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Atorvastatin attenuates diet-induced non-alcoholic steatohepatitis in APOE*3-Leiden mice by reducing hepatic inflammation

ABSTRACT: Abstract: Patients with metabolic syndrome are often prescribed statins to prevent development of 17 cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) 18 are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) 19 with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma 20 cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin sig-21 nificantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver 22 (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated 23 mice showed less immunohistochemically stained area of inflammation markers. Atorvastatin pre-24 vented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crys-25 tals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its acti-26 vation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1β 27 (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin 28 on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates 29 that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crys-30 tal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin 31 treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in 32 dyslipidemic patients.

ORGANISM(S): Mus musculus

PROVIDER: GSE229188 | GEO | 2023/05/17

REPOSITORIES: GEO

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Project description:Background and Aims: The activation of stimulator of interferon genes (STING) and NOD-like receptors protein 3 (NLRP3) inflammasomes-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnection between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown and is the focus of this study. Approach and Results: Liver fibrosis was induced in Sting knockout, Gasdermin D (Gsdmd) knockout mice, and in mice with hepatocyte-specific Nlrp3 deletion. RNA-sequencing, metabolomics, epigenetic compound screening system, and chromatin immunoprecipitation were utilized. STING and NLRP3 inflammasome signaling pathways were activated in cirrhotic livers but were suppressed by Sting knockout. Sting knockout also ameliorated hepatic pyroptosis, inflammation, and fibrosis in the murine cirrhotic model. In vitro, STING induced pyroptosis in primary murine hepatocytes via activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) were identified to regulate NLRP3 expression in STING-overexpressed AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhanced interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promoted STING-induced Nlrp3 transcription in hepatocytes. The RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes showed that metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gsdmd knockout attenuated hepatic pyroptosis, inflammation, and fibrosis in murine cirrhotic models. Conclusions: This study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis.

Project description:It has been recently reported that the patients with non-alcoholic fatty liver diseases (NAFLD) had accompanied with cardiac dysfunction. However, an animal model of steatohepatitis related cardiomyopathy has not been reported. In the current study, we have developed a mouse model of steatohepatitis-related cardiomyopathy and examined whether a selective PPARα modulator, pemafibrate would improve both steatohepatitis and cardiomyopathy. C57Bl/6 male mice were fed 1. normal chow diet (C group), 2. high fat/high cholesterol/high sucrose/bile acid diet (N group), or 3. N with pemafibrate 0.1 mg/kg (NP group) for three or eight weeks, respectively. Cardiac function was evaluated by ultra-high magnetic field 7-Tesla magnetic resonance imaging (7T-MRI). Even at the eight weeks, we have detected cholesterol crystals, free cholesterol accumulation in liver, followed by macrophage infiltration and fibrosis as wells as activation of NLRP3, caspase-1 and IL-1βmRNA. At the same time in heart, the ratio of heart to body was increased in N group compared to C group. Left ventricular (LV) ejection fraction (LVEF) was moderately decreased in N group compared to C group (50.2±2.8 vs 59.8±2.1, p<0.05). LV circumferential strain and radial strain are focally attenuated in N group, suggesting the early lesion of myocardial damages. mRNA expression of Nlrp3, Caspase1 and Il1b were enhanced in N group, followed by increased protein levels of Caspase-1 and Asc. Rna-sequence analysis revealed that NOD-like receptor and PI3 kinase-AKT pathways were up-regulated, suggesting hypertrophic phenotypes. in N group. Importantly, NLRP3 inflammasome activation were suppressed both in liver and heart in NP group, suggesting pemafibrate had the beneficial effect on steatohepatitis and cardiomyopathy. In addition, four week treatment by pemafibrate improved steatohepatitis and cardiomyopathy after development for eight week. We have first developed the animal model of steatohepatitis-related cardiomyopathy, characterized by the activation of NLRP3 inflammasome pathway both in liver and heart. Steatohepatitis-related cardiomyopathy displayed moderate reduction of LVEF and focal impairment of LV strain by 7T-MRI. Pemafibrate suppressed steatohepatitis, hepatic fibrosis and cardiomyopathy.

Project description:Inflammasome, activated by pathogen-derived and host-derived danger signals, constitutes a multimolecular signaling complex that serves as a platform for caspase-1 (CASP1) activation and interleukin-1beta (IL1B) maturation. The activation of NLRP3 inflammasome requires two-step signals. The first “priming” signal (Signal 1) enhances gene expression of inflammasome components. The second “activation” signal (Signal 2) promotes the assembly of inflammasome components. Deregulated activation of NLRP3 inflammasome contributes to the pathological processes of Alzheimer’s disease (AD) and multiple sclerosis (MS). However, at present, the precise mechanism regulating NLRP3 inflammasome activation and deactivation remains largely unknown. By genome-wide gene expression profiling, we studied the molecular network of NLRP3 inflammasome activation-responsive genes in a human monocyte cell line THP-1 sequentially given two-step signals. We identified the set of 83 NLRP3 inflammasome activation-responsive genes. Among them, we found the NR4A nuclear receptor family NR4A1, NR4A2, and NR4A3, the EGR family EGR1, EGR2, and EGR3, the IkappaB family NFKBIZ, NFKBID, and NFKBIA as a key group of the genes that possibly constitute a negative feedback loop for shutting down inflammation following NLRP3 inflammasome activation. By molecular network analysis, we identified a complex network of NLRP3 inflammasome activation-responsive genes involved in cellular development and death, and immune and inflammatory responses, where transcription factors AP-1, NR4A, and EGR serve as a hub. Thus, NLRP3 inflammasome activation-responsive genes constitute the molecular network composed of a set of negative feedback regulators for prompt resolution of inflammation. To load the Signal 1 (S1), THP-1 cells were incubated for 3 hours in the culture medium with or without inclusion of 0.2 microgram/ml lipopolysaccharide (LPS). To load the Signal 2 (S2), they were incubated further for 2 hours in the culture medium with inclusion of 10 microM nigericin sodium salt dissolved in ethanol or the equal v/v% concentration of ethanol (vehicle), followed by processing for microarray analysis on Human Gene 1.0 ST Array (Affymetrix).

Project description:Background & Aims: Overnutrition is one of the major causes of non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH). Besides the quantity of consumed calories, distinct dietary components are increasingly recognized as important contributor to the pathogenesis of NASH. We aimed to develop and characterize a hitherto missing murine model which resembles both the pathology and nutritional situation of NASH-patients in Western societies. Methods: We developed a NASH-inducing diet (ND) enriched with sucrose, cholesterol and a high concentration of fats rich in saturated fatty acids in a composition which mimics Western food. C57Bl6/N mice were fed with the ND or control chow for 12 weeks. Biochemical, real-time polymerase chain reaction, Western Blot and immunohistochemical analyses were performed to characterize systemic and hepatic changes induced by ND-feeding. Immunohistochemistry was used to assess c-Jun levels and activation in 110 human NAFLD and control liver specimens applying tissue micro array technology. Results: ND-fed mice showed significant body weight gain, impaired glucose tolerance, elevated fasting blood glucose levels as well as decreased adiponectin and increased leptin serum levels compared to control mice. In the liver, ND-feeding led to marked steatosis, enhanced cholesterol levels, distinct signs of oxidative stress, hepatocellular damage, inflammation, activation of hepatic stellate cells, and beginning fibrosis. Transcriptome-wide hepatic gene expression analysis comparing ND-fed mice and control mice indicated main alterations in lipid metabolism and inflammatory processes. Search for over-represented transcription factor target sites among the differentially expressed genes identified AP-1 as the most likely factor to cause the transcriptional changes in ND-livers. Combining differentially expressed gene and protein-protein interaction network analysis identified c-Jun (a component of the AP-1 complex) as hub in the largest connected deregulated sub-network in ND-livers. In accordance, ND-livers revealed c-Jun-phosphorylation and nuclear translocation. Moreover, hepatic c-Jun RNA and protein expression was enhanced in ND-fed compared to control mice. Also NAFLD-patients showed enhanced hepatic c-Jun levels, which correlated with inflammation, and notably, with the degree of hepatic steatosis. Conclusions: The new dietary mouse-model shows important pathological changes also found in human NASH and indicates c-jun/AP-1 activation as critical regulator of hepatic alterations. Abundance of c-jun in NAFLD likely facilitates development and progression of NASH, and thus, c-jun appears as attractive prognostic and therapeutic target of NAFLD progression. 14-weeks old male C57BL/6N mice were fed with either regular diet or a newly designed NASH-inducing diet for 12 weeks. Hepatic gene expression levels were measured thereafter.

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Atorvastatin attenuates diet-induced non-alcoholic steatohepatitis in APOE*3-Leiden mice by reducing hepatic inflammation

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