High-fat And High-cholesterol Research Articles

Remodeling Intestinal Microbiota Alleviates Severe Combined Hyperlipidemia-Induced Nonalcoholic Steatohepatitis and Atherosclerosis in LDLR-/- Hamsters.

Combined hyperlipidemia (CHL) manifests as elevated cholesterol and triglycerides, associated with fatty liver and cardiovascular diseases. Emerging evidence underscores the crucial role of the intestinal microbiota in metabolic disorders. However, the potential therapeutic viability of remodeling the intestinal microbiota in CHL remains uncertain. In this study, CHL was induced in low-density lipoprotein receptor-deficient (LDLR-/-) hamsters through an 8-week high-fat and high-cholesterol (HFHC) diet or a 4-month high-cholesterol (HC) diet. Placebo or antibiotics were administered through separate or cohousing approaches. Analysis through 16S rDNA sequencing revealed that intermittent antibiotic treatment and the cohousing approach effectively modulated the gut microbiota community without impacting its overall abundance in LDLR-/- hamsters exhibiting severe CHL. Antibiotic treatment mitigated HFHC diet-induced obesity, hyperglycemia, and hyperlipidemia, enhancing thermogenesis and alleviating nonalcoholic steatohepatitis (NASH), concurrently reducing atherosclerotic lesions in LDLR-/- hamsters. Metabolomic analysis revealed a favorable liver lipid metabolism profile. Increased levels of microbiota-derived metabolites, notably butyrate and glycylglycine, also ameliorated NASH and atherosclerosis in HFHC diet-fed LDLR-/- hamsters. Notably, antibiotics, butyrate, and glycylglycine treatment exhibited protective effects in LDLR-/- hamsters on an HC diet, aligning with outcomes observed in the HFHC diet scenario. Our findings highlight the efficacy of remodeling gut microbiota through antibiotic treatment and cohousing in improving obesity, NASH, and atherosclerosis associated with refractory CHL. Increased levels of beneficial microbiota-derived metabolites suggest a potential avenue for microbiome-mediated therapies in addressing CHL-associated diseases.

Uric Acid Elevation by Fructose Overload Exacerbates Nash and Atherosclerosis via Oxidative Stress

Background: Nonalcoholic steatohepatitis (NASH) is well associated with an increased risk of cardiovascular disease (CVD), regardless of risk factors for metabolic syndrome. However, intermediary factors between NASH and CVD remain unknown. In recent years, hyperuricemia has been associated not only with gout but also with several other organ diseases, such as hypertension, chronic renal failure, and metabolic syndrome. In addition, hyperuricemia was shown to frequently occur in patients with NASH and could be a risk factor for CVD. Furthermore, serum uric acid (UA) levels have been linked with fructose intake. Objectives: We hypothesized that fructose loading elevates UA levels and exacerbates NASH and atherosclerosis via oxidative stress. Methods: Stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr), between 14 to 24 weeks of age, were divided into two groups and fed a high-fat and high-cholesterol (HFC) diet. In addition to the HFC diet, the fructose group was subjected to 10% fructose loading. The oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed at 25-week-old, followed by blood sampling, animal sacrifice, endothelial function test, blood biochemistry, histopathological staining, xanthine oxidase activity test, and genetic analysis performed at 26-week-old. Results: Fructose loading increased UA and oxidative stress levels. In addition, fructose loading induced insulin resistance. The fructose group exhibited aggravated hepatic fibrosis and lipid deposition, as well as enhanced lipid accumulation in the mesenteric arteries. Conclusion: In the SHRSP5/Dmcr rat model, elevated UA levels were a risk factor for the exacerbation of NASH and atherosclerosis via oxidative stress.

A High-Fat, High-Cholesterol Diet Promotes Intestinal Inflammation by Exacerbating Gut Microbiome Dysbiosis and Bile Acid Disorders in Cholecystectomy.

Patients with post-cholecystectomy (PC) often experience adverse gastrointestinal conditions, such as PC syndrome, colorectal cancer (CRC), and non-alcoholic fatty liver disease (NAFLD), that accumulate over time. An epidemiological survey further revealed that the risk of cholecystectomy is associated with high-fat and high-cholesterol (HFHC) dietary intake. Mounting evidence suggests that cholecystectomy is associated with disrupted gut microbial homeostasis and dysregulated bile acids (BAs) metabolism. However, the effect of an HFHC diet on gastrointestinal complications after cholecystectomy has not been elucidated. Here, we aimed to investigate the effect of an HFHC diet after cholecystectomy on the gut microbiota-BA metabolic axis and elucidate the association between this alteration and the development of intestinal inflammation. In this study, a mice cholecystectomy model was established, and the levels of IL-Iβ, TNF-α, and IL-6 in the colon were increased in mice fed an HFHC diet for 6 weeks. Analysis of fecal BA metabolism showed that an HFHC diet after cholecystectomy altered the rhythm of the BA metabolism by upregulating liver CPY7A1, CYP8B1, and BSEP and ileal ASBT mRNA expression levels, resulting in increased fecal BA levels. In addition, feeding an HFHC diet after cholecystectomy caused a significant dysbiosis of the gut microbiota, which was characterized by the enrichment of the metabolic microbiota involved in BAs; the abundance of pro-inflammatory gut microbiota and related pro-inflammatory metabolite levels was also significantly higher. In contrast, the abundance of major short-chain fatty acid (SCFA)-producing bacteria significantly decreased. Overall, our study suggests that an HFHC diet after cholecystectomy promotes intestinal inflammation by exacerbating the gut microbiome and BA metabolism dysbiosis in cholecystectomy. Our study also provides useful insights into the maintenance of intestinal health after cholecystectomy through dietary or probiotic intervention strategies.

Selective autophagy associated with iron overload aggravates non-alcoholic steatohepatitis via ferroptosis.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that causes cirrhosis and hepatocellular carcinoma. Iron is an essential trace element in the body; however, excess iron can cause tissue damage and dysfunction. Iron overload is often observed in patients with NASH, and the amount of iron accumulated in the liver positively correlates with the histological severity of NASH. Ferroptosis, a novel form of iron-dependent cell death, is caused by the accumulation of lipid peroxidation and oxidative stress and is related to NASH. In addition, ferroptosis is closely related to autophagy, an intracellular self-degradation process. Although autophagy has many beneficial effects, it may also be harmful to the organism, for example, inducing ferroptosis. It is unclear whether iron overload aggravates NASH via autophagy. The aim of this research is to determine the mechanism by which iron overload induces ferroptosis via autophagy and aggravates NASH. Stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr) were divided into two groups and fed a high-fat and high-cholesterol (HFC) diet for eight weeks. Iron dextran was administered to the Fe group in addition to the HFC diet. Blood analysis, histological staining, calcineurin activity assay, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence staining, and electron microscopy were performed. The results showed that iron overload promoted autophagy via nuclear translocation of transcription factor EB (TFEB) and induced ferritinophagy, which is the autophagic degradation of ferritin. In addition, the HFC diet induced lipophagy, the autophagic degradation of lipid droplets. The Fe group also exhibited promoted ferroptosis and aggravated hepatic inflammation and fibrosis. In conclusion, iron overload accelerates ferritinophagy and lipophagy, aggravating NASH pathology via ferroptosis. These findings indicate the therapeutic potential of inhibiting autophagy and ferroptosis for treating NASH.

Stigmasterol Protects Against Steatohepatitis Induced by High-Fat and High-Cholesterol Diet in Mice by Enhancing the Alternative Bile Acid Synthesis Pathway

BackgroundHepatic cholesterol accumulation is a significant risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis. However, the precise mechanism by which stigmasterol (STG) mitigates this process remains unclear. ObjectivesThis study aimed to investigate the potential mechanism underlying the protective effect of STG in mice with NAFLD progressing to steatohepatitis while being fed a high-fat and high-cholesterol (HFHC) diet. MethodsMale C57BL/6 mice were fed an HFHC diet for 16 wk to establish the NAFLD model. Subsequently, the mice received STG or a vehicle via oral gavage while continuing the HFHC diet for an additional 10 wk. The study evaluated hepatic lipid deposition and inflammation as well as the expression of key rate-limiting enzymes involved in the bile acid (BA) synthesis pathways. BAs in the colonic contents were quantified using ultra-performance liquid chromatography-tandem mass spectrometry. ResultsCompared with the vehicle control group, STG significantly reduced hepatic cholesterol accumulation (P < 0.01) and suppressed the gene expression of NLRP3 inflammasome and interleukin-18 (P < 0.05) in the livers of HFHC diet-fed mice. The total fecal BA content in the STG group was nearly double that of the vehicle control group. Additionally, the administration of STG increased the concentrations of representative hydrophilic BAs in the colonic contents (P < 0.05) along with the upregulation of gene and protein expression of CYP7B1 (P < 0.01). Furthermore, STG enhanced the α-diversity of the gut microbiota and partially reversed the alterations in the relative abundance of the gut microbiota induced by the HFHC diet. ConclusionsSTG mitigates steatohepatitis by enhancing the alternative pathway for BA synthesis.

Intermittent fasting alleviates non-alcoholic steatohepatitis by regulating bile acid metabolism and promoting fecal bile acid excretion in high-fat and high-cholesterol diet fed mice.

Intermittent fasting (IF) has a protective role across a wide range of chronic disorders, including obesity, diabetes and cardiovascular disease, but its protection against non-alcoholic steatohepatitis (NASH) is still lacking. This study sought to investigate how IF alleviates NASH by regulating gut microbiota and bile acids (BAs) composition. Male C57BL/6 mice were fed a high-fat and high-cholesterol (HFHC) diet for 16 weeks to establish a NASH model. Mice then continued HFHC feeding and were treated with or without every other day fasting for 10 weeks. Hepatic pathology was assessed using hematoxylin-eosin staining. Gut microbiota of the cecum were profiled using 16S rRNA gene sequencing and the levels of BAs in serum, colon contents, and feces were measured using UPLC-MS/MS. Results indicated that IF significantly decreased murine body weight, insulin resistance, hepatic steatosis, ballooning, and lobular inflammation. IF reshaped the gut microbiota, reduced the accumulation of serum BAs, and increased total colonic and fecal BAs. Moreover, IF increased the expression of cholesterol 7α-hydroxylase 1 in liver, but decreased the expressions of both farnesoid-X-receptor and fibroblast growth factor 15 in the ileum. IF alleviated NASH by regulating bile acid metabolism and promoting fecal bile acid excretion. This article is protected by copyright. All rights reserved.

Antioxidant action of xanthine oxidase inhibitor febuxostat protects the liver and blood vasculature in SHRSP5/Dmcr rats.

Xanthine oxidase (XO) generates reactive oxygen species during uric acid production. Therefore, XO inhibitors, which suppress oxidative stress, may effectively treat non-alcoholic steatohepatitis (NASH) and atherosclerosis via uric acid reduction. In this study, we examined the antioxidant effect of the XO inhibitor febuxostat on NASH and atherosclerosis in stroke-prone spontaneously hypertensive 5 (SHRSP5/Dmcr) rats. SHRSP5/Dmcr rats were divided into three groups: SHRSP5/Dmcr + high-fat and high-cholesterol (HFC) diet [control group, n = 5], SHRSP5/Dmcr + HFC diet + 10% fructose (40 ml/day) [fructose group, n = 5], and SHRSP5/Dmcr + HFC diet + 10% fructose (40 ml/day) + febuxostat (1.0 mg/kg/day) [febuxostat group, n = 5]. Glucose and insulin resistance, blood biochemistry, histopathological staining, endothelial function, and oxidative stress markers were evaluated. Febuxostat reduced the plasma uric acid levels. Oxidative stress-related genes were downregulated, whereas antioxidant factor-related genes were upregulated in the febuxostat group compared with those in the fructose group. Febuxostat also ameliorated inflammation, fibrosis, and lipid accumulation in the liver. Mesenteric lipid deposition decreased in the arteries, and aortic endothelial function improved in the febuxostat group. Overall, the XO inhibitor febuxostat exerted protective effects against NASH and atherosclerosis in SHRSP5/Dmcr rats.

Rapid and Reliable Steatosis Rat Model Shrsp5-Dmcr for Cold Storage Experiment

Interventions for liver grafts with moderate macrovesicular steatosis have been important in enlarging donor pools. Here, we tested a high-fat and cholesterol (HFC) diet to create a steatosis model for cold hepatic preservation and reperfusion experiments. The aim of the present study was to assess the steatosis model's reliability and to show the resulting graft's quality for cold preservation and reperfusion experiment. Male SHRSP5-Dmcr rats were raised with an HFC diet for up to 2 weeks. The fat content was evaluated using magnetic resonance imaging (MRI) proton density fat fraction (PDFF). The nonalcoholic fatty liver disease activity score (NAS) was evaluated after excision. Steatosis created by 2 weeks of HFC diet was subjected to 24-hour cold storage in the University of Wisconsin and the original test solution (new sol.). Grafts were applied to isolated perfused rat livers for simulating reperfusion. The NAS were 2.2 (HFC 5 days), 3.3 (HFC 1 week), and 5.0 (HFC 2 weeks). Ballooning and fibrosis were not observed in any group. An MRI-PDFF showed 0.2 (HFC 0 days), 12.0 (HFC 1 week), and 18.9 (HFC 2 weeks). The NAS and MRI-PDFF values correlated. Many indices in the isolated perfused rat liver experiment tended to improve in the new sol. group but were insufficient. Although the new sol. failed to be effective, it acted at multiple sites under difficult conditions. In conclusion, the HFC diet for 2 weeks in SHRSP5-Dmcr rats, together with MRI-PDFF evaluation, is a reliable method for creating simple steatosis and provides good-quality cold preservation and reperfusion experiments.

In Vivo Hypolipidemic Effects and Antioxidant Capacity of Pinus morrisonicola Hay Extracts by Supercritical Carbon Dioxide Extraction

Pinus morrisonicola hay (PM) is a pine tree unique to Taiwan, whose needles are used as traditional medicine and as functional drink. PME3-1 was made using supercritical extraction to evaluate the prevention of hyperlipidemia. This study explored the hypolipidemic effect of PME3-1 on hamsters on a high fat and cholesterol (HFC) diet. Three groups of hamsters were fed with PME3-1 (0.2, 1.0, and 5.0 mg/kg bw). After feeding for eight weeks, PME3-1 reduced the serum cholesterol, triglyceride levels, the low-density lipoprotein cholesterol/high-density lipoprotein cholesterol (LDL/HDL) ratio, and the swelling of the liver and kidney significantly (p &lt; 0.05). In addition, feeding the hamsters with 5.0 mg/kg bw of PME3-1 could significantly reduce their total lipid (TL) content, total cholesterol (TC) content, total triglyceride (TG) content, and the HMG-CoA reductase activity in the liver (p &lt; 0.05). Meanwhile, the antioxidant enzymes in the liver, glutathione peroxidase (GPx) and glutathione S-transferase (GST), can also improve, promoting the excretion of triglycerides (TG) and total cholesterol (TC) in the feces (p &lt; 0.05). Therefore, these results confirm that PME3-1 hypolipidemic and antioxidant regulating functions in vivo.

Sodium cholate ameliorates nonalcoholic steatohepatitis by activation of FXR signaling.

Non-alcoholic steatohepatitis (NASH) has become a major cause of liver transplantation and liver-associated death. The gut-liver axis is a potential therapy for NASH. Sodium cholate (SC) is a choleretic drug whose main component is bile acids and has anti-inflammatory, antifibrotic, and hepatoprotective effects. This study aimed to investigate whether SC exerts anti-NASH effects by the gut-liver axis. Mice were fed with an high-fat and high-cholesterol (HFHC) diet for 20 weeks to induce NASH. Mice were daily intragastric administrated with SC since the 11th week after initiation of HFHC feeding. The toxic effects of SC on normal hepatocytes were determined by CCK8 assay. The lipid accumulation in hepatocytes was virtualized by Oil Red O staining. The mRNA levels of genes were determined by real-time quantitative PCR assay. SC alleviated hepatic injury, abnormal cholesterol synthesis, and hepatic steatosis and improved serum lipid profile in NASH mice. In addition, SC decreased HFHC-induced hepatic inflammatory cell infiltration and collagen deposition. The target protein-protein interaction network was established through Cytoscape software, and NR1H4 [farnesoid x receptor (FXR)] was identified as a potential target gene for SC treatment in NASH mice. SC-activated hepatic FXR and inhibited CYP7A1 expression to reduce the levels of bile acid. In addition, high-dose SC attenuated the abnormal expression of cancer markers in NASH mouse liver. Finally, SC significantly increased the expression of FXR and FGF15 in NASH mouse intestine. Taken together, SC ameliorates steatosis, inflammation, and fibrosis in NASH mice by activating hepatic and intestinal FXR signaling so as to suppress the levels of bile acid in NASH mouse liver and intestine.

Protective and Adverse Roles of DDX3X in Different Cell Types in Nonalcoholic Steatohepatitis Progression

Persistent hepatic cellular metabolic stress and liver inflammatory stimuli are key signatures of nonalcoholic steatohepatitis (NASH). DDX3X is a vital molecule involved in cell fate decisions in both pro-survival stress granule (SG) and pro-death NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome assembly in response to stress signals. However, the role of DDX3X in NASH remains unclear. We characterized the cell type-specific roles of DDX3X in NASH. Human liver tissues from NASH patients and normal control subjects were collected to assess DDX3X expression and distribution. Nutritional steatohepatitis models were constructed by feeding macrophage-specific DDX3X knockout (DDX3X ΔMφ ), hepatocyte-specific DDX3X knockout (DDX3X Δhep ), and wild-type control (DDX3X fl/fl ) mice a high-fat and high-cholesterol (HFHC) diet, a methionine- and choline-deficient (MCD) diet, and a high-fat/high-iron/high-fructose/high-cholesterol, low-methionine, and choline-deficient (HFHIHFHC-MCD) diet. The study demonstrated that DDX3X was predominantly expressed in macrophages and hepatocytes in control liver tissues, and its expression was down-regulated in patients or mice with NASH. Compared to DDX3X fl/fl littermates, DDX3X ΔMφ mice showed improved liver histology in nutritional steatohepatitis models. Loss of macrophage DDX3X inhibited NLRP3 inflammasome-mediated pyroptosis, causing anti-inflammatory M2 polarization and alleviating hepatocyte steatohepatitic changes. DDX3X Δhep mice developed marked steatohepatitis in multiple nutritional steatohepatitis models compared to DDX3X fl/fl littermates. DDX3X-deleted hepatocytes showed impaired SG assembly, leading to increased sensitivity and intolerance to metabolic stimulation and resultant steatohepatitis. In conclusion, DDX3X plays opposite roles in different cell types during the progression of NASH. A better understanding of the cell-specific differences in the crosstalk between SG formation and NLRP3 activation is crucial for developing prospective targeted DDX3X inhibitors for the treatment of NASH.

Dietary Cholic Acid Exacerbates Liver Fibrosis in NASH Model of Sprague-Dawley Rats Fed a High-Fat and High-Cholesterol Diet.

Background: Recently, we established a novel rodent model of nonalcoholic steatohepatitis (NASH) with advanced fibrosis induced by a high-fat and high-cholesterol (HFC) diet containing cholic acid (CA), which is known to cause hepatotoxicity. The present study aimed to elucidate the direct impact of dietary CA on the progression of NASH induced by feeding the HFC diet. Methods: Nine-week-old male Sprague–Dawley rats were randomly assigned to receive a normal, HFC, or CA-supplemented (0.1%, 0.5% or 2.0%, w/w) HFC diet for 9 weeks. Results: Histopathological assessment revealed that the supplementation of CA dose-dependently aggravated hepatic steatosis, inflammation, and fibrosis, reaching stage 4 cirrhosis in the 2.0% CA diet group. In contrast, the rats that were fed the HFC diet without any added CA developed mild steatosis and inflammation without fibrosis. The hepatic cholesterol content and mRNA expression involved in inflammatory response and fibrogenesis was higher in a CA dose-dependent manner. The hepatic chenodeoxycholic acid levels were higher in 2.0% CA diet group than in the control, although hepatic levels of total bile acid and CA did not increase dose-dependently with CA intake. Conclusion: Adding CA to the HFC diet altered bile acid metabolism and inflammatory response and triggered the development of fibrosis in the rat liver.

Is a Ketogenic Diet Superior to a High-Fat, High-Cholesterol Diet Regarding Testicular Function and Spermatogenesis?

The study aimed to determine effects of a ketogenic diet on metabolic dysfunction, testicular antioxidant capacity, apoptosis, inflammation, and spermatogenesis in a high-fat and high-cholesterol diet-induced obese mice model. Forty-two male C57BL/6 mice were fed either a normal diet (NC group) or a high-fat and high-cholesterol (HFC) diet (HFC group) for 16 weeks, and mice from the HFC group were later randomly divided into two groups: the first were maintained on the original HFC diet, and the second were fed a medium-chain triacylglycerol (MCT)-based ketogenic diet for 8 weeks (KD group). A poor semen quality was observed in the HFC group, but this was eliminated by the ketogenic diet. Both the HFC and KD groups exhibited enhanced apoptosis protein expressions in testis tissue, including caspase 3 and cleaved PARP, and higher inflammation protein expressions, including TNF-α and NF-κB. However, the KD group exhibited a statistically-significant reduction in lipid peroxidation and an increased glutathione peroxidase level as compared with the HFC group. The HFC diet induced obesity in mice, which developed body weight gain, abnormal relative organ weights, metabolic dysfunction, and liver injury. Overall, the results showed that a ketogenic diet attenuated oxidative stress and improved the semen quality reduced by the HFC diet.

Cholic acid supplementation accelerates the progression of nonalcoholic fatty liver disease to the procarcinogenic state in mice fed a high-fat and high-cholesterol diet

Nonalcoholic fatty liver disease (NAFLD) is one of the major causes of hepatocellular carcinoma (HCC). Although the intracellular cholesterol accumulation has been demonstrated to regulate the gene expression responsible for steatohepatitis, the role played by cholesterol in the development of NAFLD-associated HCC has not been fully elucidated. In this study, using microarray analysis, we investigated the molecular mechanisms governing cholesterol-mediated progression of NAFLD. To ensure hepatic cholesterol accumulation, either a high-fat and high-cholesterol (HFHC) diet or a high-fat and high-cholesterol with cholic acid (HFHCCA) diet was fed to diethylnitrosamine (DEN)-injected C57BL/6J mice for 10 weeks. While an HFHC diet increased hepatic triglyceride levels, an HFHCCA diet induced hepatic cholesterol accumulation by reducing bile acid biosynthesis in DEN-injected mice. Livers from both HFHC and HFHCCA groups exhibited increases in steatosis and necrosis; however, histological features of HCC were not observed in any of the experimental groups. Hepatic gene expression profile of the HFHCCA group was different from those of other groups. Functional analysis showed that cholic acid supplementation upregulated differentially expressed genes (DEGs) associated with inflammation, proliferation, apoptosis, chemical drug response, and cancer signaling pathway. Downregulated DEGs were associated with steroid metabolism, mitochondrial function, and oxidative phosphorylation pathway. Furthermore, hepatic cholesterol accumulation lowered the expression of DEGs associated with energy and macronutrient metabolism, especially amino acid metabolism. In this study, the results of a global gene expression profile demonstrated that feeding the HFHCCA diet to DEN-injected mice accelerated the carcinogenic progression of NAFLD, implicating the critical role played by hepatic accumulation of cholesterol.

A radioiodinated FR-β-targeted tracer with improved pharmacokinetics through modification with an albumin binder for imaging of macrophages in AS and NAFL.

The formation of advanced plaques, which is characterized by the uninterrupted aggregation of macrophages with high expression of folate receptor-β (FR-β), is observed in several concomitant metabolic syndromes. The objective of this study was to develop a novel FR-β-targeted single-photon emission computed tomography (SPECT) radiotracer and validate its application to the noninvasive detection of atherosclerosis (AS) plaque and non-alcoholic fatty liver (NAFL). Two radioiodinated probes, [131I]IPBF and [131I]IBF, were developed, and cell uptake studies were used to identify their specific targets for activated macrophages. Biodistribution in normal mice was performed to obtain the pharmacokinetic information of the probes. Apolipoprotein E knockout (ApoE-/-) mice with atherosclerotic aortas were induced by a high-fat and high-cholesterol (HFHC) diet. To investigate the affinity of radiotracers to FR-β, Kd values were determined using in vitro assays. In addition, the assessments of the aorta in the ApoE-/- mice at different stages were performed using in vivo SPECT/CT imaging, and the findings were compared by histology. Both [131I]IPBF and [131I]IBF were synthesized with > 95% radiochemical purity and up to 3MBq/nmol molar activity. In vitro assay of [131I]IPBF showed a moderate binding affinity to plasma proteins and specific uptake in activated macrophages. The prolonged blood elimination half-life (t1/2z) of [131I]IPBF (8.14h) was observed in a pharmacokinetic study of normal mice, which was significantly longer than that of [131I]IBF (t1/2z = 2.95h). As expected, the Kd values of [131I]IPBF and [131I]IBF in the Raw 264.7 cells were 43.94 ± 9.83nM and 61.69 ± 15.19nM, respectively. SPECT imaging with [131I]IPBF showed a high uptake in advanced plaques and NAFL. Radioactivity in excised aortas examined by ex vivo autoradiography further confirmed the specific uptake of [131I]IPBF in high-risk AS plaques. In summary, we reported a proof-of-concept study of an albumin-binding folate derivative for macrophage imaging. The FR-β-targeted probe, [131I]IPBF, significantly prolongs the plasma elimination half-life and has the potential for the monitoring of AS plaques and concomitant fatty liver.

A Combination of Geniposide and Chlorogenic Acid Combination Ameliorates Nonalcoholic Steatohepatitis in Mice by Inhibiting Kupffer Cell Activation.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing worldwide. Activation of Kupffer cells (KCs) is central to the development of diet-induced NASH. We investigated whether a combination of two active chemical components, geniposide and chlorogenic acid (GC), at a specific ratio (67 : 1), ameliorates diet-induced NASH and the underlying mechanisms involved. C57BL/6J mice exposed to a high-fat and high-cholesterol (HFHC) diet containing cholesterol, choline, and high-sugar drinking water, as well as RAW264.7 cells stimulated with lipopolysaccharide (LPS) were studied. The combination exerted a therapeutic effect on HFHC-induced NASH in mice. Simultaneously, GC was found to reduce the expression of cytokines secreted by hepatic macrophages, including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, monocyte chemotactic protein 1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, GC reduced the number of KCs expressing F4/80. Furthermore, TNF-α, inducible nitric oxide synthase (INOS), IL-1β, and IL-6 mRNA and TNF-α protein expression levels were suppressed upon GC treatment in RAW264.7 cells. Our findings suggest that GC has a strong anti-inflammatory effect in NASH, and this effect can be attributed to the suppression of KC activity in the liver.

Olive leaf powder prevents nonalcoholic steatohepatitis in Sprague–Dawley rats fed a high-fat and high-cholesterol diet

<h2>Summary</h2><h3>Background and aims</h3> The pathogenesis of nonalcoholic steatohepatitis (NASH) is multifactorial, and oxidative stress and the activation of inflammatory cytokines may play an important role in the progression of NASH. Olive leaves contain polyphenols, such as oleuropein, and have been reported to possess a wide range of pharmacological properties, including anti-oxidative and anti-inflammatory effects. Thus, we hypothesized that olive leaf extract might have a preventive effect on NASH progression. <h3>Methods</h3> Nine-week-old male Sprague–Dawley rats were fed a high-fat and high-cholesterol (HFC) diet with or without olive leaf powder (OLP; n = 6–7/group) for 9 weeks. The histopathology, serology, and expression of cholesterol/lipid metabolism-, fibrosis-, inflammation-, and oxidative stress-related genes in the liver were evaluated. <h3>Results</h3> The rats fed a HFC diet supplemented with 0.8% (w/w) of OLP showed lower levels of serum total cholesterol and alanine aminotransferase, a lower concentration of hepatic cholesterol, and a significantly higher level of adiponectin than the rats fed a HFC diet without OLP. Histopathologically, rats fed a HFC diet supplemented with 0.8% (w/w) of OLP showed less hepatic inflammation, fibrosis, and thioredoxin than rats fed a HFC diet without OLP. <h3>Conclusions</h3> Our results suggest that OLP possesses a preventive effect against NASH progression, presumably due to the elevated serum adiponectin levels and its anti-oxidative properties. Because this preventive effect was not dose-dependent, further studies are needed to investigate the optimal dose of OLP for obtaining the maximum preventive effects against NASH, and to elucidate the detailed pharmacological mechanism(s) of OLP.

Beneficial effects of elafibranor on NASH in E3L.CETP mice and differences between mice and men

Non-alcoholic steatohepatitis (NASH) is the most rapidly growing liver disease that is nevertheless without approved pharmacological treatment. Despite great effort in developing novel NASH therapeutics, many have failed in clinical trials. This has raised questions on the adequacy of preclinical models. Elafibranor is one of the drugs currently in late stage development which had mixed results for phase 2/interim phase 3 trials. In the current study we investigated the response of elafibranor in APOE*3Leiden.CETP mice, a translational animal model that displays histopathological characteristics of NASH in the context of obesity, insulin resistance and hyperlipidemia. To induce NASH, mice were fed a high fat and cholesterol (HFC) diet for 15 weeks (HFC reference group) or 25 weeks (HFC control group) or the HFC diet supplemented with elafibranor (15 mg/kg/d) from week 15–25 (elafibranor group). The effects on plasma parameters and NASH histopathology were assessed and hepatic transcriptome analysis was used to investigate the underlying pathways affected by elafibranor. Elafibranor treatment significantly reduced steatosis and hepatic inflammation and precluded the progression of fibrosis. The underlying disease pathways of the model were compared with those of NASH patients and illustrated substantial similarity with molecular pathways involved, with 87% recapitulation of human pathways in mice. We compared the response of elafibranor in the mice to the response in human patients and discuss potential pitfalls when translating preclinical results of novel NASH therapeutics to human patients. When taking into account that due to species differences the response to some targets, like PPAR-α, may be overrepresented in animal models, we conclude that elafibranor may be particularly useful to reduce hepatic inflammation and could be a pharmacologically useful agent for human NASH, but probably in combination with other agents.

Therapeutic roles of a natural herbal product on ameliorating hepatic steatosis via gut dysbacteriosis modulating in treatment of nonalcoholic fatty liver disease in mice

BackgroundThe gut microbiota has well documented as an important player in mediating the development of nonalcoholic fatty liver disease (NAFLD). A patent natural herbal product, compound (Fufang) Zhenzhu Tiaozhi Capsule (FTZ), composed of eight Chinese medical herbs, has prescribed for the prevention and treatment of hyperlipidemia and NAFLD in China for over a decade. Aim of the studyTo investigate gut microbiota-modulated therapeutic potential of FTZ on mice models of NAFLD. MethodsHPLC analysis performed for identifying and quantifying phytochemical constituents of FTZ. The therapeutic potentials on NAFLD were assessed by hepatic histological examination and biochemical assays of liver enzymes. The composition of the gut microbiota was analyzed by 16S rRNA sequencing. Lipid synthesis-related genes were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Correlations differentially regulated bacterial taxa mediated by FTZ intervention and the clinical parameters of NAFLD obtained by Spearman's correlation analysis. ResultsSignificantly therapeutic potentials on dysbiosis of the gut microbiota modulating in high-fat and high-cholesterol (HFHC) diet-induced mice models of NAFLD revealed after FTZ intervention. Downtrends of abnormal parameters of NAFLD characterized by increased body weight (BW), fat contents, levels of total cholesterol (TC), triglyceride (TG) both the serum and liver tissue of the mice, and the activities of alanine aminotransferase (ALT), aspartate transaminase (AST) shown after FTZ intervention. Meanwhile, the over expressions of lipid synthesis-related genes in mice were down-regulated after FTZ treatment. FTZ modulated the gut microbiota composition, which increased the abundance of Bacteroidetes and reduced the ratio of Firmicutes/Bacteroidales indicated by 16S rRNA sequence data analysis. Parallelly, the increased abundance of Bacteroidetes significantly correlated with down-regulated expressions of lipogenic genes of SCD1, FAS, CD36 and C/EBP-α by FTZ intervention. ConclusionNatural herbal product of FTZ attenuated hepatic steatosis via modulating the gut microbiota in mice models of NAFLD, the pathway of FTZ down-regulated expressions of lipogenic genes of SCD1, FAS, CD36 and C/EBP-α, thereby inhibiting liver fat biosynthesis, is considered one of the important mechanisms of FTZ in preventing and treating NAFLD.

Gut Akkermansia muciniphila ameliorates metabolic dysfunction-associated fatty liver disease by regulating the metabolism of L-aspartate via gut-liver axis

ABSTRACT The gut bacterium Akkermansia muciniphila has been increasingly recognized for its therapeutic potential in treating metabolic disorders, including obesity, diabetes, and metabolicdysfunction-associated fatty liver disease (MAFLD). However, its underlying mechanism involved in its well-known metabolic actions needs further evaluation. The present study explored the therapeutic effect and mechanism of A. muciniphila in intervening MAFLD by using a high-fat and high-cholesterol (HFC) diet induced obese mice model. Mice treated with A. muciniphila efficiently reversed MAFLD in the liver, such as hepatic steatosis, inflammatory, and liver injury. These therapeutic effects persisted after long-term drug withdrawal and were slightly weakened in the antibiotics-treated obese mice. A. muciniphila treatment efficiently increased mitochondrial oxidation and bile acid metabolism in the gut-liver axis, ameliorated oxidative stress-induced cell apoptosis in gut, leading to the reshaping of the gut microbiota composition. These metabolic improvements occurred with increased L-aspartate levels in the liver that transported from the gut. The administration of L-aspartate in vitro or in mice displayed the similar beneficial metabolic effects mentioned above and efficiently ameliorated MAFLD. Together, these data indicate that the anti-MAFLD activity of A. muciniphila correlated with lipid oxidation and improved gut–liver interactions through regulating the metabolism of L-aspartate. A. muciniphila could be a potential agent for clinical intervention in MAFLD.