Synergistic inhibitory effects of acacetin and eleven other flavonoids

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Bioactive Constituents, Metabolites, and Functions

Synergistic inhibitory effects of acacetin and eleven other flavonoids isolated from Artemisia sacrorum on lipid accumulation in 3T3-L1 cells Qianqian Ma, Yunlong Cui, Siyuan Xu, Yiyao Zhao, Haidan Yuan, and Guangchun Piao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04683 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Journal of Agricultural and Food Chemistry

Synergistic inhibitory effects of acacetin and eleven other flavonoids isolated from Artemisia sacrorum on lipid accumulation in 3T3-L1 cells Qianqian Ma,1 Yunlong Cui,1 Siyuan Xu,1Yiyao, Zhao,1 Haidan Yuan,1,2,* and Guangchun Piao 1, 2,*

College of Pharmacy, Yanbian University; Yanji, Jilin,133002, China.

1

Key Laboratory of Natural Resources of Changbai Mountain & Functional

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Molecules (Yanbian University), Ministry of Education, China. *Corresponding

authors:

Guangchun

Piao,

Tel:

86-433-2436008;

Fax:

86-433-2435026; E-mail: [email protected]; Haidan Yuan, Tel: 86-433-2435062; Fax: 86-433-2435026; E-mail: [email protected]

1

ACS Paragon Plus Environment


1 2

ABSTRACT: Artemisia sacrorum Ledeb., a Compositae forage plant in China, has

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been found to have an inhibitory effect on lipid accumulation. We selected twelve

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flavonoids, which we had isolated from Artemisia sacrorum and had the potential to

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inhibit lipid accumulation in the literature or in our preliminary experiments, and

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grouped them into eleven compound combinations; we investigated their synergistic

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inhibitory effects on lipid accumulation in 3T3-L1 cells. In screening experiments,

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Oil-Red O staining, triglyceride levels, and lipid accumulation levels all indicated that

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combined acacetin and apigenin displayed a significant synergistic inhibitory effect

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and the best repeatability. Subsequent research showed that this combination could

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synergistically promote the phosphorylations of AMPK and ACC. Furthermore, to a

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different extent, that combination had significant synergistic inhibitory effects on

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various genes or proteins related to adipogenesis and lipogenesis. Thus, that

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combination could significantly reduce triglyceride levels and lipid accumulation

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compared with acacetin or apigenin acting alone.

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KEY WORDS: Artemisia sacrorum Ledeb., AMP-activated protein kinase, lipid

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accumulation, acacetin, apigenin, synergistic effect

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INTRODUCTION

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Artemisia sacrorum Ledeb. is a semi-shrub herbaceous plant that grows on hillsides

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and in thickets, forests, and grasslands at middle and low elevations. In plant

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communities in local areas on sunny mountain slopes, it is often the dominant species

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or main companion species.1 In the Yili area (Xinjiang, China), the protein and crude

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fat contents in Compositae forage plants, such as Artemisia sacrorum, are higher than

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in Gramineae. Use of Compositae forage plants is seasonal, and they are mainly eaten

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by young animals; the plants are most commonly consumed in early spring, autumn,

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and winter.2 Furthermore, in the Yanbian area (Jilin, China) and in Korea, Artemisia

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sacrorum has a long history of folk application for the prevention and treatment of

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various liver diseases. In recent years, we have demonstrated experimentally that

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Artemisia sacrorum can inhibit lipid accumulation and adipocyte differentiation by

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activating AMP-activated protein kinase (AMPK) in 3T3-L1 cells and HepG2 cells.3-5

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We are also conducting research into the use of Artemisia sacrorum in treating

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alcoholic fatty liver (Figure 1).

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Excessive lipid accumulation is associated with many health problems, such as

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obesity, fatty liver diseases, hyperlipidemia, hypertension, and diabetes mellitus.6-8

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Lipid accumulation, such as obesity, is closely related to lipid metabolism, such as

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adipogenesis and lipogenesis. AMPK adjusts the metabolism of fats and

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carbohydrates to regulate cellular energy homeostasis.9 Upon activation, AMPK

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inhibits the expressions of Sterol regulatory element binding protein (SREBP1c),

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CCAAT/enhancer-binding protein

(C/EBPα),

peroxisome proliferator-activated

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receptor γ (PPARγ), and their downstream genes, and it thereby suppresses

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adipogenesis.10 SREBP1c is one of the transcription factors involved in adipogenesis

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and lipogenesis; it activates genes and enzymes, such as fatty acid synthase (FAS),

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stearoyl-CoA desaturase-1 (SCD1), which are related to adipocyte maturation and

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biosynthesis of triglycerides and fatty acids; they thus regulate lipogenesis

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preferentially.9 At the same time, it is important to note that mechanisms of adipocyte

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differentiation have been extensively studied using in vitro systems in which 3T3-L1

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is one of the most frequently employed cell lines. 3T3-L1 cells were clonally isolated

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from Swiss 3T3 cells derived from disaggregated mouse embryos for the past 40

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years.11

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In our previous experiment, by using different chromatographic methods (such as a

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silica gel column, macroporous adsorbent resin, ODS, Sephadex, and Prep-HPLC),

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we isolated over fifty compounds from Artemisia sacrorum and elucidated their

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structures by their physical and chemical properties and nuclear magnetic resonance

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(NMR) techniques. Among them, more than twenty were flavonoids some of which

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had inhibitory effects on lipid accumulation as reported in the literature.12-15 For

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example, luteolin treatment was found to inhibit adipogenic, lipogenic, and GLUT4

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genes and thereby reduced the levels of triglyceride in 3T3-L1 cells during

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adipogenesis.12 In another study, quercetin was observed to suppress lipid

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accumulation in 3T3-L1 cell, zebrafish, and mouse models by inhibiting the

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expressions of lipogenic and adipogenic cytokines.13 At the same time, some of these

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flavonoids had inhibitory effects on lipid accumulation in our preliminary

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experiments. So there were twelve compounds that had the potential to inhibit lipid

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accumulation in the literature or in our previous experiments. Especially, acacetin had

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strong activity and good reproducibility. Many monomer compounds exist in

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traditional medicinal plants or prescriptions, and they form the material basis for drug

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effectiveness. In many cases, there may be synergy between two or more monomers

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in effecting a cure or reducing toxicity.16 For example, both in vivo and in vitro,

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co-treatment of the monomer compound choline and ferulic acid from the traditional

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herb Rhizoma coptidis demonstrated less toxicity and stronger anti-hyperglycemic

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effects synergistically than the original herbal extract.17 Furthermore, in the treatment

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of acute promyelocytic leukemia, indirubin, tetraarsenic tetrasulfide and tanshinone

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IIA from the Realgar-Indigo naturalis formula demonstrated a synergistic effect in

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vitro and in vivo models.18 Various constituents in a herb or formula may improve the

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bioavailability, or they may act differently on target genes, proteins and channels to

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effect a cure or reduce toxicity so as to produce synergistic effects.16

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So we wonder if acacetin and eleven other flavonoids have synergistic inhibitory

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effects on lipid accumulation. If so, how does it relate to the protein and gene

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expressions mentioned above? In the present study, we aimed to identify if there are

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any synergistic effects in inhibiting lipid accumulation between acacetin and each of

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eleven other flavonoids isolated from Artemisia sacrorum.

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MATERIALS AND METHODS

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Chemicals and Reagents. Twelve compounds: jaceosidin (compound 1),

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kaempferol (compound 2), chrysoeriol (compound 3), quercetin (compound 4),

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apigenin (compound 5), hispidulin (compound 6), luteolin (compound 7), quercitrin

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(compound 8), rutin (compound 9), isorhamnetin (compound 10), genkwanin

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(compound 11), acacetin (compound 12), were all isolated from Artemisia sacrorum

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in our previous study (Figure 1). The quantities of these compounds ranged from

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several milligrams to dozens of milligrams. After multiple purification, the purity of

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each of these compounds was more than 98% respectively. 3T3-L1 cells were

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purchased from ATCC (Manassas, VA). Dulbecco's modified Eagle's medium

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(DMEM),

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penicillin-streptomycin were purchased from Gibco by Life Technologies (Grand

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Island, NY). TG assay Kit was purchased from Nanjing Jiancheng Bioengineering

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Institute, China. Protein extraction, EASY BLUE total RNA extraction, and

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ECL-reagent kits were from Intron Biotechnology Inc (Beverly, MA, USA). Bio-Rad

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protein assay kit was from Bio-Rad Laboratories (Hercules, CA, USA). Acetyl-CoA

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carboxylase (ACC), phospho-ACC (pACC), AMPK, and phospho-AMPK (pAMPK)

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antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-actin was

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from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Bovine

Serum

(FCS),

Fetal

Bovine

Serum

(FBS)

and

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Cell culture and cytotoxicity assay. 3T3-L1 cells were cultured in DMEM

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containing 10% fetal calf serum (FCS), 100 unit/mL penicillin and 100 μg/mL

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streptomycin at 37 ℃ in an atmosphere of 5% CO2. For cytotoxicity assay, 3×104

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3T3-L1 cells per well were cultured in 96-well plates and treated with twelve

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compounds at the concentrations of 0, 10, 20, 40 or 80 µM for 96 h, respectively. The

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cytotoxicities of these compounds were determined by the MTT assay.19

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Adipocyte differentiation and treatments. 3T3-L1 cells were divided into normal

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control (CON) group, differentiated control treated with differentiation medium (DM)

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group, differentiated positive control treated with differentiation medium plus

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pioglitazone (PIO) group, and twelve individual treatment groups treated with twelve

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compounds alone respectively, and eleven combined treatment groups treated with

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combinations of compound 12 plus compound X (X=1-11) respectively. Individual

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treatment groups were treated with 20 µM of compounds 2, 3, 5-12, respectively; 10

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µM of compounds 1, 4, respectively. Combined treatment groups were treated with

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combinations of compound 12 (10 µM) plus compounds 2, 3, 5-11 (10 µM)

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respectively, and combinations of compound 12 (10 µM) plus compounds 1, 4 (5 µM)

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respectively.

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5×105 3T3-L1 cells per well were cultured in 6-well culture plates. When the cells

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were incubated until confluence (day 0), they were exposed to differentiation medium

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I (DM)(DMEM, 5% FBS, 10 µg/mL insulin, 1 mM dexamethasone and 0.5 mM

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3-isobutyl-1-methylxanthine) for 4 days (day 4); and then, except CON group, the

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cells were exposed to differentiation medium II (DMEM containing 5% FBS and 10

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µg/mL insulin) for 2 more days (day 6); and then, except CON group, the cells were

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exposed to differentiation medium III (DMEM containing 5% FBS) for 2 more days

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(day 8). During adipocyte differentiation, the cells were treated with corresponding

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compounds for corresponding concentrations from day 0 to day 4.

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Oil-Red O staining. The differentiated cells were mildly washed twice with

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phophate-buffered saline (PBS), and fixed with 10% formalin for 1 h. Then the cells

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were stained with Oil-Red O Solution for 2 h, after that the solution was removed.

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Next the plates were washed with water three times. The pictures were taken by an

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Olympus microscope. Then 6-well plates were treated with isopropanol and lipid

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accumulation was determined using the absorbance at 540 nm.

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Measurement of triglyceride level. To measure triglyceride (TG) content, the

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3T3-L1 cells were lysed in lysis buffer (25 mM sucrose, 20 mM Tris-HCl, 1 mM

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EDTA and 1 mM EGTA) and the lysis buffer were collected and centrifugated at

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13,000 rpm for 15 min. And the levels of TG were measured by using TG assay Kit in

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accordance with the instructions of the manufacturer. The concentration of protein

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was determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories,

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Hercules, CA, USA) in accordance with the manufacturer's instructions.

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To prove the synergy in our experiments, here we designed another validation

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experiment of TG determination in which two doses (20 μM and 40 μM) were set for

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compounds 12 and 5, respectively; in addition, when the total dose was 20 μM or 40

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μM, three combinations were set according to the dose-ratio of the two

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drugs(compound 12: compound 5=3:1, 1:1, and 1:3). Then combination index (CI)

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and drug reduction index (DRI) were calculated using CompuSyn software

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(ComboSyn Inc, Paramus, NJ, USA).

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Western blot analysis.

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The 3T3-L1 cells were washed twice with ice-cold PBS and a protein extraction

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kit(Intron biotechnology, USA) was used to extract total protein which was

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centrifuged at 13,000 rpm for 20 min to remove insoluble protein.

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The supernatant was collected from the lysates and protein concentrations were

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determined using a Bio-Rad protein assay reagent following the manufacturer's

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instructions. A Bio-Rad protein assay kit was used to measure protein concentrations

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in cell lysates. After separation with 8% SDS-polyacrylamide gel electrophoresis

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(SDS-PAGE), equal amounts of proteins (40 μg) proteins were transferred to

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polyvinylidene difluoride membranes(Millipore, Bedford, MA).

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The membranes were further incubated for 1 h with blocking solution

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(Tris-buffered saline/Tween 20 [TBST] containing 5% skin milk (wt/vol)) at room

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temperature, and then incubated with primary antibodies over night at 4°C. Then the

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membranes were washed 4 times with 0.1% TBST and incubated with secondary

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antibody for 1 h at room temperature. An enhanced chemiluminescence Western

166

blotting detection kit was used to detect the protein bands which were then exposed to

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X-ray film.

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RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR).

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An EASY-BLUE total RNA extraction kit was used to isolate the total RNA in

170

accordance with the manufacturer`s instructions. 5 μg of RNA, oligo (15) dT primers,

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and reverse transcriptase in total volume of 50 μL were used to achieve the

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First-strand cDNA synthesis. PCR reactions were done in a total volume of 20 μL

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consisted of 2 μL of cDNA product, 0.8 unit of Taq polymerase, 20 pmol of each

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primer, and 0.2 mM of each dNTP. In order to amplify the cDNA fragments, PCR was

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performed at 95 ℃ for 30 sec, followed by 50 ℃ (FAS, ACC, C/EBPα), 56 ℃

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(SCD1), 58 ℃ (SREBP1c, Actin, glycerol-3-phosphate acyltransferase (GPAT)),

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55 ℃ (PPARγ), respectively for 30 s, and 72 ℃ for 1 min. The RT-PCR products were

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electrophoresed in 1% agarose gels under 100 V and were stained with 0.5 µg/mL

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ethidium bromide. The primers were shown in Table 1.

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Statistical analysis. All data were expressed as a mean±standard error and

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differences between groups were analyzed by one- way ANOVA analysis followed by

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Student Newman Keuls. Each value was the mean of at least three separate

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experiments in each group and mean values were considered significantly different

184

when p

Synergistic inhibitory effects of acacetin and eleven other flavonoids

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