Rat Model Of Non-alcoholic Fatty Liver Disease Research Articles

Green tea polyphenols attenuate hepatic steatosis, and reduce insulin resistance and inflammation in high-fat diet-induced rats.

Non‑alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation; however, the exact pathogenesis of NAFLD is not fully understood. Green tea polyphenols (GTP) exhibit beneficial effects against metabolic syndrome. However, the effect of GTP on NAFLD remains largely unknown. The aim of the present study was to investigate the effects of GTP on NAFLD in high‑fat diet (HFD)‑induced rats. The NAFLD rat model was induced with a HFD for 8weeks. A total of 30adult male Sprague Dawley rats were randomly divided into three groups: i) Normal control group; ii) HFD group; and iii) HFD with GTP group. Hematoxylin and eosin and OilRedO analyses were performed. The levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST) and inflammatory cytokines in the serum, as well as oxidative stress markers and hepatic lipids in the liver were measured. In addition, parameters associated with glucose metabolism were also assessed. Western blotting and RT‑qPCR were used to determine the expression levels of 5'adenosine monophosphate‑activated protein kinase(AMPK). HFD‑induced rats exhibited features associated with NAFLD. GTP intervention significantly reduced serum ALT and AST levels. Fasting serum glucose, insulin resistance and hepatic lipid levels were all decreased in the GTP‑treated rats. GTP also significantly decreased the levels of TNF‑α, IL‑6 and malondialdehyde. In contrast, superoxide dismutase levels were increased in the liver. Furthermore, GTP also significantly increased phosphorylation of AMPK and attenuated histopathological changes indicative of injury in liver tissue. GTP has a protective effect on HFD‑induced hepatic steatosis, insulin resistance and inflammation, and the underlying mechanism may involve the AMPK pathway.

Resveratrol Reduces Glucolipid Metabolic Dysfunction and Learning and Memory Impairment in a NAFLD Rat Model: Involvement in Regulating the Imbalance of Nesfatin-1 Abundance and Copine 6 Expression.

Resveratrol (RES) is a polyphenolic compound, and our previous results have demonstrated its neuroprotective effect in a series of animal models. The aim of this study was to investigate its potential effect on a nonalcoholic fatty liver disease (NAFLD) rat model. The parameters of liver function and glucose and lipid metabolism were measured. Behavior performance was observed via the open field test (OFT), the sucrose preference test (SPT), the elevated plus maze (EPM), the forced swimming test (FST), and the Morris water maze (MWM). The protein expression levels of Copine 6, p-catenin, catenin, p-glycogen synthase kinase-3beta (GSK3β), GSK3β, and cyclin D1 in the hippocampus and prefrontal cortex (PFC) were detected using Western blotting. The results showed that RES could reverse nesfatin-1-related impairment of liver function and glucolipid metabolism, as indicated by the decreased plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), glucose, insulin, and nesfatin-1; increase the plasma level of high-density lipoprotein cholesterol (HDL-C); and reduce hepatocyte steatosis in NAFLD rats. Although there was no significant difference among groups with regard to performance in the OFT, EPM, and FST tasks, RES-treated NAFLD rats showed an increased sucrose preference index in the SPT and improved learning and memory ability in the MWM task. Furthermore, the imbalanced protein expression levels of Copine 6, p-catenin, and p-GSK3β in the hippocampus and PFC of NAFLD rats were also restored to normal by treatment with RES. These results suggested that four consecutive weeks of RES treatment not only ameliorated glucolipid metabolic impairment and liver dysfunction in the NAFLD rat model but also mitigated the attendant behavioral and cognitive impairments. In addition to the mediating role of nesfatin-1, the mechanism underlying the therapeutic effect of RES on NAFLD might be associated with its ability to regulate the imbalanced expression level of Copine 6 and the Wnt signaling pathway in the hippocampus and PFC.

Mitochondrial dysfunction in high-fat diet-induced nonalcoholic fatty liver disease: The alleviating effect and its mechanism of Polygonatum kingianum

BackgroundMitochondrial dysfunction is an important mechanism of non-alcoholic fatty liver disease (NAFLD). Developing mitochondrial regulators/nutrients from natural products to remedy mitochondrial dysfunction represent attractive strategies for NAFLD therapy. In China, Polygonatum kingianum (PK) has been used as a herb and food nutrient for centuries. So far, studies in which the effects of PK on NAFLD are evaluated are lacking. Our study aims at identifying the effects and mechanism of action of PK on NAFLD based on mitochondrial regulation. MethodsA NAFLD rat model was induced by a high-fat diet (HFD) and rats were intragastrically given PK (1, 2 and 4 g/kg) for 14 weeks. Changes in body weight, food intake, histological parameters, organ indexes, biochemical parameters and mitochondrial indicators involved in oxidative stress, energy metabolism, fatty acid metabolism, and apoptosis were investigated. ResultsPK significantly inhibited the HFD-induced increase of alanine transaminase, aspartate transaminase, total cholesterol (TC), and low density lipoprotein cholesterol in serum, and TC and triglyceride in the liver. In addition, PK reduced high density lipoprotein cholesterol and liver enlargement without affecting food intake. PK also remarkably inhibited the HFD-induced increase of malondialdehyde and the reduction of superoxide dismutase, glutathione peroxidase, ATP synthase, and complex I and II, in mitochondria. Moreover, mRNA expression of carnitine palmitoyl transferase-1 and uncoupling protein-2 was significantly up-regulated and down-regulated after PK treatment, respectively. Finally, PK notably inhibited the HFD-induced increase of caspase 9, caspase 3 and Bax expression in hepatocytes, and the decrease of expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. ConclusionPK alleviated HFD-induced NAFLD by promoting mitochondrial functions. Thus, PK may be useful mitochondrial regulators/nutrients to remedy mitochondrial dysfunction and alleviate NAFLD.

Metabolomics Characterizes the Effects and Mechanisms of Quercetin in Nonalcoholic Fatty Liver Disease Development.

As metabolomics is widely used in the study of disease mechanisms, an increasing number of studies have found that metabolites play an important role in the occurrence of diseases. The aim of this study is to investigate the effects and mechanisms of quercetin in high-fat-sucrose diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) development using nontargeted metabolomics. A rat model of NAFLD was established by feeding with an HFD for 30 and 50 days. The results indicated quercetin exhibited hepatoprotective activity in 30-day HFD-induced NAFLD rats by regulating fatty acid related metabolites (adrenic acid, etc.), inflammation-related metabolites (arachidonic acid, etc.), oxidative stress-related metabolites (2-hydroxybutyric acid) and other differential metabolites (citric acid, etc.). However, quercetin did not improve NAFLD in the 50-day HFD; perhaps quercetin was unable to reverse the inflammation induced by a long-term high-fat diet. These data indicate that dietary quercetin may be beneficial to NAFLD in early stages. Furthermore, combining metabolomics and experimental approaches opens avenues to study the effects and mechanisms of drugs for complex diseases.

Protective effect and mechanism of Qiwei Tiexie capsule on 3T3-L1 adipocytes cells and rats with nonalcoholic fatty liver disease by regulating LXRα, PPARγ, and NF-κB-iNOS-NO signaling pathways

Ethnopharmacological relevanceQiwei Tiexie capsule (QWTX) is a representative prescription of Tibetan medicine, which is widely used for long-term treatment of chronic liver disease and nonalcoholic fatty liver disease (NAFLD). Aim of the studyThis study explored the effects and mechanism of QWTX on 3T3-L1 adipocytes and NAFLD. Materials and methodsThe 3T3-L1 preadipocytes and NAFLD rat model were used in the study. In 3T3-L1 cells, the cytotoxicity of QWTX was tested by CKK-8, and glucose uptake and fat acid oxidation were assessed by 2-deoxy-D-[3H] glucose and [1–14C] palmitic acid, respectively. The expression levels of carnitine palmitoyltransferase-1 (CPT-1), liver X receptor α (LXRα), peroxisome proliferator-activated receptor (PPAR) γ, inducible nitric oxide synthase (iNOS), ikappa B α (IκBα), and AKT were determined by PCR and western blot. NAFLD was established by the administration of fat emulsion and sucrose for 9 weeks. The effects of QWTX on lipid metabolism, liver function, and hepatic morphology were observed in NAFLD rats by HE and transmission electron microscope. Serum level of nitric oxide (NO) and fee fatty acid (FFA), superoxide dismutase (SOD) and malondialdehyde (MDA) contents in the liver, as well as the expression levels of Cytochrome P450 2E1 (CYP2E1), NF-κB, monocyte chemoattractant protein 1 (MCP-1), CPT-1, LXRα, PPARα, PPARβ/δ, PPARγ, and iNOS were all detected. ResultsQWTX showed no cell cytotoxicity in 3T3-L1 preadipocyte cells, and increased the 14CO2 production rate to 4.15, which indicated the reducing the fatty accumulation. In NAFLD, QWTX attenuated liver steatosis, fat vacuoles and inflammation from the HE staining and electron micrograph tests. For the oxidative stress biomarkers, serum FFA level was reduced and serum NO level was enhanced after QWTX treatment. In liver tissue, SOD was decreased and MDA was significantly increased in NAFLD, and both of them were restored by QWTX. NF-κB and CYP2E1 were also upregulated in NAFLD, while downregulated by QWTX. Downregulation of LXRα, PPARγ and iNOS by QWTX were both observed in the 3T3-L1 adipocytes and NAFLD model. ConclusionsQWTX protected the liver injury in differentiated 3T3-L1 adipocytes and NAFLD by regulating the LXRα, PPARγ, and NF-κB-iNOS-NO signal pathways.

Berberine Ameliorates High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Rats via Activation of SIRT3/AMPK/ACC Pathway

This study aimed to verify the effects of berberine (BBR) on the fat metabolism proteins involved in the sirtuin 3 (SIRT3)/adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway in the liver tissues of rats with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). Forty-eight rats were randomly divided into the normal control (NC) group, HFD group or BBR group, with 16 rats in each group. After 8 and 16 weeks of treatment, serum and liver samples were collected. Subsequently, body parameters, biochemical parameters and liver pathology were examined. The expression levels of proteins involved in the SIRT3/AMPK/ACC pathway in the liver were detected by Western blotting. After 8 and 16 weeks of a HFD, the successful establishment of rat models with different degrees of NAFLD was confirmed by hematoxylin and eosin (H&E) and Oil Red O staining. NAFLD rat models exhibited obesity and hyperlipidemia, and the protein expression levels of SIRT3, p-AMPK, p-ACC, and CPT-1A in the liver were significantly decreased compared to those in the NC group. The concurrent administration of BBR with the HFD effectively improved serum and liver lipid profiles and ameliorated liver injury. Furthermore, the protein expression levels of SIRT3, p-AMPK, p-ACC, and CPT-1A in the liver were significantly increased in the BBR group as compared with those in the HFD group. In conclusion, our data suggest that the mechanism by which BBR ameliorates HFD-induced hepatic steatosis may be related to the activation of the SIRT3/AMPK/ACC pathway in the liver.

Untargeted metabonomics reveals intervention effects of chicory polysaccharide in a rat model of non-alcoholic fatty liver disease.

In the current study, serum metabolomics techniques were used to evaluate the potential mechanism of the effect of chicory polysaccharides in non-alcoholic fatty liver disease (NAFLD) rats. A rat model of NAFLD was constructed according to the histopathological data and biochemical parameters, while the underlying mechanisms of high-fat diet (HFD) induced NAFLD and the therapeutic effects of chicory polysaccharides (CP) were studied by the adoption of serum metabolomics. The serum metabolites were analyzed by GC/MS. Multivariate statistical approaches such as principal component analysis, revealed significant differences with HFD model and CP groups against the control. Results indicated that CP plays a regulatory role in the occurrence of NAFLD. Meantime, a total of 65 candidate biomarkers were screened and identified. Cluster analysis, enrichment analysis and metabolic pathway analysis of differential metabolites also indicated that amino acid metabolism and fatty acid biosynthesis in NAFLD rats, the β-oxidation and urea cycle of very long-chain fatty acids were mainly disturbed when compared against the control group. The corresponding metabolic pathways in the CP group were relieved compared against the NAFLD rats. These results showed that untargeted metabonomics helps to explain intervention effects of chicory polysaccharide with the rat model of NAFLD.

Protection of hepatocyte mitochondrial function by blueberry juice and probiotics via SIRT1 regulation in non-alcoholic fatty liver disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Our previous study has firstly reported that blueberry juice and probiotics (BP) effectively protect liver function in NAFLD. However, the role of BP in hepatic mitochondria is unknown. Here, we aimed to investigate the effects and mechanisms of BP on the mitochondrial function and oxidative stress of rats with NAFLD. The NAFLD rat models were established and treated with BP and SIRT1 siRNA. The mitochondrial ultrastructure was analyzed by electron microscopy, reactive oxygen species (ROS) was detected by immunofluorescence, and biomarkers of mitochondrial function and oxidative stress were examined via quantitative reverse transcription-PCR, western blot, and immunohistochemistry. Results revealed that BP significantly reversed the NAFLD-induced hepatic mitochondrial damage, mitochondrial swelling, and hepatic necrosis. In particular, BP significantly restored the mitochondrial respiratory function of NAFLD rats by decreasing state 4 and 3 respiration rates, by increasing the respiration control ratio (RCR) and the ADP/O ratio, and by enhancing ATP, ADP, AMP, and EC syntheses. Moreover, BP reduced mitochondrial oxidative stress in NAFLD by decreasing the MDA level, elevating the GSH and SOD levels, and suppressing the ROS activity. Importantly, SIRT1 deficiency significantly abolished the effects of BP on the mitochondria and oxidative stress. Furthermore, BP reversed the decline of PGC-1α expression induced by NAFLD, while SIRT1 silencing significantly suppressed the effects of BP on PGC-1α. In conclusion, BP might effectively protect rats against mitochondrial dysfunction during NAFLD as potential ingredients of functional food, by modulating the SIRT1 expression. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide novel tools for therapeutic intervention.

Inhibition of CCL19 benefits non‑alcoholic fatty liver disease by inhibiting TLR4/NF‑κB‑p65 signaling.

Non‑alcoholic fatty liver disease (NAFLD), which affects approximately one‑third of the general population, has become a global health problem. Thus, more effective treatments for NAFLD are urgently required. In the present study, high levels of C‑C motif ligand 19 (CCL19), signaling pathways such as Toll‑like receptor 4 (TLR4)/nuclear factor‑κB (NF‑κB), and proinflammatory factors including interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) were detected in NAFLD patients, thereby indicating that there may be an association between CCL19 and these factors in NAFLD progression. Using a high‑fat diet (HFD), the present study generated a Sprague‑Dawley rat model of NAFLD, which displayed dyslipidemia with increased levels of plasma aspartate aminotransferase, alanine aminotransferase, total cholesterol and triglyceride. Dyslipidemia, liver histopathology and gene expression analyses indicated that the NAFLD model was successfully induced by HFD, and metformin and berberine (BBR) were effective treatments for NAFLD. HFD‑induced CCL19 levels and associated factors were markedly reduced by the two drug treatments. In addition, metformin or BBR alone significantly promoted adenosine monophosphate‑activated protein kinase (AMPK) phosphorylation, which was inhibited by HFD. These results demonstrated that metformin and BBR could improve NAFLD, which may be via the activation of AMPK signaling, and the high expression of CCL19 in NAFLD was significantly reduced by metformin and BBR. It could be inferred that inhibition of CCL19 may be an effective treatment for NAFLD.

Effects of extracts from soothing-liver and invigorating-spleen formulas on the injury induced by oxidative stress in the hepatocytes of rats with non-alcoholic fatty liver disease induced by high-fat diet

OBJECTIVETo investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress, mediated by the Nuclear factor-like 2 (Nrf2)-Antioxidant response element (ARE) pathway, in the hepatocytes of rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat diet. METHODSSoothing-liver and invigorating-spleen formula mixtures were prepared for five groups: normal, model, soothing-liver formula group (SLG), invigorating-spleen formula group (ISG), integrated formula group (IG). The rat model of NAFLD was induced by feeding rats a high-fat diet (HFD). After 16 weeks, the hepatic tissue was examined following Haematoxylin-Eosin (H&E) staining and with Transmission electron microscopy (TEM). Levels of hepatic lipids, serum lipids and hepatic functions were measured using a biochemical analyser. Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry, cellular immunofluorescence and flow cytometry. The expression levels of Nrf2, Kelch-like epichlorohydrin-associated protein 1 (Keap-1), haeme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNAs were assessed by real-time fluorescence quantitative PCR. Nrf2, Keap-1, HO-1 and NQO1 proteins were measured by Western blotting. Finally, the levels of oxidative stress factors Superoxide Dismutase (SOD), malonaldehyde (MDA) and Glutathione peroxidase (GSH-Px) in hepatocytes were measured by WST-1, TBA and colorimetry. RESULTSThe H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features. The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased. Compared with the model group, the levels of hepatic lipids, serum lipids and hepatic function in the treatment groups were ameliorated to different degrees. The yields of purified hepatocytes in each rat were 4-5 × 108. The viability of the isolated hepatocytes was higher than 95%, with a purity over 93.2%. Cellular immunohistochemistry analysis showed that the hepatocytes were brown, while in the cellular immunofluorescence analysis, the hepatocytes showed green fluorescence. The expression levels of Nrf2, Keap-1, HO-1 and NQO1 mRNA and protein in the hepatocytes were significantly higher in the model group than in the normal group (P < 0.05, P < 0.01). Compared with the model group, the expression of Nrf2, Keap-1, HO-1 and NQO-1 mRNAs and proteins in all treatment groups increased, especially in the IG (P <0.01). CONCLUSIONThe extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway, which may be the mechanism having the potential for prevention and treatment of NAFLD.

Effect of Fuzi Lizhong decoction in reducing liver injury of rats with non-alcoholic fatty liver via activating AMPK and suppressing NF-κBp65 pathway

To investigate the protective effect and relevant mechanism of Fuzi Lizhong decoction (FZLZD) on liver of rats with non-alcoholic fatty liver disease (NAFLD), totally 32 male SPF Wistar rats were randomly divided into 4 groups: control group, model group, Yishanfu (YSF) group (200 mg·kg⁻¹·d⁻¹) and FZLZD group (10 g·kg⁻¹·d⁻¹), with 8 rats in each group. Rat model of NAFLD was prepared through the intragastric administration with fat emulsion for 4 weeks. After the successful modeling, rats in each administration group were continuously administered for 4 weeks. After 8 weeks, the rats in each group were put to death, and the pathological changes in liver tissue were detected by HE staining. Automatic biochemical analyzer was used to detect fasting serum lipid levels (T-Chol, TG, LDL-C, HDL-C) and liver functions (ALT, TP, ALB) of rats in each group. The rat liver index was calculated by weighing method. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of inflammatory cytokines TNF-α and IL-6 in liver tissue. Real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of fat metabolism-related factors SREBP-1c and FASN in liver tissue. Western blot was used to detect the p-AMPK and p-NF-κBp65 protein expressions in liver tissue. The results of HE staining showed that compared with the control group, the pathological changes in liver tissue in the model group rats were obvious; specifically, the outline of hepatic lobule was unclear, the hepatic cells showed diffuse steatosis of adipose tissue, and were accompanied by inflammatory infiltration, nuclear condensation, coloring deep; compared with the model group, liver lesions of all of the treatment groups were significantly alleviated; especially, the FZLZD group showed the most significant degree of remission. The results of serum test showed that the levels of serum lipids (T-Chol, TG, LDL-C, HDL-C), liver functions (ALT, TP, ALB) and liver index in model group were significantly higher than those in control group (P<0.01); compared with the model group, the indexes of serum lipid and liver function of rats in each treatment group were significantly decreased (P<0.01), and those in FZLZD group were significantly decreased (P<0.05), while those in YSF group were not significantly changed. The results of ELISA and qRT-PCR showed that compared with the control group, the secretion levels of TNF-α, IL-6 and the mRNA levels of SREBP-1c and FASN in the liver tissue of model group rats were significantly increased (P<0.01); compared with model group, the secretion levels of TNF-α, IL-6 and the mRNA levels of SREBP-1c, FASN in liver tissue of rats in each treatment group were significantly decreased (P<0.01); compared with YSF group, the secretion levels of TNF-α and IL-6 and the mRNA levels of SREBP-1c and FASN in FZLZD group were significantly different (P<0.01). Western blotting showed that compared with the model group, the protein expression of p-AMPK in liver tissue of rats in FZLZD group was significantly increased (P<0.01), while the protein expression of p-NF-κBp65 was significantly decreased (P<0.01). FZLZD can significantly improve hepatic pathological changes, reduce serum lipid levels, promote liver function and liver index in NAFLD rats, which may be associated with the activation of the AMPK pathway and thereby the inhibition of the expressions of SREBP-1c and FASN, and the inhibition of the NF-κBp65 pathway and thereby the reduction of the release of inflammatory factors.

Tangganjian decoction ameliorates type 2 diabetes mellitus and nonalcoholic fatty liver disease in rats by activating the IRS/PI3K/AKT signaling pathway

AimPrevious clinical studies have demonstrated that tangganjian (TGJ), a modern Chinese prescribed medicine, has a clinical effect in the treatment of type 2 diabetes mellitus (T2DM) with nonalcoholic fatty liver disease (NAFLD). Our study aimed to investigate whether the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is involved in this therapeutic effect. Materials and methodsT2DM and NAFLD rat models were constructed and treated with three different concentrations of TGJ. Pioglitazone was used as a positive control, along with the model and normal groups. For analyses, blood and livers were collected. Levels of glucose and lipid metabolism indicators, including fasting insulin and total cholesterol, were determined. The expression levels of insulin receptor substrate (IRS), PI3K, and AKT were also determined by western blotting and immunohistochemistry. Liver tissues were stained with hematoxylin & eosin. ResultsIn the high-dose TGJ-treated and positive groups, there was a significant increase in the HDL-C level and decreases in the levels of the fasting blood glucose, 2 h postprandial blood glucose, fasting insulin, triglyceride, total cholesterol, and low-density lipoprotein cholesterol, along with a significant increase in the expression of IRS, PI3K, and AKT in the liver. TGJ could also attenuate or counteract the effects of T2DM and NAFLD in the liver lobules. ConclusionA high concentration of TGJ can improve glucose and lipid metabolism by activating the IRS/PI3K/AKT signaling pathway.

Fatty liver mediated by peroxisome proliferator-activated receptor-α DNA methylation can be reversed by a methylation inhibitor and curcumin.

Our studies in vitro and in vivo aimed to investigate the influence of DNA methylation of peroxisome proliferator activated receptor-α (PPAR-α) gene in non-alcoholic fatty liver disease (NAFLD) pathogenesis and to observe whether the DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) and the herbal medicine curcumin might reverse the effect both in vivo and in vitro. Steatotic hepatocyte model of cell lines and NAFLD rat models were established following protocols documented in previous studies. Subsequently, the models received 5-Aza-CdR and curcumin treatment. Morphological, histological and laboratory variables in each group were determined by routine methods, including PPAR-α mRNA expression by polymerase chain reaction (PCR), PPAR-α protein expression by Western blot and DNA methylation by pyrosequencing. The steatotic hepatocyte model and NAFLD rat model were completely established. The expressions of PPAR-α mRNA and protein were significantly lower in the steatotic hepatocyte and NAFLD rat model groups than in the controls (P < 0.05). The mean DNA methylation levels of the PPAR-α gene were significantly higher in the two steatotic model groups than in the controls, especially at several CpG sites (P < 0.05). 5-Aza-CdR and curcumin treatment significantly reversed the DNA methylation levels, increased PPAR-α mRNA and protein expression, and improved lipid accumulation in the two steatotic models (P < 0.05). DNA methylation at the PPAR-α gene is involved in the pathogenesis of NAFLD and is possibly reversible by 5-Aza-CdR and curcumin. Curcumin may be a promising candidate for NAFLD therapy.

Beneficial effect of apple vinegar on reproductive parameters in male rat model of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is associated with low testosterone levels in serum. The aim of this study was to evaluate the effect of apple vinegar on fertility indices in a rat model of NAFLD. To study this effect, 32 adult male rats were divided into four groups: A-normal diet, B-high-fat diet (HFD), C-apple vinegar and D-HFD plus apple vinegar. At the end of the week 22, the Lee index, serum lipid profiles, liver enzymes, glucose and total antioxidant levels (TAC) in serum were determined. In addition, liver and testis tissue homogenate, histopathology, serum testosterone and sperm parameters were measured and HOMA-IR calculated. Significant reduction in Lee index, serum triglyceride, cholesterol, liver enzymes and glucose levels was observed in vinegar treated group compared with HFD group. Vinegar lowered insulin resistance compared with HFD (p<0.01). Steatosis in hepatocytes reduced from 56% in HFD group to 20% in group D (p<0.05). Vinegar caused a significant increase in serum testosterone, improvement in sperm parameters and a reduced germ cell apoptosis (p<0.05). There was an insignificant increase in TAC levels in the serum and homogenate tissue of liver and testis compared with HFD. This study reports apple vinegar has beneficial effects on male rat fertility indices in an in vivo model of NAFLD.

Chlorogenic acid alleviates autophagy and insulin resistance by suppressing JNK pathway in a rat model of nonalcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases around the world and commonly associated with insulin resistance and hyperlipidemia. Chlorogenic acid (CG) was reported to have insulinsensitizing activity and exert hypocholesterolemic and hypoglycemic effect. However, the involvement of CG in NAFLD remains far from being addressed. In this study, a high-fat diet-induced NAFLD rat model was used to investigate the biological roles and underlying mechanism of CG in NAFLD. The results showed that high-fat diet-fed rats exhibited an increase in body weight, glucose tolerance, liver injury, insulin resistance, as well as autophagy and C-Jun N-terminal kinase (JNK) pathway. Nevertheless, all these effects were alleviated by CG treatment. Moreover, angiotensin treatment in CG group activated the JNK pathway, and promoted autophagy, insulin resistance, and liver injury. In conclusion, our findings demonstrated that CG ameliorated liver injury and insulin resistance by suppressing autophagy via inactivation of JNK pathway in a rat model of NAFLD. Therefore, CG might be a potential application for the treatment of NAFLD.

Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5-trisphosphate receptor.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It may result in several types of liver problems, including impaired liver regeneration (LR), but the mechanism for this is unknown. Because LR depends on calcium signaling, we examined the effects of NAFLD on expression of the type II inositol 1,4,5-trisphosphate receptor (ITPR2), the principle calcium release channel in hepatocytes. ITPR2 promoter activity was measured in Huh7 and HepG2 cells. ITPR2 and c-Jun protein levels were evaluated in Huh7 cells, in liver tissue from a rat model of NAFLD, and in liver biopsy specimens of patients with simple steatosis and nonalcoholic steatohepatitis (NASH). LR was assessed in wild-type and Itpr2 knockout (Itpr2-/- ) mice following 67% hepatectomy. Cell proliferation was examined in ITPR2-knockout HepG2 cells generated by the CRISPR/Cas9 system. c-Jun dose dependently decreased activity of the human ITPR2 promoter. c-Jun expression was increased and ITPR2 was decreased in fat-loaded Huh7 cells and in livers of rats fed a high-fat, high-fructose diet. Overexpression of c-Jun reduced protein and mRNA expression of ITPR2 in Huh7 cells, whereas knockdown of c-Jun prevented the decrease of ITPR2 in fat-loaded Huh7 cells. ITPR2 expression was decreased and c-Jun was increased in liver biopsies of patients with steatosis and NASH compared to controls. ITPR2-knockout cells exhibited less nuclear calcium signaling and cell proliferation than control cells. LR assessed by Ki-67 and proliferating cell nuclear antigen was markedly decreased in Itpr2-/- mice. Conclusion: Fatty liver induces a c-Jun-mediated decrease in ITPR2 in hepatocytes. This may account for the impaired LR that occurs in NAFLD. (Hepatology 2018;67:560-574).

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics for the investigation of the effect of Hugan Qingzhi on non-alcoholic fatty liver disease in rats

Ethnopharmacological relevanceHugan Qingzhi tablet (HQT), a traditional Chinese medicine formula has been adopted for preventing and treating nonalcoholic fatty liver disease (NAFLD). AimIn order to explore the anti-NAFLD mechanisms of HQT, iTRAQ-based proteomic was employed to investigate the expression profiles of proteins in NAFLD rats induced by high-fat diet after HQT treatment. Materials and methodsThe NAFLD rat model was administrated with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, liver histology, pro-inflammatory cytokines, and oxidative stress/antioxidant biomarkers were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the combined strategy of iTRAQ labeling with strong cation exchange-non-liquid chromatography-tandem mass spectrometry (SCX-non-LC-MS/MS) analysis were employed to explore the mechanisms of HQT's protective effect against NAFLD in rats. Western blotting was performed to verify the proteomic results. ResultsThe histopathologic characteristics and biochemical data showed that HQT exhibited protective effects on HFD-induced NAFLD rats. After being analyzed by the combined strategy of iTRAQ with LC-MS/MS and subsequent investigation, we identified 275 differentially expressed proteins in the HFD group, compared to the control; 207 altered proteins in the HQT high dose + HFD group, compared to the HFD group; and 316 altered proteins in the HQT high dose + HFD group, compared to the control. Based on the Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway mapping, the conclusion has reached that several pathways including microbial metabolism in diverse environments, fatty acid metabolism, inflammatory response, oxidative stress, bile secretion, and peroxisome proliferator activated receptor (PPAR) signaling pathway were closely related to the effects of HQT in HFD-induced NAFLD in rats. Furthermore, several differentially expressed proteins, including phytanoyl-CoA 2-hydroxylase (PHYH), acyl-CoA synthetase 1 long chain (ACSL1), hemopexin, Alpha-1-acid glycoprotein (ORM1), fatty acid binding protein 4 (FABP4), soluble sulphotransferase 2a1 (Sult2a1), and argininosuccinate synthase 1 (ASS1) were verified by the western blotting analysis and these results were consistent with the data obtained from the proteomics analysis. ConclusionsOur results not only confirm that Hugan Qingzhi exhibits a significant protective effect in HFD-induced NAFLD rats but also provide a better understanding for the treatments of NAFLD.

SERUM NESFATIN-1 LEVELS IN RAT MODEL OF NON-ALCOHOLIC FATTY LIVER DISEASE

Background: Non-alcoholic fatty liver disease (NAFLD) is a frequent progressive disorder manifested by fat accumulation in the liver and usually related to obesity and insulin resistance, but its pathogenesis is still uncertain. Nesfatin-1 is a polypeptide derived from nucleobindin-2 and involved in regulation of food intake and glucose homeostasis. The relationship between nesfatin-1 and NAFLD is still controversial. Objective: To evaluate serum levels of nesfatin-1 in NAFLD model induced by high fat diet (HFD) in male albino rats. Material and methods: Forty eight male adult albino rats were divided into four equal groups: 2 control groups that were fed ordinary diet for 4 weeks (group IA) and 12 weeks (group IB), and 2 HFD groups that were fed HFD for 4 weeks (group IIA) and 12 weeks (group IIB). In all groups, abdominal circumference, body weight, serum levels of nesfatin-1, insulin, glucose, C-reactive protein (CRP), lipid profile parameters, and liver enzymes (ALT & AST) were measured. BMI and HOMA-IR were calculated, and isolated liver tissues were examined histopathologically. Results: After 4-week and 12-week-HFD feeding, the rats developed simple steatosis and steatohepatitis, respectively. These were proved by the progressive rise of liver enzymes and the histopathological findings. Besides, there was a significant progressive rise in BMI, HOMA-IR, serum levels of nesfatin-1, glucose, insulin, CRP, and all lipid profile parameters except high density lipoprotein that significantly decreased in HFD groups in comparison to control groups. Moreover, nesfatin-1 correlated positively with all measured parameters in HFD groups except for HDL that showed negative correlation with nesfatin-1. Conclusion: Serum levels of nesfatin-1 increased in NAFLD rat model induced by HFD. This rise may be attributed to feeding rats with HFD, hyperglycemia or may compensate for the inflammation and disturbed metabolism.

Down‐regulation of lncRNA‐NEAT1 alleviated the non‐alcoholic fatty liver disease via mTOR/S6K1 signaling pathway

Without effective medical interventions for complete reverse of NAFLD, it needs to urgently explore the underlying molecular mechanisms of non-alcoholic fatty liver disease (NAFLD) to offer a novel therapeutic strategy for people suffering from NAFLD. Sprague-Dawley (SD) rats were used to establish the NAFLD animal model. Lipofectamine 2000 was used to silence or over-express NEAT1. The expression of NEAT1 and the mRNA levels of ACC and FAS were determined by qRT-PCR. Western blot assays were performed to detect the expression of ACC and FAS at protein levels and the related protein levels of mTOR/S6K1 signaling pathway. The levels of liver triglyceride (TG), serum total cholesterol (TC), ALT, and AST were assessed by an automatic biochemistry analyzer. The levels of liver TG and serum cholesterol were obviously up-regulated in NAFLD rat model. The level of NEAT1 expression and the mRNA levels of ACC and FAS were obviously enhanced in NAFLD model both in vivo and in vitro. Knockdown of NEAT1 could also reduce the elevation of ACC and FAS induced by FFA in liver cells. Moreover, inhibition of mTOR/S6K1 pathway presented with the same effect with knockdown of NEAT1 on the expression of ACC and FAS mRNA levels. The injection of si-NEAT1 lentivirus was performed to treat NAFLD of rats and the obvious efficacy for NAFLD rats was achieved. In a word, the down-regulated level of NEAT1 could remit the non-alcoholic fatty liver disease through mTOR/S6K1 signaling pathway in rats.

Chinese herb extract improves liver steatosis by promoting the expression of high molecular weight adiponectin in NAFLD rats.

High molecular weight (HMW) adiponectin (APN) is closely correlated with the development of fatty liver and is modulated by the Akt/forkhead box protein O1 (FOXO1) pathway through disulfide‑bond A oxidoreductase‑like protein (DsbA‑L). The Chinese herb extract, QSHX, is used to treat liver diseases. The present study investigated the effects of QSHX on non‑alcoholic fatty liver disease (NAFLD) and its underlying mechanism. A rat model of NAFLD was established by feeding of a high‑fat and high‑sugar diet for 20weeks. From week 13, the rats were administered with QSHX, or saline as a control, for 8weeks. The liver function, blood fat and plasma APN were measured using a radioimmunoassay. The hepatic tissue score was measured following staining for pathology. The expression and activities of Akt, FOXO1, DsbA‑L and HMW APN in the adipose tissue and primary adipocytes of the rats were measured using western blot analysis. It was found that QSHX significantly decreased the body weight, liver index, and serum levels of aspartate aminotransferase, alanine aminotransferase and triglyceride; and increased the serum level of APN in the NAFLD rats. Following 8weeks of treatment with QSHX, the hepatic steatosis in the liver tissue improved and the score of hepatic steatosis was significantly decreased. The results of the western blot analysis indicated that QSHX promoted the expression of DsbA‑L and HMW APN, and reduced the expression levels of phosphorylated FOXO1 and FOXO1 in adipose tissue and primary adipocytes. It was concluded that QSHX reduced hepatic steatosis by promoting the expression of HMW APN and DsbA‑L, which may have been induced by inhibiting the activation and expression of FOXO1 in adipocytes.