Rat Model Of Non-alcoholic Fatty Liver Disease Research Articles

Study on the mechanism of Shuganzhi Tablet against nonalcoholic fatty liver disease and lipid regulation effects of its main substances in vitro

Ethnopharmacological relevanceShuganzhi Tablet (SGZT) originates from a famous traditional Chinese herbal formula Chaihu Decoction which can be applied to treat liver diseases, however, the pharmacodynamic mechanism of SGZT needs to be evaluated. Aim of this studyTo study the mechanism of SGZT in the treatment of non-alcoholic fatty liver disease (NAFLD), and screen out its effective ingredients. Materials and methodsIn this study, firstly, the main components of SGZT were analyzed qualitatively. And a rat model of NAFLD was established by feeding high-fat diet. Serum biochemical indexes and liver pathological analysis were used to evaluate the pharmacodynamic effect of SGZT in the treatment of NAFLD. In order to explore the pharmacodynamic mechanism, proteomics and metabolomics analysis were used. Western blotting was used to verify the expression of important differential proteins. And L02 cells were treated with free fatty acids (FFA) and the main substances of SGZT to establish the cell model of NAFLD in vitro and to reveal the pharmacodynamic substance of SGZT. ResultsTwelve components were detected in SGZT, and according to the results of serum biochemical indexes and liver pathological analysis, SGZT could effectively treat NAFLD. Combined with the results of bioinformatics analysis, we found that 133 differentially expressed proteins were reversed in liver samples of rats treated with SGZT. The important proteins in PPAR signaling pathway, steroid biosynthesis, cholesterol metabolism and fatty acid metabolism were mainly regulated to maintain cholesterol homeostasis and improve lipid metabolism. SGZT also affected various metabolites in rat liver, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and taurine. In addition, the main components contained in SGZT (hesperidin, polydatin, naringin, emodin, specnuezhenide, saikosaponin A) and a metabolite (resveratrol) could significantly reduce FFA-induced intracellular lipid accumulation. ConclusionSGZT effectively treated NAFLD, and PPAR-γ, Acsl4, Plin2 and Fads1 may be the main targets of SGZT. And Fads1-EPA/DHA-PPAR-γ may be the potential pharmacodynamic pathway. Cell experiments in vitro revealed that the main components of SGZT and their metabolites, such as hesperidin, polydatin, naringin, emodin, specnuezhenide, saikosaponin A and resveratrol may be the main components of its efficacy. Further research is needed to reveal and validate the pharmacodynamic mechanism.

Lapachol treats non-alcoholic fatty liver disease by modulating the M1 polarization of Kupffer cells via PKM2

AimThis study investigated the mechanism of action of lapachol (LAP) against non-alcoholic fatty liver disease (NAFLD). MethodsPrimary Kupffer cells (KCs) of rats were used for in-vitro experiments. The proportion of M1 cells was assayed by flow cytometry, the levels of M1 inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA) combined with real-time quantitative fluorescence PCR (RT-qPCR), the expression of p-PKM2 was detected by Western-Blotting. A SD rat model of NAFLD was established with high-fat diet. Following LAP intervention, the changes in blood glucose/lipid, insulin resistance and liver function were detected, and the hepatic histopathologic changes were examined by histological staining. ResultsThe results showed that LAP could inhibit the M1 polarization of KCs, lower the levels of inflammatory cytokines, and suppress the activation of PKM2. The effect of LAP could be counteracted after using PKM2 inhibitor PKM2-IN-1 or knocking out PKM2. Small molecule docking revealed that LAP could inhibit the phosphorylation process of PKM2 by binding to ARG-246, the phosphorylation site of PKM2. In rat experiments, LAP could ameliorate the liver function and lipid metabolism of NAFLD rats, and inhibit the hepatic histopathologic changes. ConclusionOur study found that LAP can inhibit the phosphorylation of PKM2 by binding to PKM2-ARG-246, thereby regulating the M1 polarization of KCs and inhibiting the inflammatory response of liver tissues to treat NAFLD. LAP has potential as a novel pharmaceutical for treating NAFLD.

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.

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.

Gut metabolomics and 16S rRNA sequencing analysis of the effects of arecoline on non-alcoholic fatty liver disease in rats.

Introduction: Non-alcoholic fatty liver disease (NAFLD) has gradually become the primary cause of fatty liver disease. Betel nuts have been used to treat gastrointestinal diseases. Methods: In the present study, we analyzed the pathology, serology, gut flora, and metabolites in a rat model of NAFLD, with and without betel nut alkaloid treatment, using an integrated approach involving pathology, serological testing, 16S rRNA gene sequencing, and ultra-performance liquid chromatography-mass spectrometry metabolomics. Results: Two rats were used for model validation. Thirty SD rats were included and divided into the normal group (C group), NAFLD model group (M group), low-dose group, medium-dose group (T group), and high-dose group with intraperitoneal injection of arecoline. The expression of blood lipids was significantly downregulated at all three arecoline concentrations (p < 0.05). Alpha-diversity analysis of the intestinal flora showed significant differences among the three groups, with a significant reduction in population diversity in the M group and a recovery of population diversity after arecoline treatment. At the phylum level, the relative abundance of Firmicutes was significantly higher in the T group and Proteobacteria in the M group. The KEGG metabolic pathways included polyketide sugar unit biosynthesis and hypertrophic cardiomyopathy. Thirty-three significantly different metabolites were identified among the groups. Significantly different metabolites between groups T and M included indolepyruvate, 2-deoxystreptamine, sakuranetin, glycyl-leucine, and riboflavin. The KEGG metabolic pathway suggested a potential role for arachidonic acid metabolism, serotonergic synapses, neuroactive ligand-receptor interactions, tyrosine metabolism, and regiomelanin. Vitamin digestion and absorption, as well as regulation of lipolysis in adipocytes, were the main metabolic pathways that distinguished the T vs. M groups. PGE2 is involved in several metabolic pathways. Correlation analysis showed that 29 bacterial species were significantly associated with PGE2 levels in the M and T groups. Vagococcus, Lawsonia, Christensenella, unidentified Erysipelotrichaceae, unidentified Coriobacteriaceae, and five other bacterial groups are unique in the PGE2 metabolic pathway regulated by arecoline. Discussion: Arecoline has lipid-lowering effects and may exert therapeutic effects in NAFLD through intestinal metabolites and intestinal flora, as well as through the Butyricicoccus/Christensenella/Coriobacteriaceae-COX2/PGE2 pathway. Thus, arecoline may represent a potential drug or target for NAFLD treatment.

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.

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.

Metabolomics analysis of the serum metabolic signature of nonalcoholic fatty liver disease combined with prediabetes model rats after the intervention of Lycium barbarum polysaccharides combined with aerobic activity.

Metabolic disorders accompany nonalcoholic fatty liver disease (NAFLD), associated with prediabetes. Lycium barbarum polysaccharides (LBP) seem to be a potential prebiotic, and aerobic exercise has shown protective effects on NAFLD with prediabetes. However, their combined effects on NAFLD and prediabetes remain unclear. This study investigated the effects of LBP and aerobic exercise alone, and their combined effects on the metabolomics of serum, and explored the potential mechanisms utilizing a high-fat diet-induced rat model of NAFLD and prediabetes. It provided the metabolic basis for the pathogenesis and early diagnosis of prediabetes complicated with NAFLD. Untargeted metabolomics profiling was performed using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry to analyze the changes in overall metabolites in each group of samples. An orthogonal partial least squares-discriminant analysis model with variable importance on projection >1 and p < 0.05 were used as the screening criteria to screen the significant differential metabolites and analyze the expression changes and functional pathways. Different intervention treatments showed clear discrimination by univariate and multivariate analyses. The model group had a high relative level of expression of lipids. Comparison between the two groups showed steroids with high expression after LBP and aerobic exercise treatment separately and alkaloids and fatty acyls with high expression after aerobic exercise and the combination intervention, respectively. Comparison of the five groups showed some of the metabolites to be differently expressed after the intervention improved lipid and fatty acid metabolism. The three types of intervention had sound effects on the changes in liver index for the diseases studied. Furthermore, the combination treatment may be a better choice for disease prevention and treatment than a single treatment. Our analysis of metabolomics confirmed that the different treatments had significant regulatory effects on the metabolic pathways. Our findings strongly support the possibility that aerobic exercise combined with LBP can be regarded as a potential therapeutic method for NAFLD in prediabetics.

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.

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.

Dietary iron overload enhances Western diet induced hepatic inflammation and alters lipid metabolism in rats sharing similarity with human DIOS

Hepatic iron overload is often concurrent with nonalcoholic fatty liver disease (NAFLD). Dysmetabolic iron overload syndrome (DIOS) is characterized by an increase in the liver and body iron stores and metabolic syndrome components. Increasing evidences suggest an overlap between NAFLD with iron overload and DIOS; however, the mechanism how iron is involved in their pathogenesis remains unclear. Here we investigated the role of iron in the pathology of a rat model of NAFLD with iron overload. Rats fed a Western (high-fat and high-fructose) diet for 26weeks represented hepatic steatosis with an increased body weight and dyslipidemia. Addition of dietary iron overload to the Western diet feeding further increased serum triglyceride and cholesterol, and enhanced hepatic inflammation; the affected liver had intense iron deposition in the sinusoidal macrophages/Kupffer cells, associated with nuclear translocation of NFκB and upregulation of Th1/M1-related cytokines. The present model would be useful to investigate the mechanism underlying the development and progression of NAFLD as well as DIOS, and to elucidate an important role of iron as one of the "multiple hits" factors.

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.

Extraction of raspberry ketone from red raspberry and its intervention in the non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by diffused hepatocyte bullous fat in the liver, which is not caused by alcohol or drugs like amiodarone and tamoxifen. Presently, no drug is approved for NAFLD treatment. Therefore, it’s important to extract effective components from natural plants to alleviate NAFLD. In this study, we extracted and purified raspberry ketone, a natural phenolic compound from red raspberry (Rubus ideaus L.) by an ultrasonic-assisted ethanol extraction method. The structure of red raspberry ketone (RRK) was determined using Fourier-transform infrared spectroscopy and the purity of RRK was found as 80.06 ± 1.19%. After 28 days of intragastric administration of RRK, the bodyweight of NAFLD model rats decreased significantly (p < 0.05). Besides, the levels of low-density lipoprotein cholesterol, total cholesterol, and total triglyceride (TG) decreased and the content of high-density lipoprotein cholesterol in serum increased drastically. Moreover, the level of liver damage indicators (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) and the levels of glucose, insulin, free-fatty acid, tumor necrotic factor-α in the liver decreased distinctly. The levels of TG and malondialdehyde in the liver decreased, whereas the levels of superoxide dismutase, total glutathione, and glutathione peroxidase drastically increased. We also found that RRK reduced the uneven size of liver cells and blurred boundaries of hepatic lobules, and alleviated hepatic steatosis and inflammation caused by NAFLD. We inferred that RRK could relieve NAFLD progression by regulating glucose and lipid metabolism and alleviating oxidative stress in vivo. This study sheds new light on the use of RRK as a functional food for NAFLD prevention.

Metformin, pioglitazone, dapagliflozin and their combinations ameliorate manifestations associated with NAFLD in rats via anti-inflammatory, anti-fibrotic, anti-oxidant and anti-apoptotic mechanisms

Non-alcoholic fatty liver disease (NAFLD) is an important health threat that is strongly linked to components of metabolic syndrome, particularly the low-grade inflammatory changes. Significantly, several of the available anti-diabetic drug classes demonstrate a considerable anti-inflammatory effect, and hence might be of benefit for NAFLD patients. In this study, we used a rat model of diet-induced NAFLD to examine the potential effect of metformin, pioglitazone, dapagliflozin and their combinations on NAFLD manifestations. Rats were fed an atherogenic diet containing 1.25 % cholesterol, 0.5 % cholic acid and 60 % cocoa butter for 6 weeks causing a number of metabolic and hepatic alterations including insulin resistance, dyslipidemia, systemic inflammation, increased hepatic oxidative stress and lipid peroxidation, hepatic steatosis, lobular inflammation, as well as increased markers of liver inflammation and hepatocyte apoptosis. Drug treatment, which started at the third week of NAFLD induction and continued for three weeks, not only ameliorated the observed metabolic impairment, but also functional and structural manifestations of NAFLD. Specifically, anti-diabetic drug treatment reversed markers of systemic and hepatic inflammation, oxidative stress, hepatic fibrosis, and hepatocyte apoptosis. Our findings propose that anti-diabetic drugs with a potential anti-inflammatory effect can ameliorate the manifestations of NAFLD, and thus may provide a therapeutic option for such a condition that is closely associated with metabolic diseases. The detailed pharmacology of these classes in aspects linked to the observed impact on NAFLD requires to be further investigated and translated into clinical studies for tailored therapy specifically targeting NAFLD.

Intracerebroventricular injection of ghrelin receptor antagonist alleviated NAFLD via improving hypothalamic insulin resistance.

Objective(s):Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of clinical metabolic syndrome. Insulin resistance is an important factor in the pathogenesis of NAFLD. Ghrelin, widely distributed in peripheral tissues and the central nervous system, plays a vital role in regulating food intake, energy balance, and substance metabolism. In this study, the effect of intracerebroventricular (ICV) injection of ghrelin receptor antagonist on NAFLD was explored.Materials and Methods:A rat model of NAFLD was established by feeding a high-fat diet, and a selective ghrelin receptor antagonist [D-Lys-3]-GHRP-6 was injected via ventricular intubation implantation. The serum total cholesterol (TC), triglycerides (TGs), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic TGs were measured using the colorimetric method. Fasting plasma glucose (FPG) and fasting plasma insulin (FPI) were determined to calculate homeostatic model assessment insulin resistance (HOMA-IR). Hematoxylin-eosin (HE) and Oil Red O staining were conducted to observe the pathological changes and lipid accumulation in the liver. Hosphatidylinositide3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway protein expressions were measured using western blot analysis.Results:ICV injection of [D-Lys-3]-GHRP-6 significantly reduced serum lipids, transaminase, and HOMA-IR, improved liver injury, and inhibited lipid accumulation in the liver of NAFLD rats. Moreover, ICV injection of [D-Lys-3]-GHRP-6 significantly up-regulated the phosphorylation levels of PI3K/Akt/mTOR signaling protein expressions in the hypothalamus, indicating a significant improvement in hypothalamic insulin resistance.Conclusion:Blockade of central ghrelin receptor can treat NAFLD possibly via the hypothalamic PI3K/Akt/mTOR signaling pathway to improve insulin resistance.

Daily Intake of Smallanthus sonchifolius (Yacon) Roots Reduces the Progression of Non-alcoholic Fatty Liver in Rats Fed a High Fructose Diet.

High-fructose diet is associated with an increased risk of dyslipidemia, metabolic syndrome, and the development of non-alcoholic fatty liver disease (NAFLD) through chronic inflammation. The present study aimed to elucidate the potential benefit of daily consumption of Smallanthus sonchifolius (yacon) roots, rich in fructooligosaccharides (FOS), on the progression to liver fibrosis, in a rat model of NAFLD induced by a high-fructose diet. Male Wistar rats were fed a standard diet (CD, n= 6) or a standard diet plus 10% fructose solution (FD; n= 18). After 20weeks, FD rats were randomly separated into the following groups (n= 6, each): FD; FD treated with yacon flour (340mg FOS/body weight; FD + Y) and FD treated with fenofibrate (30mg/kg body weight; FD + F), for 16weeks. Daily intake of yacon flour significantly reduced body weight gain, plasma lipid levels, transaminase activities, and improved systemic insulin response in FD rats. In the liver, yacon treatment decreased fructose-induced steatosis and inflammation, and reduced total collagen deposition (64%). Also, yacon decreased TGF-β1 mRNA expression (78%), followed by decreased nuclear localization of p-Smad2/3 in liver tissue. Yacon significantly reduced the expression of α-smooth muscle actin (α-SMA), Col1α1, and Col3α1 mRNAs (85, 44, and 47%, respectively), inhibiting the activation of resident hepatic stellate cells (HSCs). These results suggested that yacon roots have the potential to ameliorate liver damage caused by long-term consumption of a high-fructose diet, being a promising nutritional strategy in NAFLD management.

Danshao Shugan Granule therapy for non-alcoholic fatty liver disease

BackgroundDanshao Shugan Granules (DSSG), a traditional Chinese medicine (TCM), is given to protect the liver. The objective is to evaluate the mechanisms of the effects of DSSG on non-alcoholic fatty liver disease (NAFLD).Methods260 patients with NAFLD were randomly allocated to positive control drugs rosiglitazone (n = 30) and Silibinin (n = 50) as well as DSSG (n = 130) and combined DSSG/Silibinin (n = 50) groups, from which 90 patients in the DSSG group were further subdivided into 3 groups (n = 30, each) depending on the severity of symptoms. In total 33 Sprague–Dawley rats were assigned to normal (n = 10) or 45% high-fat diet (n = 23) groups, from which 9 rats served as negative controls, 10 as model controls and 10 were treated with DSSG.ResultsDSSG medications had significantly highest effects on B-ultrasonography finding improvements, and reductions of total cholesterol, triglyceride, aspartate transaminase and γ-glutamyl transpeptidase in NAFLD patients. Silibinin application only led to significantly highest alanine transaminase reductions and rosiglitazone medication to significantly highest fasting plasma glucose reductions. In a murine in vivo NAFLD model glucose (GLU), total cholesterol (TC) triacylglycerol (TG) as well as glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and gamma-glutamyl transferase (GGT) serum concentrations were all significantly reduced (P < 0.001) and the expression of nuclear factor-κB (NF‑κB) was significantly decreased in DSSG treated compared to untreated NAFLD animals (P < 0.001). In addition, the DSSG treated rats exhibited increased superoxide dismutase activity and reduced malondialdehyde values.ConclusionsDSSG was effective for treating NAFLD patients, which could be attributed to increased activity of superoxide dismutase, a decrease of malondialdehyde as well as reduced NF‑κB activity in a NAFLD rat model.

Effects of Resveratrol Administration in Liver Injury Prevention as Induced by an Obesogenic Diet: Role of Ruminococcaceae.

Gut microbiota dysbiosis has been described in several metabolic disruptions, such as non-alcoholic fatty liver disease (NAFLD). Administration of resveratrol has been claimed to elicit benefits against NAFLD along with modulating gut microbiota composition. This investigation aims to study the putative mediating role of gut microbiota in the potential hepato-protective effects of resveratrol in a diet-induced NAFLD rat model. The involvement of bacteria from the Ruminococcaceae family in such effects was also addressed. Resveratrol administration resulted in lowered liver weight and serum total and non-HDL cholesterol concentrations, as well as in increased serum HDL cholesterol levels. The administration of this polyphenol also prevented obesogenic diet-induced serum transaminase increases. In addition, histopathological analysis revealed that resveratrol administration ameliorated the dietary-induced liver steatosis and hepatic inflammation. Gut microbiota sequencing showed an inverse relationship between some bacteria from the Ruminococcaceae family and the screened hepatic markers, whereas in other cases the opposite relationship was also found. Interestingly, an interaction was found between UBA-1819 abundance and resveratrol induced liver weight decrease, suggesting that for this marker resveratrol induced effects were greater when the abundance of this bacteria was high, while no actions were found when UBA-1819 abundance was low.

Mechanical Study of Jian-Gan-Xiao-Zhi Decoction on Nonalcoholic Fatty Liver Disease Based on Integrated Network Pharmacology and Untargeted Metabolomics.

Jian-Gan-Xiao-Zhi decoction (JGXZ) has demonstrated beneficial effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanisms by which JGXZ improve NAFLD are still unclear. Methods. In this study, we first used a high-fat diet (HFD) to establish a NAFLD rat model to clarify the therapeutic effect of JGXZ on NAFLD. Secondly, we used network pharmacology to predict the potential targets of JGXZ on NAFLD, and then the key targets obtained from network pharmacology were verified. Finally, we used untargeted metabolomics to study the metabolic regulatory mechanism of JGXZ. Results. JGXZ treatment could decrease body weight and ameliorate dyslipidemia in NAFLD model rats. H&E and oil red O staining indicated that JGXZ reduced steatosis and infiltration of inflammatory cells in the liver. In addition, network pharmacology research found that the potential targets of JGXZ on NAFLD pathway were mainly associated with improving oxidative stress, apoptosis, inflammation, lipid metabolism disorders, and insulin resistance. Further experimental verification confirmed that JGXZ could inhibit inflammation and improve oxidative stress, insulin resistance, and lipid metabolism disorders. Serum untargeted metabolomics analyses indicated that the JGXZ in the treatment of NAFLD may work through the linoleic acid metabolism, alpha-linolenic acid metabolism, tryptophan metabolism, and glycerophospholipid metabolism pathways. Conclusions. In conclusion, this study found that JGXZ has an ameliorative effect on NAFLD, and JGXZ alleviates the inflammatory response and oxidative stress and lipid metabolism disorders in NAFLD rats. The mechanism of action of JGXZ in the treatment of NAFLD may be related to the regulation of linoleic acid metabolism, tryptophan metabolism, alpha-linolenic acid metabolism, and glycerophospholipid metabolism.