FL83B Cells Research Articles

Lactoferrin as a therapeutic agent for attenuating hepatic stellate cell activation in thioacetamide-induced liver fibrosis

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF’s protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF’s mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-β1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF’s ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1β and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-β1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis.

An alleviative effect of Lonicerae japonicae flos water extract against liver fibrogenesis in vitro and in vivo.

Lonicerae japonicae (L. japonicae) flos is a medical and food homology herb. This study investigated the phenolic acid and flavonoid contents in L. japonicae flos water extract solution (LJWES) and the preventive effects of LJWES against liver fibrogenesis via FL83B cells and rats. LJWES contains many polyphenols, such as chlorogenic acid, morin, and epicatechin. LJWES increased cell viability and decreased cytotoxicity in thioacetamide (TAA)-treated FL83B cells (75 mM) (p < .05). LJWES decreased (p < .05) gene expressions of Tnf-α, Tnfr1, Bax, and cytochrome c but upregulated Bcl-2 and Bcl-xl in TAA-treated cells; meanwhile, increased protein levels of P53, cleaved caspase 3, and cleaved caspase 9 in TAA treated cells were downregulated (p < .05) by LJWES supplementation. In vivo, results indicated that TAA treatment increased serum liver damage indices (alanine aminotransferase [ALT]and alkaline phosphatase [ALP])and cytokines (interleukin-6 and transforming growth factor-β1) levels and impaired liver antioxidant capacities (increased thiobarbituric acid reactive substance value but decreased catalase/glutathione peroxidase activities) in rats (p < .05) while LJWES supplementation amended (p < .05) them. Liver fibrosis scores, collagen deposition, and alpha-smooth muscle actin deposition in TAA-treated rats were also decreased by LJWES supplementation (p < .05). To sum up, LJWES could be a potential hepatoprotective agent against liver fibrogenesis by enhancing antioxidant ability, downregulating inflammation in livers, and reducing apoptosis in hepatocytes.

Heat-Killed Enterococcus faecalis Inhibit FL83B Hepatic Lipid Accumulation and High Fat Diet-Induced Fatty Liver Damage in Rats by Activating Lipolysis through the Regulation the AMPK Signaling Pathway

Continuous consumption of high-calorie meals causes lipid accumulation in the liver and liver damage, leading to non-alcoholic fatty liver disease (NAFLD). A case study of the hepatic lipid accumulation model is needed to identify the mechanisms underlying lipid metabolism in the liver. In this study, the prevention mechanism of lipid accumulation in the liver of Enterococcus faecalis 2001 (EF-2001) was extended using FL83B cells (FL83Bs) and high-fat diet (HFD)-induced hepatic steatosis. EF-2001 treatment inhibited the oleic acid (OA) lipid accumulation in FL83B liver cells. Furthermore, we performed lipid reduction analysis to confirm the underlying mechanism of lipolysis. The results showed that EF-2001 downregulated proteins and upregulated AMP-activated protein kinase (AMPK) phosphorylation in the sterol regulatory element-binding protein 1c (SREBP-1c) and AMPK signaling pathways, respectively. The effect of EF-2001 on OA-induced hepatic lipid accumulation in FL83Bs enhanced the phosphorylation of acetyl-CoA carboxylase and reduced the levels of lipid accumulation proteins SREBP-1c and fatty acid synthase. EF-2001 treatment increased the levels of adipose triglyceride lipase and monoacylglycerol during lipase enzyme activation, which, when increased, contributed to increased liver lipolysis. In conclusion, EF-2001 inhibits OA-induced FL83B hepatic lipid accumulation and HFD-induced hepatic steatosis in rats through the AMPK signaling pathway.

Development of Functional Fermented Dairy Products Containing Taiwan Djulis (Chenopodium formosanum Koidz.) in Regulating Glucose Utilization

Taiwan djulis (Chenopodium formosanum Koidz.) is a plant native to Taiwan and is a grain rich in nutrients, vitamins, and minerals with antioxidant properties. This paper aimed to use appropriate processing technology and incorporate probiotics, thus combining Taiwan’s high-quality milk sources to develop Taiwan djulis fermented dairy products. Later, FL83B cells have used to evaluate the glucose utilization ability after the administration of djulis. We first screened Lactiplantibacillus plantarum and combined it with the traditional yogurt strains Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for cultivation. Further, the fermentation process was optimized where 7.5% djulis and an inoculum of 107 colony forming unit/mL were fermented at 40 °C for 18 h. Compared to fermented milk without djulis, the analysis of various nutrients and active ingredients showed that free radical scavenging abilities of DPPH and ABTS reached 2.3 and 2.0 times (752.35 ± 29.29 µg and 771.52 ± 3.79 µg TE/g, respectively). The free phenol content increased 2.5 times (169.90 ± 14.59 mg gallic acid/g); the total flavonoid content enhanced 4.8 times (3.05 ± 0.03 mg quercetin/g), and the gamma-aminobutyric acid content was 3.07 ± 0.94 mg/g. In a co-culture of mouse liver cells with fermented products, 100 ppm ethanol extract of fermented products effectively improved glucose utilization with increased glucose transporter expression. This functional fermented dairy product can be developed into the high value added local agricultural products and enhance multiple applications including medical and therapeutic fields.

Mangosteen xanthone γ-mangostin exerts lowering blood glucose effect with potentiating insulin sensitivity through the mediation of AMPK/PPARγ

Diabetes mellitus (DM) is concomitant with significant morbidity and mortality and its prevalence is accumulative in worldwide. The conventional antidiabetic agents are known to mitigate the symptoms of diabetes; however, they may also cause side and adverse effects. There is an imperative necessity to conduct preclinical and clinical trials for the discovery of alternative therapeutic agents that can overcome the drawbacks of current synthetic antidiabetic drugs. This study aimed to investigate the efficacy of lowering blood glucose and underlined mechanism of γ-mangostin, mangosteen (Garcinia mangostana) xanthones. The results showed γ-Mangostin had a antihyperglycemic ability in short (2 h)- and long-term (28 days) administrations to diet-induced diabetic mice. The long-term administration of γ-mangostin attenuated fasting blood glucose of diabetic mice and exhibited no hepatotoxicity and nephrotoxicity. Moreover, AMPK, PPARγ, α-amylase, and α-glucosidase were found to be the potential targets for simulating binds with γ-mangostin after molecular docking. To validate the docking results, the inhibitory potency of γ-mangostin againstα-amylase/α-glucosidase was higher than Acarbose via enzymatic assay. Interestingly, an allosteric relationship between γ-mangostin and insulin was also found in the glucose uptake of VSMC, FL83B, C2C12, and 3T3-L1 cells. Taken together, the results showed that γ-mangostin exerts anti-hyperglycemic activity through promoting glucose uptake and reducing saccharide digestion by inhibition of α-amylase/α-glucosidase with insulin sensitization, suggesting that γ-mangostin could be a new clue for drug discovery and development to treat diabetes.

Death-Associated Protein 6 (Daxx) Alleviates Liver Fibrosis by Modulating Smad2 Acetylation.

Transforming growth factor-β (TGF-β) has been identified as an inducer of hepatocyte epithelial–mesenchymal transition (EMT), which triggers liver fibrosis. Death-associated protein 6 (Daxx) is known to be associated with the TGF-β-induced apoptotic pathway, but the function of Daxx in liver fibrosis remains unknown. This study aimed to elucidate the role of Daxx in liver fibrosis. We used liver fibrosis tissues from humans and mice to assess Daxx expression. EMT properties and TGF-β signaling pathway activation were investigated in the Daxx-overexpressing FL83B cell line. The therapeutic effect of Daxx was investigated in a mouse model of liver fibrosis by the hydrodynamic injection of plasmids. The expression of Daxx was markedly decreased in hepatocytes from fibrotic human and mouse livers, as well as in hepatocytes treated with TGF-β in vitro. The overexpression of Daxx inhibited the EMT process by interfering with the TGF-β-induced phosphorylation of Smad2. Coimmunoprecipitation analysis confirmed that Daxx reduced the transcriptional activity of Smad2 by binding to its MH1 domain and interfering with Smad2 acetylation. In addition, the therapeutic delivery of Daxx alleviated liver fibrosis in a thioacetamide-induced fibrosis mouse model. Overall, our results indicate that Daxx could be a potential therapeutic target to modulate fibrogenesis, as well as a useful biomarker for liver fibrosis.

Alleviative Effect of Ruellia tuberosa L. on Insulin Resistance and Abnormal Lipid Accumulation in TNF-α-Treated FL83B Mouse Hepatocytes.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease, and most patients with T2DM develop nonalcoholic fatty liver disease (NAFLD). Both diseases are closely linked to insulin resistance (IR). Our previous studies demonstrated that Ruellia tuberosa L. (RTL) extract significantly enhanced glucose uptake in the skeletal muscles and ameliorated hyperglycemia and IR in T2DM rats. We proposed that RTL might be via enhancing hepatic antioxidant capacity. However, the potent RTL bioactivity remains unidentified. In this study, we investigated the effects of RTL on glucose uptake, IR, and lipid accumulation in vitro to mimic the T2DM accompanied by the NAFLD paradigm. FL83B mouse hepatocytes were treated with tumor necrosis factor-α (TNF-α) to induce IR, coincubated with oleic acid (OA) to induce lipid accumulation, and then, treated with RTL fractions, fractionated with n-hexane or ethyl acetate (EA), from column chromatography, and analyzed by thin-layer chromatography. Our results showed that the ethyl acetate fraction (EAf2) from RTL significantly increased glucose uptake and suppressed lipid accumulation in TNF-α plus OA-treated FL83B cells. Western blot analysis showed that EAf2 from RTL ameliorated IR by upregulating the expression of insulin-signaling-related proteins, including protein kinase B, glucose transporter-2, and peroxisome proliferator-activated receptor alpha in TNF-α plus OA-treated FL83B cells. The results of this study suggest that EAf2 from RTL may improve hepatic glucose uptake and alleviate lipid accumulation by ameliorating and suppressing the hepatic insulin signaling and lipogenesis pathways, respectively, in hepatocytes.

Pepsin-digested chicken-liver hydrolysate attenuates hepatosteatosis by relieving hepatic and peripheral insulin resistance in long-term high-fat dietary habit.

This study aims to clarify the effects of chicken liver hydrolysates (CLHs) on long-term high-fat diet (HFD)-induced insulin resistance (IR) and hepatosteatosis in mice. In vitro, the 400 μM oleic acid (OA)-added medium successfully stimulated the cellular steatosis on FL83B cells, and the cellular steatosis was attenuated ( p < 0.05) by supplementing with CLHs (4 mg/L). In vivo, the effects of CLHs on IR and hepatosteatosis development were tested in 20-week HFD-fed mice. HFD-induced increases in final body weight, but body weight gains of mice were decreased ( p < 0.05) by supplementing CLHs. Elevated ( p < 0.05) serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), free fatty acids (FFAs), triglyceride (TG), total cholesterol (TC), and fasted glucose values in HFD-fed mice decreased ( p < 0.05) by supplementing CLHs. Both results of hepatic steatosis and fibrotic scores also indicated the retardation ( p < 0.05) of the hepatosteatosis in cotreated groups. Moreover, the CLH supplementation sustained ( p < 0.05) hepatic and peripheral insulin signal sensitivity in HFD-fed mice. CLH supplementation could ameliorate hepatic lipid deposition, hepatic/peripheral IR in a long-term high-fat dietary habit, and also improve the universal glucose homeostasis by upregulating hepatic and peripheral insulin sensitivities.

TNF Alpha-Induced SOX2 Expression Promotes Hepatic Steatois in Diet-Induced Obesity Model

Diet-induced obesity can cause metabolic or inflammatory damage on liver. Nonalcoholic fatty liver disease (NAFLD) begins with the fat accumulation in hepatocyte, but can lead to hepatocellular carcinoma (HCC). Sex-determining region Y-box 2 (SOX2) is a critical transcription factor involving regeneration and pluripotency. The expression level of Sox2 is correlated with progression of HCC, and anti-inflammatory effects of Sox2 in mesenchymal stem cells have been found. However, the expression of Sox2 by inflammatory cytokines in hepatocyte in NAFLD or the role of SOX2 in fat accumulation has been rarely reported. Here, we found that high-fat diet feeding, with or without high fructose in drinking water, significantly upregulated SOX2 in the livers of mice. In vitro, treatment with free fatty acids (FFAs) and fructose increased SOX2 expression in FL83B cells compared with the vehicle-treated group. Furthermore, overexpression or knockdown of SOX2 in FL83B cells promoted or suppressed, respectively, triglyceride synthesis and lipid accumulation after FFAs stimulation. The expression levels of several lipogenesis-related molecules were found to be altered by SOX2 expression. In addition, among several cytokines, only the treatment of tumor necrosis factor-alpha (TNFα) increased the SOX2 expression compared with the vehicle-treated control. Further, upregulation of (TNFα) by FFA/fructose was observed, and TNFα and FFA/fructose induced SOX2 expression was abolished by pretreatment of a TNFα inhibitor. Collectively, our findings suggest that TNFα-SOX2 signaling pathway in hepatocyte may be one of targets for early prevention of the development of NAFLD.

Acetaminophen Induces DNA Damage and ATM Mediated DNA Damage Repair in Mouse Hepatocytes

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. While safe at therapeutic doses, it presents a risk for acute liver failure at higher dosages. Accounting for 50% of the cases of liver failure, drug-induced liver toxicity is the most common cause of liver damage in western societies. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then contributes to the further progression of APAP-induced liver injury. Methods Mouse hepatocytes (FL86B cells) were treated with APAP (1mM-100mM) and viability was assessed. FL83B cells were then treated for 24 hours with 5mM APAP, a dose that gave 70% of viability after 24 hr. A DNA comet assay was performed on the treated cells to assay DNA damage. ATM, phospho-ATM, phospho-Chk2 and gH2AX were assessed via immunofluorescence. ATM, Chk2, and BP53 were assess via qPCR. To assess the effects that the removal of ATM has on APAP exposure, the FL86B cells were transfected with ATM siRNA for 48 hours prior to 24-hour exposure of 5mM APAP. ATM and Chk2 were assessed via qPCR and ATM, phospho-ATM, Chk2 and phospho-Chk2 were assessed via immunofluorescence. Results APAP exposure to mouse hepatocytes showed a marked increase in the accumulation of DNA damage when compared to untreated hepatocytes. An increase in the markers for DNA double strand breaks (DSB), gH2AX and 53BP, as well the comet assay confirmed the accumulation of DNA damage after exposure to APAP. An increase of ATM and phosphor-ATM and its downstream effector Chk2 and active form phospho-Chk2 were increased after exposure to APAP. The removal of ATM attenuated the phosphorylation of ATM and Chk2 and enhanced cell viability with exposure to APAP. Conclusion Exposure to APAP induces DNA damage within hepatocytes. This elevated amount of DNA damage results in the activation of ATM and the DDR through Chk2. ATM-mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM-mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP-induced liver damage.

Identification of Bioactive Components from Ruellia tuberosa L. on Improving Glucose Uptake in TNF-α-Induced Insulin-Resistant Mouse FL83B Hepatocytes.

Ruellia tuberosa L. (RTL) has been used as a folk medicine to cure diabetes in Asia. RTL was previously reported to alleviate hyperglycemia, insulin resistance (IR), abnormal hepatic detoxification, and liver steatosis. However, the potential bioactive compounds of RTL have still not been identified. The aim of this study was to investigate the bioactive compounds in RTL ethyl acetate (EA) fractions by using a glucose uptake assay in TNF-α-treated mouse FL83B hepatocytes to discover a mechanism by which to improve IR. The bioactive compounds were identified by the high-performance liquid chromatography (HPLC) assay. Using the Sephadex LH20 gel packing chromatography column, the EAF5 fraction was isolated from RTL and significantly increased glucose uptake in TNF-α-treated FL83B cells. Moreover, the MCI gel packing chromatography column separated EAF5 into five subfractions and had no significant cytotoxic effect in FL83B cells when treated at the concentration of 25 μg/ml. Among the subfractions, EAF5-5 markedly enhanced glucose uptake in TNF-α-treated FL83B cells. The possible bioactive compounds of the EAF5-5 fraction that were identified by the HPLC assay include syringic acid, p-coumaric acid, and cirsimaritin. The bioactive compound with the best effect of increasing glucose uptake was p-coumaric acid, but its effect alone was not as good as the combined effect of all three compounds of the EAF5-5 fraction. Thus, we speculate that the antidiabetic effect of RTL may be the result of multiple active ingredients.

Extracellular vesicle-mediated macrophage activation: An insight into the mechanism of thioredoxin-mediated immune activation

Extracellular vesicles (EVs) generated from redox active anticancer drugs are released into the extracellular environment. These EVs contain oxidized molecules and trigger inflammatory responses by macrophages. Using a mouse model of doxorubicin (DOX)-induced tissue injury, we previously found that the major sources of circulating EVs are from heart and liver, organs that are differentially affected by DOX. Here, we investigated the effects of EVs from cardiomyocytes and those from hepatocytes on macrophage activation. EVs from H9c2 rat cardiomyocytes (H9c2 EVs) and EVs from FL83b mouse hepatocytes (FL83 b EVs) have different levels of protein-bound 4-hydroxynonenal and thus different immunostimulatory effects on mouse RAW264.7 macrophages. H9c2 EVs but not FL83 b EVs induced both pro-inflammatory and anti-inflammatory macrophage activation, mediated by NFκB and Nrf-2 pathways, respectively. DOX enhanced the effects of H9c2 EVs but not FL83 b EVs. While EVs from DOX-treated H9c2 cells (H9c2 DOXEVs) suppressed mitochondrial respiration and increased glycolysis of macrophages, EVs from DOX-treated FL83b cells (FL83b DOXEVs) enhanced mitochondrial reserve capacity. Mechanistically, the different immunostimulatory functions of H9c2 EVs and FL83 b EVs are regulated, in part, by the redox status of the cytoplasmic thioredoxin 1 (Trx1) of macrophages. H9c2 DOXEVs lowered the level of reduced Trx1 in cytoplasm while FL83b DOXEVs did the opposite. Trx1 overexpression alleviated the effect of H9c2 DOXEVs on NFκB and Nrf-2 activation and prevented the upregulation of their target genes. Our findings identify EVs as a novel Trx1-mediated redox mediator of immune response, which greatly enhances our understanding of innate immune responses during cancer therapy.

Anti-oxidative and protective effect of soy tempeh on di(2-ethylhexyl)-phthalate (DEHP) injured FL83B mice liver cells

The interest on Tempeh, a well known Rhizopus oligosporus-fermented food from Indonesia, is keep increasing as it possess potential bioactivities. Previous research on co-inoculation of Rhizopus oligosporus together with lactic acid bacteria in tempeh making showed promising results as lactic acid bacteria did not inhibit the growth of Rhizopus oligosporus. The aims of this study are to analyze the bioactivities (antioxidative and hepatoprotective activities) of single- and co-inoculated tempeh compared to pre-fermented soybean. Experiments was conducted by observing the effect of different lactic acid bacteria (Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei) on antioxidant capacities. Afterwards, one of lactic acid bacteria strain will be used to produce tempeh and the protective activity against di(2-ethylhexyl)-phthalate (DEHP)-injured FL83B cell was observed. The result showed that Lactobacillus plantarum co-inoculated tempeh had the highest ferrous ion chelating activity, while Lactobacillus casei co-inoculated tempeh had the highest radical scavenging acticity and reducing power. Moreover, genistein and daidzein, aglycone isoflavone, found highest in Lactobacillus bulgaricus co-inoculated tempeh. For hepatoprotective activity, Lactobacillus plantarum co-inoculated and single-inoculated tempeh had protective activities against DEHP-injured FL83B cell, while soybean has no significant protective effect.

Abstract 5747: Lack of FGF21 accelerates the Th17-IL-17 axis-mediated transition from nonalcoholic steatohepatitis to hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) has been increasingly recognized in nonalcoholic steatohepatitis (NASH) before cirrhosis stage and the estimated annual HCC incidence in NASH patients is about 0.3%. Accumulating evidence shows that T helper (Th) 17-Interleukin (IL)-17 axis mediates the transition from NASH to HCC, while fibroblast growth factor 21 (FGF21) exerts pharmacologic efficacy in downregulation of Th17- IL-17 axis. In fact, FGF21 has recently emerged as a promising therapeutic candidate for the treatment of obesity and diabetes. However, the NASH-related carcinogenesis and role of FGF21 played during NASH-HCC transition are largely unknown. Aim: To investigate the effect of FGF21 on Th17-IL-17 axis during the NASH-HCC carcinogenetic transformation and to reveal the potential NASH-HCC mechanism. Methods: NASH-HCC was induced in FGF21 knockout (KO) mice treated with DEN at age 2 weeks at 100 mg/kg body weight (i.p.), and followed feeding with high-fat diet (HFD,60% kcal% fat) or methionine/choline-deficient diet (MCD). In an orthotopic HCC model with inoculation of 106 hepal-6 cells into 10 week-old male C57L/J mice, fenofibrate (50 mg/kg) was administered to induce FGF21. In vitro studies were performed using shRNA-FGF21 knockdown hepatocyte and HCC cell lines. The cellular and molecular events, in turn, white adipose tissue (WAT) lipolysis, insulin resistance (IR), release of free fatty acids (FFAs), Th17 cell infiltration and IL-17A production, and NASH-HCC initiation, were determined both in vivo and in vitro. Results: Early HCC detection and increased HCC incidence were found in the FGF21 knockout (KO) mice (P&amp;lt;0.05 versus wild-type controls). Increased WAT lipolysis, hepatic FFAs uptake, Th17 cells/IL-17A, and aberrant NF-κB and p53 signaling were found in NASH and HCC tissues (adipose, spleen and liver) from in FGF21-KO mice by flow cytometry, immunohistochemistry (IHC) and Western blot. IL-17A significantly induced FFAs uptake in the shRNA-FGF21 knockdown hepal-6 cells and FL83B cells. IL-17A promoted the shRNA-FGF21 knockdown hepal-6 cells spheroid formation through Wnt/β-catenin pathway. Fenofibrate treatment significantly increased PPAR-α and native FGF21 protein levels in the liver tissues, and decreased the tumor volumes. Conclusions: 1) Lack of FGF21 played a key role in hepatic lipid accumulation and subsequent upregulated Th17-IL-17 signaling in liver; 2) lack of FGF21 accelerated the Th17-IL-17 axis promoted NASH-HCC transition; 3) upregulation of native FGF21 not only attenuates NASH, but also prevents HCC carcinogenetic transformation via downregulation of Th17-IL-17 axis. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Citation Format: Qianqian Zheng, Harshul Pandit, Xingkai Liu, Youxi Yu, Suping Li, Shelby Turner, Xin Meng, Robert Martin, Yan Li. Lack of FGF21 accelerates the Th17-IL-17 axis-mediated transition from nonalcoholic steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5747.

Fisetin Protects Against Hepatic Steatosis Through Regulation of the Sirt1/AMPK and Fatty Acid β-Oxidation Signaling Pathway in High-Fat Diet-Induced Obese Mice

Background/Aims: Fisetin is a naturally abundant flavonoid isolated from various fruits and vegetables that was recently identified to have potential biological functions in improving allergic airway inflammation, as well as anti-oxidative and anti-tumor properties. Fisetin has also been demonstrated to have anti-obesity properties in mice. However, the effect of fisetin on nonalcoholic fatty liver disease (NAFLD) is still elusive. Thus, the present study evaluated whether fisetin improves hepatic steatosis in high-fat diet (HFD)-induced obese mice and regulates lipid metabolism of FL83B hepatocytes in vitro. Methods: NAFLD was induced by HFD in male C57BL/6 mice. The mice were then injected intraperitoneally with fisetin for 10 weeks. In another experiment, FL83B cells were challenged with oleic acid to induce lipid accumulation and treated with various concentrations of fisetin. Results: NAFLD mice treated with fisetin had decreased body weight and epididymal adipose tissue weight compared to NAFLD mice. Fisetin treatment also reduced liver lipid droplet and hepatocyte steatosis, alleviated serum free fatty acid, and leptin concentrations, significantly decreased fatty acid synthase, and significantly increased phosphorylation of AMPKα and the production of sirt-1 and carnitine palmitoyltransferase I in the liver tissue. In vitro, fisetin decreased lipid accumulation and increased lipolysis and β-oxidation in hepatocytes. Conclusion: This study suggests that fisetin is a potential novel treatment for alleviating hepatic lipid metabolism and improving NAFLD in mice via activation of the sirt1/AMPK and β-oxidation pathway.

Comparative Proteomic Identification of Protein Disulphide Isomerase A6 Associated with Tert-Butylhydroperoxide-Induced Liver Injury in Rat Hepatocytes

Background/Aims: Oxidants are important human toxicants. They have been implicated in the occurrence and development of liver diseases. Increased intracellular tert-butylhydroperoxide (t-BHP) may be critical for oxidant toxicity, and is commonly used for evaluating mechanisms involving oxidative stress, but the method remains controversial. Methods: Primary cultures of hepatocytes as well as human Hep G2 and mouse FL83B liver cells were obtained. Cell viability was measured by annexin V–FITC/propidium iodide and DAPI staining to determine the effects of t-BHP treatment on acute liver injury. A proteomic assay provided information that was used to identify the differentially expressed proteins following t-BHP treatment; immunohistochemistry and western blotting were performed to detect the expression of PDIA6 activity in apoptotic and endoplasmic reticulum (ER) stress pathways. Results: Our results demonstrate that t-BHP treatment of liver cells increased cell cytotoxicity and the generation of reactive oxygen species. This treatment also increased the level of PDIA6; this was validated in vitro and in vivo based on a comparison of t-BHP-treated and -untreated groups. Treatment of mouse liver FL83B cells with t-BHP activated caspase 3, increased the expression of apoptotic molecules, caused cytochrome c release, and induced Bcl-2, Bax and IRE1α/TRAF2 complex formation. t-BHP-dependent induction of apoptosis was accompanied by sustained phosphorylation of the IRE1α/ASK1/JNK1/2/p38 pathways and PDIA6 expression. Furthermore, t-BHP induced liver FL83B cell viability and apoptosis by upregulating the levels of PDIA6; this process could be involved in the activation of the IRE1α/ASK1/JNK1/2/p38 signalling pathways. Conclusions: We conclude that t-BHP induced an apoptosis cascade and ER stress in hepatocytes by upregulation of PDIA6, providing a new mechanism underlying the effects of t-BHP on liver injury.

Comparison of Anti-Inflammatory Activities of Structurally Similar Triterpenoids Isolated from Bitter Melon

Momordica charantia L., or bitter melon, has been suggested to exhibit anti-inflammatory activity. In a previous study, three structurally similar triterpenes, namely 5β,19-epoxy-25-methoxycucurbita-6,23-diene-3β,19-diol (EMCD), 5β,19-epoxy-25-methoxycucurbita-6,23-dien-3β-ol (EMCO), and 5β,19-epoxy-19,25-dimethoxycucurbita-6,23-dien-3β-ol (EDMO), were isolated from bitter melon. EMCD has been shown to exhibit in vitro anti-inflammatory activity. In this study, the anti-inflammatory activities of EMCD, EMCO, and EDMO were compared. All three compounds were toxic to the RAW 264.7 macrophage cell line but not the FL83B cells. EMCD and EMCO inhibited tumor necrosis factor (TNF)-α-induced inducible nitric oxide synthase (iNOS) expression in FL83B cells, and the IC50 values were 19.8 and 25.7 μM, respectively. By contrast, EDMO did not effectively reduce iNOS expression. Furthermore, EMCD and EMCO suppressed other TNF-α-induced proinflammatory signals including the activation of inhibitor kappa B kinase complex, the phosphorylation of inhibitor of nuclear factor-κB, and the activation of c-Jun kinase. EMCD consistently exhibited a higher efficacy than did EMCO in these assays. Hence, the in vivo anti-inflammatory activity of EMCD was tested. EMCD clearly repressed 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. In conclusion, differences in the functional group on carbon 19 do affect the anti-inflammatory activities of EMCD, EMCO, and EDMO. EMCD exhibited the highest anti-inflammatory activity among these molecules, and its in vivo anti-inflammatory activity was confirmed.

Di-N-octylphthalate acts as a proliferative agent in murine cell hepatocytes by regulating the levels of TGF-β and pro-apoptotic proteins

Di-n-octylphthalate (DNOP) is a phthalate used in the manufacturing of a wide variety of polyvinyl chloride-containing medical and consumer products. A study on chronic exposure to DNOP in rodents showed the development of pre-neoplastic hepatic lesions following exposure to a tumor initiator. The objective of this study was to identify the mechanisms by which DNOP leads to pre-neoplastic hepatic lesions. Mouse hepatocyte AML-12 and FL83B cells were treated with DNOP. The rate of cell proliferation was increased in treated cells in a concentration-dependent manner. DNOP increased the expression of transforming growth factor-β (tgf-β) in both cell lines, and primary culture mouse hepatocytes. The TGF-β receptor inhibitor LY2109761 impaired the effect of DNOP. The presence of pro-apoptotic proteins decreased in the presence of DNOP. Our observation indicates that DNOP, through an increase in the expression of tgf-β and a decrease in the levels of pro-apoptotic proteins, acts as a proliferative agent in normal mouse hepatocytes. We also studied the morphological and functional changes of the mouse liver upon a short-term treatment of DNOP. Mice exposed to DNOP displayed an epithelial-to-mesenchymal transition and cholestasis, which was reflected in an increase in hepatic bile acids and glutathione levels.

Fucoxanthin attenuates fatty acid-induced lipid accumulation in FL83B hepatocytes through regulated Sirt1/AMPK signaling pathway

The fucoxanthin, isolated from brown algae, was reported to have multiple biological functions to anti-inflammation, anti-tumor, and ameliorated obesity in mice. In this study we investigated whether fucoxanthin could inhibit lipids accumulation in FL83B hepatocytes. FL83B cells were induced as fatty liver cell model by 0.5 mM oleic acid for 48 h, and treated with various concentration of fucoxanthin for 24 h. The results demonstrated that fucoxanthin significantly suppressed lipid accumulation and decreased lipid peroxidation in hepatocytes. Fucoxanthin could decrease lipogenesis-related transcription factor expression, including sterol regulatory element-binding proteins 1c and peroxisome proliferator-activated receptor γ. It also reduced fatty acid synthase expression and increased adipose triglyceride lipase and the phosphorylation of hormone-sensitive lipase production for lipolysis. Furthermore, fucoxanthin significantly increased phosphorylation of AMP-activated protein kinase (AMPK), and decreased activity of acetyl-CoA carboxylase for regulating fatty acid synthesis. The results suggest that fucoxanthin is an effective marine nature compound for increasing lipolysis and inhibiting lipogenesis in oleic acid induced fatty liver cells through promoted Sirt1/AMPK pathway.

Fatsia polycarpa Triterpenoids and Acetylated Derivatives Thereof Inhibit Tumor Necrosis Factor-α-Induced Inflammation

Fatsia polycarpa Hayata is an evergreen shrub endemic to Taiwan and used locally to treat several inflammatory disorders. The crude extract of F. polycarpa has been proven to exhibit an anti-inflammatory effect in vitro and in vivo; however, which constituents of the extract confer the anti-inflammatory function remains unclear. Fatsicarpain D (Fat D) and fatsicarpain F (Fat F) are oleanane-type triterpenoids and two of the feature constituents of the F polycarpa extract. Ester substitution on C-3 has been proposed to enhance the activities of triterpenoids. Thus, this study compared and characterized the anti-inflammatory activities of Fat D, Fat F, and the C-3-acetylated derivatives thereof. These compounds were toxic to RAW 264.7 cells, but not to FL83B cells. The compounds dose-dependently inhibited tumor necrosis factor-α (TNF-α)-induced expression of inducible nitric oxide synthase in FL83B cells, with the IC50 values being Fat D 8.5 μM, acetylated Fat D 10.4 μM, acetylated Fat F 10.7 μM, and Fat F 27.4 μM, respectively. Thus, acetylation of C-3 improved the activity of Fat F. Moreover, the compounds suppressed TNF-α-induced expression of nuclear factor-κB (NF-κB) p65 subunit and protein tyrosine phosphatase-1B. Furthermore, they inhibited TNF-α-mediated activation of the inhibitor kappa B kinase (IKK), and that of the mitogen-activated protein kinases (MAPKs) extracellular-signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), but promoted the activation of MAPK p38, which was found to be anti-inflammatory in certain cell types. Thus, Fat D and Fat F exhibited obvious anti-inflammatory activities in vitro and inhibited ERK, JNK, and the IKK/NF-κB pathway.