Liver Of Non-alcoholic Fatty Liver Disease Mice Research Articles

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 μM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

Xiaozhi formula attenuates non-alcoholic fatty liver disease by regulating lipid metabolism via activation of AMPK and PPAR pathways

BackgroundXiaozhi formula (XZF) is a practical Chinese herbal formula for the treatment of non-alcoholic fatty liver disease (NAFLD), which possesses an authorized patent certificate issued by the State Intellectual Property Office of China (ZL202211392355.0). However, the underlying mechanism by which XZF treats NAFLD remains unclear. PurposeThis study aimed to explore the main component of XZF and its mechanism of action in NAFLD treatment. MethodsUHPLC-Q-Orbitrap HRMS was used to identify the components of the XZF. A high-fat diet (HFD)-induced NAFLD mouse model was used to demonstrate the effectiveness of XZF. Body weight, liver weight, and white fat weight were recorded to evaluate the therapeutic efficacy of XZF. H&E and Oil Red O staining were applied to observe the extent of hepatic steatosis. Liver damage, lipid metabolism, and glucose metabolism were detected by relevant assay kits. Moreover, the intraperitoneal insulin tolerance test and the intraperitoneal glucose tolerance test were employed to evaluate the efficacy of XZF in insulin homeostasis. Hepatocyte oxidative damage markers were detected to assess the efficacy of XZF in preventing oxidative stress. Label-free proteomics was used to investigate the underlying mechanism of XZF in NAFLD. RT-qPCR was used to calculate the expression levels of lipid metabolism genes. Western blot analysis was applied to detect the hepatic protein expression of AMPK, p-AMPK, PPARɑ, CPT1, and PPARγ. Results120 compounds were preliminarily identified from XZF by UHPLC-Q-Orbitrap HRMS. XZF could alleviate HFD-induced obesity, white adipocyte size, lipid accumulation, and hepatic steatosis in mice. Additionally, XZF could normalize glucose levels, improve glucolipid metabolism disorders, and prevent oxidative stress damage induced by HFD. Furthermore, the proteomic analysis showed that the major pathways in fatty acid metabolism and the PPAR signaling pathway were significantly impacted by XZF treatment. The expression levels of several lipolytic and β-oxidation genes were up-regulated, while the expression of fatty acid synthesis genes declined in the HFD + XZF group. Mechanically, XZF treatment enhanced the expression of p-AMPK, PPARɑ, and CPT-1 and suppressed the expression of PPARγ in the livers of NAFLD mice, indicating that XZF could activate the AMPK and PPAR pathways to attenuate NALFD progression. ConclusionXZF could attenuate NAFLD by moderating lipid metabolism by activating AMPK and PPAR signaling pathways.

MiR-192-5p Ameliorates Hepatic Lipid Metabolism in Non-Alcoholic Fatty Liver Disease by Targeting Yy1.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in the liver. Clarifying the molecular mechanism of lipid metabolism is crucial for the treatment of NAFLD. We examined miR-192-5p levels in the livers of mice in which NAFLD was induced via a high-fat diet (HFD), as well as in mouse primary hepatocytes and human HepG2 cells treated with free fatty acids (FFAs). MiR-192-5p inhibitor was administered to NAFLD mice and hepatocytes to verify the specific function of miR-192-5p in NAFLD. We validated the target gene of miR-192-5p and further illustrated the effects of this miRNA on the regulation of triglyceride (TG) metabolism. We found that miR-192-5p was significantly increased in the livers of NAFLD mice and FFA-treated hepatocytes. Inhibition of miR-192-5p increased the accumulation of hepatic TGs and aggravated hepatic steatosis in NAFLD mice. In FFA-treated hepatocytes, miR-192-5p inhibitors markedly increased TG content, whereas overexpression of miR-192-5p reduced TG levels. Yin Yang 1 (Yy1) was identified as the target gene of miR-192-5p, which regulates TG synthesis via the YY1/fatty-acid synthase (FASN) pathway. Our results demonstrated that miR-192-5p should be considered a protective regulator in NAFLD that can inhibit hepatic TG synthesis by targeting Yy1.

The transcription factor T-bet promotes the pathogenesis of nonalcoholic fatty liver disease by upregulating intrahepatic inflammation

ObjectiveTo investigate the effect of the transcription factor T-bet on the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the regulation of the intrahepatic immune microenvironment. MethodsWild-type and T-bet knockout NASH mouse models were constructed. The effect of T-bet knockout on the pathogenesis of NAFLD was observed by histochemical staining. The expression of T-bet in immune cells in the liver and the effect of T-bet knockout on the proportion and function of immune cell subsets in the liver were determined by flow analysis. ResultsFlow cytometry results indicated that T-bet expression was increased in immune cells, especially NKT cells, in the livers of NAFLD mice. Knocking out the transcription factor T-bet reduced intrahepatic inflammation, reduced lipid accumulation, and ameliorated the pathogenesis of NAFLD. Based on the analysis of immune cell subsets, knocking out the transcription factor T-bet decreased the proportion, survival, and degree of activation of NK, NKT, and CD8 T cells in NAFLD liver; additionally, it decreased the secretion of IFN-γ by T cells and NKT cells but had no effect on the proportion of Th17 cells and Treg cells. Knocking out the transcription factor T-bet also reduced the proportion of proinflammatory myeloid-derived macrophages (MoMFs) in NAFLD liver, mainly the proportion of proinflammatory Ly6Chigh MoMFs. Furthermore, knocking out the transcription factor T-bet had no significant effect on the secretion of TNF-α from MoMFs but significantly reduced the expression of MHC class II molecules. Further analysis showed that the transcription factor T-bet may directly affect the expression of MHC class II molecules H2-AB1 and H2-Dmb1 through transcriptional regulation. ConclusionsKnocking out the transcription factor T-bet reduced the proinflammatory effect of innate immune cells (MoMFs, NK cells, and NKT cells) and T lymphocytes in NAFLD liver, thereby reducing intrahepatic inflammation and delaying the pathogenesis of NAFLD.

Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy

BackgroundCurrent evidence indicates a rising global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD), which is closely associated to conditions such as obesity, dyslipidemia, insulin resistance, and metabolic syndrome. The relationship between the gut microbiome and metabolites in NAFLD is gaining attention understanding the pathogenesis and progression of dysregulated lipid metabolism and inflammation. The Xie Zhuo Tiao Zhi (XZTZ) decoction has been employed in clinical practice for alleviating hyperlipidemia and symptoms related to metabolic disorders. However, the pharmacological mechanisms underlying the effects of XZTZ remain to be elucidated. PurposeThe objective of this study was to examine the pharmacological mechanisms underlying the hypolipidemic and anti-inflammatory effects of XZTZ decoction in a mouse model of NAFLD, as well as the effects of supplementing exogenous metabolites on PO induced cell damage and lipid accumulation in cultured hepatocytes. MethodsA high-fat diet (HFD) mouse model was established to examine the effects of XZTZ through oral gavage. The general condition of mice and the protective effect of XZTZ on liver injury were evaluated using histological and biochemical methods. Hematoxylin and eosin staining (H&E) staining and oil red O staining were performed to assess inflammatory and lipid accumulation detection, and cytokine levels were quantitatively analyzed. Additionally, the study included full-length 16S rRNA sequencing, liver transcriptome analysis, and non-targeted metabolomics analysis to investigate the relationship among intestinal microbiome, liver metabolic function, and XZTZ decoction. ResultsXZTZ had a significant impact on the microbial community structure in NAFLD mice. Notably, the abundance of Ileibacterium valens, which was significantly enriched by XZTZ, exhibited a negative correlation with liver injury biomarkers such as, alanine transaminase (ALT) and aspartate transaminase (AST) activity. Moreover, treatment with XZTZ led to a significant enrichment of the purine metabolism pathway in liver tissue metabolites, with inosine, a purine metabolite, showing a significant positive correlation with the abundance of I. valens. XZTZ and inosine also significantly enhanced fatty acid β-oxidation, which led to a reduction in the expression of pro-inflammatory cytokines and the inhibition of liver pyroptosis. These effects contributed to the mitigation of liver injury and hepatocyte damage, both in vivo and vitro. Furthermore, the utilization of HPLC fingerprints and UPLC-Q-TOF-MS elucidated the principal constituents within the XZTZ decoction, including naringin, neohesperidin, atractylenolide III, 23-o-Acetylalisol B, pachymic acid, and ursolic acid which are likely responsible for its therapeutic efficacy. Further investigations are imperative to fully uncover and validate the pharmacodynamic mechanisms underlying these observations. ConclusionThe administration of XZTZ decoction demonstrates a protective effect on the livers of NAFLD mice by inhibiting lipid accumulation and reducing hepatocyte inflammatory damage. This protective effect is mediated by the upregulation of I.valens abundance in the intestine, highlighting the importance of the gut-liver axis. Furthermore, the presesnce of inosine, adenosine, and their derivatives are important in promoting the protective effects of XZTZ. Furthermore, the in vitro approaching, we provide hitherto undocumented evidence indicating that the inosine significantly improves lipid accumulation, inflammatory damage, and pyroptosis in AML12 cells incubated with free fatty acids.

Erchen Decoction improves iron homeostasis in mice with non-alcoholic fatty liver disease by regulating iron transport capacity in the spleen

To investigate the effect of Erchen Decoction on iron homeostasis in mice with nonalcoholic fatty liver disease (NAFLD) and its mechanism for regulating iron transport in spleen cells. Thirty male C57BL/6J mice were given a high-fat diet for 12 weeks and randomized (n=6) at the 7th week for gavage (3 times a week) of drinking water (NAFLD model group), Erchen Decoction at low, medium and high doses (7.5, 15, and 30g/kg, respectively), or polyene phosphatidyl choline (PPC; 9.12 mg/kg), with another 6 mice with low-fat and low-sugar feeding as the control group. The active components of Erchen Decoction were determined by HPLC-MS. Lipid accumulation in the liver was evaluated by HE staining and Nile red staining. Prussian blue staining was used to observe iron content in the spleen. The iron ion content in the liver tissue was detected using a detection kit. The expressions of ferroportin1 (Fpn1), transferrin receptor (TfR), Steap3, HO-1, Ter-119, CD163 and CD68 were detected using Western blotting, immunohistochemistry and immunofluorescence staining. Medium- and high-dose Erchen Decoction partially reversed the increase of lipid accumulation in the liver of NAFLD mice and showed better lipid-lowering effect than PPC. The NAFLD mice showed significantly decreased iron ion content in the spleen with increased hepatic and serum iron contents (P < 0.05), decreased TfR protein expression (P < 0.05), and increased Fpn1 and Steap3 protein expressions (P < 0.05), and these changes were significantly improved by the drug interventions. Erchen Decoction also improved the function of CD163 macrophages in the spleen of NAFLD mice by up-regulating the expression of HO-1 (P < 0.05). Erchen Decoction can alleviate high-fat diet-induced iron metabolism disorder by improving the iron ion transport ability of the spleen cells to delay the progression of NAFLD.

Identification of key modules and driving genes in nonalcoholic fatty liver disease by weighted gene co-expression network analysis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive liver fat deposition, and progresses to liver cirrhosis, and even hepatocellular carcinoma. However, the invasive diagnosis of NAFLD with histopathological evaluation remains risky. This study investigated potential genes correlated with NAFLD, which may serve as diagnostic biomarkers and even potential treatment targets. The weighted gene co-expression network analysis (WGCNA) was constructed based on dataset E-MEXP-3291. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to evaluate the function of genes. Blue module was positively correlated, and turquoise module negatively correlated with the severity of NAFLD. Furthermore, 8 driving genes (ANXA9, FBXO2, ORAI3, NAGS, C/EBPα, CRYAA, GOLM1, TRIM14) were identified from the overlap of genes in blue module and GSE89632. And another 8 driving genes were identified from the overlap of turquoise module and GSE89632. Among these driving genes, C/EBPα (CCAAT/enhancer binding protein α) was the most notable. By validating the expression of C/EBPα in the liver of NAFLD mice using immunohistochemistry, we discovered a significant upregulation of C/EBPα protein in NAFLD. we identified two modules and 16 driving genes associated with the progression of NAFLD, and confirmed the protein expression of C/EBPα, which had been paid little attention to in the context of NAFLD, in the present study. Our study will advance the understanding of NAFLD. Moreover, these driving genes may serve as biomarkers and therapeutic targets of NAFLD.

Prediction and verification of the active ingredients and potential targets of Erhuang Quzhi Granules on non-alcoholic fatty liver disease based on network pharmacology.

Erhuang Quzhi Granules (EQG) is a compound composed of 13 traditional Chinese medicines developed by the First Affiliated Hospital of Shihezi University. In clinical practice, EQG has been applied to the treatment of hyperlipidemia and non-alcoholic fatty liver disease (NAFLD), and could significantly improve the serum biochemical indicators of NAFLD patients. This study aims to explore the bioactive compounds, potential targets, and molecular mechanisms of EQG against NAFLD through network pharmacology, molecular docking, and experimental verification. The chemical components of EQG came from the literature and quality standard. Bioactive compounds were screened based on the absorption, distribution, metabolism, and excretion (ADME) feature, and their potential targets were predicted using the substructure-drug-target network-based inference (SDTNBI). The core targets and signaling pathways were obtained through the analysis of protein-protein interaction (PPI), gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway. The results were further confirmed by literature retrieval, molecular docking, and in vivo experiments. The results of network pharmacology showed 12 active ingredients and 10 core targets for EQG in treating NAFLD. And EQG mainly regulates lipid and atherosclerosis-related pathways to improve NAFLD. The collected literature verified the regulatory effect of the active components of EQG on core targets TP53, PPARG, EGFR, HIF1A, PPARA, and MTOR. Molecular docking results showed that Aloe-Emodin (AE), Emodin, Physcion, and Rhein (RH) had stable binding structures with the core targets HSP90AA1. In vivo experiment showed that AE and RH reduced aspartate transaminase (AST), alanine aminotransferase (ALT), interleukin (IL)-1β, IL-6, IL18, and tumor necrosis factor α (TNF-α) in the serum or liver of NAFLD mice, improved liver lipid deposition and fibrosis, and inhibit gene expression of nuclear factor kappa B (NF-κB), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), IL-1β, TNF-α and protein expression of HSP90, NF-κB and Cleaved caspase-1. This study comprehensively revealed the biological compounds, potential targets, and molecular mechanisms of EQG in the treatment of NAFLD, providing a reference basis for the promotion of EQG in the clinic.

Transcriptome Sequencing Reveals Autophagy Networks in Rat Livers during the Development of NAFLD and Identifies Autophagy Hub Genes

(1) Autophagy is an important biological process in cells and is closely associated with the development and progression of non-alcoholic fatty liver disease (NAFLD). Therefore, this study aims to investigate the biological function of the autophagy hub genes, which could be used as a potential therapeutic target and diagnostic markers for NAFLD. (2) Male C57BL/6J mice were sacrificed after 16 and 38 weeks of a high-fat diet, serum biochemical indexes were detected, and liver lobules were collected for pathological observation and transcriptome sequencing. The R software was used to identify differentially expressed autophagy genes (DEGs) from the transcriptome sequencing data of mice fed with a normal diet for 38 weeks (ND38) and a high-fat diet for 38 weeks (HFD38). Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the DEGs, a protein-protein interaction (PPI) network of the DEGs was established using the STRING data website, and the results were visualized through Cytoscape. (3) After 16 weeks and 38 weeks of a high-fat diet, there was a significant increase in body weight, serum total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and triglycerides (TG) in mice, along with lipid accumulation in the liver, which was more severe at 38 weeks than at 16 weeks. The transcriptome data showed significant changes in the expression profile of autophagy genes in the livers of NAFLD mice following a long-term high-fat diet. Among the 31 differentially expressed autophagy-related genes, 13 were upregulated and 18 were downregulated. GO and KEGG pathway analysis revealed that these DEGs were primarily involved in autophagy, cholesterol transport, triglyceride metabolism, apoptosis, the FoxO signaling pathway, the p53 signaling pathway and the IL-17 signaling pathway. Four hub genes were identified by the PPI network analysis, of which Irs2, Pnpla2 and Plin2 were significantly downregulated, while Srebf2 was significantly upregulated by the 38-week high-fat diet. (4) The hub genes Irs2, Pnpla2, Srebf2 and Plin2 may serve as key therapeutic targets and early diagnostic markers in the progression of NAFLD.

Fu brick tea alleviates high fat induced non-alcoholic fatty liver disease by remodeling the gut microbiota and liver metabolism.

Fu brick tea (FBT) and its extracts have good lipid-lowering effects and have been used in the treatment of obesity in previous studies. Unfortunately, the therapeutic effect of FBT on non-alcoholic fatty liver disease (NAFLD) has not been thoroughly studied. In this study, we explored the mechanism by which FBT alleviates NAFLD from the perspective of the gut microbiota and liver metabolites. The results showed that FBT could reduce the body weight, liver weight and abdominal fat of NAFLD mice, and improve liver pathological morphology, liver lipid deposition, blood lipids and liver function. Moreover, FBT improved the diversity of the gut microbiota and changed the profile of liver metabolism in NAFLD mice. Further studies showed that FBT could ameliorate the cecum barrier, and regulate the effects of factors related to lipid synthesis in the cecum and liver of NAFLD mice. In conclusion, the present study confirmed that FBT can alleviate high fat induced NAFLD by regulating the homeostasis of the gut microbiota and liver metabolites.

Exploring the Therapeutic Effects of Black Ginseng on Non-Alcoholic Fatty Liver Disease by Using Network Pharmacology and Molecular Docking.

This study aimed to investigate the therapeutic effect of black ginseng (BG) on non-alcoholic fatty liver disease (NAFLD) using network pharmacology combined with the molecular docking strategy. The saponin composition of BG was analyzed by liquid chromatography-mass spectrometry (LC/MS) instrument. Then the network pharmacology was applied to explore the potential targets and related mechanisms of BG in the treatment of NAFLD. After screening out key targets, molecular docking was used to predict the binding modes between ginsenoside and target. Finally, a methionine and choline deficiency (MCD) diet-induced NAFLD mice model was established to further confirm the therapeutic effect of BG on NAFLD. Twenty-four ginsenosides were annotated based on the MS and tandem MS information. Ten proteins were screened out as key targets closely related to BG treatment of NAFLD. The molecular docking showed that most of the ginsenosides had good binding affinities with AKT1. The validation experiment revealed that BG administration could reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the MCD diet-induced histological changes in liver tissue. Moreover, BG could upregulate the phosphorylation level of AKT in the liver of NAFLD mice, thereby exerting the therapeutic effect on NAFLD. Further studies on the active ginsenosides as well as their synergistic action on NAFLD will be required to reveal the underlying mechanisms in-depth. This study demonstrates that network pharmacological prediction in conjunction with molecular docking is a viable technique for screening the active chemicals and related targets of BG, which can be applied to other herbal medicines.

Polarity-sensitive and lipid droplet-specific red emission fluorophore for identifying fatty liver of living mice through in vivo imaging

The illustration of the correlation between lipid droplets (LDs) variation and nonalcoholic fatty liver disease (NAFLD) is a challenging and important work in biomedical research. Herein, a red emission fluorophore LD-HW containing donor-π-bridge-acceptor (D-π-A) structure was readily constructed and systematically investigated. It was found that LD-HW could selectively identify polarity variation accompanying with an obvious blue-shift (around 80 nm) in fluorescence spectra, and a sharp enhancement (about 440-fold) in fluorescence quantum yield (QY) over the solvent polarity ranging from water (polarity parameter Δf = 0.3200) to 1,4-dioxane (Δf = 0.0205). In addition, probe LD-HW could precisely light up LDs within a short time (≤5 min) through a wash-free procedure and real-time monitor the dynamic behavior of cellular LDs. More importantly, LD-HW exhibited an excellent performance in differentiating fatty liver through in vivo imaging the change of cellular LDs. The in situ fluorescence spectra of corresponding tissue section proved that polarity level in the liver of NAFLD mice was lower than that in normal mice. Taken together, probe LD-HW presented great potential in non-invasive diagnosis of fatty liver through in vivo imaging.

Tetrahydrocurcumin protects against nonalcoholic fatty liver disease by improving lipid metabolism and redox homeostasis

• Tetrahydrocurcumin (THC) improves glucose and lipid metabolism disorders in nonalcoholic fatty liver disease (NAFLD) in mice. • THC regulates the expression of oxidoreductase genes and lipid-metabolizing enzyme genes in the liver of NAFLD mice. • THC increases the antioxidant capacity and inhibits lipid deposition in HepG2 cells by activating the NRF2 signaling pathway. Nonalcoholic fatty liver disease (NAFLD) is the most common liver metabolic disease in the world. In this study we investigated the effect of tetrahydrocurcumin (THC), a potent antioxidant, on NAFLD. We found that THC significantly reduced the body weight, serum and liver lipids, serum malondialdehyde (MDA) and fasting blood glucose (FBG) of male NAFLD mice induced by feeding a high-fat diet (HFD) for 8 weeks. A RNA-seq analysis revealed that THC also affected the expression of many genes involved in lipid metabolism in the liver of NAFLD mice, including increasing the mRNA expression of CYP51 and FOXQ1 . Moreover, THC significantly reduced sodium oleate-induced lipid accumulation, activated the NRF2 pathway, and up-regulated the mRNA expression of FGF21 in HepG2 cells. Overall, our study showed that THC can improve the antioxidant capacity of liver, which suggests that dietary THC may be used to treat NAFLD.

Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy–lysosomal pathway

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy–lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1–TFEB–ALP axis could represent a novel pharmacological strategy to combat NAFLD.

Inositol hexanicotinate self-micelle solid dispersion is an efficient drug delivery system in the mouse model of non-alcoholic fatty liver disease

Inositol hexanicotinate (IHN) self-micelle solid dispersion (SD) with glycyrrhizic acid (GA) and arabic gum (AG) was prepared by mechanical ball milling process to improve the solubility, stability of amorphous state, and bioavailability of IHN, which enhanced the treatment of IHN on hyperlipidemia and nonalcoholic fatty liver disease (NAFLD). The physicochemical properties of IHN/GA/AG SDs in solid state were characterized by differential scanning calorimetry, X-ray diffraction studies, and scanning electron microscopy. The characteristics of the sample solutions were analyzed by reverse-phase HPLC, particle characterization, critical micelle concentration, and transmission electron microscopy. Further pharmacokinetic study of this SD formulation in rats showed a significant 3.3-fold increase in bioavailability compared to pure IHN. Moreover, biomarkers in serum and liver of NAFLD mice were significantly ameliorated after oral administration of IHN/GA/AG SDs for 15 days. Altogether, these results establish the mechanochemically prepared IHN/GA/AG SDs as an efficacious formulation for the treatment of hyperlipidemia and NAFLD.

The protective effects of trelagliptin on high-fat diet-induced nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) occurs in patients with type 2 diabetes mellitus (T2DM). Trelagliptin is an important member of the Gliptins family, which has been recently licensed for the treatment of T2DM. However, the pharmacological function of trelagliptin in NAFLD has not been previously reported. In this study, we aimed to investigate the roles of trelagliptin in the development of NAFLD in a mouse model. To induce NAFLD disease, C57BL/6 mice were fed a high-fat diet for 10 weeks. Our results indicate that trelagliptin reduced plasma lipid levels in NAFLD mice by reducing triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. Treatment with trelagliptin exhibited an improvement in insulin resistance. More important, trelagliptin improved liver function by reducing alanine transaminase, aspartate transaminase, lactate dehydrogenase, and total bile acid. In addition, trelagliptin ameliorated oxidative stress in the liverof NAFLD mice by reducing malondialdehyde and increasing the levels of reduced glutathione and superoxide dismutase activity. Also, the enzyme-linked immunosorbent assayresultsindicate that trelagliptin-treated mice displayed anti-inflammatory properties by reducing the levels of interleukin 1β(IL-1β), IL-6, and tumor necrosis factor-α. Hematoxylin and eosinand Oil red O staining show that trelagliptin treatment ameliorates liver tissue damage and hepatic lipid deposition. Mechanistically, we found that the administration of trelagliptin reduced the activity of hepatic nuclear factor-κB but increased the activity of AMP-activated protein kinase. These findings suggest that trelagliptin might become a promising therapeutic agent for the treatment of NAFLD.

Withdrawal Notice: Therapeutic Effect of Prdx1 on NAFLD Mice May be Related to the Activation of Nrf-2/HO-1 Pathway

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Abstract 991: Platelets inhibit hepatocellular carcinoma growth in the context of nonalcoholic fatty liver disease

Abstract Purpose: Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HCC) in the US and globally. Platelets have been recognized as an important element of immune system and recently, platelets have been shown to be activated and aggerate in the livers of mice and humans with NAFLD. However, the role of platelets in HCC disease progression in NAFLD is still unknown. Methods: In this study, mice underwent an intrahepatic injection of an HCC cell line and were fed a methionine choline deficient (MCD) diet to generate an HCC NAFLD mouse model. The mouse model was then used to interrogate the interplay between platelets, immune responses and HCC growth within a liver with NAFLD. Anti-GP1bα or clopidogrel were used to deplete or inhibit platelet function and effects on tumor growth and immune cell activation were studied. Platelets express leukotriene receptors, and the possible involvement of leukotrienes was tested with zileuton, a leukotriene inhibitor. Finally, antibody dependent T cell depletion was used to identify the effector cells controlling HCC tumor development. Results: NAFLD mice with HCC treated with clopidogrel showed a close to 3-fold increase in tumor weight compared to vehicle control (50.6g vs 147.8g P&amp;lt;0.05) as well as tumor-to-liver weight ratio (0.05 vs 0.13 P&amp;lt;0.05). CD4+ and CD8+ T-cells from the livers of NAFLD mice showed a 2- to 3-fold increase in CD69+ T-cells compared to non-NAFLD mice (24.2% vs 9.1% P&amp;lt;0.05 in CD4+ and 23.7% vs 8.9% P&amp;lt;0.05 in CD8+, respectively). Interestingly, in NAFLD mice treated with clopidogrel, there was a decrease in CD69+ compared to NAFLD mice treated with control in both CD4+ (18.3% vs 24.2% P&amp;lt;0.05, respectively) and CD8+ (15.32% vs 23.7% P&amp;lt;0.05, respectively) T-cells. Consistently, when NAFLD mice were depleted of platelets with anti-GP1bα, there was a significant decreased in T cell activation compared to IgG control. Similarly, when NAFLD mice were treated with zileuton, there was a decrease in IFN-gamma production in liver isolated CD4+ (5.4% vs 3.1% P=0.12) and CD8+ (23.7% vs 16.7% P&amp;lt;0.05) T-cells compared to vehicle control treated mice. Finally, the clopidogrel treatment increased HCC tumor-to-liver weight ratio was abolished in mice treated with anti-CD8 (0.29 vs 0.23 P&amp;gt;0.05) but not in mice treated with anti-CD4 (0.11 vs 0.16 P&amp;lt;0.05). Conclusion: The results of this study show that in NAFLD livers, platelets increase immune cell activation and inhibit HCC in a CD8+ T-cell dependent manner. The results also suggest that leukotrienes may play a key role in modulating this response, but further studies are required to better characterize this observation. Citation Format: John C. McVey, Chi Ma, Laurence Diggs, Qiong Fu, Tim F. Greten. Platelets inhibit hepatocellular carcinoma growth in the context of nonalcoholic fatty liver disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 991.

Liraglutide Attenuates Non-Alcoholic Fatty Liver Disease in Mice by Regulating the Local Renin-Angiotensin System.

The renin-angiotensin system (RAS) is involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and represents a potential therapeutic target for NAFLD. Glucagon-like peptide-1 (GLP-1) signaling has been shown to regulate the RAS within various local tissues. In this study, we aimed to investigate the functional relationship between GLP-1 and the local RAS in the liver during NAFLD. Wild-type and ACE2 knockout mice were used to establish a high-fat-induced NAFLD model. After the mice were treated with liraglutide (a GLP-1 analogue) for 4 weeks, the key RAS component genes were up-regulated in the liver of NAFLD mice. Liraglutide treatment regulated the RAS balance, preventing a reduction in fatty acid oxidation gene expression and increasing gluconeogenesis and the expression of inflammation-related genes caused by NAFLD, which were impaired in ACE2 knockout mice. Liraglutide-treated HepG2 cells exhibited activation of the ACE2/Ang1-7/Mas axis, increased fatty acid oxidation gene expression, and decreased inflammation, which could be reversed by A779 and AngII. These results indicate that the local RAS in the liver becomes overactivated in response to NAFLD. Moreover, ACE2 knockout increases the severity of liver steatosis. Liraglutide has a negative and antagonistic effect on the ACE/AngII/AT1R axis, a positive impact on the ACE2/Ang1-7/Mas axis, and is mediated through the PI3K/AKT pathway. This may represent a potential new mechanism by which liraglutide improves NAFLD.

Leucine‐Rich Repeat Kinase 2 Promotes β‐Oxidation via the Regulation of CPT1a in Nonalcoholic Fatty Liver Disease

Nonalcoholic steatohepatitis (NASH) occurs in about 30% patients of the nonalcoholic fatty liver disease (NAFLD), and these patients have high risk to liver cirrhosis and liver cancer. However, the etiology of the NASH development in NAFLD patients has not been clearly elucidated. In the NAFLD mice induced by high‐fat diet, we identified that leucine‐rich repeat kinase 2 (LRRK2) was down‐regulated in the livers accompanied with inflammation. Moreover, the treatment of palmitic acid (PA), a fatty acid that induces inflammation, led to down‐regulation of LRRK2 in HepG2 cells. The in vivo and in vitro data suggested the down‐regulation of LRRK2 may be correlated to liver inflammation. In addition, in PA‐treated HepG2 cells, the overexpression of LRRK2 significantly decreased the intracellular level of non‐esterified fatty acids as a result of the up‐regulation of CPT1a, which acts as the rate limiting step of beta‐oxidation. Accumulated studies have demonstrated the increase of CPT1a can enhance the beta‐oxidation to inhibit the PA‐induced inflammation. Interestingly, the overexpression of LRRK2 suppressed the NF‐kB‐mediated pro‐inflammatory pathway and inhibited the secretion of the pro‐inflammatory cytokine TNFa induced by PA treatments. Therefore, our results suggested the anti‐inflammatory effect of LRRK2 may also work through the promotion of beta‐oxidation in the liver. In this study, we firstly observed that LRRK2 was down‐regulated in the livers of NAFLD mice. Furthermore, the overexpression of LRRK2 enhanced the beta‐oxidation and inhibited the inflammation in HepG2 cells. Conversely, the decrease of LRRK2 resulted in the inflammation by suppressing beta‐oxidation in the livers. Therefore, LRRK2 may be a therapy target to improve NAFLD in human.