Rat Model Of Non-alcoholic Fatty Liver Disease Research Articles

The hypolipidemic, anti-inflammatory and antioxidant effect of Kavolì® aqueous extract, a mixture of Brassica oleracea leaves, in a rat model of NAFLD

Herein we characterized the bioactive metabolites of the aqueous extract of Kavolì®, a commercial product composed of a mixture of Brassica oleracea leaves, and assessed its potential ameliorating effects in a rat model of non-alcoholic fatty liver disease (NAFLD). Kavolì® extract showed high levels of bioactive compounds and strong in vitro antioxidant activities. Chlorogenic and neochlorogenic acids were identified as the most representative polyphenols. The administration of brassica extract to steatotic rats significantly ameliorated the levels of blood lipids and transaminases, and lipid content and inflammatory markers in liver. Oxidative stress parameters were significantly improved in both liver and brain of steatotic rats. Moreover, plasma and feces levels of short chain fatty acids (SCFAs) were bring back close to control values by Kavolì® treatment, in spite of high fat diet/streptozotocin (HFD/STZ)-induced alterations. The efficacy of Kavolì® in treating hypercholesterolemia, reducing the level of inflammation and cardiovascular disease biomarkers, steatosis and oxidative stress parameters, as well as the ability in modulating SCFAs levels is probably related to the bioactive compounds of the water extract administered to the rat model of NAFLD. In particular, the ameliorating effects are largely attributable to the high content in polyphenols observed in our study.

Effects of 8 Weeks Feeding Diet with Low and High Isoflavones Soy Protein Concentrate on Serum Isoflavones in Non-alcoholic Fatty Liver Disease (NAFLD) Rat Model

ObjectivesTo examine the effects of 8 weeks feeding of low and high isoflavones levels in soy protein concentrate on the total serum isoflavones in a NAFLD obese rat model.Methods42 six-week old lean (L, n = 21) and obese (O, n = 21) Zucker rats were randomly assigned to 1 of 3 dietary groups: casein diet (C = control), soy protein with low isoflavones (LIF), or soy protein with high isoflavones (HIF) for 8 weeks (n = 7 rats/group). Rats were weighed twice weekly. After 8 weeks, all rats were sacrificed and serum for total isoflavones (genistein, daidzein and equol) were measured using a validated UPLC-MS/MS method and quantified utilizing stable-labeled internal standards.ResultsObese rats gained significantly more weight (P < 0.0001) than lean rats for all three diet groups. There was no detectable serum IF for both LC and OB groups. The mean ± SD for LIF vs HIF serum total IF (uM) for genistein were; (0.11 + 0.04 vs 2.55 + 1.04; p < 0.001), daidzein (0.07 + 0.02 vs 1.14 + 0.45; p < 001) and equol (0.07 + 0.03 vs 1.84 + 0.8; p < 0.001). For OLIF vs OHIF serum genistein were; (0.22 + 0.08 vs 3.69 + 1.52; p < 0.001), daidzein (0.12 + 0.04 vs 1.58 + 0.70; p < 001) and equol (0.06 + 0.03 vs 2.40 + 0.7; p < 0.001). Comparing serum genistein LLIF vs OLIF; p < 0.05 for Daidzein p < 0.05 and equol p = 0.86.ConclusionsHigh isoflavones soy protein concentrate compared to low isoflavones increased serum genistein and daidzein but had no effects on serum equol in a NAFLD rat model.Funding SourcesThis project is support USDA #12889791 to RH.

Gynostemma pentaphyllum saponins alleviate non-alcoholic fatty liver disease in rats by regulating intestinal flora and short-chain fatty acid metabolism

This study aimed to explore the effects of Gynostemma pentaphyllum saponins(GPs) on non-alcoholic fatty liver disease(NAFLD) induced by high-fat diet in rats and reveal the underlying mechanism. The NAFLD model rats were prepared with high-fat diet. Forty male Sprague Dawley(SD) rats were randomly assigned into the control group, model group, and low-, moderate-, and high-dose GPs(50, 100, and 150 mg·kg~(-1), respectively) groups. After intragastric administration for 8 continuous weeks, we determined the body weight, liver weight, the levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-c), high-density lipoprotein cholesterol(HDL-c), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum, and the levels of TC, TG, malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), and interleukin 6(IL-6) in the liver. Furthermore, we observed the pathological changes of liver tissue by oil red O staining and hematoxylin-eosin(HE) staining, sequenced the 16 S rRNA of the intestinal flora in rat feces, and determined the content of short-chain fatty acids in rat feces. The results showed that GPs inhibited the excessive weight gain of high-fat diet-induced NAFLD in rats, reduced the liver weight, lowered the TC, TG, LDL-c, AST, and ALT levels in serum(P&lt;0.05), and rose the HDL-c level in serum(P&lt;0.01). GPs relieved the liver damage caused by high-fat diet, mainly manifested by the lowered levels of TC, TG, MDA, and IL-6 in the liver(P&lt;0.01) and elevated levels of CAT and SOD in the liver. Furthermore, GPs reversed the intestinal flora disorder caused by high-fat diet, restored the diversity of intestinal flora, increased the relative abundance of Bacteroides, and reduced the relative abundance of Firmicutes and the ratio of Firmicutes to Bacteroides. Moreover, GPs promoted the proliferation of beneficial bacteria such as Akkermansia, Bacteroides, and Parabacteroides, and inhibited the growth of harmful bacteria such as Desulfovibrio, Escherichia-Shigella, and Helicobacter. GPs increased the content of short-chain fatty acids(acetic acid, propionic acid, and butyric acid)(P&lt;0.01). These findings indicate that GPs can alleviate the high-fat diet-induced NAFLD in rats via regulating the intestinal flora and short-chain fatty acid metabolism.

Hibiscus sabdariffa extract improves hepatic steatosis, partially through IRS-1/Akt and Nrf2 signaling pathways in rats fed a high fat diet

Non-alcoholic fatty liver disease (NAFLD) has become a major world-wide health problem and is characterized by lipid accumulation in the liver induced by high fat diet (HFD) consumption. It is usually associated with inflammation, oxidative stress, and insulin resistance. Roselle extract (Hibiscus sabdariffa) is an herb which is used in traditional medicine. However, further study is necessary to represent the mechanism of NAFLD and find new preventive strategies. This study aims to investigate the protective effects of roselle extract on NAFLD rat models. Male Sprague–Dawley rats (n = 35) were divided into 5 groups, control, HFD, HFD + Simvastatin (HFD + SIM), HFD + 250 mg/kg BW, and HFD + 500 mg/kg BW of roselle extract (HFD + R250 and HFD + R500, respectively). The results showed that roselle extract reduced hepatic lipid contents, de novo lipogenesis enzymes, microsomal triglyceride transfer protein, inflammatory cytokines, malondialdehyde, and increased antioxidant properties, transporter related with lipoprotein uptake, and insulin signal proteins. Comparing to SIM, the HFD + R500 group exhibited the greater benefit in terms of anti-hepatic steatosis, antioxidant properties, and an ability to improve insulin resistance. This study demonstrates that roselle extract improved antioxidant properties and attenuated hepatic steatosis, liver inflammation, oxidative stress, and insulin resistance in HFD-induced NAFLD in rats, which could be used for NAFLD prevention.

Protective Roles of Shilajit in Modulating Resistin, Adiponectin, and Cytokines in Rats with Non-alcoholic Fatty Liver Disease.

To evaluate the effect of Shilajit, a medicine of Ayurveda, on the serum changes in cytokines and adipokines caused by non-alcoholic fatty liver disease (NAFLD). After establishing fatty liver models by feeding a high-fat diet (HFD) for 12 weeks, 35 Wistar male rats were randomly divided into 5 groups, including control (standard diet), Veh (HFD + vehicle), high-dose Shilajit [H-Sh, HFD + 250 mg/(kg·d) Shilajit], low-dose Shilajit [L-Sh, HFD + 150 mg/(kg·d) Shilajit], and pioglitazone [HFD + 10 mg/(kg·d) pioglitazone] groups, 7 rats in each group. After 2-week of gavage administration, serum levels of glucose, insulin, interleukin 1beta (IL-1β), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), adiponectin, and resistin were measured, and insulin resistance index (HOMA-IR) was calculated. After NAFLD induction, the serum level of IL-10 significantly increased and serum IL-1β, TNF-α levels significantly decreased by injection of both doses of Shilajit and pioglitazone (P<0.05). Increases in serum glucose level and homeostasis model of HOMA-IR were reduced by L-Sh and H-Sh treatment in NAFLD rats (P<0.05). Both doses of Shilajit increased adiponectin and decreased serum resistin levels (P<0.05). The probable protective role of Shilajit in NAFLD model rats may be via modulating the serum levels of IL-1β, TNF-α, IL-10, adipokine and resistin, and reducing of HOMA-IR.

Water Specific MRI T1 Mapping for Evaluating Liver Inflammation Activity Grades in Rats With Methionine-Choline-Deficient Diet-Induced Nonalcoholic Fatty Liver Disease.

Early detection and grading of liver inflammation are important for the management of nonalcoholic fatty liver disease (NAFLD) patients. There is still lack of a noninvasive way for the inflammation characterization in NAFLD. To assess liver inflammation grades by water specific T1 (wT1) in a rat model. Prospective. A total of 65 male rats with methionine-choline-deficient diet-induced NAFLD and 15 male normal rats as control. A 3 T; multiecho variable flip angle gradient echo sequence. The wT1 and proton density fat fraction were quantified. Inflammation and fibrosis were assessed histologically with H&E and Sirius red stained slices according to the nonalcoholic steatohepatitis scoring system. Inflammation grade was scored with G0/G1/G2/G3 as none/mild/moderate/severe inflammation in NALFD rats. G0 + G1 and G2 + G3 were combined as none-to-mild grade (GL) and moderate-to-severe grade (GH) inflammation groups. Analysis of variance (ANOVA), Mann-Whitney U test, Spearman's correlation, and receiver operating characteristic (ROC) analysis were performed. The areas under ROC (AUROC) was used for the diagnostic performance of wT1 in discriminating GH and GL. A P value < 0.01 was considered statistically significant. Seventy-six rats were included in the analysis. The numbers in G0-G3 groups were 5, 16, 13, and 27. wT1 of G0-G3 was 568.55 ± 63.93 msec, 582.53 ± 62.98 msec, 521.21 ± 67.31 msec, and 508.79 ± 60.53 msec. A moderate but significant negative correlation between wT1 and histopathological inflammation grades was observed (rs =-0.42). The wT1 of GH (512.80 ± 62.22 msec) was significantly lower than GL (579.20 ± 61.89 msec). The AUROC of wT1 was 0.79, and the optimal cut-off of wT1 was 562.64 msec (sensitivity: 90%, specificity: 76%), for the discrimination of GL and GH. wT1 could differentiate none-to-mild inflammation from moderate-to-severe inflammation in the early stage of the NAFLD rat model. 1 TECHNICAL EFFICACY: Stage 1.

RMRP inhibition prevents NAFLD progression in rats via regulating miR-206/PTPN1 axis.

This study aimed to investigate the regulatory function of lncRNA RMRP in non-alcoholic fatty liver disease (NAFLD). In vitro and in vivo NAFLD models were constructed. Hematoxylin & Eosin (H&E) and Oil-Red O staining assays were conducted to observe the morphology and lipid accumulation in liver tissues. Triglycine (TG) secretion was detected by ELISA assay. The expression levels of RMRP, microRNA-206, PTPN1 (protein tyrosine phosphatase, non-receptor type 1), and their downstream genes were assessed by qRT-PCR and Western blot. The regulatory relationship among these molecules was determined by luciferase reporter and RNA pull-down assays. RMRP and PTPN1 were up-regulated, while miR-206 was down-regulated in the liver tissues of NAFLD patients and rat model. RMRP inhibition improved the pathological state and liver function-related indexes of liver lipid deposition in the liver tissues of NAFLD rats. RMRP inhibition alleviated steatosis and TG secretion in free fatty acids (FFA)-treated AML-12 cells. RMRP could bind to miR-206 and downregulate its expression. Meanwhile, RMRP inhibition attenuated lipid accumulation by downregulating the PTPN1-PP2ASP1-SREBP1C pathway. Furthermore, RMRP inhibited the miR-206/PTPN1-SREBP1C signaling pathway in NAFLD rats and FFA-treated AML-12 cells. RMRP inhibition prevented NAFLD progression in rats via targeting the miR-206/PTPN1 axis.

Reduction of Hepatic Steatosis, Oxidative Stress, Inflammation, Ballooning and Insulin Resistance After Therapy with Safranal in NAFLD Animal Model: A New Approach.

IntroductionNon-alcoholic fatty liver disease (NAFLD) is intimately linked to hepatic steatosis, inflammation, insulin resistance (IR), oxidative stress (OS), and ballooning. A high fat diet (HFD) is considered a major etiological factor that primarily covers the numerous features of NAFLD.MethodsThe present study aimed to evaluate the protective effect of safranal on hepatic steatosis, OS, liver index, IR index, liver function enzymes, plasma lipids, TNF-α, malondialdehyde (MDA), advanced oxidation protein products (AOPPs) and nitrite (NO2–) levels in a NAFLD rat model fed with a HFD for 12 weeks. The ELISA kits were used to measure TNF-α and insulin in serum and plasma, respectively.ResultsHFD significantly induced hepatic steatosis, OS, IR, liver, and oxidative enzyme elevation and inflammation in experimental animals. Rats treated with safranal in ascending order of doses 250 and 500 mg/kg orally for 4-weeks showed a reduction in hepatic lipid’s accumulation, liver index, hepatic enzymes, collagen, hepatic oxidonitrative stress markers (like AOPP, MDA and NO2–), and raised the levels of catalase (CAT) and superoxide dismutase (SOD) enzymes. Glutathione system components, namely glutathione (GSH), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) levels were also restored in the safranal-treated groups. The reduction in serum TNF-α and IR provided further support to the anti-NAFLD effect of safranal. Moreover, the histopathological images indicated reverse of NAFLD activity score (NAS) through mild fatty degeneration, ballooning and inflammation in hepatocytes of treated groups.ConclusionFindings of blood and tissue analysis concluded that safranal can be a good choice in the management and cure of NAFLD.

Protective effect of quercetin against the metabolic dysfunction of glucose and lipids and its associated learning and memory impairments in NAFLD rats

BackgroundQuercetin (QUE) is a flavonol reported with anti-inflammatory and antioxidant activities, and previous results from the group of this study have demonstrated its neuroprotective effect against lipopolysaccharide-induced neuropsychiatric injuries. However, little is known about its potential effect on neuropsychiatric injuries induced or accompanied by metabolic dysfunction of glucose and lipids.MethodsA nonalcoholic fatty liver disease (NAFLD) rat model was induced via a high-fat diet (HFD), and glucolipid parameters and liver function were measured. Behavioral performance was observed via the open field test (OFT) and the Morris water maze (MWM). The plasma levels of triggering receptor expressed on myeloid cells-1 (TREM1) and TREM2 were measured via enzyme-linked immunosorbent assay (ELISA). The protein expression levels of Synapsin-1 (Syn-1), Synaptatogmin-1 (Syt-1), TREM1 and TREM2 in the hippocampus were detected using western blotting. Morphological changes in the liver and hippocampus were detected by HE and Oil red or silver staining.ResultsCompared with the control rats, HFD-induced NAFLD model rats presented significant metabolic dysfunction, hepatocyte steatosis, and impaired learning and memory ability, as indicated by the increased plasma concentrations of total cholesterol (TC) and triglyceride (TG), the impaired glucose tolerance, the accumulated fat droplets and balloon-like changes in the liver, and the increased escaping latency but decreased duration in the target quadrant in the Morris water maze. All these changes were reversed in QUE-treated rats. Moreover, apart from improving the morphological injuries in the hippocampus, treatment with QUE could increase the decreased plasma concentration and hippocampal protein expression of TREM1 in NAFLD rats and increase the decreased expression of Syn-1 and Syt-1 in the hippocampus.ConclusionsThese results suggested the therapeutic potential of QUE against NAFLD-associated impairment of learning and memory, and the mechanism might involve regulating the metabolic dysfunction of glucose and lipids and balancing the protein expression of synaptic plasticity markers and TREM1/2 in the hippocampus.

Xanthohumol alleviates T2DM-induced liver steatosis and fibrosis by mediating the NRF2/RAGE/NF-κB signaling pathway.

Hyperglycemia-associated advanced glycation end products (AGEs)and thereceptor for AGE (RAGE) contributeto nonalcoholic fatty liver disease (NAFLD). Xanthohumol (XH) exhibits protective activities against liver diseases. Aim: To investigate the effects of XH on Type IIdiabetes mellitus(T2DM)-induced liver steatosis and fibrosis. Methods: NAFLD rat models were duplicated. Biomolecular markers were detected. Quantitative real-time PCR(RT-PCR) and western blot were used to detect mRNA and protein expression. Immunofluorescence assays were employed to identifythe subcellular locations. Results: XH significantly ameliorated hyperglycemia and hyperlipidemia in rats. XH attenuated the expression of RAGE and NF-κB signaling. XH significantly alleviated inflammation and oxidation by upregulating NRF2 expression. Knockdown of NRF2 blocked XH protection in hepatocytes. Conclusion: XH protected against T2DM-induced liver steatosis and fibrosis by mediating NRF2/AGE/RAGE/NF-κB signaling.

Human umbilical cord-derived mesenchymal stem cell-exosomal miR-627-5p ameliorates non-alcoholic fatty liver disease by repressing FTO expression.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disorders. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)-based therapy is currently considered to be an effective treatment for NAFLD. The present study aimed to determine whether hUC-MSCs-exosomes have a hepatoprotective effect on NAFLD. We constructed NAFLD rat model by high-fat high-fructose feeding. Liver cells (L-O2) were treated with palmitic acid (PA) to mimic NAFLD model. NAFLD rats and PA-treated L-O2 cells were treated with hUC-MSCs-exosomes, and then we determined the influence of exosomes on liver damage and glucose and lipid metabolism in vivo and in vitro. We found that hUC-MSCs-exosomes exhibited an up-regulation of miR-627-5p. Exosomal miR-627-5p promoted cell viability and repressed apoptosis of PA-treated L-O2 cells. Exosomal miR-627-5p also enhanced the expression of G6Pc, PEPCK, FAS and SREBP-1c and suppressed PPARα expression in PA-treated L-O2 cells. Moreover, miR-627-5p interacted with fat mass and obesity-associated gene (FTO) and inhibited FTO expression in L-O2 cells. MiR-627-5p-enriched exosomes improved glucose and lipid metabolism in L-O2 cells by targeting FTO. In vivo, exosomal miR-627-5p ameliorated insulin tolerance, liver damage, glucose and lipid metabolism and reduced lipid deposition in NAFLD rats. Exosomal miR-627-5p also reduced body weight, liver weight, and liver index (body weight/liver weight) in NAFLD rats. In conclusion, these data demonstrate that HUC-MSCs-derived exosomal miR-627-5p improves glucose and lipid metabolism and alleviateliver damage by repressing FTO expression, thereby ameliorating NAFLD progression. Thus, hUC-MSCs-exosomes may be a potential treatment for NAFLD.

Probiotics Alleviated Nonalcoholic Fatty Liver Disease in High-Fat Diet-Fed Rats via Gut Microbiota/FXR/FGF15 Signaling Pathway

Gut microbiota (GM) dysbiosis and bile acid (BA) metabolism disorder play an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Probiotics had a beneficial effect on NAFLD, but further study is needed to explore probiotics as a potential therapeutic agent to NAFLD. The aim of this study was to investigate the regulatory effect of probiotics on gut microbiota in NAFLD rats and to explore the possible mechanism of probiotics regulating the bile acid receptor farnesoid X receptor/growth factor 15 (FXR/FGF15) signaling pathway in rats. We established a rat model of NAFLD fed with a high-fat diet (HFD) for 14 weeks, which was given different interventions (312 mg/kg/day probiotics or 10 mg/kg/day atorvastatin) from the 7th week. Serum lipids and total bile acids (TBA) were biochemically determined; hepatic steatosis and lipid accumulation were evaluated with HE staining. The expression levels of FXR, FGF15 mRNA, and protein in rat liver were detected. 16S rDNA was used to detect the changes of gut microbiota in rats. Compared with the HFD group, probiotics and atorvastatin significantly reduced serum lipids and TBA levels. And probiotics increased dramatically the expression of FXR, FGF15 mRNA, and protein in the liver. But there were no significant changes in the atorvastatin group. Probiotics and atorvastatin can upregulate the diversity of gut microbiota and downregulate the abundance of pathogenic bacteria in NAFLD model rats. In summary, probiotics alleviated NAFLD in HFD rats via the gut microbiota/FXR/FGF15 signaling pathway.

Thymosin beta 4 alleviates non-alcoholic fatty liver by inhibiting ferroptosis via up-regulation of GPX4

Thymosin beta 4 (Tβ4) can improve the liver fibrosis and reduce inflammation, while the role of Tβ4 in non-alcoholic fatty liver disease (NAFLD) whether mediated by ferroptosis remains unclear. A rat model of NAFLD was established on a high-fat diet (HFD), and rats were assigned ferroptosis inducer erastin and inhibitor Ferrostatin 1 (Fer-1). Subsequently, histopathology of the liver and the expression of ferroptosis-related genes in rat liver were detected. The steatosis of LO2 cells was induced by palmitic acid (PA) to reproduce the results of the rat experiment. The small interfering RNA (siRNA) was used to interfere with GPX4 expression to explore the influence on Tβ4 function. Tβ4 improved the inflammation, biochemical and lipid metabolism indexes, increased the antioxidant level, and inhibited abnormal accumulation of intracellular reactive oxygen species in HFD-induced NAFLD rats. Also, Tβ4 improved PA-induced LO2 damage and inhibited apoptosis of PA-induced LO2 cells. Both in vivo and in vitro, Tβ4 regulated expression of genes associated with ferroptosis, and Fer-1 treatment exaggerated the above effects of Tβ4, while erastin attenuated the protective effect of Tβ4. Moreover, siRNA GPX4 attenuated the protective effect of Tβ4 on the rat liver and on the mitochondrial membrane integrity of LO2 cells. Interfered expression of GPX4 with siRNA also regulated the expression of Bcl-2, Bax, Caspase-3 and SOD1, which attenuated therapeutic effect of Tβ4 on rat liver and LO2 cells. This study revealed that Tβ4 protects hepatocytes by inhibiting the GPX4-mediated ferroptosis pathway, which provides a new strategy and target for the treatment of NAFLD.

Biochanin A Regulates Cholesterol Metabolism Further Delays the Progression of Nonalcoholic Fatty Liver Disease

PurposeTo discover the possible target of biochanin A (BCA) in the lipid metabolism pathway and further explore its mechanism to nonalcoholic fatty liver disease (NAFLD).MethodsWe adopted a high-fat and high-glucose diet for 12 weeks to build the NAFLD rat model, which was then treated with different proportions of BCA for 4 weeks. General condition, body weight, Lee index, and liver index were then evaluated. Furthermore, blood lipid level and insulin resistance (IR) were detected. Moreover, hematoxylin and eosin and oil red O staining were used to observe the pathological changes in the liver. Finally, Western blotting was used to detect the protein expression levels of CYP7A1, HMGCR, LDLR, PPAR-α, PPAR-γ, and SREBP-1c in the liver.ResultsThe vital signs of rats in each group were stable. The treatment with BCA effectively reduced Lee index and liver index (F = 104.781, P < 0.05); however, the weight was not effected in each group. Additionally, BCA effectively reduced the related lipid metabolism indexes of NAFLD, such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), blood glucose, insulin, IR (F =12.463 (TC), 6.909 [TG], and 15.3 effected 75 [LDL], P < 0.05), and increased HDL (F = 11.580, P < 0.05). We observed that BCA could significantly improve steatosis and inflammatory cell infiltration in liver slices. Furthermore, BCA significantly increased the CYP7A1, LDLR, and PPAR-α protein expression in the liver and downregulated the HMGCR, SREBP-1c, and PPAR-γ protein expression.ConclusionBCA could delay the liver damage of NAFLD induced by a high-fat diet, regulate the blood lipid level, and improve the expression of lipid metabolism-related genes in rats.

Changes in digoxin pharmacokinetics associated with hepatic P-glycoprotein upregulation in rats with non-alcoholic fatty liver disease.

Upregulation of hepatic P-glycoprotein (P-gp) expression has been reported in patients with non-alcoholic fatty liver disease (NAFLD) and rodent models thereof. Here, we explored the changes hepatic P-gp expression and activity in a NAFLD rat model and the effects thereof on the pharmacokinetics of digoxin (a probe substrate of P-gp). Rats were fed a 1% (w/w) orotic acid-containing diet for 20days to induce NAFLD; control rats received a normal diet. P-gp expression and biliary digoxin excretion were examined. The pharmacokinetics of digoxin were evaluated after it had been administered intravenously (10μg·kg-1 ) and orally (200μg·kg-1 ) to control and NAFLD rats. The total areas under the plasma concentration-time curves (AUCs) of digoxin after intravenous and oral administration were significantly smaller (by 39.1% and 73.0%, respectively) in NAFLD rats because of faster biliary digoxin excretion, reflecting elevations of hepatic P-gp expression and activity. Notably, the steady-state volume of distribution rose by 98.2%, while extent of oral bioavailability fell by 55.5% in NAFLD rats. This is the first study to report digoxin pharmacokinetic changes caused by hepatic P-gp upregulation in NAFLD. Further studies are needed to explore the clinical impact of enhanced P-gp-mediated biliary excretion on pharmacotherapies using P-gp substrates in patients with NAFLD.

Mechanism of Huo-Xue-Qu-Yu Formula in Treating Nonalcoholic Hepatic Steatosis by Regulating Lipid Metabolism and Oxidative Stress in Rats.

Huo-Xue-Qu-Yu formula (HXQYF) is a prescription consisting of Ginkgo biloba leaf and Paeonia lactiflora Pall. for treating hyperlipidemia and NAFLD in China. Here, we investigated the hepatic and renal function, oxidative stress and lipid metabolism, and potential mechanisms of HXQYF on nonalcoholic fatty liver disease (NAFLD) rat models. NAFLD rat models were induced with high-fat diet (HFD) and 10% fructose water for 18 weeks and orally administered with or without HXQYF simultaneously. The results showed that HXQYF (22.5, 45, 90 mg/kg) significantly improved blood lipid levels via reducing serum TC, TG, LDL-C, and APOB values and elevating HDL-C and APOA1 levels in NAFLD rats. The higher levels of ALT, AST, CR, and BUN in serum induced by HFD were reduced by HXQYF. HE staining showed that HXQYF (90 mg/kg) reduced the accumulation of fat droplets and alleviated inflammatory response in liver cells. Three doses of HXQYF exhibited notable antioxidant effects by elevating SOD, GSH, and CAT activities and decreasing MDA and OH-1 levels in the liver. Furthermore, abnormal lipid metabolism caused by HFD was alleviated by HXQYF, which was associated with the upregulation of PPAR-α, AdipoR2, and CPT1 mRNAs as well as the downregulation of CYP2E1 and SREBP-1c mRNAs in liver tissue. In conclusion, our work verified that HXQYF could reduce the degree of hepatic steatosis, suppress oxidative stress, and attenuate lipid metabolism, thus preventing NAFLD.

Jian-Gan-Xiao-Zhi Decoction Alleviates Inflammatory Response in Nonalcoholic Fatty Liver Disease Model Rats through Modulating Gut Microbiota

Background Jian-Gan-Xiao-Zhi decoction (JGXZ), composed of Salvia miltiorrhiza Bunge, Panax notoginseng, Curcuma zedoaria, and other 9 types of herbs, has demonstrated beneficial effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanisms behind JGXZ's impact on NAFLD remain unknown. Methods In this study, a NAFLD rat model induced by a high-fat diet (HFD) received oral treatment of JGXZ (8 or 16 g crude herb/kg) for 12 weeks. The therapeutic effects of JGXZ on NAFLD model rats were investigated through blood lipid levels and pathological liver changes. 16S rRNA analysis was used to study the changes in gut microbiota after JGXZ treatment. The expressions of occludin and tight junction protein 1 (ZO-1) in the colon were investigated using immunostaining to study the effects of JGXZ on gut permeability. The anti-inflammatory effects of JGXZ were also studied through measuring the levels of IL-1β, IL-6, and TNF-α in the serum and liver. 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. 16S rRNA analysis showed that JGXZ impacted the diversity of gut microbiota, decreasing the Firmicutes–to-Bacteroidetes ratio, and increasing the relative abundance of probiotics, such as Alloprevotella, Lactobacillus, and Turicibacter. Gut permeability evaluation found that the expressions of ZO-1 and occludin in the colon were increased after JGXZ treatment. Moreover, JGXZ treatment could decrease the levels of IL-1β, IL-6, and TNF-α in the serum and liver. Conclusions Our study illustrated that JGXZ could ameliorate NAFLD through modulating gut microbiota, decreasing gut permeability, and alleviating inflammatory response.

Astragaloside IV Improves High-Fat Diet-Induced Hepatic Steatosis in Nonalcoholic Fatty Liver Disease Rats by Regulating Inflammatory Factors Level via TLR4/NF-κB Signaling Pathway.

Objective: Astragaloside IV (AS-IV) is the primary bioactive component purified from Astragalus membranaceus which is one of the traditional Chinese medicines. Research studies found that AS-IV has significant pharmacological effects on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver cirrhosis, and diabetic nephropathy, but little is known about the effects of AS-IV on nonalcoholic fatty liver disease (NAFLD). In this study, we investigated whether AS-IV has beneficial effects on NAFLD in rats and its potential mechanisms. Methods: Male SD rats were fed with high-fat diet (HFD) for 12 weeks to establish NAFLD rat model, and then, the rats were divided into five groups. The control group rats were fed with normal diet for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The model group rats were fed with HFD for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The AS-IV-L, AS-IV-M, and AS-IV-H groups were treated with 20, 40, and 80 mg kg−1 day−1 of AS-IV by intragastric administration for 4 weeks and given HFD diet. Then, we detected serum transaminase (ALT, AST), blood lipid (TG, TC), inflammatory cytokines (IL-6, IL-8 and TNF-α), liver histology(NAFLD activity score), TLR4/MyD88 signaling pathway in liver tissue. Results: We found AS-IV significantly reduced serum levels of AST, ALT, TG, TNF-α, IL-6, and IL-8 in NAFLD rats and downregulate the expression of TLR4 mRNA, MyD88 mRNA, NF-κB mRNA, and proteins in liver tissue. Moreover, AS-IV could significantly reduce the NAFLD activity score of NAFLD rat liver. Conclusion: In this study, we demonstrated that AS-IV have a protective effect on NAFLD by inhibiting TNF-α, IL-6 and IL-8 levels and down-regulating TLR4, MyD88 and NF-κB expression in rat liver tissues.

The Therapeutic Efficacy of Curcumin vs. Metformin in Modulating the Gut Microbiota in NAFLD Rats: A Comparative Study

Structural disruption of gut microbiota is closely related to the occurrence of non-alcoholic fatty liver disease (NAFLD). Previous research has demonstrated that both curcumin (CUR) and metformin (MET) have a therapeutic effect against NAFLD and play a role in modulating the gut microbiota. However, there is a lack of direct comparison between the two medications in terms of the therapeutic efficacy and the regulatory effect on gut microbiota. In this study, we administered either CUR or MET to rats with high-fat diet (HFD)-induced obesity to observe changes in body parameters, biochemical parameters, liver, and ileum pathology and gut microbiota, and used next generation sequencing and multivariate analysis to evaluate the structural changes of gut microbiota in a NAFLD rat model before and after CUR and MET intervention. It was found that both CUR and MET attenuated hepatic ectopic fat deposition, alleviated inflammatory factors, and improved intestinal barrier integrity in HFD-fed rats. More importantly, CUR and MET reduced the Firmicutes/Bacteroidetes ratio and reverted the composition of the HFD-disrupted gut microbiota. Both CUR and MET treatments effectively modified the gut microbiome, enriched the abundance of beneficial bacteria and reduced opportunistic pathogens in obese rats. The abundance of Butyricicoccus was increased while the abundance of Dorea was decreased in HFD + CUR group. Besides, some beneficial bacteria such as Prevotella were increased in MET-treated animals. Spearman’s correlation analysis showed that Helicobacter, Akkermansia, Desulfovibrio, Romboutsia, Corynebacterium, Lactobacillus, Ruminococcaceae_unclassified, Lachnospiraceae_unclassified, and Clostridiales_unclassified showed significantly positive correlations with TG, TC, LDL-C, GLU, IL-6, IL-1β, and TNF-α, and negative correlations with HDL-C (both p < 0.05). However, Prevotella and Stomatobaculum showed an opposite trend. In summary, CUR and MET showed similar effects in alleviating hepatic steatosis, improving intestinal barrier integrity and modulating gut microbiota in HFD-induced obesity rats, and therefore may prove to be a novel adjunctive therapy for NAFLD.

Hesperetin ameliorates hepatic oxidative stress and inflammation via the PI3K/AKT-Nrf2-ARE pathway in oleic acid-induced HepG2 cells and a rat model of high-fat diet-induced NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disease. Dietary supplementation has become a promising strategy for managing NAFLD. Hesperetin, a citrus flavonoid, is mainly found in citrus fruits (oranges, grapefruit, and lemons) and possesses multiple pharmacological properties, including anti-cancer, anti-Alzheimer and anti-diabetic effects. However, the anti-NAFLD effect and mechanisms of hesperetin remain unclear. In this study, we investigated the therapeutic effect of hesperetin against NAFLD and the underlying mechanism in vitro and in vivo. In oleic acid (OA)-induced HepG2 cells, hesperetin upregulated antioxidant levels (SOD/GPx/GR/GCLC/HO-1) by triggering the PI3 K/AKT-Nrf2 pathway, alleviating OA-induced reactive oxygen species (ROS) overproduction and hepatotoxicity. Furthermore, hesperetin suppressed NF-κB activation and reduced inflammatory cytokine secretion (TNF-α and IL-6). More importantly, we revealed that this anti-inflammatory effect is attributed to reduced ROS overproduction by the Nrf2 pathway, as pre-treatment with Nrf2 siRNA or an inhibitor of superoxide dismutase (SOD) or/and glutathione peroxidase (GPx) abolished hesperetin-induced NF-κB inactivation and reductions in inflammatory cytokine secretion. In a rat model of high-fat diet (HFD)-induced NAFLD, we confirmed that hesperetin relieved hepatic steatosis, oxidative stress, inflammatory cell infiltration and fibrosis. Moreover, hesperetin activated the PI3 K/AKT-Nrf2 pathway in the liver, increasing antioxidant expression and inhibiting NF-κB activation and inflammatory cytokine secretion. In summary, our results demonstrate that hesperetin ameliorates hepatic oxidative stress through the PI3 K/AKT-Nrf2 pathway and that this antioxidative effect further suppresses NF-κB-mediated inflammation during NAFLD progression. Thus, our study suggests that hesperetin may be an effective dietary supplement for improving NAFLD by suppressing hepatic oxidative stress and inflammation.