Steatosis In HepG2 Cells Research Articles

Pterostilbene and Its Derivative 3'-Hydroxypterostilbene Ameliorated Nonalcoholic Fatty Liver Disease Through Synergistic Modulation of the Gut Microbiota and SIRT1/AMPK Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) is a recent chronic liver disease common in many developed countries and is closely associated with metabolic syndrome, such as obesity and insulin resistance. The present study was performed to investigate the effects of pterostilbene (Pt) and its derivative 3'-hydroxypterostilbene (OHPt) on free fatty acids (FFA)-induced lipid accumulation in HepG2 cells and high-fat diet (HFD)-induced NAFLD in C57BL/6J mice. The results showed that Pt and OHPt significantly ameliorated FFA-induced steatosis in HepG2 cells and enhanced lipolysis through the upregulation of SIRT1/AMPK and insulin signaling pathways. In the in vivo study, Pt and OHPt treatment resulted in reduced hepatic lipid droplets accumulation. The data showed that Pt and OHPt upregulated the SIRT1/AMPK pathway and subsequently downregulated the protein expression of SREBP-1 to activate fatty acid (FA) β-oxidation to inhibit FA synthesis. Pt and OHPt administration activated the insulin signaling pathway and further ameliorated the insulin resistance and liver function in the HFD-fed mice. Furthermore, Pt and OHPt markedly increased the numbers of Oscillospira and decreased the numbers of Allobaculum, Phascolarctobacterium, and Staphylococcus compared with those in the HFD group. These robust results indicate that Pt and OHPt are able to possess potential health benefits in improving insulin resistance and hepatic steatosis by promoting healthy populations or abundances of considered vital microbiota. Besides, OHPt is more effective than Pt, which might have promising chemotherapeutic effects for future clinical application.

Chitosan oligosaccharide attenuates hepatic steatosis in HepG2 cells via the activation of AMP-activated protein kinase.

Chitosan oligosaccharides (COSs), oligomers of decomposed chitosan possess many biological functions including immunomodulatory, antitumor, and antiinflammation. The aim of this study was to investigate the protective effects of COS against free fatty acid (FFA)-induced cellular hepatic steatosis and underlying mechanisms in HepG2 cells. Results showed that COS significantly reduced the lipid contents and elevated the activities of antioxidant enzymes including total-superoxide dismutase, glutathione peroxidase, and catalase in FFA-stimulated HepG2 cells. COS phosphorylated the acetyl-CoA carboxylase and reduced both mRNA and protein levels of lipogenesis markers including fatty acid synthase and sterol regulatory element-binding protein 1c. COS also significantly increased the expression levels of fatty acid oxidation-related factors including carnitine palmitoyltransferase 1A, acyl-coenzyme A oxidase 1, and peroxisome proliferators-activated receptor α. Besides, COS markedly phosphorylated AMP-activated protein kinase (AMPK). The inhibition of lipogenesis and the enhancement of fatty acid oxidation induced by COS were all blocked by AMPK antagonist (compound C), showing that the attenuation of hepatic steatosis by COS was dependent on AMPK activation. In conclusion, COS attenuated hepatic steatosis via suppressing lipid synthesis and enhancing fatty acid oxidation. AMPK was also involved in the alleviation of hepatic steatosis by COS. These results indicated that COS might be used as a potential ingredient to ameliorate nonalcoholic fatty liver disease. PRACTICAL APPLICATIONS: Nonalcoholic fatty liver disease (NAFLD) has been regarded as pathological fat deposition in the liver, which includes a range of pathologies, from steatosis to steatohepatitis, fibrosis, and cellular carcinoma. Our findings demonstrated that Chitosan oligosaccharides (COS) attenuated steatosis via improving lipid metabolism. COS suppressed lipogenesis and also enhanced fatty acid oxidation. Besides, the underlying molecular mechanism whereby COS elicited these beneficial effects has also been proved to be through the modulation of upstream protein kinase, AMP-activated protein kinase. This study provides new knowledge to support that COS might be used as a food supplement for the prevention of NAFLD.

Exendin-4 alleviates steatosis in an in vitro cell model by lowering FABP1 and FOXA1 expression via the Wnt/-catenin signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Agonists of the glucagon-like peptide-1 receptor (GLP-1R), currently approved to treat type 2 diabetes, hold promise to improve steatosis and even steatohepatitis. However, due to their pleiotropic effects, the mechanisms underlying their protective effect on NAFLD remain elusive. We aimed to investigate these mechanisms using an in vitro model of steatosis treated with the GLP-1R agonist Exendin-4 (Ex-4). We established steatotic HepG2 cells by incubating the cells with 400 µM oleic acid (OA) overnight. Further treatment with 200 nM Ex-4 for 3 h significantly reduced the OA-induced lipid accumulation (p < 0.05). Concomitantly, Ex-4 substantially reduced the expression levels of Fatty Acid-Binding Protein 1 (FABP1) and its primary activator, Forkhead box protein A1 (FOXA1). Interestingly, the silencing of β-catenin with siRNA abolished the effect of Ex-4 on these genes, suggesting dependency on the Wnt/β-catenin pathway. Additionally, after β-catenin silencing, OA treatment significantly increased the expression of nuclear transcription factors SREBP-1 and TCF4, whereas Ex-4 significantly decreased this upregulation. Our findings suggest that direct activation of GLP-1R by Ex-4 reduces OA-induced steatosis in HepG2 cells by reducing fatty acid uptake and transport via FABP1 downregulation.

Dulaglutide Ameliorates Palmitic Acid-Induced Hepatic Steatosis by Activating FAM3A Signaling Pathway.

BackgroundDulaglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), has been shown to reduce body weight and liver fat content in patients with type 2 diabetes. Family with sequence similarity 3 member A (FAM3A) plays a vital role in regulating glucose and lipid metabolism. The aim of this study was to determine the mechanisms by which dulaglutide protects against hepatic steatosis in HepG2 cells treated with palmitic acid (PA).MethodsHepG2 cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide for 24 hours. Hepatic lipid accumulation was determined using Oil red O staining and triglyceride (TG) assay, and the expression of lipid metabolism-associated factor was analyzed using quantitative real time polymerase chain reaction and Western blotting.ResultsDulaglutide significantly decreased hepatic lipid accumulation and reduced the expression of genes associated with lipid droplet binding proteins, de novo lipogenesis, and TG synthesis in PA-treated HepG2 cells. Dulaglutide also increased the expression of proteins associated with lipolysis and fatty acid oxidation and FAM3A in PA-treated cells. However, exendin-(9–39), a GLP-1R antagonist, reversed the expression of FAM3A, and fatty acid oxidation-associated factors increased due to dulaglutide. In addition, inhibition of FAM3A by siRNA attenuated the reducing effect of dulaglutide on TG content and its increasing effect on regulation of fatty acid oxidation.ConclusionThese results suggest that dulaglutide could be used therapeutically for improving nonalcoholic fatty liver disease, and its effect could be mediated in part via upregulation of FAM3A expression through a GLP-1R-dependent pathway.

Electrophilic thymol isobutyrate from Inula nervosa Wall. (Xiaoheiyao) ameliorates steatosis in HepG2 cells via Nrf2 activation

• Electrophilic components from Xiaoheiyao are knocked out using magnetic GSH probe. • Electrophilic components have the potential to ameliorate steatosis in HepG2 cells. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate is discovered as a bioactive compound. • 10-Isobutyryloxy-8,9-epoxythymol isobutyrate improves steatosis via Nrf2 activation. Xiaoheiyao , the rhizomes of Inula nervosa Wall., is a culinary spice known to contain some electrophilic Nrf2 activators that could potentially intervene in nonalcoholic fatty liver disease (NAFLD). In this study, the electrophilic components from Xiaoheiyao were annotated by comprehensive cheminformatics analyses on UHPLC-MS/MS data after being knocked out with glutathione (GSH)-immobilized magnetic microspheres. Their intervention effects against hepatic steatosis were investigated in free fatty acid (FFA)-treated HepG2 cells. The electrophilic components in Xiaoheiyao were found to be the major bioactive components preventing hepatic steatosis in FFA-treated HepG2 cells. In particular, 10-isobutyryloxy-8,9-epoxythymol isobutyrate (XHY-1) improved hepatic lipid metabolism disorder, Krebs cycle activity, oxidative phosphorylation, and the cellular and mitochondrial redox status. Moreover, XHY-1 activates the Nrf2-ARE signaling pathway through the up-regulation of Nrf2 expression and promotion of Nrf2 nuclear translocation. Molecular docking further showed that XHY-1 could conjugate with Cys151 in the BTB domain of Keap1 through the epoxy group. In conclusion, the electrophilic components of Xiaoheiyao , especially XHY-1, attenuated hepatic steatosis in FFA-treated HepG2 cells through activation of the Nrf2-ARE signaling pathway.

Pink Lotus Essential Oil and Alleviates on Free Fatty Acid Induced Steatosis in HepG2 Cells via PI3K/Akt and NF-κB Pathways.

Pink lotus essential oil (PLEO) is the volatile components extracted from lotus flowers and there are few relevant research. The purpose of this study was to observe the effect of PLEO on NAFLD in vitro model and its possible mechanism. The ingredients of PLEO were determined by gas chromatography-mass spectrometry (GS-MS) and its lipid-lowering and hepatoprotective activities were investigated. HepG2 cells were treated with free fatty acid (FFA) to establish a cell model of NAFLD. Cell viability was evaluated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Total cholesterol (TC), triglyceride (TG), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were determined by Enzyme-Linked Immune Sorbent Assay (ELISA). Oil red O staining was performed to observe the lipid accumulation in the HepG2 cells. Lipid metabolism enzymes including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD-1), and carnitine palmitoyltransferase-1 (CPT-1), insulin signaling pathways including phosphatidylinositol 3 kinase (PI3K) and protein kinase B Akt, inflammatory signaling pathways such as nuclear factor kappa-B (NF-κB), were determined by Western blotting. There were 46 components determined in PLEO with many terpenoids compounds. PLEO decreased TC and TG contents in the FFA-treated HepG2 cells. Furthermore, PLEO inhibited TNF-α, IL-6 and IL-1β excretion, decreased NF-κB, FAS, ACC and SCD-1 while increased phosphorylation of NF-κB, PI3K, Akt, and CPT-1 expression. It is the first time to reveal that PLEO alleviates FFA-induced steatosis in HepG2 cells by regulating lipid metabolism, inhibiting inflammatory response, and improving insulin sensitivity.

Steatosis induced by nonylphenol in HepG2 cells and the intervention effect of curcumin.

Non-alcoholic fatty liver disease (NAFLD) has increasingly become a serious public health problem. There is growing evidence that nonylphenol (NP) exposure may cause steatosis, but the underlying mechanism is not fully understood. Curcumin (CUR) improves NAFLD-related lipid metabolism disorders and oxidative stress, but its preventive and therapeutic effects on NP-induced steatosis have not been reported. The objective of this investigation was to determine the capability and potential mechanism of NP to induce steatosis in vitro and the intervention of curcumin. HepG2 cells were treated with 0 μM, 20 μM, 30 μM, 40 μM NP for 24 h. Lipid droplets accumulated significantly in HepG2 cells after NP treatment, and the concentration of triglyceride (TG) and total cholesterol (T-CHO) increased significantly. Simultaneously, lipogenesis gene expression was up-regulated significantly, fatty acid oxidation (FAO) gene expression was significantly down-regulated, and reactive oxygen species (ROS) were overproduced. Meanwhile, the expression of p-AMPK/AMPK in the AMPK/mTOR signaling pathway was significantly down-regulated and the expression of p-mTOR/mTOR was markedly up-regulated. However, blocking ROS production with N-acetyl-L-cysteine (NAC) can reverse these phenomena. In addition, our study found that curcumin effectively ameliorated the effects of NP-induced steatosis. Our study indicates that NP can induce steatosis in HepG2 cells, and may be implicated in inhibiting the ROS-dependent AMPK/mTOR pathway, and that curcumin ameliorates the NAFLD-like changes induced by NP in HepG2 cells.

Berberine mitigates nonalcoholic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2 pathway for cholesterol synthesis

ObjectiveTo investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms. MethodsA steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation. ResultsFFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation. ConclusionBBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.

In vitro effects of peanut skin polyphenolic extract on oxidative stress, adipogenesis, and lipid accumulation

The in vitro bioactivity of peanut skin was evaluated on its ability to protect RAW264.7 macrophages against the formation of reactive oxygen species (ROS), the occurrence of inflammatory responses, and the ability to prevent differentiation in 3T3-L1 preadipocytes and the lipid accumulation in HepG2 hepatocytes. Here, high-performance liquid chromatography coupled with ion-trap tandem mass spectrometry identified 18 flavonoids in peanut skin extract (PSE) including condensed tannins and flavones. The PSE showed strong antioxidant potential against 2-diphenyl-1-picrylhydrazyl, peroxyl radicals, and antioxidant activity by inhibiting the ROS formation by 60%–80% in RAW264.7 macrophages. The effects of PSE flavonoids against inflammation induced by lipopolysaccharide resulted in the reduced expression of biomarkers IL-6 and TNF-α and the decreased production of nitric oxide. The administration of PSE decreased the differentiation of 3T3-L1 adipocytes by approximately 60%–72% and ameliorated the hepatic steatosis in HepG2 cells by decreasing the lipid concentration by up to 85%. Novelty impact statement Phenolic compounds are widely studied because of their ability to prevent and treat the risk factors related to the onset of several diseases including cancer, diabetes, and obesity. Peanut skin is an agroindustry waste from which phenolic compounds can be processed and reused as a low-cost raw material to formulate new products. Peanut skin extract contained 18 flavonoids that justified the high total phenolic content and antioxidant capacity, the in vitro potential to prevent oxidative stress via reactive oxygen species scavenging, to suppress adipogenesis in 3T3-L1 preadipocytes and the hepatic steatosis in the HepG2 cell model.

Olanzapine leads to nonalcoholic fatty liver disease through the apolipoprotein A5 pathway

The antipsychotic drug olanzapine was reported to induce nonalcoholic fatty liver disease (NAFLD), whereas the underlying mechanism remains incompletely understood. This study investigated whether apolipoprotein A5 (apoA5) and sortilin, two interactive factors involved in NAFLD pathogenesis, are implicated in olanzapine-induced NAFLD. In our study, at week 8, olanzapine treatment successfully induced hepatic steatosis in female C57 BL/6 J mice, which was independent of body weight gain. Likewise, olanzapine effectively mediated hepatocyte steatosis in HepG2 cells characterized by substantially elevated intracellular lipid droplets. Increased plasma triglyceride concentration and decreased plasma apoA5 levels were observed in mice treated with 8-week olanzapine. Surprisingly, olanzapine markedly enhanced hepatic apoA5 protein levels in mice, without a significant effect on rodent hepatic ApoA5 mRNA. Our in vitro study showed that olanzapine reduced apoA5 protein levels in the medium and enhanced apoA5 protein levels in hepatocytes, whereas this drug exerted no effect on hepatocyte APOA5 mRNA. By transfecting APOA5 siRNA into HepG2 cells, it was demonstrated that APOA5 knockdown effectively reversed olanzapine-induced hepatocyte steatosis in vitro. In addition, olanzapine drastically increased sortilin mRNA and protein levels in vivo and in vitro. Interestingly, SORT1 knockdown reduced intracellular apoA5 protein levels and increased medium apoA5 protein levels in vitro, without affecting intracellular APOA5 mRNA levels. Furthermore, SORT1 knockdown greatly ameliorated hepatocyte steatosis in vitro. This study provides the first evidence that sortilin inhibits the hepatic apoA5 secretion that is attributable to olanzapine-induced NAFLD, which provides new insight into effective strategies against NAFLD for patients with schizophrenia administered olanzapine.

Comprehensive analysis of LncRNAs expression profiles in an in vitro model of steatosis treated with Exendin-4

Background and aimsThe hallmark of non-alcoholic fatty liver disease (NAFLD) is the excessive hepatic lipid accumulation. Currently, no pharmacotherapy exists for NAFLD. However, the glucagon-like peptide-1 receptor agonists have recently emerged as potential therapeutics. Here, we sought to identify the long non-coding RNAs (LncRNAs) associated with the steatosis improvement induced by the GLP-1R agonist Exendin-4 (Ex-4) in vitro.MethodsSteatosis was induced in HepG2 cells with oleic acid. The transcriptomic profiling was performed using total RNA extracted from untreated, steatotic, and Ex-4-treated steatotic cells. We validated a subset of differentially expressed LncRNAs with qRT-PCR and identified the most significantly enriched cellular functions associated with the relevant LncRNAs.ResultsWe confirm that Ex-4 improves steatosis in HepG2 cells. We found 379 and 180 differentially expressed LncRNAs between untreated and steatotic cells and between steatotic and Ex-4-treated steatotic cells, respectively. Interestingly, 22 upregulated LncRNAs in steatotic cells became downregulated with Ex-4 exposure, while 50 downregulated LncRNAs in steatotic cells became upregulated in the presence of Ex-4. Although some LncRNAs, such as MALAT1, H19, and NEAT1, were previously associated with NAFLD, the association of others with steatosis and the positive effect of Ex-4 is being reported for the first time. Functional enrichment analysis identified many critical pathways, including fatty acid and pyruvate metabolism, and insulin, PPAR, Wnt, TGF-β, mTOR, VEGF, NOD-like, and Toll-like receptors signaling pathways.ConclusionOur results suggest that LncRNAs may play essential roles in the mechanisms underlying steatosis improvement in response to GLP-1R agonists and warrant further functional studies.

Livogrit ameliorates acetaldehyde-induced steatosis in HepG2 cells through modulation of lipogenesis and β-oxidation pathways

BackgroundFrequent alcohol consumption leads to development of alcoholic liver steatosis. Onset of steatosis is caused by alcohol metabolite ‘acetaldehyde’ causing intracellular accumulation of lipid bodies due to abnormal metabolism. Livogrit is a tri-herbal Ayurveda formulation prepared using Boerhavia diffusa L., Phyllanthus niruri L., and Solanum nigrum L. plant extracts. The formulation has earlier proven effective against carbon tetrachloride-induced toxicity and non-alcoholic fatty acid induced liver steatosis. PurposeIn present study, we evaluated efficacy of Livogrit in ameliorating ‘acetaldehyde’ induced steatosis and assessed its mode of action. MethodHepG2 cells were exposed to varying concentrations of acetaldehyde and assessed for lipid body accumulation and cytosafety. An inhibition concentration of 50% for acetaldehyde was observed at 71.42 mM in HepG2 cells. 1 mM acetaldehyde was selected as a non-cytotoxic steatosis-stimulating dose. The acetaldehyde-stimulated HepG2 cells were used for studying the disease-reversing efficacy of Livogrit. Efficacy of the formulation was analyzed at cellular, biochemical, and molecular levels. ResultsAcetaldehyde stimulation caused an increase in intracellular lipid and triglyceride accumulation, release of ALT and AST enzymes, ROS generation, and decrease in extracellular release of glycerol molecules in HepG2 cells. Livogrit treatment of the stimulated-HepG2 cells showed a reversal of steatosis through normalization of observed intra- and extracellular parameters. At gene level, Livogrit-modulated the acetaldehyde-stimulated upregulation of SREBP1c, FAS, PLIN2, TNF-α, NF-kB, and LC3A genes associated with lipogenesis, inflammation, and autophagy. At protein level, the protective role of Livogrit was observed in acetaldehyde-stimulated HepG2 cells through modulation of TNF-α, PLIN2, and LC3 proteins. Livogrit provided further protection against steatosis through stimulation of fatty acid β-oxidation associated with SIRT1, CPT1A, and PPAR-α genes. ConclusionIn conclusion, Livogrit recovered HepG2 cells from alcoholic steatosis at biochemical and molecular levels through multifaceted modulation of lipogenesis, inflammation, autophagy, and β-oxidation. Hence, Livogrit was found to be a potential candidate for the amelioration of alcohol-induced steatosis.

Delphinidin-3-sambubioside from Hibiscus sabdariffa. L attenuates hyperlipidemia in high fat diet-induced obese rats and oleic acid-induced steatosis in HepG2 cells

ABSTRACT Hibiscus sabdariffa. L is folk medicine that is often used for its hypolipidemic and antihypertensive effects; however, the active compound responsible for its anti-obesity effect is presently unknown. Delphinidin-3-sambubioside (Dp3-Sam) is an anthocyanin, was extracted from Hibiscus sabdariffa L. The present research aimed to investigate the role of Dp3-Sam in the prevention of hyperlipidemia in vivo and in vitro. Rats were fed with a standard chow diet (Control group) or high-fat diet (HFD and DP group) for eight weeks. Besides, HepG2 cells were stimulated with 0.2 mM oleic acid, with or without Dp3-Sam (100–200 µg/ml). Lipid profiles were measured by commercial kits. Oil Red O staining was performed to measure the hepatic and intracellular lipid levels. The key genes of lipid metabolism were measured by RT-PCR. In HFD-fed rats, Dp3-Sam reduced the body weight gain, visceral fat, and abdominal fat and decreased hepatic lipid deposits. Dp3-Sam decreased intracellular TG levels and lipid accumulation in oleic acid-treated HepG2 cells. Besides, Dp3-Sam downregulated the mRNA expression of HMG-CoA reductase (HMGCR), sterol regulatory element-binding protein-1 c (SREBP-1 C), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) and upregulated the mRNA expression of cholesterol 7α-hydroxylase (CYP7A1), carnitine palmitoyltransferase1 (CPT1), acyl-coenzyme A oxidase (ACOX), and peroxisome proliferator-activated receptor alpha (PPARα). Dp3-Sam up-regulated the expression of phosphorylation of AMP-activated protein kinase (pAMPK) in HFD-fed rats. Our findings indicated that Dp3-Sam possesses the potential to improve lipid metabolism dysfunction and our results offered evidence for the use of Dp3-Sam as therapy for the prevention of obesity and dyslipidemia.

Optimized preparation of eugenol microcapsules and its effect on hepatic steatosis in HepG2 cells

This study was aimed at evaluating the potential of peach gum (PG) and gelatin in the microencapsulation of eugenol and the intervention of eugenol microcapsules on hepatic steatosis in vitro. Response surface method (RSM) was used to optimize the encapsulation conditions of eugenol microcapsules. The microcapsules were characterized by scanning electron microscopy (SEM), dynamic Light Scattering (DLS), Fourier transform infrared spectroscopy (FT-IR) and release behavior in vitro was determined. The effect of eugenol microcapsules on free fatty acids (FFA) treated hepatocellular cells (HepG2) cells was evaluated by oil red O staining and intracellular total cholesterol (TC) and triglyceride (TG) determination. The results showed that the optimal encapsulation conditions were as follows: the PG-gelatin ratio was 1.6:1.4, the core-wall ratio was 1.6:1.4, the pH was 4 and the emulsification speed was 9000 r/min. The optimized microcapsules were smooth spherical with a size of about 3.09 ± 0.58 μm and the encapsulation was confirmed by FT-IR. In vitro release behavior showed that eugenol microcapsules could be released stably in a neutral environment for 72 h. Oil red O staining showed that 50 and 100 μM eugenol microcapsules could significantly inhibit the lipid accumulation and reduce the TC and TG in steatotic HepG2 cells induced by FFA. Therefore, PG and gelatin can be used as excellent carriers for the microencapsulation of volatile compounds in the field of biomedical industry, and eugenol microcapsules is a promising preparation for the treatment of nonalcoholic fatty liver disease (NAFLD).

Effect and mechanism of ginsenoside Rg1-regulating hepatic steatosis in HepG2 cells induced by free fatty acid

Ginsenoside Rg1 (G-Rg1) is a bioactive phytochemical that has been found to be beneficial for the treatment of several diseases including nonalcoholic fatty liver disease (NAFLD). But there is a lack of literature reporting the effect of G-Rg1 on lipid metabolism balance in NAFLD. We investigated the effect and mechanism of G-Rg1 on lipid metabolism in vitro. We found that G-Rg1 decreased the levels of TG, TC, and MDA, and increased activity of SOD. Results of RT-PCR and western blotting showed that supplementation with G-Rg1 downregulated the expression of PPAR γ, FABP1, FATP2/5, CD36, SREBP1c, and FASN, while the expression of PPAR ɑ, CPT1, ACOX1, MTTP, and ApoB100 was upregulated, after induction by a free fatty acid. Taken together, we conclude that G-Rg1 inhibits lipid synthesis and lipid uptake, and enhances lipid oxidation and lipid export to reduce hepatic steatosis of HepG2 cells by regulating PPAR ɑ and PPAR γ expression.

Amelioration by Idesia polycarpa Maxim. var. vestita Diels. of Oleic Acid-Induced Nonalcoholic Fatty Liver in HepG2 Cells through Antioxidant and Modulation of Lipid Metabolism.

Idesia polycarpa Maxim. var. vestita Diels (I. polycarpa) is well known as an edible oil plant which contains abundant linoleic acid and polyphenols. The objective of this study was to maximize the by-product of defatted fruit of I. polycarpa. We found that the fraction D of ethyl acetate extract (EF-D) contained more polyphenols, which contribute to its strong antioxidant activity by antioxidant assays (DPPH, ABTS, and FRAP). Meanwhile, EF-D showed a significant lipid-lowering effect on oleic acid- (OA-) induced hepatic steatosis in HepG2 cells through enhancing antioxidant activity, reducing liver damage, and regulating lipid metabolism, antioxidant, and inflammation-related gene expression. The SOD and T-AOC levels significantly increased, but the levels of MDA, AST, and ALT decreased obviously when treated with EF-D. In general, EF-D improved the antioxidant enzyme activities and decreased the hepatic injury activities. Besides, treatment with EF-D for NAFLD influenced lipid metabolism and inflammation by activating PPARα which was associated with the increased expression of CPT1 and decreased expression of SCD, NF-κB, and IL-1. Moreover, EF-D improved the oxidative stress system through activation of the Nrf2 antioxidant signal pathways and upregulated its target genes of HO-1, NQO1, and GSTA2. The results highlighted the EF-D from the defatted fruit of I. polycarpa regarding lipid-lowering, proving it to be a potential drug resource of natural products for treating the nonalcoholic fatty liver disease (NAFLD).

Fructose-palmitate based high calorie induce steatosis in HepG2 cells via mitochondrial dysfunction: An in vitro approach

A proper in vitro model for conducting research on high energy food induced steatosis via defective energy metabolism in the liver is not visible in the literature. The present study developed an in vitro model in HepG2 cell line to mimic high energy diet induced steatosis in liver via mitochondrial dysfunction. For this, HepG2 cells were treated with fructose (100 mM) and palmitate (100 μM) for about 24 h and subjected for biochemical analysis relevant to lipogenesis and mitochondrial biology. Our findings showed that fructose-palmitate treatment caused significant lipid accumulation and rise in lipogenic proteins. Further studies showed alteration in mitochondrial integrity, dynamics and oxidative phosphorylation. Mitochondrial integrity was affected by the dissipation of trans-membrane potential, surplus mitochondrial superoxide with calcium overload. Similarly, mitochondrial dynamics were altered with up regulation of mitochondrial fission proteins: DRP1 and FIS1, cytochrome c release, caspase-3 activity and apoptosis. Various components of the electron transport chain: complex I, II, III and IV were altered with significant depletion in oxygen consumption. Overall our findings illustrate the dominant role of mitochondria in the genesis of high fructose-palmitate induced steatosis in HepG2 cells. Since continuous high energy food consumption is the main inducer of steatosis, this model is found to be an ideal one for preliminary and basic research in the area of liver disease via mitochondrial dysfunction.

Aflatoxin B1-Induced COX-2 Expression Promotes Mitophagy and Contributes to Lipid Accumulation in Hepatocytes In Vitro and In Vivo.

Aflatoxin B1 (AFB1) is hepatotoxic. Numerous studies have shown that mitochondria play an essential role in AFB1-induced steatosis. However, the mechanisms of AFB1-induced steatosis via mitochondria are still obscure. The present study aimed to confirm that AFB1 causes hepatocyte steatosis regulated by cyclooxygenase-2 (COX-2)-induced mitophagy, both in vivo and in vitro. Adult male C57BL/6 mice were randomly divided into control group with the same volume of peanut oil and exposure group administered 0.6 mg/kg AFB1 once in 2 days for 1 month. HepG2 and Cas9-PTGS2 cells were treated with 5 μM AFB1 for 48 hours. Then, various indicators were evaluated. Aflatoxin B1 causes liver injury and steatosis with increased alanine aminotransferase, aspartate aminotransferase, total cholesterol, total triglyceride levels in vivo and in vitro, and elevated lipid droplets in HepG2 cells. Cyclooxygenase-2 and mitophagy pathway were induced by AFB1 in both liver tissues and cultured HepG2 cells. Further studies have shown that knockout of COX-2 with the CRISPR/Cas9 system inhibited the AFB1-induced mitophagy and steatosis in HepG2 cells. Also, the inhibition of PTEN-induced putative kinase with RNA interference attenuated the AFB1-induced steatosis. The results of the current study suggested that AFB1 increases the expression of COX-2, which, in turn, elevates the level of mitophagy, thereby disrupting the normal mitochondrial lipid metabolism and causing steatosis. Thus, this study implies that COX-2 may be a potential target for therapy against AFB1-induced steatosis.

Growth hormone ameliorates high glucose-induced steatosis on in vitro cultured human HepG2 hepatocytes by inhibiting de novo lipogenesis via ChREBP and FAS suppression

Objective: Growth hormone (GH) deficiency has been associated with increased steatosis but the molecular mechanism has not been fully elucidated. We investigated the effect of GH on lipid accumulation of HepG2 cells cultured on an in vitro steatosis model and examined the potential involvement of insulin-like growth factor 1 (IGF-1) as well as lipogenic and lipolytic molecules.Methods: Control and steatosis conditions were induced by culturing HepG2 cells with 5.5 or 25 mmol/l glucose for 24 h, respectively. Afterward, cells were exposed to 0, 5, 10 or 20 ng/ml GH for another 24 h. Lipid content was quantified as well as mRNA and protein levels of IGF-1, carbohydrate responsive element-binding protein (ChREBP), sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS), carnitine palmitoyltransferase 1A (CPT1A), and peroxisome proliferator-activated receptor alpha (PPAR-alpha) by qPCR and western blot, respectively. Data were analyzed by one-way ANOVA and the Games-Howell post-hoc test.Results: In the steatosis model, HepG2 hepatocytes showed a significant 2-fold increase in lipid amount as compared to control cells. IGF-1 mRNA and protein levels were significantly increased in control cells exposed to 10 ng/ml GH, whereas high glucose abolished this effect. High glucose also significantly increased both mRNA and protein of ChREBP and FAS without having effect on SREBP1c, CPT1A and PPAR-alpha. However, GH inhibited ChREBP and FAS production, even in HepG2 hepatocytes cultured under steatosis conditions.Conclusions: Growth hormone ameliorates high glucose-induced steatosis in HepG2 cells by suppressing de novo lipogenesis via ChREBP and FAS down-regulation.

Octyl gallate induces hepatic steatosis in HepG2 cells through the regulation of SREBP-1c and PPAR-gamma gene expression.

Octyl gallate (OG) is an antioxidant commonly used in food, although there is no definition of its acceptable daily intake. There are reports in vitro and in vivo showing that food additives and drugs can alter lipid metabolism. Lipid droplet accumulation in hepatic cells is one of the main findings in the unregulated lipid metabolism and is strongly related to the development of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of OG on lipid metabolism in the hepatocellular carcinoma cell line (HepG2). The results have shown, for the first time, that treatment with OG increased the overall amount of lipids, the triglyceride concentration, the lipid droplet area, and SREBP-1c and PPAR-γ gene expression. Taken together, the findings indicate that OG induces lipid droplet accumulation in HepG2 cells through the regulation of SREBP-1c and PPAR-γ gene expression without involving mTOR/S6K1 and may contribute to NAFLD when used as a food additive.