD Diet-Induced NASH by Inhibiting the p53/mTOR Pathway.

Nonalcoholic steatohepatitis (NASH) is a form of chronic liver disease that is characterized by liver inflammation and steatosis, with possible progression to fibrosis. Currently, no drugs have been approved for the treatment of NASH. In this study, we isolated a polysaccharide from Atractylodes lancea rhizome (AP) and established a methionine- and choline-deficient (MCD) diet -induced NASH mouse model to investigate the preventive effect and potential mechanism of AP on NASH. The results showed that AP effectively reduced liver lipid accumulation and inflammation and reduced autophagy and ferroptosis in hepatocytes, thereby preventing the development of NASH. These findings suggest that AP may be a promising natural candidate for the treatment of NASH.

The Nr4a family regulates intrahepatic Treg proliferation and liver fibrosis in MASLD models.

Nonalcoholic steatohepatitis (NASH) is a chronic progressive liver disease and highly prevalent worldwide. NASH is characterized by hepatic steatosis, inflammation, fibrosis and liver damage, which eventually results in liver dysfunction due to cirrhosis or hepatocellular carcinoma. However, the cellular and molecular mechanisms underlying NASH progression remain largely unknown. Here, we found an increase of Nr4a family of orphan nuclear receptors expression in intrahepatic T cells from mice with diet-induced NASH. Loss of Nr4a1 and Nr4a2 in T cell (dKO) ameliorated liver cell death and fibrosis, thereby mitigating liver dysfunction in NASH mice. dKO resulted in reduction of infiltrated macrophages and Th1/Th17 cells, whereas massive accumulation of T regulatory (Treg) cells in the liver of NASH mice. Combined single-cell RNA transcriptomic and TCR sequencing analysis revealed that intrahepatic dKO Tregs exhibited enhanced TIGIT and IL10 expression and were clonally expanded during NASH progression. Mechanistically, we found that dKO Tregs expressed high levels of Batf which promotes Treg cell proliferation and function upon TCR stimulation. Collectively, our findings not only provide an insight into the impact of intrahepatic Treg cells on NASH pathogenesis, but also suggest a therapeutic potential of targeting of Nr4a family to treat the disease.

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

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

Chronic Peroxisome Proliferator-Activated Receptor α/γ and Cannabinoid Receptor 2 Agonist Treatments Attenuated Visceral Adipose Tissue (VAT)–Derived Extracellular Vesicle–Related VAT and Intestinal Abnormalities in Nonalcoholic Steatohepatitis Mice

This study explores the mechanisms and combined effects of chronic peroxisome proliferator-activated receptor (PPAR)α/γ and cannabinoid receptor 2 (CB2R) agonists on visceral adipose tissue (VAT)-derived extracellular vesicle (EVs) release and associated systemic/VAT inflammation, decreased VAT capillary density/fibrosis, and intestinal inflammation/hyperpermeability in nonalcoholic steatohepatitis (NASH) mice. NASH mice receiving 1 month of PPARα/γ agonist aleglitazar (10 mg/kg/day), CB2R agonist JWH015 (3 mg/kg/day) alone or combined. High EV release from VAT of NASH mice was associated with severe systemic/VAT/intestinal inflammation, reduced capillary network of VAT, and intestinal hyperpermeability. Combined JWH015 with aleglitazar treatment significantly suppressed HFD-induced obesity/adiposity, inhibited VAT expansion, reduced VAT inflammation/fibrosis, normalized VAT capillary network, and attenuated intestinal mucosal injury, inflammation, and hyperpermeability in NASH+aleg+JWH015 mice. The inhibition of AT-derived EV release and hypoxia inducible factor (HIF)1α levels in AT-derived EV, normalization of CB2R, PPARα, PPARγ, PPARγ1, PPARγ2, tight junction proteins, VEGF/CD31 expression, and downregulations of HIF1α, MCP-1 and TGFβ1 were observed in the VAT and intestine of the NASH+aleg+jwh015 group. In vitro experiments revealed that PPARα/γ and CB2R activation attenuated NASH AT-derived EV (EVnash)-induced pathogenic changes in the J774/SVEC4-10/Caco2 /3T3-L1 cell system. This study suggested that VAT-derived EVs contribute to the pathogenesis of NAFLD and that combined PPARα/γ and CB2R agonist treatment reduces VAT-released EV release and HIF1/MCP-1 signals to ameliorate hepatic steatosis and VAT/intestine abnormalities of NASH mice.

Exercise training alleviates cholesterol and lipid accumulation in mice with non-alcoholic steatohepatitis: Reduction of KMT2D-mediated histone methylation of IDI1

Exercise training is a cornerstone treatment for non-alcoholic fatty liver disease (NAFLD). This study aims to investigate the effects of exercises on lipid accumulation in non-alcoholic steatohepatitis (NASH) and to explore the molecular mechanism. Established NASH mice were remained sedentary or subjected to moderate-intensity continuous training or high-intensity interval training (HIIT). The two training regimens, especially the latter one, reduced liver weight, steatosis, inflammation, lipid accumulation, collagen deposition, and cholesterol content in the mouse liver. Similarly, the HIIT regimen improved clinical presentation of NAFLD patients. RNA sequencing analysis revealed lysine methyltransferase 2D (Kmt2d) and isopentenyl-diphosphate delta isomerase 1 (Idi1) as two important genes downregulated in mice underwent HIIT. By using mouse hepatocytes AML12, we found that KMT2D promoted Idi1 expression by catalyzing H3K4me1 modification near its promoter. Upregulation of either KMT2D or IDI1 blocked the ameliorating effects of HIIT on mice. Meanwhile, in AML12 cells modeled by palmitic acid and oleic acid treatment, KMT2D and IDI1 were found to be correlated with lipid accumulation, cholesterol content, inflammation, and cell death and senescence. In conclusion, this study demonstrates that the ameliorating effects of exercise training on NASH might involve the downregulation of the KMT2D/IDI1 axis.

HIF-2α drives hepatic Kupffer cell death and proinflammatory recruited macrophage activation in nonalcoholic steatohepatitis.

Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow-derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)- and extracellular signal-regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α-dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage-specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α-specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype-specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH.

Deciphering the mechanism of Chaihu Shugan San in the treatment of nonalcoholic steatohepatitis using network pharmacology and molecular docking.

In China, there is a long history and rich clinical experience in treating nonalcoholic steatohepatitis (NASH) with traditional Chinese herbal medicines, including Chai Hu Shu Gan San. This study aims to investigate the potential regulatory effects of Chaihu Shugan San (CSS) on liver lipid metabolism and inflammatory damage in mice with experimental nonalcoholic steatohepatitis (NASH) induced by a choline-deficient high-fat diet (CDHFD). Utilizing network pharmacology, we systematically explore the mechanisms of action and therapeutic potential of CSS against NASH. Potential targets in CSS and targets for NASH were identified using online databases. Functional enrichment and protein-protein interaction analyses were conducted to identify hub-targeted genes and elucidate the underlying molecular mechanisms. The affinities of active compounds in CSS with hub-targeted genes were evaluated using molecular docking. Finally, hub-targeted genes were validated through real-time polymerase chain reaction, western blotting, and immunofluorescence in choline-deficient high-fat diet mice, both with and without CSS treatment. CSS reduces serum ALT and AST levels in NASH mice(P < 0.05) and ameliorates ballooning degeneration in the livers of NASH mice, thereby lowering the NAS score(P < 0.05). Including naringenin, high-performance liquid chromatography/mass spectrometrys identified 12 chromatographic peaks. Based on network pharmacology analysis, CSS contains a total of 103 active compounds and 877 target genes. Transferase activity represents a potential mechanism for therapeutic intervention of CSS in NASH. The transcriptional levels and protein expression of the SIRT1 gene in NASH mice are significantly increased by CSS (P < 0.05). Naringenin is probable active compound in CSS and SIRT1 is the hub gene by which CSS is involved in NASH treatment.

Excretory/secretory antigens from Trichinella spiralis muscle larvae ameliorate HFD-induced non-alcoholic steatohepatitis via driving macrophage anti-inflammatory activity

No approved effective therapy for non-alcoholic steatohepatitis (NASH) is currently available. Trichinella spiralis (T. spiralis) infection and their products have positive impact on several metabolic diseases. Considering, we firstly investigated the effects of the T. spiralis-derived Excretory-Secretory antigens (ESA) on high fat diet (HFD)-induced NASH mouse models. To further elucidate the mechanism of action, HepG2 cells were incubated with palmitic acid (PA) to construct NASH-like cell model, and then the culture medium supernatant collected from ESA-treated macrophages was applied to intervene the cell model in vitro. In NASH mouse models, ESA significantly alleviated hepatic steatosis and hepatic inflammation, as reflected by reducing pro-inflammatory cytokines and inactivating TLR4/MYD88/NF-κB pathway and NLRP3 inflammasome. Meanwhile, the HFD-induced oxidative stress was restored by ESA through lessening the level of MDA, increasing the activity of T-SOD and enhancing Nrf2 signaling-related proteins, including p-Nrf2, NQO1, HO-1, GPX4, and p-AMPK. Notably, ESA preferentially promoted macrophages polarization toward M2 anti-inflammatory phenotype in vivo and vitro. Moreover, in vitro, intervention of PA-treated HepG2 cells with medium supernatant of ESA-treated macrophages attenuated lipid accumulation, inflammation, as well as oxidative stress. In conclusion, T. spiralis-derived ESA may serve as a novel promising candidate for the treatment of NASH via its properties of driving macrophage anti-inflammatory activity.

Collagen-targeted protein nanomicelles for the imaging of non-alcoholic steatohepatitis

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding – thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. Statement of significanceDirect molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. NIR-Col1-TRAP clears from the serum faster in these mice compared to control mice, and accumulates significantly more in fibrotic livers.This work advances the development of targeted protein probes for in vivo fibrosis imaging.

GTPBP8 mitigates nonalcoholic steatohepatitis (NASH) by depressing hepatic oxidative stress and mitochondrial dysfunction via PGC-1α signaling

Nonalcoholic steatohepatitis (NASH) is emerging as a major cause of liver transplantation and hepatocellular carcinoma (HCC). Regrettably, its pathological mechanisms are still not fully comprehended. GTP-binding protein 8 (GTPBP8), belonging to the GTP-binding protein superfamily, assumes a crucial role in RNA metabolism, cell proliferation, differentiation, and signal transduction. Its aberrant expression is associated with oxidative stress and mitochondrial dysfunctions. Nevertheless, its specific functions and mechanisms of action, particularly in NASH, remain elusive. In our current study, we initially discovered that human hepatocytes L02 displayed evident mitochondrial respiratory anomaly, mitochondrial damage, and dysfunction upon treatment with palmitic acids and oleic acids (PO), accompanied by significantly reduced GTPBP8 expression levels through RNA-Seq, RT-qPCR, western blotting, and immunofluorescence assays. We then demonstrated that GTPBP8 overexpression mediated by adenovirus vector (Ad-GTPBP8) markedly attenuate lipid accumulation, inflammatory response, and mitochondrial impair and dysfunction in hepatocytes stimulated by PO. Conversely, adenovirus vector-mediated GTPBP8 knockdown (Ad-shGTPBP8) significantly accelerated lipid deposition, inflammation and mitochondrial damage in PO-treated hepatocytes in vitro. Furthermore, we constructed an in vivo NASH murine model by giving a 16-week high fat high cholesterol diet (HFHC) diet to hepatocyte specific GTPBP8-knockout (GTPBP8HKO) mice. We firstly found that HFHC feeding led to metabolic disorder in mice, including high body weight, blood glucose and insulin levels, and liver dysfunctions, which were accelerated in these NASH mice with GTPBP8 deficiency in hepatocytes. Consistently, GTPBP8HKO remarkably exacerbated the progression of NASH phenotypes induced by HFHC, as proved by the anabatic lipid accumulation, inflammation, fibrosis and reactive oxygen species (ROS) production in liver tissues, which could be largely attributed to the severe mitochondrial damage and dysfunction. Mechanistically, we further identified that GTPBP8 interacted with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in hepatocytes. Importantly, the hepaprotective effects of GTPBP8 against mitochondrial dysfunction, oxidative stress and inflammation was largely dependent on PGC-1α expression. Collectively, GTPBP8 may exert a protective role in the progression of NASH, and targeting the GTPBP8/PGC-1α axis may represent a potential strategy for NASH treatment by improving mitochondrial functions.

Sulforaphane ameliorates non-alcoholic steatohepatitis by KLF4-mediated macrophage M2 polarization

Non-alcoholic fatty liver disease (NAFLD) has become a global issue and a severe threat to public health. However, to date, no approved therapeutic drugs have been developed. Dietary interventions with natural products have shown promise in preventing and treating NAFLD. Sulforaphane (SFN) is a phytocompound with antioxidant and anti-inflammatory properties, and previous research has demonstrated that SFN can ameliorate hepatic lipid accumulation and inflammation. However, the molecular mechanisms underlying these beneficial effects remain unclear. In this study, we confirmed the protective effects of SFN on excessive lipid accumulation and inflammatory injury in a high-fat, high-fructose diet-induced non-alcoholic steatohepatitis (NASH) mouse model. We found that SFN attenuates the inflammatory injury in a macrophage cell line and the liver of NASH mice, owing to the promotion of M1-type macrophage polarization toward the M2-type and the regulation of inflammatory mediators. Further analysis demonstrated that this SFN-induced macrophage M2-type polarization occurs in a Krüppel-like factor 4 (KLF4)-dependent manner. In summary, we uncovered a new mechanism of action underlying SFN activity and provide evidence that dietary intervention with SFN might be protective against NASH.

Tanshinone IIA Inhibits the Endoplasmic Reticulum Stress-Induced Unfolded Protein Response by Activating the PPARα/FGF21 Axis to Ameliorate Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a critical stage in the progression of nonalcoholic fatty liver disease (NAFLD). Tanshinone IIA (TIIA) is a tanshinone extracted from Salvia miltiorrhiza; due to its powerful anti-inflammatory and antioxidant biological activities, it is commonly used for treating cardiovascular and hepatic diseases. A NASH model was established by feeding mice a methionine and choline-deficient (MCD) diet. Liver surface microblood flow scanning, biochemical examination, histopathological examination, cytokine analysis through ELISA, lipidomic analysis, transcriptomic analysis, and Western blot analysis were used to evaluate the therapeutic effect and mechanism of TIIA on NASH. The results showed that TIIA effectively reduced lipid accumulation, fibrosis, and inflammation and alleviated endoplasmic reticulum (ER) stress. Lipidomic analysis revealed that TIIA normalized liver phospholipid metabolism in NASH mice. A KEGG analysis of the transcriptome revealed that TIIA exerted its effect by regulating the PPAR signalling pathway, protein processing in the ER, and the NOD-like receptor signalling pathway. These results suggest that TIIA alleviates NASH by activating the PPARα/FGF21 axis to negatively regulate the ER stress-induced unfolded protein response (UPR).

Linking antibody against apolipoprotein A-1 to nonalcoholic steatohepatitis in mice

Linking antibody against apolipoprotein A-1 to nonalcoholic steatohepatitis in mice

Amelioration of nonalcoholic fatty liver disease by inhibiting the deubiquitylating enzyme RPN11

Nonalcoholic fatty liver disease (NAFLD), including its more severe manifestation nonalcoholic steatohepatitis (NASH), is a global public health challenge. Here, we explore the role of deubiquitinating enzyme RPN11 in NAFLD and NASH. Hepatocyte-specific RPN11 knockout mice are protected from diet-induced liver steatosis, insulin resistance, and steatohepatitis. Mechanistically, RPN11 deubiquitinates and stabilizes METTL3 to enhance the m6A modification and expression of acyl-coenzyme A (CoA) synthetase short-chain family member 3 (ACSS3), which generates propionyl-CoA to upregulate lipid metabolism genes via histone propionylation. The RPN11-METTL3-ACSS3-histone propionylation pathway is activated in the livers of patients with NAFLD. Pharmacological inhibition of RPN11 by Capzimin ameliorated NAFLD, NASH, and related metabolic disorders in mice and reduced lipid contents in human hepatocytes cultured in 2D and 3D. These results demonstrate that RPN11 is a novel regulator of NAFLD/NASH and that suppressing RPN11 has therapeutic potential for the treatment.

A novel experimental mouse model of diabetic nonalcoholic steatohepatitis: A critical role for acid-sensitive Ion Channel 1a

BackgroundA two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model. MethodsThe combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model. ResultsThe mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy. ConclusionA FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH.

Tranilast Inhibits TRPV2 and Suppresses Fibrosis Progression and Weight Gain in a NASH Model Mouse.

This study aimed to investigate the role of transient receptor potential vanilloid 2 (TRPV2) in a mouse model with non-alcoholic steatohepatitis (NASH) and to examine the effects of tranilast on TRPV2 and fibrosis-related cytokines. C57BL/6N mice were fed a Gubra-Amylin NASH (GAN) diet for 20 weeks to induce NASH. The tranilast groups received oral administration of tranilast at doses of 300, 400 and 500 mg/kg/day, five days per week for 20 weeks, in addition to the GAN diet. The effects of tranilast were assessed based on the dosage of food intake, changes in body weight, liver weight, blood biochemical parameters, histopathological examination, and expression of TRPV2 and inflammatory cytokines. Hepatic expression of TRPV2 was observed in the GAN-fed NASH mouse model. The tranilast groups showed significantly suppressed increases in body and liver weights. The development of intrahepatic fat deposition and liver fibrosis, assessed histopathologically, was inhibited. Tranilast administration improved the expression of TRPV2 and inflammatory cytokines in the liver. Additionally, blood tests indicated a reduction in elevated liver enzyme levels. In GAN diet NASH models, TRPV2 was up-regulated in the liver and tranilast inhibited TRPV2 and suppressed fibrosis. Therefore, it might prevent the incidence of hepatocellular carcinoma associated with NASH.

The role of C5aR1-mediated hepatic macrophage efferocytosis in NASH

Non-alcoholic fatty liver disease (NAFLD) has become the first major chronic liver disease in developed countries. 10–20% of NAFLD patients will progress to non-alcoholic steatohepatitis (NASH), and up to 25% of NASH patients may develop cirrhosis within 10 years. Therefore, it is critical to find key targets that may treat this disease. Here, we identified C5aR1 as a highly-expressed gene in NASH mouse model through analyzing Gene Expression Omnibus (GEO) database and confirmed its higher expression in livers of NASH patients than that of NAFL patients. Meanwhile, we verified its positive correlation with patients’ serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. In vivo and in vitro experiments revealed that knocking down C5aR1 in liver significantly reduced liver weight ratio and serum ALT and AST levels and attenuated inflammatory cell infiltration and cell apoptosis in the liver of NASH mice as well as enhanced the efferocytotic ability of liver macrophages, suggesting that C5aR1 may play a crucial role in the efferocytosis of liver macrophages. Furthermore, we also found that the expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, IL-1β and other inflammation-related factors in the liver were significantly reduced. Our work demonstrates a potential mechanism of how C5aR1 deficiency protects against diet-induced NASH by coordinating the regulation of inflammatory factors and affecting hepatic macrophage efferocytosis.