Non-alcoholic Fatty Liver Disease In Rats Research Articles

Effect of Atractylodes lancea on nonalcoholic fatty liver disease rat induced by high fat - high fructose diet

Non-alcoholic fatty liver disease (NAFLD) induced by long term high-fat and high-fructose diet has led to serious medical problems such as non-alcoholic steatohepatitis or cirrhosis. NAFLD has related to obesity, insulin resistance or type Ⅱ diabetes induced by western diet. With the increasing the incidence of obesity and NFALD due to western diet consisting of high-fat and high-fructose diets, research of the treatment or prevention of NFALD due to western diets is urgently needed. Atractylodes lancea (AL) is traditional Chinese medicine and has been used for diuretic, sedation or antibacterial effect. To investigate effect of AL on NAFLD rat model induced by high-fat and high fructose diet, present study was performed. To induce NAFLD, wistar rats, which were 9-weeks-old, were fed with 45 kcal% high fat diet and 10% fructose water daily for 16 weeks. The olmesartan and atractylodes lancea were treated by oral administration for 8 weeks every day. The groups were composed of 5 groups; control (CON, n=9) fed with 10 kcal% general diet, non-alcoholic fatty liver disease group (NFD, n=9) induced by 45 kcal% high fat diet and 10% fructose diet, NFD group with 10 mg/kg/day Olmesartan orally (OLM, n=9), NFD group with 100 mg/kg/day AL (ALL, n=9) and NFD group with 200 mg/kg/day AL (ALH, n=9). AL decreased body weight, liver weight and epididymal fat pads weight compared with NFD group induced by high-fat and high fructose diet. In serum biomarkers, glucose level of plasma, triglyceride level and total cholesterol level in plasma were increased in NFD group compared with CON. AL restored the serum biomarkers including glucose, or triglycerides and total cholesterol level in serum. The plasma HDL-Cholesterol level, which was decreased in NFD group, attenuated by AL treatment. The atherogenic coeffient was upregulated by chronic high-fat and high-fructose diet, however, the atherogenic coeffient was significantly restored in AL treatment group. Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly deteriorated by long term high-fat and high-fructose diet. However, the GOT and GPT level in serum were alleviated in AL treatment groups. Oil red O staining showed that the lipid accumulation in the liver was increased in NFD group compared with CON. However, the lipid accumulation was restored by AL treatment groups. The fibrosis in liver was deteriorated in NFD induced by chronic high-fat and high fructose diet, however, the AL inhibited collagen deposition in liver. Also, AL suppressed the mRNA expressions of TGF-β, collagen Ⅰ and collagen Ⅲ in liver compared with NFD group. In liver tissue, the LXR and SREBP-1c mRNA expressions were downregulated in ALH group. Taken together, these results showed that AL could be potential supplements for NAFLD induced by long term high-fat and high-fructose diet by regulating fibrosis and LXR/SREBP-1c signaling pathway in liver. This study was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIP) (2017R1A5A2015805) (2022R1A6A3A01087272). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Positive effect of Limosilactobacillus plantarum grx16 on nonalcoholic fatty liver disease in rats

We aimed to investigate the potential and mechanism of probiotics as digestive enzyme inhibitors to alleviate nonalcoholic fatty liver disease (NAFLD). We compared the level of NAFLD in rats before and after Limosilactobacillus plantarum grx16 intervention, as well as changes in gut digestive enzymes and inflammatory factors. L. plantarum grx16 inhibited the absorption of starch and triglycerides by inhibiting the activity of gut amylase and lipase to alleviate insulin resistance and reduce lipid accumulation in the body, thereby alleviating the inflammatory response, and thus alleviating NAFLD in rats.

In silico and in vivo demonstration of the regulatory mechanism of Qi-Ge decoction in treating NAFLD

Background Nonalcoholic fatty liver disease (NAFLD), a chronic and progressive liver disease, often causes steatosis and steatohepatitis. Qi-Ge decoction (QGD) shows a good effect against NAFLD in the clinic. But the molecular mechanism for QGD in improving NAFLD is unknown. Purpose This study explored the molecular mechanism of QGD in NAFLD model rats using comprehensive network pharmacology, molecular docking and in vivo verification strategies. Methods Active components and targets of QGD were obtained from public database. The overlapped genes between QGD and NAFLD targets were analyzed by enrichment analysis. Active components and targets were used to predict molecular docking analysis. Finally, seven key targets were screened out and the gene expression were verified in the NAFLD rat’s liver tissues after QGD treatment. Results Fifty-eight common QGD therapeutic targets were associated with NAFLD. Molecular docking demonstrated that seven targets had strong binding ability for the corresponding active ingredients. GO analysis identified 18 biological process entries, which were mainly related to regulation of lipid storage, lipid localization and peptide transport. KEGG analysis identified multiple signaling pathways, which were mainly associated with tumor necrosis factor signaling and NAFLD. In vivo data confirmed that the effect of QGD in the treatment of NAFLD was mainly exerted through improving liver steatosis and inflammatory cell infiltration. Additionally, QGD upregulated the expression of MAPK8 and ESR1 and downregulated the transcriptional expression of IL6, VEGFA, CASP3, EGFR and MYC. These targets may affect lipid metabolism by regulating lipid storage and inflammation. Conclusion The integration of results obtained in silico and in vivo indicated that QGD regulates multiple targets, biological processes and signaling pathways in NAFLD, which may represent a complex molecular mechanism by which QGD improves NAFLD. Key messages QGD intervention is related to multiple biological processes such as inflammation, oxidation and cell apoptosis in NAFLD. Lipid and atherosclerosis, TNF signaling pathway, IL-17 signaling pathway, non-alcoholic fatty liver disease and AGE-RAGE signaling pathway in diabetic complications are the main pathways for QGD intervention NAFLD. The active components of QGD can form good binding with relevant target proteins through intermolecular forces, exhibiting excellent docking activity.

Combined therapy with pioglitazone and FGF21 mRNA synergistically ameliorates metabolic disorders in NAFLD rats

BackgroundCurrent study aims to investigate the ameliorative effect of pioglitazone (PIO) combined with mRNA encoding FGF21 (termed mFGF21) on the metabolic disorders in rats with nonalcoholic fatty liver disease (NAFLD) and its potential mechanism. MethodsIn vitro functional activity of FGF21 protein expressed by mFGF21 was evaluated in human adipose-derived stem cells (hASCs). The pharmacokinetic profiles of FGF21 protein expressed by mFGF21 were investigated in normal SD rats and NAFLD rats, respectively. ResultsAs the results, it showed that the PIO could enhanced in vitro functional activity of FGF21 protein expressed from mFGF21 in hASCs. Not only that, mFGF21 turns the body into a processing plant for endogenous protein expression, which enhanced the pharmacokinetic profiles of FGF21 proteins. Combined treatment with PIO and mFGF21 significantly reduced body weight, fasting blood glucose levels, insulin levels and lipid metabolism in NAFLD rats compared with control or both two monotherapy groups. The results of H&E staining and Western blot revealed that combined treatment with PIO and mFGF21 significantly decreased hepatic fat accumulation in NAFLD rats by activating the SHP1/AMPK signaling pathway. ConclusionsOur finding collectively demonstrated that PIO and mFGF21 combination therapy could synergistically ameliorate metabolic disorders in NAFLD rats.

Vitamin D improves hepatic steatosis in NAFLD via regulation of fatty acid uptake and β-oxidation.

The study aimed to explore the association of serum 25(OH)D3 and hepatic steatosis in non-alcoholic fatty liver disease (NAFLD) patients and to determine whether the effect of vitamin D (VD) is mediated by activation of the peroxisome proliferator-activated receptor α (PPARα) pathway. The study contained a case-control study, in vivo and in vitro experiments. A case-control study was conducted to compare serum parameters between NAFLD patients and controls and to evaluate the association of 25(OH)D3 and NAFLD. In vivo study, male Wistar rats were randomly divided into control and model groups, fed a standard chow diet and a high-fat diet (HFD), respectively, for 7 weeks to generate an NAFLD model. Then, the rats were treated with VD and a PPARα antagonist (MK886) for 7 weeks. Tissue and serum were collected and assessed by biochemical assays, morphological analysis, histological analysis, and western blot analysis. In vitro, HepG2 cells were incubated with oleic acid (OA) to induce steatosis, which was evaluated by staining. HepG2 cells were pretreated with MK886 followed by calcitriol treatment, and differences in lipid metabolism-related proteins were detected by western blot. NAFLD patients were characterized by impaired liver function, dyslipidemia, and insulin resistance. Serum 25(OH)D3 was negatively associated with alanine aminotransferase (ALT) in NAFLD. VD deficiency was a risk factor for patients with no advanced fibrosis. Adequate VD status (25(OH)D3 >20 ng/mL) had a protective effect in patients after adjustment for confounding variables. NAFLD rats showed hyperlipidemia with severe hepatic steatosis, systematic inflammation, and lower serum 25(OH)D3. VD treatment ameliorated hepatic steatosis both in NAFLD rats and OA-induced HepG2 cells. Further, MK886 inhibited the anti-steatosis effect of VD. The study revealed that an adequate VD level may act as a protective factor in NAFLD and that VD may alleviate hepatic steatosis via the PPARα signaling pathway.

The Effect of the Saffron Intervention on NAFLD Status and Related Gene Expression in a Rat Model.

According to the worldwide increasing prevalence of non-alcoholic fatty liver disease (NAFLD), the present study aimed to investigate the mechanism effects of saffron consumption on preventing NAFLD in a rat model. In an experimental study, 12 rats were randomly divided into 2 groups to be evaluated in the prevention phase for 7 weeks. In the prevention phase, the animals were randomly assigned to either fed HFHS + 250 mg/kg saffron (S) or fed with HFHS. Afterward, parts of the liver were excised for histopathologic examination. Plasma concentrations of ALT, AST, GGT, ALP, serum lipids, insulin concentrations, plasma glucose, hs-CRP, and TAC were measured. Moreover, Also, the gene expression of 6 target genes was evaluated, including FAS, ACC1, CPT1 ،PPARα ،DGAT2, and SREBP 1-c at the beginning and end of the study. Also, the differences among groups were evaluated by the Mann-Whitney test for non-normal data and the independent t test for normal data. The prevention phase groups have a significant elevation in body weight ( P = 0.034) and food intake (P = 0.001) of the HFHS group versus HFHS + 250 mg/kg S group. Also, there was a significant difference between groups 1 and 2 for ALT (P = 0.011) and AST (P = 0.010), and TG (P = 0.040). The HFHS group had higher plasma levels of FBS (P = 0.001), insulin (P = 0.035), HOMA-IR (P = 0.032), and lower TAC (P = 0.041) versus the HFHS+ S group. Also, the difference between HFHS + 250 mg/kg S and HFHS for PPARα gene expression was significant (P = 0.030). The present study showed that consumption of saffron could prevent developing NAFLD in rats at least partially through modulation in gene expression of PPARα.

Acupuncture improved hepatic steatosis in HFD-induced NAFLD rats by regulating intestinal microbiota.

Intestinal dysbiosis has been increasingly implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Acupuncture has been shown to have beneficial effects on NAFLD, but the mechanism is not yet clear. This study explores the potential beneficial effects of acupuncture on intestinal microbiota in NAFLD. An NAFLD model in Sprague Dawley rats was established using a high-fat diet (HFD) for 10 weeks. NAFLD rats were randomly divided into control, model, and acupuncture groups. Following acupuncture treatment over 6 weeks, automated biochemical analysis was used to measure serum lipid metabolism parameters, including levels of alanine transferase, aspartate transferase, alkaline phosphatase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The level of serum inflammatory factors interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) were measured by enzyme-linked immunosorbent assay. The characteristics of steatosis were evaluated using quantitative computed tomography, hematoxylin and eosin staining, and Oil Red O staining in the liver, while the intestinal microbiota was determined using 16S rRNA gene sequencing. Acupuncture decreased the systemic inflammatory response, ameliorated dyslipidemia, and improved liver function indexes in NAFLD model rats. Tomography and staining indicated that acupuncture reduced steatosis and infiltration of inflammatory cells in the liver. 16S rRNA analysis showed that acupuncture reduced the Firmicutes to Bacteroidetes (F/B) ratio, increased the abundance of microbiota, including Bacteroidales_S24-7_group, Prevotellaceae, Bacteroidaceae, Blautia, norank_f_Bacteroidales_S24-7_group, Bacteroides, and Prevotella_9, and decreased the abundance of Ruminococcaceae_UCG-014. Correlation analysis suggested a close correlation between lipid metabolism, inflammation factors, hepatic steatosis, and the changed intestinal microbiota. Acupuncture can significantly improve lipid metabolism and the systemic inflammatory response in HFD-induced NAFLD rats, potentially by regulating intestinal microbiota composition.

Untargeted metabonomic analysis of non-alcoholic fatty liver disease with iron overload in rats via UPLC/MS

Background/Aims In recent years, many metabolites specific to nonalcoholic fatty liver disease (NAFLD) have been identified thanks to the application of metabolomics techniques. This study aimed to investigate the candidate targets and potential molecular pathways involved in NAFLD in the presence of iron overload. Methods Male Sprague Dawley rats were fed with control or high-fat diet with or without excess iron. After 8, 16, 20 weeks of treatment, urine samples of rats were collected for metabolomics analysis using ultra-performance liquid chromatography/mass spectrometry (UPLC-MS). Blood and liver samples were also collected. Results High-fat, high-iron diet resulted in increased triglyceride accumulation and increased oxidative damage. A total of 13 metabolites and four potential pathways were identified. Compared to the control group, the intensities of adenine, cAMP, hippuric acid, kynurenic acid, xanthurenic acid, uric acid, and citric acid were significantly lower (p < 0.05) and the concentration of other metabolites was significantly higher in the high-fat diet group. In the high-fat, high-iron group, the differences in the intensities of the above metabolites were amplified. Conclusion Our findings suggest that NAFLD rats have impaired antioxidant system and liver function, lipid disorders, abnormal energy, and glucose metabolism, and that iron overload may further exacerbate these disorders.

Integrated Gut Microbiota and Urine Metabolite Analyses of T2DM with NAFLD Rat Model.

Globally 80% type 2 diabetes mellitus (T2DM) patients suffer nonalcoholic fatty liver disease (NAFLD). The interplay of gut microbiota and endogenous metabolic networks has not yet been reported in the setting of T2DM with NAFLD. As such, this study utilized 16S rRNA gene sequencing to assess the changes in intestinal flora and nuclear magnetic resonance spectroscopy (1H NMR) to identify potential metabolites in a T2DM with NAFLD rat model. Spearman correlation analysis was performed to explore the relationship between gut microbiota and metabolites. Results revealed that among T2DM with NAFLD rats, diversity indexes of intestinal microbiota were distinctly decreased while levels of 18 bacterial genera within the intestinal tract were significantly altered. In addition, levels of eight metabolites mainly involved in the synthesis and degradation of ketone bodies, the TCA cycle, and butanoate metabolism were altered. Correlation analysis revealed that gut bacteria such as Blautia, Ruminococcus torques group, Allobaculum, and Lachnoclostridium strongly associate with 3-hydroxybutyrate, acetone, acetoacetate, 2-oxoglutarate, citrate, creatinine, hippurate, and allantoin. Our findings can provide a basis for future development of targeted treatments.

Protective effect of corn peptide on nonalcoholic fatty liver disease in rats induced by high fat diet

To explore the dose-effect and mechanism of the protective effect of corn peptide(CP) on nonalcoholic fatty liver disease(NAFLD). Seventy male SD rats were randomly divided into control group, model group, CP group, puerarin positive control group, low-, medium-, high-dose corn peptide group, the experiment started after 1 week of adaptive feeding. The control group and CP group were fed with ordinary feed and others were fed with high-fat feed, while the control group was given intragastric administration of purified water, the CP group and the low-, medium-, high-dose corn peptide group were given intragastric administration of corn peptide(800, 400, 600 and 800 mg/(kg·d), respectively), and the puerarin positive control group was given intragastric administration of 300 mg/(kg·d) of puerarin, for 10 weeks. The body weight and general condition of rats were recorded every week, after 10 weeks, the indexes of 7 groups were compared. Compared with the model group, the liver index(3.67, 3.29, 3.19 and 3.04, P&lt;0.05) of the rats in the three doses of corn peptide groups decreased. The homeostasis model assessment of insulin resistance(13.66, 11.82, 11.55 and 10.53, P&lt;0.05) decreased. The serum triglyceride(TG)(0.59, 0.42, 0.34 and 0.28, P&lt;0.05), total cholesterol(TC)(2.57, 2.10, 1.67 and 1.46, P&lt;0.05), free fatty acid(FFA)(0.66, 0.44, 0.40 and 0.36, P&lt;0.05), and leptin(10.64, 9.87, 9.43 and 9.05, P&lt;0.05) decreased. The serum interleukin-6(74.61, 61.66, 51.12 and 35.68, P&lt;0.05) and tumor necrosis factor-α(100.18, 74.13, 67.44 and 48.46, P&lt;0.05) decreased. The serum aspartate aminotransferase(41.38, 19.41, 14.98 and 8.44, P&lt;0.05) and alanine aminotransferase(25.14, 13.10, 11.57 and 7.48, P&lt;0.05) activity decreased. The liver TG(9.89, 5.55, 4.73 and 3.13, P&lt;0.05), TC(1.73, 0.97, 0.80 and 0.50, P&lt;0.05) and FFA(1.81, 1.11, 0.77 and 0.70, P&lt;0.05) decreased. The serum malondialdehyde(MDA)(21.36, 15.24, 12.75 and 10.87, P&lt;0.05) level and liver MDA(11.87, 8.27, 6.33 and 4.98, P&lt;0.05) level decreased. The serum and liver superoxide dismutase and glutathione peroxidase activity increased(P&lt;0.05), and the effect is related to the intervention dose of corn peptide. The size of the liver became smaller, the liver color gradually changed from light yellow to light red. Corn peptide has a certain preventive effect on NAFLD in rats, and the preventive effect of corn peptide is related to its intervention dose.

Raman imaging of rat nonalcoholic fatty liver tissues reveals distinct biomolecular states.

An essential challenge in diagnosing states of nonalcoholic fatty liver disease (NAFLD) is the early prediction of progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) before the disease progresses. Histological diagnoses of NAFLD rely on the appearance of anomalous tissue morphologies, and it is difficult to segment the biomolecular environment of the tissue through a conventional histopathological approach. Here, we show that hyperspectral Raman imaging provides diagnostic information on NAFLD in rats, as spectral changes among disease states can be detected before histological characteristics emerge. Our results demonstrate that Raman imaging of NAFLD can be a useful tool for histopathologists, offering biomolecular distinctions among tissue states that cannot be observed through standard histopathological means.

The Anti-Obesity and Anti-Steatotic Effects of Chrysin in a Rat Model of Obesity Mediated through Modulating the Hepatic AMPK/mTOR/lipogenesis Pathways

Obesity is a complex multifactorial disease characterized by excessive adiposity, and is linked to an increased risk of nonalcoholic fatty liver disease (NAFLD). Flavonoids are natural polyphenolic compounds that exert interesting pharmacological effects as antioxidant, anti-inflammatory, and lipid-lowering agents. In the present study, we investigated the possible therapeutic effects of the flavonoid chrysin on obesity and NAFLD in rats, and the role of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathways in mediating these effects. Thirty-two Wistar male rats were divided into two groups: the control group and the obese group. Obesity was induced by feeding with an obesogenic diet for 3 months. The obese rats were subdivided into four subgroups, comprising an untreated group, and three groups treated orally with different doses of chrysin (25, 50, and 75 mg/kg/day for one month). Results revealed that chrysin treatment markedly ameliorated the histological changes and significantly and dose-dependently reduced the weight gain, hyperglycemia, and insulin resistance in the obese rats. Chrysin, besides its antioxidant boosting effects (increased GSH and decreased malondialdehyde), activated the AMPK pathway and suppressed the mTOR and lipogenic pathways, and stimulated expression of the genes controlling mitochondrial biogenesis in the hepatic tissues in a dose-dependent manner. In conclusion, chrysin could be a promising candidate for the treatment of obesity and associated NAFLD, aiding in attenuating weight gain and ameliorating glucose and lipid homeostasis and adipokines, boosting the hepatic mitochondrial biogenesis, and modulating AMPK/mTOR/SREBP-1c signaling pathways.

Lipid-droplet associated mitochondria promote fatty-acid oxidation through a distinct bioenergetic pattern in male Wistar rats

Mitochondria empower the liver to regulate lipid homeostasis by enabling fatty acid oxidation during starvation and lipogenesis during nutrient-rich conditions. It is unknown if mitochondria can seamlessly regulate these two distinct processes or if two discrete populations of mitochondria achieve these two functions in the liver. For the first time in the liver, we report the isolation of two distinct populations of mitochondria from male Wistar rats on an ad-libitum diet: cytoplasmic mitochondria and lipid droplet-associated mitochondria. Our studies show that while lipid droplet mitochondria exhibit higher fatty acid oxidation and are marked by enhanced levels of pACC2, MFN2, and CPT1 activity, cytoplasmic mitochondria are associated with higher respiration capacity. Notably, lipid droplet-associated mitochondria isolated from a non-alcoholic fatty liver disease (NAFLD) rat model are compromised for fatty acid oxidation. We demonstrate the importance of functional segregation of mitochondria as any aberration in lipid droplet-associated mitochondria may lead to NAFLD.

Exploring the molecular mechanism of Gan Shuang granules for the treatment of non-alcoholic steatohepatitis using network pharmacology, molecular docking, and experimental verification.

Background: With the gradual increase in prevalence in recent years, non-alcoholic steatohepatitis (NASH) has become one of the significant health problems that urgently needs to be addressed worldwide. GanShuang Granules (GSG) is derived from the classical Chinese formula Xiaoyao San and mainly used in the clinical treatment of chronic liver diseases. Objective: In this study, we aim to gain a deeper insight into the inhibiting effects of GSG on non-alcoholic fatty liver disease (NAFLD) rats and preliminarily elucidate the underlying intervention mechanisms. Methods: First, High performance liquid chromatography (UHPLC-Q/Orbitrap-MS/MS) was used for the active compounds prediction in GSG. Then the data was mapped to mzCloud database. The targets corresponding to GSG compounds were collected from public databases, along with disease genes for NAFLD. The core targets and molecular mechanisms of GSG for NAFLD treatment were predicted by protein-protein interaction (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Molecular docking of the core target-component interactions was simulated using AutoDock Vina software. The effect of GSG on NASH rats was evaluated by pathological staining and analysis of various index results. Finally, the candidate targets were further validated by ELISA and western blot (WB) analyses. Results: Combining UHPLC-Q/Orbitrap-MS/MS data analysis and public database data, a total of 346 cross-targets were obtained, corresponding to 81 compounds. The subnetwork with an MCODE score of 53.623 is a potential core target group for this study. GO and KEGG enrichment analyses showed that the targets of GSG in NAFLD were mostly related to oxidative stress, the NF-κB signaling pathway, and the apoptosis signaling pathway. By integrating the results of network pharmacology analysis, the core objectives of this study mainly include AKT1, CASP9, TNF, and CASP8. The core ingredients are related to resveratrol and fisetin. The molecular docking results indicated key binding activity between AKT1-fisetin, AKT1-Resveratrol, and CASP8-fisetin. Moreover, GSG could improve the inflammatory status and restore the abnormal lipid accumulation of NAFLD/NASH liver, and these levels are further verified by pathological staining and detection of related indicators. Mechanistically, GSG could regulate protein expression levels in the liver for P65, p-P65, IKB, p-IKB, IKK, caspase-3, -8, -9, and cytochrome C, etc. It reflects the inhibitory effect of GSG on the NF-κB/IκB signaling pathway. Conclusion: Our results suggested that GSG demonstrated therapeutic effects on NAFLD/NASH rats, and these may be mainly reflected in the inhibitory effects on the NF-κB/IκB signaling pathway and its downstream inflammation and apoptosis signals.

THE THERAPEUTIC EFFECT OF ALLOPURINOL IN FATTY LIVER DISEASE IN RATS.

Hyperuricemia is associated with non-alcoholic fatty liver disease (NAFLD). We therefore aimed at evaluating the influence of allopurinol on the course of NAFLD in rats. We divided 21 mature albino Sprague Dawley rats into three groups: controls (n = 7, normal diet for 12 weeks); NAFLD rat models (by feeding water containing 30% fructose for first 8 weeks) treated with allopurinol subsequently for the next 4 weeks (n = 7); and similar case treated with placebo (saline) subsequently for the next 4 weeks (n = 7). We compared the histopathological scores, IL-1 and IL-2 immunoexpression levels across the groups. Liver histopathological score was determined by observing the steatosis (the percentage of liver cells containing fat): <25% = 1+, 25% - 50% = 2+, 51% - 75% = 3+, >75% = 4+; inflammation and necrosis: 1 focus per low-power field = 1+; and 2 or more foci = 2+. The number of liver IL-1 and IL-2 positive cells was measured by systematically scoring at least 100 hepatocyte cells per field in 10 fields of tissue sections by a magnification of 100. Xanthine oxidase (XO) activity and lipid peroxidation was significantly different in the allopurinol group compared to the saline group (XO; 0.098 ± 0.006 mU/mg vs. 0.162 ± 0.008 mU/mg, p = 0.01, 0.116 ± 0.040 nmol malondialdehyde/mg versus 0.246 ± 0.040 nmol malondialdehyde /mg, p = 0.01). The allopurinol group had lower histopathological scores, IL-1 and IL-2 immunoexpression levels in the liver compared to the saline group (2.13 ± 0.35 against 5.45 ± 0.24, p = 0.003, IL-1; 5.76 ± 0.43 against 12.85 ± 3.26, p = 0.023, IL-2; 8.55 ± 1.14 against 56.23 ± 7.12, p = 0.002). Allopurinol has a therapeutic role against the progression of NAFLD of the rats.

UPLC-QTOF-MS Based Comparison of Rotundic Acid Metabolic Profiles in Normal and NAFLD Rats.

Rotundic acid, the principal bioactive constituent of the herbal remedy "Jiubiying", has been considered as a candidate compound for treating non-alcoholic fatty liver disease (NAFLD). However, the in vivo and in vitro metabolism of rotundic acid has remained unclear. With the aim of elucidating its metabolic profile, a reliable approach that used ultra-high performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was applied for screening and identifying rotundic acid in vivo (plasma, feces, urine, and liver tissue of normal and NAFLD model rats) and in vitro (rat liver microsomes) metabolites. Herein, 26 metabolites of rotundic acid were identified, including 22 metabolites in normal rats, 20 metabolites in NAFLD model rats, and eight metabolites in rat liver microsomes. Among them, 17 metabolites were identified for the first time. These data illustrate that the pathological status of NAFLD affects the metabolism of rotundic acid. Furthermore, the major pathways of metabolism included phase Ⅰ (demethylation, desaturation, etc.) and phase Ⅱ (sulfation and glucuronidation) reactions, as well as a combined multiple-step metabolism. This work provides important information on the metabolism of rotundic acid and lays the foundation for its future clinical application.

The Synergistic Protective Effect of γ-Oryzanol (OZ) and N-Acetylcysteine (NAC) against Experimentally Induced NAFLD in Rats Entails Hypoglycemic, Antioxidant, and PPARα Stimulatory Effects.

This study estimated that the combined effect of γ-Oryzanol and N-acetylcysteine (NAC) against high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats also estimated some of their mechanisms of action. Adult male rats were divided into seven groups (n = 8 each) as control, control + NAC, control + γ-Oryzanol, HFD, HFD + NAC, HFD + γ-Oryzanol, and HFD + NAC + γ-Oryzanol. NAC was administered orally at a final concentration of 200 mg/kg, whereas γ-Oryzanol was added to diets at a concentration of 0.16. All treatments were conducted for 17 weeks and daily. Both NAC and γ-Oryzanol were able to reduce final body weights, fat weights, fasting glucose, fasting insulin, serum, and serum levels of liver function enzymes as well as the inflammatory markers such as tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6), and leptin in HFD-fed rats. They also improved hepatic structure and glucose tolerance, increased adiponectin levels, and reduced serum and hepatic levels of triglycerides (TGs) and cholesterol (CHOL) in these rats. These effects were concomitant with a reduction in the hepatic levels of lipid peroxides (MDA) and serum levels of LDL-C, but also with an increment in the hepatic levels of superoxide dismutase (SOD) and glutathione (GSH). Interestingly, only treatment with γ-Oryzanol stimulated the mRNA levels of proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) in the liver and white adipose tissue (WAT) of rats. Of note, the combination therapy of both drugs resulted in maximum effects and restored almost normal liver structure and basal levels of all the above-mentioned metabolic parameters. In conclusion, a combination therapy of γ-Oryzanol and NAC is an effective therapy to treat NAFLD, which can act via several mechanisms on the liver and adipose tissue.

Yinchen Linggui Zhugan decoction ameliorates high fat diet-induced nonalcoholic fatty liver disease by modulation of SIRT1/Nrf2 signaling pathway and gut microbiota.

Yinchen Linggui Zhugan decoction (YLZD) is an effective and classical traditional herbal prescription for treating the nonalcoholic fatty liver disease (NAFLD) and has been proven to be effective in the regulation of lipid metabolism disorder and attenuate inflammation for a NAFLD rat model. However, the exact underlying mechanism has not been elucidated. In the current study, a NAFLD rat model was established using a high-fat diet (HFD) for 10 weeks, followed by YLZD treatment with 1.92 g/kg/day for 4 weeks to explore the mechanisms of YLZD. Our results showed that YLZD decreased the hepatic lipid deposition, restored the liver tissue pathological lesions, inhibited the expression of oxidative stress, and decreased the inflammatory cytokines levels. Meanwhile, the genes and proteins expressions of SIRT1/Nrf2 signaling pathway together with downstream factors including HO-1 and NQO1 were elevated in the YLZD treated NAFLD rats. For further elaborating the upstream mechanism, short-chain fatty acids (SCFAs) in serum and feces were measured by liquid chromatograph mass spectrometer and gas chromatograph mass spectrometer, and the differences in gut microbiota of rats in each group were analyzed through high-throughput sequencing of 16S rRNA. The results demonstrated that the contents of butyric acid (BA) and total SCFAs in YLZD-treated NAFLD rats were significantly increased in serum and feces. 16S rRNA sequencing analysis illustrated that YLZD intervention led to a modification of the gut microbiota composition, with a decrease of Oribacterium, Lactobacillus and the ratio of Firmicutes/Bacteroides, as well as the increase in SCFAs-producing bacteria such as Christensenellaceae, Clostridia, Muribaculaceae, and Prevotellaceae. Spearman rank correlation analysis indicated that BA and total SCFAs were negatively co-related with oxidative stress-related factors and inflammatory cytokines, while they were positively co-related with SIRT1/Nrf2 pathway related genes and proteins. Furthermore, in vitro study confirmed that BA effectively reduced oxidative stress by activating SIRT1/Nrf2 signaling pathway in L02 cells. Together, the present data revealed YLZD could ameliorate HFD-induced NAFLD in rats by the modulation of SIRT1/Nrf2 signaling pathway and gut microbiota.

Inulin intervention attenuates hepatic steatosis in rats via modulating gut microbiota and maintaining intestinal barrier function

Increasing evidence has suggested the mitigatory efficacy of prebiotic inulin on nonalcoholic fatty liver disease (NAFLD), nevertheless, its action mechanisms remain elusive. Herein, inulin consumption effectively ameliorated high-sucrose diet-induced hepatic steatosis and inflammation, and rehabilitated liver lipogenesis regulators, including carbohydrate response element-binding protein, stearoyl-CoA desaturase-1 and peroxisome proliferator-activated receptor alpha. Furthermore, inulin supplementation restored the intestinal barrier integrity and function by up-regulating expressions of tight junction proteins (zonula occludens-1, claudin-1 and occludin). High-throughput sequencing demonstrated that inulin administration regulated the gut microbiota composition, wherein abundance of short-chain fatty acid (SCFA)-producers, including Bifidobacterium, Phascolarctobacterium and Blautia, was significantly enhanced in the inulin-treated rats, conversely, opportunistic pathogens, such as Acinetobacter and Corynebacterium_1, were suppressed. SCFA quantitative analysis showed that dietary inulin suppressed faecal acetate levels, but improved propionate and butyrate concentrations in rats with NAFLD. Functional prediction showed that tryptophan metabolism was one of the key metabolic pathways affected by gut microbiota changes. A targeted metabolomics profiling of tryptophan metabolism demonstrated that inulin intervention up-regulated faecal contents of indole-3-acetic acid and kynurenic acid, whereas down-regulated levels of kynurenine and 5-hydoxyindoleacetic acid in NAFLD rats. Therefore, this study demonstrated that inulin intake alleviated hepatic steatosis likely by regulating the gut microbiota composition and function and restoring the intestinal barrier integrity, which may provide a novel notion for the prevention and treatment of NAFLD in future.

Epigenetic association study uncovered H3K27 acetylation enhancers and dysregulated genes in high-fat-diet-induced nonalcoholic fatty liver disease in rats.

Aim: To evaluate the regulatory landscape underlying the active enhancer marked by H3K27ac in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) in rats. Materials & methods: H3K27ac chromatin immunoprecipitation and high-throughput RNA sequencing to construct regulatory profiles and transcriptome of liver from NAFLD rat model induced by HFD. De novo motif analysis for differential H3K27ac peaks. Functional enrichment, Kyoto Encyclopedia of Genes and Genomes pathway and protein-protein interaction network were examined for differential peak-genes. The mechanism was further verified by western blot, chromatin immunoprecipitation-quantitativePCR and real-timePCR. Results:A total of1831 differential H3K27ac peaks were identified significantly correlating with transcription factors and targetgenes (CYP8B1, PLA2G12B, SLC27A5, CYP7A1 and APOC3) involved in lipid and energy homeostasis. Conclusion: Altered acetylation induced by HFD leads to the dysregulation of gene expression, further elucidating the epigenetic mechanism in the etiology of NAFLD.