Osthole attenuates lipid accumulation, regulates the expression of inflammatory mediators, and increases antioxidants in FL83B cells

Biomedicine & Pharmacotherapy 91 (2017) 78–87

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Original article

Osthole attenuates lipid accumulation, regulates the expression of inflammatory mediators, and increases antioxidants in FL83B cells Wen-Chung Huanga,b,1, Po-Chen Liaob,c,1, Chun-Hsun Huangd,e , Sindy Hue , Shih-Chun Huangb , Shu-Ju Wub,e,* a Graduate Institute of Health Industry Technology, Research Center for Industry of Human Ecology, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, No.261, Wenhua 1st Rd., Guishan Dist., Taoyuan City 33303, Taiwan b Department of Nutrition and Health Sciences, Research Center for Food and Cosmetic Safety, and Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, No.261, Wenhua 1st Rd., Guishan Dist., Taoyuan City 33303, Taiwan c Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan d Department of Cosmetic Science, Chang Gung University of Science and Technology, No.261, Wenhua 1st Rd., Guishan Dist., Taoyuan City 33303, Taiwan e Aesthetic Medical Center, Department of Dermatology, Chang Gung Memorial Hospital, Guishan Dist., Taoyuan 33303, Taiwan

A R T I C L E I N F O

Article history: Received 6 January 2017 Received in revised form 12 April 2017 Accepted 13 April 2017 Keywords: Nonalcoholic fatty liver disease p38 MAPK NF-kB Antioxidant Osthole

A B S T R A C T

Osthole is found in Cnidium monnieri (L.) and has anti-inflammatory and anti-oxidative properties. It also inhibits the proliferation of hepatocellular carcinoma cells. This study aimed to evaluate the osthole suppressive nonalcoholic fatty liver disease effects in oleic acid (OA)-induced hepatic steatosis and if it can modulate inflammatory responses and oxidative stress. FL83B cells were pretreated with OA (250 mM) for 24 h, and then added different concentrations of osthole (3–100 mM) for 24 h. Subsequently, lipolysis and transcription factors of adipogenesis and phosphorylation of AMP-activated protein kinase proteins were measured. In addition, cells with OA-induced steatosis were H2O2–stimulated, and then incubated with osthole to evaluated if it could suppress its progression to steatohepatitis. Osthole significantly enhanced glycerol release and lipolysis protein expression. Osthole also promoted phosphorylation of AMP-activated protein kinases and increased the activity of triglyceride lipase and hormone- sensitive lipase. Osthole suppressed the nuclear transcription factor kappa-B and the p38 mitogen-activated protein kinase pathway, and decreased the malondialdehyde concentration in FL83B cells with OA-induced steatosis that were treated with H2O2. These results suggest that osthole might suppress nonalcoholic fatty liver disease by decreasing lipid accumulation, and through its anti-oxidative and anti-inflammatory effects via blocked NF-kB and MAPK signaling pathways. © 2017 Published by Elsevier Masson SAS.

1. Introduction Nonalcoholic fatty liver disease (NAFLD) is a serious and widespread metabolic disorder and encompasses a wide spectrum of liver conditions ranging from simple steatosis, to steatohepatitis and fibrosis, and end-stage liver diseases, including cirrhosis and hepatocellular carcinoma [1,2]. Many recent studies have found that NAFLD is associated with serious cardiometabolic

* Corresponding author at: Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, No.261, Wenhua 1st Rd., Guishan Dist., Taoyuan City 33303, Taiwan. E-mail address: [email protected] (S.-J. Wu). 1 Wen-Chung Huang and Po-Chen Liao are equal contributors to the work. http://dx.doi.org/10.1016/j.biopha.2017.04.051 0753-3322/© 2017 Published by Elsevier Masson SAS.

abnormalities, including type 2 diabetes mellitus, metabolic syndrome, obesity, and dysregulated insulin action in the liver [3]. The hallmark of NAFLD is excessive lipid accumulation in the liver, mainly triacylglycerol, in the absence of significant ethanol consumption. This accumulation interferes with the signaling pathways involved in the normal metabolism of hepatocytes, causes insulin resistance, and may even lead to metabolic syndrome abnormalities [4]. Hepatic lipid accumulation results from an imbalance between lipid availability and lipid disposal, and eventually triggers lipid peroxidative stress and hepatic injury [5]. Current treatments for patients with fatty liver disease include change of lifestyle and improvement of insulin sensitivity to alleviate the associated metabolic syndrome [6]. The AMPactivated protein kinase (AMPK) pathway is important in lipid metabolism. Studies indicated that phosphorylated acetyl-CoA carboxylase-1 (ACC-1) activated by phosphorylation of AMPK,

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which can suppress acetyl-CoA catalyze to malonyl-CoA during fatty acid (FA) and triglyceride synthesis [7–9]. Osthole, 7-methoxy-8-(3-methyl-2-butenyl) coumarin, is an active constituent of Cnidium monnieri (L.) that has been extracted from many medicinal plants. Osthole has long been used in traditional Chinese medicine for the treatment of eczema, cutaneous pruritus, Trichomonas vaginalis infection, and sexual dysfunction [10]. Recent studies have revealed that osthole may have anti-proliferative, vasorelaxant, anti-inflammatory, antimicrobacterial, anti-allergic, and prophylactic effects in hepatitis [11]. Furthermore, anti-cancer effects have been reported for osthole [12–14]. However, the effects of osthole on lipid metabolism and anti-oxidation remain unclear. A few experiments indicated that osthole treatment attenuated liver steatosis by decreasing triglyceride synthesis and had nominal effects on insulin resistance and liver inflammation in histological sections and an animal model [15–17]. The aims of the present study were to examine osthole-induced AMPK signaling, the potential mechanisms for controlling hepatocellular lipid metabolism, and to test the effects of osthole on intrahepatic fatty acid synthesis and inflammation. 2. Materials and methods

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was spectrophotometrically measured at 570 nm using a microplate reader (Gene5, Synergy HT, BioTek, USA). 2.4. Oil red O staining FL83B cells treated drug in 6-well plates for 24 h and fixed using 10% formalin for 30 min. Cells stained with oil red O (Sigma Chemical, St. Louis, MO, USA) for 1 h at room temperature. To remove excess dye cells were washed three times with 1 mL of PBS and dipped in 100% isopropanol. The OD value was measured at 490 nm using a microplate reader (Gene5, Synergy HT, BioTek, USA) to quantify the lipid accumulation. 2.5. Measurement of glycerol production FL83B cells treated with osthole in OA microenvironment for 24 h, and glycerol levels of culture medium quantified using glycerol quantification kit (GPO-Trinder) according to the manufacturer’s instructions (Sigma-Aldrich). The results quantified at 570 nm using a microplate reader (Gene5, Synergy HT, BioTek, USA). 2.6. Antioxidant assay: malondialdehyde concentration and superoxide dismutase activity

2.1. Materials Osthole (Fig. 1A) purchased from the ChromaDex (Irvine, CA, USA). The purity was >97.9% as determined by HPLC. Stock solution (100 mM) prepared by dissolving osthole in DMSO and stored at
20  C, as previously described [10]. The final concentration in culture medium of DMSO was 0.1%.

FL83B cells treated with OA for 24 h and H2O2 (500 mM) for 2 h; then, the cells treated with osthole for 24 h. Superoxide dismutase (SOD) and malondialdehyde (MDA) measured according to the commercial kit instructions (Sigma-Aldrich), and the absorbance at 450 and 532 nm with a spectrophotometer, respectively. 2.7. Preparation of total and nuclear proteins

2.2. Cell line and treatment FL83B cells purchased from the Bioresource Collection and Research Center (BCRC, Taiwan). Cells were cultured in F12 medium (Invitrogen-GibcoTM, Paisley, Scotland) added with 10% FBS, 2 mM glutamine, 1% penicillin and streptomycin at 37  C in a humidified atmosphere containing 5% CO2. Cells exposed to oleic acid (OA) 250 mM for 24 h, then treated with osthole (3–100 mM) in OA microenvironment for 24 h.

FL83B cells treated with OA for 24 h and stimulated with H2O2 for 2 h. Then, cells treated with osthole in 6 well plates. Cells were harvested with 300 mL protein lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM DTT, 50 mM Tris–HCl, pH 7.4, 0.5% NP40, and 0.1% sodium dodecyl sulfate [SDS]) containing protease inhibitor cocktail and phosphatase inhibitors (Sigma). Cytoplasm and nuclear proteins were using the NE-PER1 extraction reagent kits (Pierce, Rockford, IL, USA). Proteins quantitated using BCA assay kit (Pierce)[18].

2.3. Cell viability assay 2.8. Western blot analysis Using MTT assay performed to evaluate the cytotoxicity of osthole. Cells were plated in 96-well plates cultured overnight and treated with osthole for 24 h. The MTT solution (5 mg/ml) was added to each well and incubated for 4 h at 37  C, followed by isopropanol dissolution of formazan crystals. The OD absorbance

An equal amount of protein was separated on polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with different primary antibodies for phosphorylated acetyl-CoA

Fig. 1. Structures of osthole (A) and the cytotoxicity of osthole in FL83B cells (B).

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carboxylase (ACC), fatty acid synthase (FAS), proliferator-activated receptor g (PPAR-g), CAAT/enhancer binding protein alpha (C/ EBPa), C/EBPb, phosphorylated hormone-sensitive lipase (HSL), and adipose triglyceride lipase (ATGL) (Epitomics, Burlingame, CA

USA); sterol regulatory element-binding protein 1c (SREBP-1c), phosphorylated AMPKa, SIRT-1, cyclooxygenase-2 (COX-2), p65, and proliferating cell nuclear antigen (Santa Cruz, CA, USA); phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2),

Fig. 2. The effects of osthole on oil red O stain (A), 100% isopropanol release of a lipid droplet (B), and the glycerol assay (C) in FL83B cells. Cells were pretreated with OA and treated with osthole for 24 h. The cells were stained with oil red O to observe triglyceride content by OD at 490 nm, and the glycerol concentrations were measured in supernatants. Data are presented as the mean  standard deviation (SD), n = 3; ***p < 0.005, **p < 0.01, *p < 0.05, compared with OA alone.

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phosphorylated p38, phosphorylated c-Jun N-terminal kinase (JNK), phosphorylated IkB-a, ERK1/2, p38, JNK, and I kappa B alpha (IkB-a) (Millipore); and b-actin (Sigma-Aldrich) overnight at 4  C. Followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. The signals were incubated in ECL kit (Millipore) and detection image in BioSpectrum 600 system (UVP, Upland, CA, USA). Working concentration of primary antibodies are 1–2 mg/ml. 2.9. RNA isolation and real-time PCR for gene expression Total RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA, USA) and PCR analyzed according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The primer sequences (sense/antisense) used were: SIRT1, forward 50 -GAC GCT GTG GCA GAT TGT TA-30 and reverse 50 -GGA ATC CCA CAG AGA CAG A-30 ; FAS, forward 50 -GGT CGT TTC TCC ATT AAA TTC TCA T-30 and reverse 50 - CTA GAA ACT TTC CCA GAA ATC TTC C-30 ; HSL, forward 50 -GCT GGG CTG TCA AGC ACT GT-30 and reverse 50 -GTA ACT GGG TAG GCT GCC AT-30 ; SREBP-1c, forward 50 -GGA GCC ATG GAT TGC ACA TT-30 and reverse 50 -GGC CCG GGA AGT CAC TGT-30 . Data were expressed as the signals of interest band to against b-actin mRNA expression. 2.10. Statistical analysis All data were presented as the mean  standard deviation (SD). Statistical analysis was performed using one-way analysis of variance and post hoc analysis with Dunnett’s test, with p < 0.05 considered significant. 3. Results 3.1. Cytotoxicity of osthole in FL83B cells The MTT assay was used to detect cytotoxicity in FL83B cells. At concentrations 100 mM, osthole did not significantly affect cell viability (Fig. 1B). Therefore, 3–100 mM osthole was used in all experiments. 3.2. The effect of osthole on lipid accumulation Previous study demonstrated that OA-induced steatosis in hepatocytes [19]. Therefore, we endeavored to examine the effect of osthole on lipogenesis. FL83B cells were pretreated with OA

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(250 mM) for 24 h, then treated with or without various concentrations of osthole for 24 h. Lipid accumulation was measured by oil red O staining (Fig. 2A) and lipid accumulation (Fig. 2B). We found that 10–100 mM osthole significantly decreased lipid accumulation compared with the OA group. In addition, treatment with 10–100 mM osthole significantly increased glycerol release (osthole 3 mM: 36.6  3.8, p = 0.38; osthole 10 mM: 40  2.3, p < 0.05; osthole 30 mM: 42.5  2.1, p < 0.05; osthole 100 mM: 40.81  2.83, p < 0.05 versus OA: 37.5  2.1; Fig. 2C). 3.3. The effect of osthole on the expression of proteins involved in lipolysis The two-hit hypothesis in the NAFLD pathogenesis considers extensive lipid accumulation is the first hit [20]. Hence, we analyzed the enzymes involved in triglyceride lipolysis. We found osthole can significantly increase the release of glycerol from hepatocytes with OA-induced lipid accumulation (Fig. 2) and treatment with osthole enhanced expression of ATGL and phosphorylated HSL (Fig. 3). Osthole concentrations 3 mM significantly increased phosphorylated HSL expression in a dose-response manner, 10 and 100 mM osthole enhanced ATGL expression compared with the OA group. This is important to induce the expression of lipolytic enzymes in hepatocytes lipid accumulation. Activated ATGL hydrolyzes triacylglycerol into diacylglycerol (DAG) and free FA, phosphorylated HSL hydrolyzes DAG into monoacylglyceroland (MAG) and one molecule of FA. Therefore, activating lipolysis and decreasing lipid accumulation may reduce the incidence of NAFLD. 3.4. The effect of osthole on FAS and transcription factors of adipogenesis The transcription factors of adipogenesis including PPARg, C/ EBPa, C/EBPb and SREBPs. SREBP1c closely mimics that of known C/EBP targets for lipid biosynthesis and plays a central role in the induction of PPARg expression. Thus, up-regulated PPAR and SREBP-1c expression can activate lipid biosynthesis, leading to excessive lipid accumulation in hepatocytes [7]. To examine the effects of osthole on adipogenesis, OA-induced steatosis in FL83B hepatocytes were treated with osthole for 24 h. We found that Treatment with 100 mM osthole decreased expression of FAS compared with the OA group. Osthole could directly regulate SREBP1c expression and at 100 mM significantly inhibit PPARg, C/

Fig. 3. Effects of osthole (OS) on lipolysis protein expression in hepatocytes. The FL83 B cells (106 cells/mL) were pretreated with OA and then treated with OS for 24 h. The ATGL and phosphorylated HSL proteins were detected by Western blots (n = 3 per group) in OS-treated cells. Expression of b-Actin was used as an internal control.

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Fig. 4. Effects of osthole on transcription factors of adipogenesis in hepatocytes. The FL83B cells (106 cells/mL) were pretreated with OA and treated with osthole (OS) for 24 h. The PPARg, SREBP-1, C/EBPa, and C/EBPb proteins were detected in OS-treated cells by Western blots (n = 3 per group). Expression of b-Actin was used as an internal control.

Fig. 5. The mRNA expression of lipolysis proteins SIRT-1 (A) and HSL (B) and the adipogenesis proteins FAS (C) and SREBP1c (D). Lipolysis and adipogenesis proteins were quantified in FL83B cells treated with different concentrations of osthole (3–100 mM) for 24 h. Data are presented as the mean  standard deviation (SD); *p < 0.05, **p < 0.01, compared with OA alone.

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EBPa, and C/EBPb protein expression compared with the OA group (Fig. 4). 3.5. The effect of osthole on the gene expression of lipolysis and adipogenesis The relative gene expression levels of lipolysis and adipogenesis were the ratio of different gene expression and b-actin. Compared with OA alone, osthole significantly enhanced the mRNA expression of SIRT-1 (3 mM osthole: 0.68  0.07; 10 mM osthole: 0.76  0.02, P < 0.05; 30 mM osthole: 0.68  0.06; 100 mM osthole: 0.72  0.08 P < 0.05 vs. OA alone: 0.62  0.02; Fig. 5A) and HSL (3 mM osthole: 0.61  0.01;10 mM osthole: 0.65  0.03; 30 mM osthole: 0.74  0.05, P < 0.05; 100 mM osthole: 1.22  0.04, P < 0.01 vs. OA alone: 0.66  0.02; Fig. 5B). In addition, osthole significantly decreased the mRNA expression of FAS (3 mM osthole: 0.34  0.01, P < 0.05; 30 mM osthole: 0.36  0.06, P < 0.05 vs. OA alone: 0.46  0.03; Fig. 5C) and SREBP1c (3 mM osthole: 0.62  0.23;10 mM osthole: 0.33  0.09, P < 0.01; 30 mM osthole: 0.38  0.05, P < 0.01; 100 mM osthole: 0.27  0.06 P < 0.01 vs. OA alone: 1.01  0.21; Fig. 5D). These results suggest that osthole can suppress the OA-induced lipid accumulation in FL83B cells. 3.6. The effect of osthole on the AMPK pathway The AMPK is a metabolic energy regulator as described previously [7]. AMPK phosphorylation stimulates ACC phosphorylated, which regulates enzymes during fatty acid synthesis for malonyl-CoA production. SIRT1 regulates hepatocyte lipid metabolism through activating AMPK [7–9]. Thus, up-regulated Sirt1 and AMPK are expected to improve lipid metabolism and decrease lipid acceleration. We also investigated whether osthole could modulate the Sirt1 and AMPK pathway in OA-induced steatosis. Osthole significantly increased SIRT1 deacetylase activity and phosphorylation of AMPK, and its downstream target ACC, in osthole concentration at 3–30 mM (Fig. 6). Our results indicated that osthole may protect against free fatty acid-induced lipid

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accumulation by activating SIRT1 and AMPK overexpression in hepatocytes. 3.7. Osthole increased the antioxidant capacity in H2O2-induced hepatic steatosis cells Oxidative stress plays a critical role in the “second hit” of the NAFLD pathogenesis, involving lipid peroxidation and inflammation, as the steatosis progresses to steatohepatitis [20]. This study to investigate osthole whether inhibited H2O2-induced lipid peroxidation and oxidative stress, MDA is a lipid peroxidation product and SOD is an antioxidant enzyme. Hence, we investigated whether osthole may decrease the MDA concentration and increase SOD activity. Compared with OA + H2O2 group, osthole significantly decreased the MDA concentration (10 mM osthole: 3.6  0.8, P < 0.05; 30 mM osthole: 2.6  0.2, P < 0.01; 100 mM osthole: 1.3  0.4, P < 0.01 vs. OA alone: 4.19  0.02) (Fig. 7A) and enhanced the SOD activity (10 mM osthole: 60.2  2.8, P < 0.05; 30 mM osthole: 63.4  2.2, P < 0.05 vs. OA alone: 56.2  7.1) (Fig. 7B). 3.8. Effects of osthole on H2O2-induced COX-2 protein expression and NF-kB activation in hepatic steatosis cells The “second hit” is believed to result from an increase in oxidative stress and expression of inflammatory mediators [20]. Previously studies indicated that improved liver inflammation could alleviate hepatic steatosis in mouse models of NASH. The NFkB pathway plays a critical role in the inflammatory response by regulating the expression of inflammatory cytokines, e.g., TNF-a [21]. Hence, we investigated whether osthole may suppress COX-2, the phosphorylation of IkB-a, and translocation of NF-kB (active subunit p65) into the nucleus. Osthole significantly suppressed COX-2 protein expression in a concentration-dependent manner compared with H2O2-stimulated cells (Fig. 8A). We observed that osthole significantly suppressed IkB-a phosphorylation and degradation compared with H2O2-induced FL83B cells (Fig. 8B), and p65 was mostly distributed in the cytoplasm and hardly

Fig. 6. Effects of osthole on the AMPK pathway in hepatocytes. The FL83B cells (106 cells/mL) were pretreated with OA and treated with osthole for 24 h. Phosphorylated and unphosphorylated AMPKa and ACC, SIRT-1, and FAS proteins were detected by Western blots (n = 3 per group) in OS-treated cells. Expression of b-Actin expression was used as an internal control.

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Fig. 7. Effects of osthole (OS) on the antioxidant activity in hepatocytes. The FL83B cells (106 cells/mL) were pretreated with OA (250 mM) for 24 h, treated with H2O2 (250 mM) for 2 h, and then with different concentrations of OS for 24 h. The MDA concentration (A) and SOD activity (B) were measured. Data are presented as the mean  standard deviation (SD); *p < 0.05, **p < 0.01, compared with OA alone.

Fig. 8. Effects of osthole (OS) on H2O2-induced production of COX-2 (A), b-actin expression was used as an internal. Phosphorylation of the active subunit of IkB-a, p65 (B), with total IkB-a levels used as internal controls. For the nuclear translocation of NF-kB (p65) (C), cells were pretreated with OA (250 mM) for 24 h and then H2O2 (250 mM) for 2 h. Then, cells were treated with different concentrations of OS for 24 h. The internal controls were a-tubulin in the nucleus and b-actin in the cytosol. The densitometry values from three independent experiments were analyzed and compared with the H2O2-treated group.

translocated into the nucleus in unstimulated cells (Fig. 8C). Interestingly, osthole suppressed the nuclear translocation of p65 in a concentration-dependent manner compared with H2O2induced cells.

cells with H2O2-activated hepatic steatosis. We found that osthole (100 mM) significantly decreased phosphorylation of p38 compared with H2O2-activated FL83B cells; however, osthole didn’t suppress phosphorylation of ERK1/2 and JNK (Fig. 9).

3.9. Effect of osthole on MAPK pathways in H2O2-activated hepatic steatosis cells

4. Discussion

Previous studies have shown that the MAPK signaling pathways are closely related with mechanisms of inflammation and are involved in the pathological process of NAFLD [22]. Here, we investigated whether osthole affects the MAPK pathways in FL83B

A “two-hit” hypothesis has been proposed in NAFLD for the progression to steatohepatitis. First, hepatic intracellular reversible deposition of triacylglycerols and development of hepatic steatosis (“first hit”) occurs; then, oxidative stress and cytokineinduced liver injury are responsible for the switch from steatosis to

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Fig. 9. Effect of osthole (OS) on H2O2-induced phosphorylation of MAPK. The FL83B cells were pretreated with OA (250 mM) for 24 h and then H2O2 (250 mM) was added for 2 h. Cells were then treated with different concentrations of OS for 24 h. Total MAPK levels were used as internal controls. The densitometry values of three independent experiments were analyzed and compared with the H2O2-treated group.

steatohepatitis (“second hit”) [20]. There is evidence that the rate of de novo lipid synthesis is elevated because of unnecessary activation of lipogenic transcription factors, as an excess supply of dietary lipids was observed in insulin resistant patients with NAFLD, compared with healthy subjects [23]. Furthermore, liver lipid synthesis is positively modulated by increased activity of the transcription factors PPAR and SREBP-1c [24]. Overexpression of genes that activate lipogenic genes, such as SREBP-1, FAS, and ACC have been found in steatotic human livers and obese mice [25,26]. Osthole, a coumarin compound, was isolated from the dried fruits of Cnidium monnieri (Umbelliferae). A previous study indicated that osthole induced a significant acceleration of betaoxidation of hepatic fatty acids, via an increase in acyl-CoA oxidase and carnitine palmitoyl transferase 1a mRNA expression, in strokeprone spontaneously hypertensive rats, suggesting osthole suppressed hepatic lipid accumulation [27]. A recent study demonstrated that osthole is a dual agonist of PPAR a/g, and decreases hepatic lipid accumulation and inflammatory cytokine production [28]. Thus, osthole may increase expression of the lipolysis proteins, pHSL and ATGL, and suppress the adipogenesis transcription factors SREBP1,PPARg, and CEBPa/b, to decrease lipid accumulation. In addition, researchers indicated that AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high free fatty acids in hepatocytes [29]. In this study, we found that osthole increased SIRT1-mediated AMPK activation in a dose-dependent manner and also enhanced the basal phosphorylation of AMPK and ACC, but suppressed expression of FAS in hepatocytes with accumulated lipids caused by high free fatty acids. The present study provided direct evidence of the beneficial effects of osthole on hepatocyte lipid metabolism. It has been proposed that oxidative stress is the main instigator triggering the progression of steatosis to steatohepatitis. The “second hit” involves lipid peroxidation, pro-inflammatory cytokines, and mitochondrial dysfunction promoting hepatic injury, inflammation, and fibrosis and leading to the development and progression of NAFLD [30]. Many studies demonstrated the

association between the levels of lipid oxidation products and the pathogenesis of NAFLD and NASH. Previously studies showed that lipid accumulation was accompanied by increased generation of oxidative stress and cellular inflammatory responses [31]. Various antioxidants have been used to treat NAFLD, including radical scavenger vitamin E and b-carotene to prevent lipid peroxidation [32]. Clinical studies indicated that dietary supplementation with vitamin E significantly improved liver transaminase and serum TNF-a levels in patients with NASH [32], and recent studies demonstrated that osthole had good antioxidant activity [33]. This study found that osthole decreased the level of the lipid peroxidation product, MDA, and improved the activity of the antioxidant enzyme, SOD, which can metabolize H2O2 to nontoxic H2O. Inflammatory reactions and oxidative stress are implicated in the pathogenesis of steatosis to steatohepatitis. Studies had shown that lipid accumulation activated MAPKs and NF-kB [34]. Therefore, fatty acids are believed to agitate the progress of steatohepatitis by inducing cellular stress and organelle toxicity [35]. A study demonstrated that osthole inhibited PPAR a/g and decreased inflammatory cytokine production [28]. The reduced activity of PPARg by osthole may have advantageous effects on hepatic lipid storage and the inflammatory response; therefore, osthole may act as a PPAR agonist to reduce hepatic steatosis. Furthermore, activation of NF-kB seems to be a major determinant for disease progression from steatosis to steatohepatitis, and can trigger intercellular cascades to induce and maintain inflammation. NF-kB (is a hetero-dimer of p65 and p50) was suppressed by IkB (a hetero dimer containing IkB-a and IkB-b subunits) in the cytoplasm. The phosphorylation of IkBa leads to the activation of NF-kB, which is rapidly degraded through protease to release NFkB from the cytoplasm into the nucleus to express proinflammatory cytokines and COX-2 [7,10]. Our results indicated osthole decrease the inflammatory mediator COX-2 expression via suppression of the NF-kB pathway. In addition, activation of MAPK-related signaling may be critical step in promoting

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Fig. 10. Model explaining the mechanism of Osthole attenuates lipid accumulation, regulates the expression of inflammatory mediators, and increases antioxidantion in FL83B Cells.

progression from lipid accumulation and steatosis to inflammation. We found that osthole may decrease inflammatory responses through suppression of NF-kB and the p38 MAPK pathway in hepatic steatosis stimulated by H2O2. In this study, MTT assay showed that osthole at concentrations 100 mM did not significantly affect cell viability. Previously studies indicated that treated with osthole (100 mM) for 24 h, inhibited proliferation in lung cancer cells (A549) [36]. In addition, hepatocellular carcinoma (HCC) cell lines including human HCC cell line (SK-HP-1, SMMC-7721, HepG-2) and murine HCC cell line (Hepa1-6) were treated with osthole at different doses (0, 41.0, 84.0, 123.0, 164.0 and 205.0 mM) for 48 h. MTT assay was measured cell growth inhibition rate at 48 h after osthole treatment. Osthole inhibited the proliferation of all four HCC cell lines in a dosedependent manner. There was no significant difference in drug sensitivity (IC50: 189.5 mM, 161.9 mM, 161.4 mM and 137.0 mM, respectively) between the four HCC cell lines. And osthole was administered at the dose of 123.0 mM, showed that SMMC-7721 and Hepa1-6 cells both exhibited increased cell percentages in G2 phase [37]. In contrast, this study we used normal hepatocytes (FL83B cells) and treated with osthole 100 mM no longer than 24 h. Hence, we confirmed osthole 100 mM have no inhibitory effect in FL83B cells growth. To summarize, osthole suppressed hepatic lipid accumulation by increasing lipolysis and decreasing adipogenesis transcription factors. Osthole also increased SIRT1, which regulates hepatic lipid metabolism through activation of AMPK. During the steatosis, osthole significantly inhibited lipid peroxidation and COX-2 expression by suppressing activation of NF-kB and the p38 MAPK pathway in H2O2-stimulated FL83B cells (Fig. 10). 5. Conclusion Osthole suppressed hepatic lipid accumulation through activated AMPK pathway improved lipolysis and suppressed adipogenesis transcription factors protein expression. Simultaneously,

osthole by intervening NF-kB and p38 MAPK pathways suppressed inflammation. We believe that osthole shows promise as a natural anti-NAFLD drug to attenuate liver inflammatory disease. Author’s disclosure The authors report no declarations of interest. Acknowledgements This study was supported in part by grants from the Chang Gung Memorial Hospital (CMRPF1F0121) and Ministry of Science and Technology in Taiwan (MOST 105-2320-B-255-004-), and Chang Gung University of Science and Technology (EZRPF3F0141). References [1] R. Gupta, A. Bhangoo, N.A. Matthews, H. Anhalt, Y. Matta, B. Lamichhane, S. Narwal, G. Wetzler, S. Ten, The prevalence of non-alcoholic fatty liver disease and metabolic syndrome in obese children, J. Pediatr. Endocrinol. Metab. 24 (2011) 907–911. [2] Y.P. Hwang, H.G. Kim, J.H. Choi, M.T. Do, Y.C. Chung, T.C. Jeong, H.G. Jeong, Sallyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway, J. Nutr. Biochem. 24 (2013) 1469–1478. [3] V. Nobili, M. Manco, R. Devito, C.V. Di, D. Comparcola, M.R. Sartorelli, F. Piemonte, M. Marcellini, P. Angulo, Lifestyle intervention and antioxidant therapy in children with nonalcoholic fatty liver disease: a randomized controlled trial, Hepatology 48 (2008) 119–128. [4] G. Musso, R. Gambino, M. Cassader, Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD), Prog. Lipid Res. 48 (2009) 1–26. [5] G.R. Romeo, J. Lee, S.E. Shoelson, Metabolic syndrome insulin resistance, and roles of inflammation-mechanisms and therapeutic targets, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 1771–1776. [6] Q. Liu, S. Bengmark, S. Qu, The role of hepatic fat accumulation in pathogenesis of non-alcoholic fatty liver disease (NAFLD), Lipids Health Dis. 9 (2010) 42. [7] W.C. Huang, W.T. Chang, S.J. Wu, P.Y. Xu, N.C. Ting, C.J. Liou, Phloretin and phlorizin promote lipolysis and inhibit inflammation in mouse 3T3-L1 cells and in macrophage-adipocyte co-cultures, Mol. Nutr. Food Res. 57 (2013) 1803–1813.

W.-C. Huang et al. / Biomedicine & Pharmacotherapy 91 (2017) 78–87 [8] R. Lage, C. Dieguez, A. Vidal-Puig, M. Lopez, AMPK: a metabolic gauge regulating whole-body energy homeostasis, Trends Mol. Med. 14 (2008) 539– 549. [9] X. Hou, S. Xu, K.A. Maitland-Toolan, K. Sato, B. Jiang, Y. Ido, F. Lan, K. Walsh, M. Wierzbicki, T.J. Verbeuren, R.A. Cohen, M. Zang, SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase, J. Biol. Chem. 29 (2008) 20015–20026. [10] S.J. Wu, Osthole attenuates inflammatory responses and regulates the expression of inflammatory mediators in HepG2Cells grown in differentiated medium from 3T3-L1 preadipocytes, J. Med. Food 18 (2015) 972–979. [11] M. Zimecki, J. Artym, W. Cisowski, I. Mazol, M. Wlodarczyk, M. Glensk, Immunomodulatory and anti-inflammatory activity of selected osthole derivatives, Z. Naturforsch. C 64 (2009) 361–368. [12] S.Y. Chou, C.S. Hsu, K.T. Wang, M.C. Wang, C.C. Wang, Antitumor effects of Osthol from Cnidium monnieri: an in vitro and in vivo study, Phytother. Res. 21 (2007) 226–230. [13] D. Ding, S. Wei, Y. Song, L. Li, G. Du, H. Zhan, Y. Cao, Osthole exhibits anti-cancer property in rat glioma cells through inhibiting PI3K/Akt and MAPK signaling pathways, Cell. Physiol. Biochem. 32 (2013) 1751–1760. [14] X. Xu, Y. Zhang, D. Qu, T. Jiang, S. Li, Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3 K/Akt pathway, J. Exp. Clin. Cancer Res. 29 (2011) 33 30. [15] V.C. Lin, C.H. Chou, Y.C. Lin, J.N. Lin, C.C. Yu, C.H. Tang, H.Y. Lin, T.D. Way, Osthole suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt/mTOR pathway, J. Agric. Food Chem. 58 (2010) 4786–4793. [16] H.H. Nam, D.W. Jun, H.J. Jeon, J.S. Lee, W.K. Saeed, E.K. Kim, Osthol attenuates hepatic steatosis via decreased triglyceride synthesis not by insulin resistance, World J. Gastroenterol. 20 (2014) 11753–11761. [17] F. Sun, M.L. Xie, L.J. Zhu, J. Xue, Z.L. Gu, Inhibitory effect of osthole on alcoholinduced fatty liver in mice, Dig. Liver Dis. 41 (2009) 127–133. [18] W.C. Huang, S.J. Wu, Y.L. Laic, C.J. Liou, Phloretin inhibits interleukin-1binduced COX-2 and ICAM-1 expression through inhibition of MAPK, Akt, and NF-kB signaling in human lung epithelial cells, Food Funct. 10 (2015) 1960– 1967. [19] Y.J. Hwang, H.R. Wi, H.R. Kim, K.W. Park, K.A. Hwang, Pinus densiflora Sieb. et Zucc. alleviates lipogenesis and oxidative stress during oleic acid-induced steatosis in HepG2 cells, Nutrients 23 (2014) 2956–2972. [20] Z. Lu, J.T. Wai, J.Y. Jin, J.Z. Bei, Signal transductions and nonalcoholic fatty liver: a mini-review, Int. J. Clin. Exp. Med. 7 (2014) 1624–1631. [21] S.M. Abd El-Kader, E.M. El-Den Ashmawy, Non-alcoholic fatty liver disease: the diagnosis and management, World J. Hepatol. 7 (2015) 846–858. [22] C. Garcia-Ruiz, J.C. Fernandez-Checa, Mitochondrial glutathione: hepatocellular survival-death switch, J. Gastroenterol. Hepatol. 21 (2006) S3–S6. [23] K.L. Donnelly, C.I. Smith, S.J. Schwarzenberg, J. Jessurun, M.D. Boldt, E.J. Parks, Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease, J. Clin. Invest. 115 (2005) 1343–1351.

87

[24] G.Y. Lee, H. Jang, J.H. Lee, J.Y. Huh, S. Choi, J. Chung, J.B. Kim, PIASy-mediated sumoylation of SREBP1c regulates hepatic lipid metabolism upon fasting signaling, Mol. Cell. Biol. 34 (2014) 926–938. [25] M. Bell, H. Wang, H. Chen, J.C. McLenithan, D.W. Gong, R.Z. Yang, D. Yu, S.K. Fried, M.J. Quon, C. Londos, C. Sztalryd, Consequences of lipid droplet coat proteins downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance, Diabetes 57 (2008) 2037–2045. [26] B.K. Straub, P. Stoeffel, H. Heid, R. Zimbelmann, P. Schirmacher, Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis, Hepatology 47 (2008) 1936–1946. [27] H. Ogawa, N. Sasai, T. Kamisako, K. Baba, Effects of osthol on blood pressure and lipid metabolism in stroke-prone spontaneously hypertensive rats, J. Ethnopharmacol. 30 (2007) 26–31. [28] X. Zhao, J. Xue, X.L. Wang, Y. Zhang, M. Deng, M.L. Xie, Involvement of hepatic peroxisome proliferator-activated receptor a/g in the therapeutic effect of osthole on high-fat and high-sucrose-induced steatohepatitis in rats, Int. Immunopharmacol. 22 (2014) 176–181. [29] M. Boutant, C. Cantó, SIRT1 metabolic actions: integrating recent advances from mouse models, Mol. Metab. 3 (2014) 5–18. [30] S. Stojsavljevi c, M. Gomer9 ci c Pal9ci c, L. Virovi c Juki c, L. Smir9 ci c Duvnjak, M. Duvnjak, Adipokines and proinflammatory cytokines, the key mediators in the pathogenesis of nonalcoholic fatty liver disease, World J. Gastroenterol. 20 (2014) 18070–18091. [31] Y. Sumida, E. Niki, Y. Naito, T. Yoshikawa, Involvement of free radicals and oxidative stress in NAFLD/NASH, Free Radic. Res. 47 (2013) 869–880. [32] C.A. Loguercio, M. Federico, C. Trappoliere, S.I. de Tuccillo, L.A. Di, M. Niosi, M.V. D’Auria, R. Capasso, C. Del Vecchio Blanco, The effect of a silybin vitamin Ephospholipid complex on nonalcoholic fatty liver disease: a pilot study, Dig. Dis. Sci. 52 (2007) 2387–2395. [33] S.M. Yang, Y.L. Chan, K.F. Hua, J.M. Chang, H.L. Chen, Y.J. Tsai, Y.J. Hsu, L.K. Chao, F.L. Yang, Y.L. Tsai, S.H. Wu, Y.F. Wang, C.L. Tsai, A. Chen, S.M. Ka, Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-kB-mediated COX-2 expression and apoptosis, Free Radic. Biol. Med. 73 (2014) 260–269. [34] J.K. Reddy, M.S. Rao, Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation, Am. J. Physiol. Gastrointest. Liver Physiol. 290 (2006) G852–G858. [35] A.M. Diehl, Lessons from animal models of NASH, Hepatol. Res. 33 (2005) 138– 144. [36] X.M. Xu, M.L. Zhang, Y. Zhang, L. Zhao, Osthole induces lung cancer cell apoptosis through inhibition of inhibitor of apoptosis family proteins, Oncol. Lett. 12 (2016) 3779–3784. [37] L. Zhang, G. Jiang, F. Yao, Y. He, G. Liang, Y. Zhang, B. Hu, Y. Wu, Y. Li, H. Liu, Growth inhibition and apoptosis induced by osthole, a natural coumarin, in hepatocellular carcinoma, PLoS One 7 (5) (2012) e37865.