Apigenin Inhibits the Expression of IL-6, IL-8, and ICAM-1in DEHP-Stimulated Human Umbilical Vein Endothelial Cellsand In Vivo

Jia Wang,1 Yanyan Liao,1 Jianglin Fan,2 Ting Ye,1 Xia Sun,1

and Sijun Dong1,3

Abstract—Di-(2-ethylhexyl) phthalate (DEHP) in house dust is associated with asthma and allergicinflammatory symptoms in children. This study aimed to examine an inhibitory effect of a flavonoidapigenin on DEHP-stimulated inflammatory responses in human umbilical vein endothelial cells(HUVECs). We found that apigenin significantly suppressed DEHP-stimulated expression of inte-rcellular adhesion molecule-1 (ICAM-1) at the mRNA and protein levels and subsequently inhibitedthe adhesion of THP-1 monocytic cells to HUVECs. Treatment with apigenin also led to a dose-dependent inhibition of mRNA and protein expression of interleukin (IL)-6 and IL-8 in DEHP-stimulated HUVECs. Moreover, pretreatment with apigenin partially inhibited the DEHP-inducedactivation of c-Jun N-terminal kinase (JNK) but not the degradation of IκBα or the phosphorylationof extracellular-regulated kinase (ERK)1/2, indicating that the inhibitory effect of apigenin on theexpression of IL-6, IL-8, and ICAM-1 may be mediated by JNK pathway but not IκBα/nuclearfactor-κB or ERK/mitogen-activated protein kinase pathway. Furthermore, apigenin reduced therelease of IL-6, IL-8, and ICAM-1 and inhibited compound 48/80-induced systemic anaphylaxis invivo. These results suggest that apigenin can be used as a therapeutic means for the treatment ofDEHP-associated allergic disorders.

KEY WORDS: interleukin-8; interleukin-6; NF-κB; ERK1/2; JNK; intercellular adhesion molecule-1.

INTRODUCTION

Recent epidemiological studies revealed that ex-cess indoor exposure to phthalates including di-(2-ethylhexyl) phthalate (DEHP) increases the allergicrespiratory diseases such as rhinitis and asthma,especially among children and young adults [1, 2]. Asthe most worldwide plasticizer, DEHP has been usedfor various plastics such as polyvinyl chloride, building

materials, and medical devices [3]. DEHP has beenreported to potentiate the inflammatory immuneresponses through the induction of pro-inflammatorymediators such as interleukin (IL)-4, IL-6, COX-2, andTNF-α in vitro and in vivo [4, 5]. Furthermore, wedemonstrated that DEHP treatment could stimulate theproduction of intercellular adhesion molecule-1 (ICAM-1)and IL-8 in human umbilical vein endothelial cells(HUVECs) [6].

Endothelial cells represent an essential part of theimmune system for the participation in immunoregula-tory and inflammatory events [7]. Pro-inflammatorycytokines and adhesion molecules expressed by endo-thelial cells are increased in response to inflammatorystimuli thereby playing an important role in thepathogenesis of allergic inflammation [8]. Previousreports showed that the TNF-α-induced expression ofcytokines and ICAM-1 was associated with an increase

1 Key Laboratory of Urban Environment and Health, Institute of UrbanEnvironment, Chinese Academy of Sciences, Xiamen 361021( FujianProvince, People’s Republic of China

2 Department of Molecular Pathology, Interdisciplinary GraduateSchool of Medicine and Engineering, University of Yamanashi,Yamanashi 409-3898, Japan

3 To whom correspondence should be addressed at Institute of UrbanEnvironment, Chinese Academy of Sciences, Xiamen 361021, FujianProvince, People’s Republic of China. E-mail: sjdong@iue.ac.cn

0360-3997/12/0400-1466/0 # 2012 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 4, August 2012 (# 2012)DOI: 10.1007/s10753-012-9460-7

1466

in the activation of nuclear factor-κB (NF-κB) andmitogen-activated protein kinases (MAPKs), includingthe extracellular-regulated kinase 1/2 (ERK1/2), p38,and c-Jun N-terminal kinase (JNK) [9–11]. Also,cytokines including IL-8 and IL-6 can further potentiateimmune responses through the subsequent induction ofother inflammatory mediators and adhesion molecules[12, 13]. Therefore, regulation of these inflammation-related mediators is believed to be an importantmechanism for mediating allergic inflammatoryresponses.

Apigenin (4′, 5, 7-trihydroxyflavone), a nonmuta-genic edible flavonoid abundantly distributed in onions,parsley, oranges, thyme, wheat sprouts, peppermint,olives, and herbs like chamomile, was reported topossess a variety of biological activities such asantioxidant, antitumor, and anti-inflammatory activities[14, 15]. Apigenin inhibits the production of pro-inflammatory mediators such as IL-6, TNF-α, and IL-8 in several cell lines through NF-κB signaling pathway[16, 17]. Apigenin also exhibits an inhibitory effect oncytokine-induced upregulation of ICAM-1 expressionand the adhesion of THP-1 cells to sites of inflammation[18]. However, there has been no report to demonstratewhether apigenin has any anti-inflammatory effect onDEHP-stimulated HUVECs. In the present study, weinvestigated the gene and protein expression of IL-6 inDEHP-stimulated HUVECs. We then examined whetherapigenin has an inhibitory effect on DEHP-inducedupregulation of the expression of pro-inflammatorycytokines (IL-6 and IL-8) and an adhesion moleculeICAM-1 in HUVECs. Furthermore, we examinedwhether NF-κB, ERK/MAPK, and JNK signaling path-ways were involved in the mechanisms underlying thebeneficial effects of apigenin for DEHP-related allergicdisorders.

MATERIALS AND METHODS

Chemicals and Reagents

Apigenin, dimethyl sulfoxide (DMSO), compound48/80, SP600125, and Calcein-AM were purchased fromSigma-Aldrich (St. Louis, MO, USA). DEHP wassupplied from Supelco (Bellefont, PA). Antibodiesagainst human ICAM-1, IκBa, phospho-p44/42 MAPK,p44/42 MAPK, phospho-SAPK/JNK(Thr183/Tyr185),β-actin, and GAPDH were obtained from Cell SignalingTechnology (Danvers, MA, USA). Horseradish peroxi-

dase-conjugated goat anti-rabbit immunoglobulin G(IgG) was from Pierce Biotechnology (Rockford, IL,USA). Fetal bovine serum (FBS), DMEM culturemedium (high glucose), and RPMI 1640 culture mediumwere from Gibco (Grand Island, NY, USA). Trypsin–EDTAwas from KeyGEN Biotech (Nanjing, China). Forcell experiments, apigenin and DEHP were dissolved inDMSO, and the final concentration of DMSO was lessthan or equal to 0.1 % in culture medium.

Animals and Cell Culture

Female BALB/c mice (6 weeks, 18–21 g, SPF grade),were purchased from Xiamen University (Xiamen, Fujian,China) and housed eight per cage in a laminar air flowroom at 22±2 °C and 60±5 % relative humidity. The careand treatment of the mice were in accordance with theinternationally accepted principles for laboratory animaluse and care as found in Xiamen University guidelines. Acommercial diet and water were available ad libitum. Allmice were kept for at least 1 week prior to the injection ofcompound 48/80.

HUVEC (KeyGEN Biotech) were cultured inRPMI 1640 medium supplemented with 10 % FBS at37 °C in a humidified atmosphere of 5 % CO2 in air.Human monocytic leukemia THP-1 cells (KeyGENBiotech) were grown in DMEM medium (high glucose)containing 10 % FBS. Cells were grown to 80–95 %confluence in all experiments.

MTT Assay for Cell Viability

Cell viability was examined by 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyl-tetrazolium bromide (thiazolylblue tetrazolium bromide, MTT) assay (KeyGENBiotech). All procedures were performed according tothe manufacturer’s instructions. All samples were deter-mined in triplicate.

Total RNA Extraction and Real-Time RT-PCRAnalysis

Total RNA from HUVECs was isolated with a RNAisolation kit (Roche, Mannheim, Germany). RNA concen-trations were determined by spectrophotometry. cDNAwassynthesized from total RNA (1 μg) with a SYBR PremixEX Taq II kit (TakaRa, Dalian, China). Real-timequantitative PCR was carried out by using a SYBR GreenI Master Mix kit (TakaRa, Dalian, China) with an ABI7500 Real-time PCR system (Applied Biosystems, FosterCity, CA). The sequences for upstream and downstream

1467Apigenin Inhibits the Expression of IL-6, IL-8, and ICAM-1

primers were as follows: 5′-CACAGACAGCCACTCACCTC-3′ and 5′-TTTTCTGCCAGTGCCTCTTT-3′for IL-6; 5′-AGATATTGCACGGGAGAATATACAAA-3′ and 5′-TCAATTCCTG-AAATTAAAGTTCGGAT-3′for IL-8; 5′-CCGGAAGGTGTATGAACTGA-3′ and 5′-GGCAGCGTAGGGTAAGGTT-3′ for ICAM-1; and 5′-GGAGAAGGCTGGGGCTCAT-3′ and 5′-TGATGGCATGGACTGTGGTC-3′ for GAPDH. GAPDHwas usedas the normalizer. The amplification conditions were 30 s at95 °C, 40 cycles of 5 s at 95 °C, 34 s at 60 °C. All sampleswere assayed in triplicate, and each experiment wasperformed at least twice.

Western Blotting Analysis

For isolation of total cell proteins, HUVECs werelysed with ice-cold RIPA lysis buffer (Boster, Huhan,China) containing cocktails of protease and phosphataseinhibitors (Roche). Cell lysates were centrifuged at12,000×g for 15 min at 4 °C, and the supernatant wascollected. Cytoplasmic extracts were prepared by using anuclear and cytoplasmic protein extraction kit (Pierce,Rockford, IL). Protein concentrations were determinedusing bicinchoninic acid method (Pierce). Protein(30 μg) was separated by SDS-PAGE through 10 %(w/v) running gel and transferred to 0.2 μm polyvinyli-dene fluoride membranes. The membranes were imme-diately blocked with 5 % BSA in Tris-buffered saline for1 h and incubated with primary antibodies againsthuman phospho-p44/42, p44/42 MAPK, ICAM-1, IκBa,phospho-SAPK/JNK, β-actin, and GAPDH, which wereused at a dilution of 1: 2,000 in TBS-T. The proteinbands were visualized with enhanced chemilumines-cence reagents (Lulong, Xiamen, China). Densitometricanalysis was performed by using Quantity One software.

Determination of IL-8, IL-6 and ICAM-1 Amountsby Enzyme-Linked Immunosorbent Assay

Concentrations of IL-8 and IL-6 in the conditionedmedia of HUVECs were measured by using commer-cially available enzyme-linked immunosorbent assay(ELISA) kits (Dakewe, Shenzhen, China). Proteinconcentrations of IL-6, ICAM-1 (BOSTER, Wuhan,China), and IL-8 (Rapidbio, Shanghai, China) in mouseserum were measured by using commercially availableELISA kits. All procedures were performed according tothe manufacturer’s instructions. All samples were deter-mined in triplicate.

THP-1 Cell Adhesion Assay

HUVECs in a monolayer were pretreated with orwithout apigenin (20 μM) for 1 h prior to induction withDEHP (25 μM) for 4 h at 37 °C. THP-1 cells werelabeled with 4 μM Calcein-AM for 30 min and washedtwice with phosphate-buffered saline (PBS). Then, thelabeled THP-1 cells (1.5×106/mL) were added to amonolayer of HUVECs and then incubated for 1 h at37 °C. Non-adherent cells were gently washed out withPBS containing 10 % FBS. Images from random fieldswere captured using a confocal laser scanningmicroscopy (LSM710, Zeiss). The numbers of adherentTHP-1 cells were counted using an image analysissoftware (ImagePro, Media Cybernetics, USA).

Compound 48/80-Induced Systemic Anaphylaxis

As a convenient in vivomodel for the immediate-typeallergic reaction, compound 48/80-induced systemic ana-phylaxis wasmeasured as previously described [4]. Briefly,the BALB/c mice (n=8/group) were administered intra-peritoneal injections of compound 48/80 (9 mg/kg BW).The mice in the control group received PBS. Apigeninwere dissolved in corn oil and intragastrically administeredat a dose of 5–20 mg/kg BW 1 h prior to the injection ofcompound 48/80. The mice in the control group receivedcorn oil. Mortality was monitored for 1 h after theinduction of anaphylactic shock. After compound 48/80injection for 10 min, the blood was collected from theeyeball of mice and centrifuged at 3,000 rpm for 20 min.The supernatant was separated and stored at −70 °C tomeasure the levels of IL-6, IL-8, and ICAM-1.

Statistical Analysis

Statistical differences were determined by Student`sttest using GraphPad Prism Program (GraphPad, SanDiego, CA, USA). A difference of P<0.05 was consideredto be significant.

RESULTS

Apigenin Inhibits the Expression of ICAM-1in DEHP-Stimulated HUVECs

Incubation with apigenin did not affect the viabilityof HUVECs (Fig. 1a). More than 90 % of the cellssurvived after the treatment of 5, 10, or 20 μM apigeninfor 24 h, which were within therapeutic dose range invivo [14]. The effect of apigenin on DEHP-stimulated

1468 Wang, Liao, Fan, Wang, Ye, Sun, Fan, and Dong

expression of ICAM-1 mRNA and protein in HUVECwasexamined using real-time RT-PCR and Western blotting.As shown in Fig. 1b, c, pretreatment of apigenin dose-dependently inhibited the expression of ICAM-1 mRNAand protein in DEHP-stimulated HUVECs. Apigeninsignificantly suppressed DEHP-induced ICAM-1 proteinexpression by 0 % (5 μM), 54 % (10 μM, P<0.05), and100 % (20 μM, P<0.01) (Fig. 1c).

Apigenin Inhibits mRNA and Protein Expressionof IL-8 and IL-6 in DEHP-Treated HUVECs

We investigated whether DEHP could affect theexpression of IL-6 and found that HUVECs treated withDEHP resulted in time- and dose-dependent increases of theexpression of IL-6 at both mRNA and protein levels (Fig. 2a,b). IL-6 protein expression reached a maximum within48-h treatment of DEHP (25 μM). To study whetherapigenin could affect mRNA and protein expression of IL-

8 and IL-6 in DEHP-treated HEVECs, we pretreated the cellswith apigenin for 2 h prior to stimulationwithDEHP (25μM)for 48 h.We found that DEHP-stimulated mRNA and proteinexpression of IL-8 and IL-6 were dose-dependently attenu-ated when cells were pre-incubated with various concen-trations (5–20 μM) of apigenin for 2 h (Fig. 3a, b).

Apigenin Inhibits the Adhesion of THP-1 Cellsto DEHP-Treated HUVECs

To investigate the effect of apigenin on DEHP-stimulated adhesion of THP-1 cells to HUVECs,fluorescence-labeled THP-1 cells were used. HUVECstreated with DEHP (25 μM) for 4 h resulted in a 1.5-foldincrease in the adhesion of THP-1 cells to the monolayerof HUVECs (Fig. 3c). However, pretreatment ofHUVECs with apigenin (20 μM) for 1 h markedlyblocked DEHP-induced adhesion of THP-1 cells toHUVECs (P<0.05).

a

c

b

ICAM-1

GAPDH

20105--Api (μM)

++++-DEHP

Fig. 1. The effect of apigenin on cell viability and the DEHP-induced elevation of ICAM-1 mRNA and protein levels. a HUVECs were treated withindicated concentrations of apigenin for 24 h, and cell growth was analyzed by MTT. b, c HUVECs were treated with or without indicatedconcentrations of apigenin for 2 h (b)/1 h (c) prior to induction with DEHP (1 μM) for 6 h (b)/15 min (c). b Total RNA was extracted and relativeamount of ICAM-1 mRNA level was determined by quantitative real-time RT-PCR. c Cell lysates were measured by Western blot using antibodiesspecific for ICAM-1 and GAPDH (left part). Densitometric analysis from the immunoblots was shown as bar chart (right part). Data were expressedas mean±SEM of triplicate experiments. #P<0.05; ##P<0.01 compared to the vehicle-treated group; *P<0.05; **P<0.01 compared to the DEHP-treated group.

1469Apigenin Inhibits the Expression of IL-6, IL-8, and ICAM-1

Apigenin Has No Inhibitory Effect on DEHP-Activated IκB/NF-κB Pathway in HUVECs

To study whether the NF-κB pathway played a rolein DEHP-stimulated expression of cytokines and ICAM-1,the expression of IκBα in the cytosolic fraction wasexamined by Western blotting. As shown in Fig. 4a,treatment with DEHP (1 μM) for indicated times caused atime-dependent degradation of IκBα, which suggests thatthe NF-κB pathway could be activated by DEHP and maybe involved in the expression of cytokines and ICAM-1 inDEHP-treated HUVECs.

DEHP-induced degradation of IκBα was notinhibited but dose-dependently aggravated by pretreat-ment of apigenin (10 and 20 μM) (Fig. 4b), whichsuggests that the inhibitory effect of apigenin on DEHP-induced expression of cytokines and ICAM-1 was inde-pendent of the NF-κB signaling pathway. Notably,apigenin (5 μM) significantly inhibited the degradation ofIκBα in HUVECs, which is in accordance with theprevious reports showing the inhibitory effect of apigenin(1–5 and 50 μM) on IκB degradation in several cell lines[16–18]. Therefore, there might be a U-shaped dose–response in the relationship between apigenin concentra-tion and IκBα degradation.

Apigenin Inhibits DEHP-Induced Phosphorylationof JNK but Not ERK1/2

In our previous study, we found that a transientphosphorylation of ERK1/2 was stimulated after treat-ment with DEHP (1 μM) for 15 min. Here, weinvestigated whether apigenin could affect DEHP-inducedphosphorylation of ERK1/2 in HUVECs using Westernblotting. HUVECs incubated with apigenin (5–20 μM) for60 min prior to the exposure to DEHP (1 μM) for 15 minshowed no inhibitory effect on DEHP-induced phosphor-ylation of ERK1/2 (Fig. 4c). In contrast, apigeninsignificantly increased the phosphorylation of ERK1/2.These results suggest that the inhibitory effect of apigeninon DEHP-induced expression of cytokines and ICAM-1was independent of the ERK/MAPK signaling pathway.

To investigate the effect of apigenin on DEHP-induced phosphorylation of JNK, HUVECs were pre-treated with apigenin for 1 h before stimulation withDEHP for 1 h. As shown in Fig. 4d, DEHP (1 μM)significantly stimulated the phosphorylation of JNKwithin 1 h which suggested that the JNK pathway couldbe activated by DEHP. However, DEHP-stimulated JNKphosphorylation was attenuated when cells were pre-incubated with apigenin (20 μM) for 2 h (Fig. 4d),

Fig. 2. DEHP-activated mRNA and protein expression of IL-6 in HUVECs. a, b HUVECs were treated with DEHP (25 μM) for indicated times (leftpart). Cells were treated with indicated concentrations of DEHP for 48 h (right part). a The induction of IL-6 mRNA was determined by real-timeRT-PCR using total RNA isolated from DEHP-treated HUVECs. b The protein secretions of IL-6 in culture supernatants of HUVECs after DEHPexposure determined by ELISA. Data were expressed as mean±SEM of triplicate experiments. #P<0.05, ##P<0.01 compared to the vehicle-treatedgroup; *P<0.05; **P<0.01 compared to the DEHP-treated group.

1470 Wang, Liao, Fan, Wang, Ye, Sun, Fan, and Dong

indicating that the JNK pathway may be involved in theanti-inflammatory effect of apigenin in HUVECs.

Effect of Apigenin on Compound 48/80-InducedSystemic Reaction

To examine whether apigenin had a prevention effecton allergic reaction, compound 48/80 as an in vivo modelfor systemic anaphylaxis shock was employed. As shownin Table 1, the injection of compound 48/80 into the miceresulted in fatal shock in 100 % of animals. However,intragastric administration of apigenin (5–20 mg/kg BW)for 1 h prior to injection of compound 48/80 (9 mg/kg BW)completely reduced the mortality induced by compound48/80. We also examined the protein expression of IL-6,IL-8, and ICAM-1 in the blood serum of mice, which weregiven apigenin (20 mg/kg BW) 1 h before compound48/80 injection. As shown in Fig. 5, the protein expressionof IL-6, IL-8, and ICAM-1 were significantly upregulatedwithin 10-min injection of compound 48/80. However, thiseffect was inhibited when mice were given apigenin before

compound 48/80 injection. These results indicated thatapigenin had the prevention effect on compound 48/80-induced systemic reaction. To study whether JNK pathwayplayed a role in apigenin-induced downregulation ofprotein expression of inflammatory mediators, 20 mg/kgBW of SP600125 (JNK pathway inhibitor) was given tomice 1 h before compound 48/80 injection. Results showedthat pretreatment with SP600125 caused a decreasedexpression of IL-6 and IL-8 proteins, indicating that theJNK pathway may be involved in the inhibitory effect ofapigenin on the protein expression of IL-6 and IL-8 inmouse serum. However, pretreatment with SP600125showed no inhibitory effect on ICAM-1 expression, whichwas consistent with previous findings [19].

To further study the anti-inflammatory effect ofapigenin, mice were intragastrically administered withapigenin (5–20 mg/kg BW) immediately after compound48/80 (9 mg/kg BW) injection. We found that all of themice died within 30 min. However, mice administeredwith higher concentration of apigenin (20 mg/kg BW)lived much longer.

ba

Control DEHP

DEHP + Api20 Api20

c

Fig. 3. The inhibitory effect of apigenin on DEHP-activated mRNA and protein expression of IL-8 and IL-6, as well as THP-1 adhesion to HUVECs.HUVECs were pretreated with or without indicated concentrations of apigenin for 2 h (a, b)/1 h (c), followed by induction with 25 μMof DEHP for 48 h (a,b)/4 h (c). a The induction of IL-6 and IL-8 mRNAwas determined by real-time RT-PCR using total RNA isolated from DEHP-treated HUVECs. b Theprotein secretions of IL-6 and IL-8 in culture supernatants of HUVECs after DEHP exposure determined by ELISA. c Cells were co-cultured with calcein-AM-labeled THP1 monocytes for 1 h. Microphotographs were obtained using a laser confocal microscope (magnification×200). Data were expressed asmean±SEMof triplicate experiments. #P<0.05, ##P<0.01 compared to the vehicle-treated group; *P<0.05; **P<0.01 compared to the DEHP-treated group.

1471Apigenin Inhibits the Expression of IL-6, IL-8, and ICAM-1

DISCUSSION

Chinese traditional medicines derived from naturalsources including fruits and vegetables are often used forthe prevention and treatment of immune disorders andinflammation [20]. Some polyphenolic flavonoids have

long been identified as reagents with pharmacologicalactivities such as antitumor, anti-ischemic, and anti-inflammatory activities [21, 22]. In the present study, wedemonstrated for the first time that apigenin flavonoideffectively attenuated DEHP-induced allergic inflamma-tion by reduction of the expression of pro-inflammatory

15 45 60301050Time(min)

Iκ Bα (cytosolic fraction)

++++++-DEHP

beta-Actin

Iκ Bα (cytosolic fraction)

20105--Api (μ M)

++++-DEHP

beta-Actin

a

b

p-ERK1/2

t-ERK1/2

20105--Api (μ M)

++++-DEHP

c

dp-JNK

beta-Actin

20--Api (μ M)

++-DEHP

Fig. 4. The effect of apigenin on DEHP-induced degradation of IκBα and phosphorylation of ERK1/2 and JNK. Cells were pretreated with or withoutindicated concentrations of apigenin for 1 h before stimulation with DEHP (1 μM) for indicated times (a)/1 h (b, d)/15 min (c). a, b Proteins fromcytosolic fractions were prepared and subjected to SDS-PAGE. The expression of IκBα was measured by Western blotting using antibodies specificfor IκBα and beta-actin (left part). c, d Cell lysates were measured by Western blot using antibodies specific for phospho-ERK1/2 (c) and phospho-JNK (d) (left part). Densitometric analysis from the immunoblots was shown as bar chart (a–d right part). Each bar represents mean±SEM oftriplicate experiments. #P<0.05, ##P<0.01 compared to the vehicle-treated group; *P<0.05; **P<0.01 compared to the DEHP-treated group.

1472 Wang, Liao, Fan, Wang, Ye, Sun, Fan, and Dong

mediators (IL-6 and IL-8) and adhesion moleculeICAM-1 in HUVECs as well as inhibition of theadhesion of THP-1 cells to HUVECs.

There were reports suggesting that phthalatesincluding DEHP may adversely affect human immunefunctions, thereby enhancing the progress of allergicinflammatory disorders such as allergic asthma [23, 24].One possible reason is that phthalates have the potentialto increase the expression of cytokines or chemokines ininflammatory cells [25]. DEHP was reported to cause anincrease in the amount of IgG, which is associated withthe development of allergic respiratory diseases [26].

Our results here along with our previous study clearlyrevealed that the expression of both IL-6 and IL-8 couldbe increased in DEHP-treated HUVECs, which mightprovide us with a better understanding of the mecha-nisms underlying DEHP-induced allergic disorders. AsIL-6 and IL-8 play important roles in regulatinginflammatory responses and vascular inflammation, theinhibition of them is believed to be a reliable strategy forattenuating allergic diseases [27]. Kang et al. demon-strated that apigenin inhibited the release of inflamma-tory mediators (IL-6, TNF-α, IL-8, and COX-2) inhuman mast cells (HMC-1) [17]. Our results showed thatapigenin dose-dependently attenuated the expression ofIL-6 and IL-8 at mRNA and protein levels in DEHP-stimulated HUVECs.

The adhesion molecule ICAM-1 plays a central roleat the early stage of inflammatory response for facilitat-ing the adhesion and transmigration of leukocytes invascular endothelial cells [28]. Furthermore, ICAM-1 isimplicated in the pathogenesis of numerous inflamma-tory diseases such as asthma, atherosclerosis, rheumatoidarthritis, congestive heart failure, inflammatory boweldisease, and respiratory distress syndrome [18, 29, 30].Modulation of ICAM-1 secretion from endothelial cellsis considered to be an effective strategy for the inhibitionof monocyte adhesion and inflammatory responses. Itwas reported that apigenin could attenuate TNF-α-induced expression of adhesion molecules and theadhesion of monocytes to endothelial cells [18, 31].Thus, we investigated whether apigenin had any inhib-itory effect on DEHP-induced expression of ICAM-1 inHUVECs. We found for the first time that pretreatmentwith apigenin (5–20 μM) dose-dependently decreasedDEHP-stimulated expression of ICAM-1 at gene andprotein levels. Our results also showed that apigenin(20 μM) significantly blocked the adhesion of THP-1cells to HUVECs.

MAPKs signaling pathways including ERK/MAPKand JNK are involved in the expression of cytokines andICAM-1 in several cell types [10, 27, 28]. The self-perpetuated ERK1/2 signal plays an important role in thepathogenesis of asthma [32]. Therefore, we studiedwhether ERK/MAPK signaling pathway had effects onmediating the expression of IL-6, IL-8, and ICAM-1 inHUVECs. Our results showed that apigenin had noinhibitory effect on DEHP-activated phosphorylation ofERK1/2 in HUVECs, which was consistent with theprevious findings [15, 33]. These results suggest that theinhibitory effect of apigenin on DEHP-induced allergicinflammation may occur through the ERK1/2-indepen-

Table 1. The Effect of Apigenin on Compound 48/80-Induced SystemicAphylactic Reaction in Mice

Apigenin (mg/kg BW)aCompound 48/80(9 mg/kg BW)b Mortality (%)c

– − 0– + 1005 + 0*

10 + 0*

20 + 0*

* P<0.01; significantly different as compared to the group treated withcompound 48/80 only

a The groups of mice (n=8) were intragastrically administered corn oilor apigenin was given at various doses 1 h before the compound48/80 injection

b The compound 48/80 solution was intraperitoneally injected to thegroups of mice

cMortality (in percent) is presented as the number of dead mice×100/total number of experimental mice

Fig. 5. The effect of apigenin and SP600125 on compound 48/80-inducedprotein expression of IL-6, IL-8, and ICAM-1 in vivo. Mice were pretreatedwith or without apigenin (20 mg/kg BW) or SP600125 (20 mg/kg BW) for1 h before stimulation with compound 48/80 (9 mg/kg BW) for 10 min. Theprotein secretions of IL-6, IL-8, and ICAM-1 in the blood serum of micewere determined by ELISA. Each bar represents mean±SEM of triplicateexperiments. #P<0.05 compared to the vehicle-treated group; *P<0.05compared to the DEHP-treated group.

1473Apigenin Inhibits the Expression of IL-6, IL-8, and ICAM-1

dent pathway. Previous studies showed that JNK pathwaywas essential for IL-6 and IL-8 expression [27]. JNKMAPK binds to the AP-1 site on the IL-8 promoter. Shan etal. found that MAPKs (ERK/2, p38, and JNK) wereinvolved in thymic stromal lymphopoietin-induced pro-inflammatory gene expression such as IL-6 and CC/CXCchemokines in human airway smooth muscle cells [34].Therefore, we investigated whether the JNK pathway hadany effect on mediating the DEHP-induced inflammationin HUVECs. We found that the phosphorylation of JNKcould be induced by DEHP and attenuated when pretreatedwith apigenin in HUVECs, indicating that the JNKpathway may be involved in the anti-inflammatory effectof apigenin in DEHP-treated HUVECs.

The production of cytokines and adhesion mole-cules during inflammation is associated with the activa-tion of a transcription factor NF-κB [35]. In the restingstate, NF-κB is present in cytosol as an inactivecomplex; for example, NF-κB p50/p65 heterodimerassociates with the IκB family. Once activated bystimulus, such as TNF-α and high glucose, IκBs wererapidly phosphorylated by IκB kinase, leading to theubiquitination and proteasomal degradation of IκBs. Thereleased NF-κB p50/p65 heterodimer translocates fromthe cytoplasm to the nucleus and binds to the promoterregions of its target genes [36]. Here, we found thatDEHP (1 μM) caused a time-dependent degradation ofIκBα within 1 h, suggesting that the NF-κB signalingpathway can be stimulated by DEHP in HUVECs. Thesuppression of NF-κB activation is considered to be arational strategy for inhibition of inflammatoryresponses. Apigenin inactivates the NF-κB signalingpathway through inhibition of the degradation of IκBαin a variety of cell lines [17, 37]. Here, we found noinhibitory effect of apigenin (10 and 20 μM) on DEHP-induced IκBα degradation in HUVECs. This result wasconsistent with the previous findings by Tago et al.,where apigenin (20 μM) failed to inhibit TNF-α-induceddegradation of IκBs in NIH-3 T3 cells [33]. Recentstudies have demonstrated a nonclassical mechanismthat the suppression effect of apigenin on NF-κBactivation lie in the inhibition of the transcriptionalactivity of NF-κB as well as the phosphorylation of p65subunit in the absence of IκBα degradation [33, 38].Our results support this non-canonical pathway; howev-er, the precise signal transduction cascades remain notfully characterized and need to be further studied. Theseresults suggest that apigenin exerts its anti-inflammatoryeffect via a IκBα degradation-independent pathway inDEHP-stimulated HUVECs.

Apigenin exhibited an inhibitory effect on allergen-induced airway inflammation in a murine model ofasthma [14]. We further examined whether apigenin hasan antiallergic effect on compound 48/80-inducedsystemic reaction in BALB/c mice. Compound 48/80 isknown to be one of the most potent secretagogues,which increases the permeability of the lipid bilayermembrane by causing a perturbation in the membrane[4]. In this study, our results showed that apigenin hadan antiallergic property for the inhibition of compound48/80-induced systemic anaphylaxis, which indicatedthat apigenin could act on the lipid bilayer membranepreventing the stimulators-induced perturbation. Previ-ous studies have revealed that compound 48/80 couldenhance the release of histamine, IL-4, ICAM-1, and IgEin vivo [39, 40]. However, whether the compound 48/80could affect the expression of inflammatory mediators(IL-6 and IL-8) is not fully elucidated. In this study, wefound that the protein expression of IL-6, IL-8, andICAM-1 in mouse serum was stimulated by compound48/80 injection and inhibited by pretreatment withapigenin, which suggested that the systemic inflamma-tion played a role in the early stage of compound 48/80-induced systemic anaphylaxis. Our results also showedthat pretreatment of mice with SP600125 (JNK pathwayinhibitor) caused a decreased expression of IL-6 andIL-8 proteins, indicating the involvement of JNKpathway in the inhibitory effect of apigenin on the proteinexpression of inflammatory mediators in vivo. Theseresults indicate that apigenin has an antiallergic effect oncompound 48/80-induced systemic reaction in mice.

In summary, our results demonstrated that apigeninmay inhibit the allergic inflammatory responses by block-ing monocyte adhesion and reducing the production ofICAM-1, IL-6, and IL-8 in DEHP-treated HUVECs, andthis effect was partially dependent on the JNK pathway butnot ERK/MAPK or IκBα/NF-κB pathway. Moreover,apigenin reduced the release of IL-6, IL-8, and ICAM-1and inhibited compound 48/80-induced systemic anaphy-laxis in vivo. We suggest that apigenin is a potentialantiallergenic agent in treating DEHP-mediated inflamma-tory diseases. Further investigations are needed to elucidatethe precise mechanism underlying antiallergenic effects ofapigenin using animal model system.

ACKNOWLEDGMENTS

We thank Professor Ryoiti Kiyama (National Instituteof Advanced Industrial Science and Technology, Ibaraki,

1474 Wang, Liao, Fan, Wang, Ye, Sun, Fan, and Dong

Japan) for correcting the manuscript and Dr. Tingdong Yan(National University of Singapore) for technical assistance.This investigation was supported by 100 Talents Programof Chinese Academy of Sciences (KZCX2-YW-BR-18)and the Science and Technology Planning Project ofXiamen City (3502Z20112019).

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