International Immunopharmacology 16 (2013) 27–34

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International Immunopharmacology

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Cucurbitacin IIa induces caspase-3-dependent apoptosis and enhancesautophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages

Jian He a,1, Yao Wang a,1, Li-hui Xu b, Jing Qiao a, Dong-yun Ouyang a, Xian-hui He a,⁎a Department of Immunobiology, Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University,Guangzhou 510630, Chinab Department of Cell Biology, College of Life Science and Technology, Jinan University,Guangzhou 510630, China

⁎ Corresponding author at: Department of Immunobioplantation and Immunology, Jinan University, 601 Hua510632, China. Tel./fax: +86 20 85220679.

E-mail address: thexh@jnu.edu.cn (X. He).1 These two authors contributed equally to this work

1567-5769/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.intimp.2013.03.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 September 2012Received in revised form 19 January 2013Accepted 13 March 2013Available online 27 March 2013

Keywords:Cucurbitacin IIaCaspase-3ApoptosisAutophagyActin cytoskeletonAnti-inflammation

Cucurbitacin IIa (CuIIa), a member of cucurbitacin family, is isolated from the root of Hemsleya amabiliswhich hasbeen used as an ancient remedy for bacillary dysentery and gastroenteritis. The anti-inflammatory properties ofCuIIa have long been recognized but the underlyingmechanism is largely unknown. In this study, we investigatedthe anti-inflammatory effect of CuIIa on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Theresults showed that CuIIa inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependentmanner.Whereas CuIIa did not cause apoptosis in unstimulatedRAW264.7 cells, it did induce a significant apopto-sis in LPS-stimulated cells, which was caspase-3-dependent and associated with downregulation of survivin.Furthermore, LPS induced autophagy in RAW 264.7 cells and this effect was further enhanced by CuIIa asevidenced by increased levels of LC3-II conjugates and formation of LC3 puncta. In addition, CuIIa disruptedactin cytoskeleton via inducing actin aggregation. However, neither the synthesis of tumor necrosis factor-α, northe activation of the mitogen-activated protein kinases and NF-κB pathways in LPS-stimulated cells wassuppressed by CuIIa treatment. Collectively, these results suggested that induction of apoptosis and enhancementof autophagy contributed to the anti-inflammatory activity of CuIIa against inflammation-related diseases.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Cucurbitacins, a large family of triterpenoid compounds isolatedfrom cucurbitaceous plants, possess a wide spectrum of pharmacologi-cal activities such as anti-cancer, anti-virus, anti-bacterium, andanti-inflammation activities [1]. A number of studies have revealedthe anti-cancer activities of several cucurbitacins including cucurbitacinB, E, I and D [2–5]. Mechanistically, these cucurbitacins have beenreported to induce cell growth arrest and apoptotic cell death throughinhibiting signal transducer and activator of transcription 3 (STAT3)phosphorylation [4,6,7]. However, although the precise mechanismfor damaging actin cytoskeleton is largely unknown, mounting evi-dence indicates that many cucurbitacins and their derivatives can dis-rupt actin cytoskeleton in a variety of cell types leading to cell cyclearrest at G2/M phase [2,3], suggesting that cucurbitacins represent agroup of novel therapeutic agents targeting actin cytoskeleton.

Apart from their anti-cancer activities, the anti-inflammatoryproperties of cucurbitacins have been revealed both in vitro and in

logy, Institute of Tissue Trans-ngpu Dadao West, Guangzhou

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rights reserved.

vivo. In the in vitro studies, cucurbitacin R reduces the proliferation ofphytohemagglutinin A-stimulated human T lymphocytes and decreasesthe production of cytokines such as interleukin (IL)-2, IL-4, IL-10, andinterferon-γ [8,9], while cucurbitacin E downregulates the level of nitricoxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macro-phages [10]. In addition, 23,24-dihydrocucurbitacin D can effectivelyblock NO production and NF-κB activation in peritoneal macrophages[11]. These anti-inflammatory activities have been confirmed inseveral in vivo studies. For instance, cucurbitacin E could markedlysuppress carrageenan-induced paw edema in rats [10], and 23,24-dihydrocucurbitacin B was able to decrease carrageenan-inducedpaw edema in mice [12]. In an adjuvant-induced arthritis model,cucurbitacin R can reduce the inflammation and bone damage inLewis rats [8]. These observations reveal the potential application ofcucurbitacins as the potent agents for anti-inflammatory therapy.

Cucurbitacin IIa (also known as 25-O-acetyl-23,24-dihydrocucurbitacin F, Fig. 1A), a member of cucurbitacin family, ispurified from the root of medicinal plant Hemsleya amabilis(Cucurbitaceae), which has been used as an ancient remedy forbacillary dysentery and gastroenteritis [13]. Like other cucurbitacins,cucurbitacin IIa (CuIIa) has been recently shown to inhibit cancercell growth both in vitro and in xenografted tumor model viadisrupting actin cytoskeleton but independent of STAT3 activity[14,15]. However, the anti-inflammatory effect of CuIIa and the under-lyingmechanism have not beenwell characterized. In this study, we ex-plored the anti-inflammatory effect of CuIIa in LPS-stimulated RAW

Fig. 1. CuIIa suppressed the proliferation and migration of RAW 264.7 macrophage cells. (A) The chemical structure of cucurbitacin IIa. (B) Cell proliferation was measured by MTSassay. Cells were incubated with the indicated doses of CuIIa for 24 h and 48 h, respectively. Values are shown as mean ± SD (n = 3). (C) Cell migration was determined by atranswell migration system. Cells were seeded in the upper chamber and treated with indicated doses of CuIIa in serum-free medium for 24 h. The lower chamber contained10% FBS complete medium. The cells on the bottom surface of the upper well were stained by crystal violet and typical images were taken under a microscope. (D) Quantitativeassessment of (C). n = 3; *, P b 0.05 vs control; **, P b 0.01 vs control.

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264.7 macrophage cell line. Our data revealed that CuIIa induced apo-ptosis and enhanced autophagy in LPS-stimulated RAW 264.7 cells,but failed to suppress the mitogen-activated protein kinases (MAPKs)and NF-κB activation and tumor necrosis factor (TNF)-α expression.These data suggest a novelmechanism for the anti-inflammation actionof CuIIa.

2. Materials and methods

2.1. Reagents and antibodies

Cucurbitacin IIa (CuIIa; purity≥98%)was obtained from the NationalInstitutes for Food and Drug Control (Beijing, China). Propidium iodide(PI), dimethyl sulfoxide (DMSO) and lipopolysaccharide (Escherichiacoli O55:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA).CuIIa was dissolved in DMSO at 20 mM, and stored at −20 °C.Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin,RNase A, L-glutamine and fetal bovine serum (FBS) were purchasedfrom Gibco/Invitrogen (Carlsbad, CA, USA). The antibodies for Westernblot and immunofluorescent staining were purchased from Cell Signal-ing Technology (Danvers, MA, USA) unless indicated otherwise.

2.2. Cell culture

The mouse macrophage cell line RAW 264.7 was obtained from theCell Bank of the Chinese Academy of Sciences (Shanghai, China),maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin,100 μg/ml streptomycin, 2 mML-glutamine at 37 °C in a humidified in-cubator of 5% CO2, and subcultured every three days.

2.3. Cell proliferation assay

Cells in log-phasewere seeded in 96-well plates (1 × 104 cells/well)for 24 h and then treated with indicated concentrations of CuIIa. Theworking concentration of DMSO did not exceed 0.2%, which had nocytotoxicity in RAW 264.7 cell line (data not shown). After 24 h and48 h, CellTiter96 AQueous One Solution Cell Proliferation Assay (MTS)kit (Promega, Madison, WI, USA) was used to determine cell viabilityas previously described [2]. In brief, 20 μl of MTS reagent was addedto each well and the plates were incubated for 2–4 h at 37 °C. The ab-sorbance at 490 nm was measured by a microplate reader (Model680, Bio-Rad; Hercules, CA, USA), and the 50% inhibition concentration(IC50) was determined from the dose–response curve.

2.4. Transwell migration assay

Cells were seeded in transwell with 8 μm porous membrane(Corning, NY, USA). 2 × 104 cells were cultured in the upper chamberin serum-free medium with different concentrations of CuIIa (2.5, 10and 40 μM), while complete medium containing 10% FBS was used inlower chamber. After cultured for 24 h, cells on the top surfaces ofupper chamber were wiped with a cotton swab and the bottomsurfaces were stained with 0.1% crystal violet in 20% methanolfor 20 min. Stained cells were photographed under a microscope(Nikon; Tokyo, Japan).

2.5. Cell cycle analysis

Cell cycle distribution was analyzed as previously described [2]. Inbrief, the cells were fixed and stained with phosphate-buffered saline(PBS) containing 50 μg/ml propidium iodide and 30 μg/ml of RNase A.

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DNA content data were acquired using CELLQuest software on a flowcytometer (FACSCalibur; Becton Dickinson, Mountain View, CA, USA).Data of at least 20,000 events were collected for each sample.

2.6. Intracellular cytokine staining

Cells were incubated in the presence or absence of CuIIa at 37 °Cfor 1 h followed by treatment with LPS (1 μg/ml) and monensin(2 μM) for another 4 h. Then cells were collected and fixed in 4%paraformaldehyde at 4 °C for 20 min. Subsequently, cells werepermeabilized in 0.1% saponin in PBS-F (PBS containing 0.1% NaN3

and 2% FBS) for 10 min and stained with PE-conjugated monoclonalanti-TNF-α (eBioscience; San Jose, CA, USA) in the dark at 4 °C. Thedata were acquired using CELLQuest software on a flow cytometer(FACSCalibur).

2.7. Immunofluorescence microscopy

RAW 264.7 cells were seeded in glass-bottom cell culture dishesand treated with CuIIa (10 and 40 μM) and then stimulated withLPS (1 μg/ml) for 24 h. Cells were then fixed in 4% paraformaldehydeprepared in PBS, permeabilized with ice-cold 100% methanol, andimmunostained with mouse anti-β-actin and rabbit anti-LC3Bantibodies (Cell Signaling Technologies, Danvers, MA), followed byCF488-conjugated goat-anti-mouse IgG or CF568-conjugated goat-anti-rabbit IgG, highly cross-absorbed (Biotium, Hayward, CA). Nucleiwere revealed by Hoechest33342 staining. Fluorescence images werecollected under a Leica DMIRB fluorescent microscope (LeicaMicrosystems, Wetzlar, Germany).

2.8. Extraction of soluble actin and insoluble actin filament

Cells were washed twice with cold (4 °C) PBS after exposure toCuIIa or vehicle (control), and the cells were kept on ice. G-actin(soluble) was extracted with soluble actin extraction solution (50 mMTris–HCl, pH 7.4, 300 mM sucrose, 25 mM NaCl, 2 mM ethylene glycoltetraacetic acid, 5 mM MgCl2, 0.2% Triton X-100, 25 mM sodiumfluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride and protease inhibitor cocktail from Roche (Mannheim,Germany). The residual pellets (containing F-actin) were further lysedwith 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) loading buffer containing 200 mM dithiothreitol.

Fig. 2. Effect of CuIIa on TNF-α expression in LPS-activated RAW 264.7 cells. Cells werepretreated with CuIIa (2.5, 10 and 40 μM) for 1 h followed by exposure to LPS (1 μg/ml)stimulation for 4 h. TNF-αwas determined by intracellular cytokine staining as describedin theMaterials andmethods. Data are shown asmean ± SD (n = 3). **, P b 0.01 vs con-trol (untreated cells).

2.9. Western blot analysis

Cells were treated with CuIIa in the presence or absence of LPS for6 h and 24 h. The cells were washed with ice-cold PBS and then lysedin 2× SDS-PAGE loading buffer. Protein concentration was measuredby SDS-PAGE with a known sample determined by a BCA proteinassay kit (Pierce, Rockford, IL, USA) according to the manufacturer'sinstructions. Samples were separated on 10%–15% SDS-PAGE. Theproteins were then transferred by electro-transfer to polyvinylidenedifluoride (PVDF) membranes (Hybond-P; GE Healthcare Life Sci-ences, Piscataway, NJ, USA) for western blot analyses. After blockedin blocking buffer (50 mM Tris-buffered saline (pH 7.4) containing5% non-fat milk and 0.1% Tween-20) for 1 h, membranes were incu-bated overnight with antibodies. After incubated in horseradishperoxidase (HRP)-conjugated-secondary antibody for 1 h, membranewas incubated with enhanced chemiluminescence kit (BeyoECL Plus;Beyotime) for 2 min and then exposed to X-ray films (Kodak;Xiamen, China). Data were quantified using FluorChem 8000(AlphaInnotech; San Leandro, Calif., USA).

Fig. 3. Effect of CuIIa on the phosphorylation of MAPKs and NF-κB in LPS-activatedRAW 264.7 cells. Cells were pretreated with CuIIa (2.5, 10 and 40 μM) for 1 h, then ex-posed to LPS (1 μg/ml) for 1 h (A) or 4 h (B). Western blot analysis was adopted to an-alyze the activation of MAPKs and NF-κB pathways. β-Tubulin was used as a loadingcontrol.

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2.10. Statistical analysis

Unless otherwise indicated, all experiments were performedin triplicate, with one representative experiment shown. Dataare expressed as mean ± SD. Statistical analysis was performedusing Graphpad Prism 4.0 (GraphPad Software Inc., San Diego, CA).One-way ANOVA, followed by Newman–Keuls's multiple compari-son tests, was used to analyze the statistical significance amongmul-tiple groups. P values b 0.05 were considered statistically significant.

3. Results

3.1. CuIIa inhibited cell proliferation and migration of RAW 264.7 cells

We initially explored the effects of CuIIa on the proliferation andmi-gration of RAW 264.7 cells. Cell proliferation assays showed that CuIIadose-dependently inhibited the proliferation of RAW264.7 cells as com-pared with untreated (control) cells (Fig. 1B). The IC50 values were122.32 μMand 6.42 μM for 24 h and 48 h, respectively. A transwell sys-temwas used to evaluate themigration ability of RAW264.7 cells in thepresence or absence of CuIIa. As shown in Fig. 1C and D, CuIIa markedlyinhibited cell migration in a dose-dependent manner.

3.2. CuIIa did not inhibit LPS-induced TNF-α expression in RAW 264.7cells

Next, we used LPS-activated RAW 264.7 cells as an in vitro modelof inflammatory response to evaluate the effect of CuIIa on cytokinesynthesis. The expression of TNF-α was assayed by intracellular cyto-kine staining method. As shown in Fig. 2, in LPS-activated cells, theproportion of TNF-α-positive cells was markedly increased to37.52% as compared with control or CuIIa-treated cells. However,CuIIa treatment had no significant effect on LPS-induced expressionof TNF-α. This result suggested that CuIIa did not inhibit the acute re-sponse of RAW 264.7 cells to LPS stimulation.

3.3. Phosphorylation of MAPKs and NF-κB in LPS-activated RAW 264.7cells was not inhibited by CuIIa

It has been reported that MAPKs play a critical role in inflammationvia activation of downstream nuclear factor kappa B (NF-κB) pathwayand production of pro-inflammatory cytokines such as TNF-α [16]. Toinvestigate whether CuIIa affected MAPKs phosphorylation, weanalyzed the levels of phosphorylated p38, ERK1/2 and JNK inLPS-activated RAW 264.7 cells. As compared with control, LPS stimula-tion markedly increased p38 and ERK1/2 phosphorylation but onlyslightly increased JNK phosphorylation levels (Fig. 3A). Unexpectedly,CuIIa did not suppress LPS-induced phosphorylation of MAPKs butslightly increased their phosphorylation levels at high doses (Fig. 3A).

NF-κB is a crucial transcription factor that activates the expressionof many genes involved in inflammation response, such as cytokinesIL-6 and TNF-α. In resting macrophages, NF-κB is associated withthe inhibitor of NF-κB proteins (IκB) in the cytoplasm. When cellsare activated by extracellular signals such as LPS, IκB will be phos-phorylated by upstream kinases and then be degraded [17]. To exam-ine the effect of CuIIa on NF-κB signaling pathway in activated RAW264.7 cells, the phosphorylation levels of IκB and RelA/p65, a criticalcomponent of the NF-κB complex, were detected by western blotanalysis. As shown in Fig. 3B, there was no significant difference in

Fig. 4. Induction of apoptosis by CuIIa in LPS-stimulated RAW 264.7 cells. Cells were treated(A) Phase-contrast images showing the morphological changes induced by LPS or CuIIa plusthe presence or absence of LPS for 24 h. (B) Flow cytometry plots showing cell cycle distripeak; M2: G0/G1 phase; M3: S phase; M4: G2/M phase. (C) Quantitative analysis of sub##, P b 0.01 vs LPS. (D) Western blot analysis of cleaved caspase-3 and survivin. β-Tubuliand survivin in (D). Data are shown as mean ± SD (n = 3). *, P b 0.05 vs control; **, P b

point, respectively.

the phosphorylation levels of IκBα and p65 between LPS alone andCuIIa plus LPS groups. These results were consistent with the data ofTNF-α expression andMAPK phosphorylation mentioned above, indi-cating that CuIIa did not suppress the acute response of RAW 264.7cells in response to LPS stimulation and that other mechanism hadbeen involved in its anti-inflammation ability.

3.4. CuIIa induced apoptosis in LPS-stimulated RAW 264.7 cells

We next tested whether CuIIa could induce apoptotic cell death inthe LPS-activated cells since apoptosis plays a critical role in dampen-ing inflammation. Morphological observation showed that whileunstimulated cells displayed epithelioid appearance, LPS-stimulatedcells became arborized morphology (Fig. 4A), a typical characteristicof macrophage activation. CuIIa significantly reduced the proportionof cells with arborization and even caused cell death as evidencedby cell fragmentation. Flow cytometry was then used to analyzeCuIIa-induced apoptotic cell death in LPS-stimulated RAW 264.7cells. LPS treatment only slightly increased the proportion of apopto-tic cells (sub-G0/G1 peak) from 0.62% (control) to 6.52%, whereasCuIIa pretreatment significantly elevated the proportions of apoptoticcells in LPS-stimulated groups up to 17.55%, 24.64% and 33.28% for2.5, 10 and 40 μM of CuIIa, respectively (Fig. 4B and C). However,CuIIa alone did not increase the proportions of cells in sub-G0/G1

peaks. In addition, although LPS alone had little effect on cell cycledistribution, CuIIa induced a significant cell cycle arrest at G2/Mphase in both LPS-stimulated cells and unstimulated cells (Fig. 4B).

To further confirm that CuIIa induced apoptosis in theLPS-activated cells, we analyzed the levels of cleaved caspase-3 andsurvivin by Western blotting. Caspases play a critical role in the pro-cess apoptosis. Among them, caspase-3 is frequently activated to be-come a death protease, catalyzing the specific cleavage of many keycellular proteins [18,19]. As shown in Fig. 4D, CuIIa treatment led toa significant increase in the level of cleaved caspase-3 in theLPS-stimulated cells as compared with untreated (control) or LPSalone group. A much higher level of cleaved caspase-3 was observedat 24 h in the cells treated with LPS plus CuIIa as compared withCuIIa alone. This result demonstrated that CuIIa induced apoptosisin LPS-activated RAW 264.7 cells in a caspase-3-dependent manner.

As CuIIa induced significantly cell cycle arrest (Fig. 4B), we rea-soned that cell cycle-related proteins might have been involved inCuIIa-induced cell apoptosis. Survivin, a member of inhibitor of apo-ptosis (IAP) protein family, participates in the regulation of cell divi-sion and apoptosis through inhibiting the activity of caspase-3[20–22]. As shown in Fig. 4D, survivin had only slightly changed at6 h. However, at 24 h, LPS plus CuIIa (2.5 and 10 μM) dramaticallydecreased the expression of survivin when compared with CuIIaalone (Fig. 4D), which was in agreement with the elevatedcaspase-3 activation. In line with its induction of potent cell cycle ar-rest (Fig. 2B), 40 μM of CuIIa alone also profoundly decreased survivinexpression. Together, these results indicated that the increasedcleaved caspase-3 and decreased survivin level might have contribut-ed to CuIIa-induced apoptosis in LPS-activated RAW 264.7 cells.

3.5. CuIIa enhanced LPS-induced autophagy in RAW 264.7 cells

Autophagy plays a crucial role in removing damaged organelles orproteins for recycling their constituents upon nutrient deprivation orstress, and thus conferring protective effect for the cells under various

with indicated doses of CuIIa for 6 or 24 h in the presence or absence of LPS (1 μg/ml).LPS. Cells were seeded in 6-well plate (3 × 105/well), and then incubated with CuIIa inbution and CuIIa-induced apoptosis in LPS-stimulated RAW264.7 cells. M1: sub-G0/G1

-G0/G1 peaks in (B). Data are shown as mean ± SD (n = 3). *, P b 0.05 vs control;n was used as a loading control. (E and F) Quantitative analysis of cleaved caspase-30.01 vs LPS; #, P b 0.05 and ##, P b 0.01 vs LPS plus CuIIa of the same dose and time

Fig. 5. Western blot analysis of autophagy-related protein LC3B in LPS-stimulated RAW 264.7 cells. Cells were incubated with the indicated doses of CuIIa in the presence of LPS(1 μg/ml) or CQ (10 μM) or both LPS and CQ for 24 h. β-Tubulin was used as a loading control. (A and C) Expression of LC3B was determined by western blot analysis. (B andD) Quantification of LC3B-II levels in (A and C) (n = 3). B) *, P b 0.05 vs control; #, P b 0.05 and ##, P b 0.01 vs LPS; (#), P b 0.05 and (##), P b 0.01 vs LPS plus CuIIa of thesame dose and time point, respectively. D) *, P b 0.05, **, P b 0.01 vs control; #, P b 0.05 vs CQ.

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cellular stresses [23,24]. On induction of autophagy, the cytosolicmicrotubule-associated protein 1 light chain 3 (LC3)-I is conjugatedto phosphatidylethanolamine (PE) to form LC3-PE conjugate (alsoknown as LC3-II), which is presented on both the inner and outermembranes of autophagosomes, and the accumulation of LC3-II isthus widely used as a marker of autophagy [25]. As recent studies in-dicate that autophagy induction also plays an important role in elim-inating intracellular infection and regulating inflammation [26], weexplored whether CuIIa enhanced autophagy in macrophages underthe stimulation of LPS. Western blot analysis showed that LC3B-IIwas significantly increased in LPS-activated RAW 264.7 cells as com-pared with control at 24 h (Fig. 5A and B), indicating that LPS inducedautophagy in the cells, consistent with previous reports [27,28]. CuIIatreatment further enhanced the LPS-induced increase of LC3B-II levelin a dose-dependent manner, indicating an enhancement of autopha-gic response. Although CuIIa alone also induced an increase in LC3-IIlevels, this effect was significantly lower than that of LPS plusCuIIa-treated cells at 24 h. The enhanced autophagy by CuIIa inLPS-stimulated macrophages was confirmed by the further increaseof LC3B-II levels in the presence of chloroquine (CQ), which raisesthe pH within the lumen of lysosomes and/or autolysosomes andtherefore compromises autophagic degradation (Fig. 5C and D).Moreover, CuIIa-enhanced autophagy was also confirmed by immu-nofluorescence microscopy showing more LC3 puncta in CuIIa plusLPS group than in LPS alone group (Fig. 6). Together, these data indi-cated that CuIIa significantly enhanced autophagy in LPS-activatedcells.

3.6. CuIIa induced actin cytoskeleton disruption in RAW 264.7 cells

As one previous study has indicated that CuIIa can disrupt actincytoskeleton and induce actin aggregation in cancer cells [14], we

tested whether CuIIa had similar effect on RAW 264.7 cells. Asexpected, CuIIa severely induced actin aggregation in RAW 264.7cells either in the presence (Fig. 6) or absence of LPS (data notshown), whereas the actin cytoskeleton was normally distributed inthe cytoplasm of control or LPS-stimulated cells. Moreover, CuIIa-induced actin aggregation was confirmed by Western blotting andSDS-PAGE analysis showing reduced G-actin and increased F-actinlevels in cells exposed to CuIIa treatment (Fig. 7). These results indi-cated that CuIIa disrupted actin cytoskeleton in RAW 264.7 cells viainducing actin aggregation.

4. Discussion

As an active component of H. amabilis which has been used asan ancient remedy for bacillary dysentery and gastroenteritis,CuIIa has long been identified as an anti-inflammatory agent, al-though the underlying mechanism of action is unclear. In thisstudy, we investigated the anti-inflammatory effect of CuIIa inLPS-activated RAW 264.7 cells. Our results indicate that CuIIahad little influence on the MAPK and NF-κB activation andTNF-α expression in RAW 264.7 cells in response to LPS stimula-tion, suggesting that it did not affect these conventional inflam-matory pathways. However, it not only suppressed the cellproliferation and migration but also markedly induced apoptoticcell death and enhanced autophagy in LPS-activated cells. Theseobservations suggest a novel mechanism of the anti-inflammatory ac-tion of CuIIa against inflammation-related diseases.

Cell migration plays an important role in the recruitment of leuko-cytes including macrophages into an inflammation site to eliminatepotential infection [29]. Consistent with previous studies [14], CuIIacould significantly induce actin aggregation leading to a reduction ofG-actin pool. Thus, the normal actin dynamics required for cell

Fig. 6. Immunofluorescence microscopy analysis of LC3B and actin cytoskeleton in LPS-activated macrophages. Cells were treated with LPS (1 μg/ml) or CuIIa plus LPS for 24 h, andthen performed as described in the Materials and methods. The β-actin (green) and LC3B (red) were revealed by double-immunofluorescent staining. Nuclei (blue) were stained byHoechst33342. Scale bar, 10 μm.

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motility was impaired by CuIIa treatment. As actin dynamics betweenG-actin and F-actin is critical for cell motility [30], CuIIa efficientlysuppressed cell migration of RAW 264.7 cells towards serum-containing medium, in agreement with its actin cytoskeletondisrupting property. It is likely that the activated macrophageswould not immigrate into the inflamed tissues to promote inflamma-tory response under CuIIa treatment. This may at least partially

Fig. 7. Effect of CuIIa on actin cytoskeleton of RAW264.7 cells. (A)Westernblot analysis ofthe levels of G-actin and F-actin. Cells were treated with 20 μM CuIIa for different times,and G-actin and F-actin were sequentially extracted by actin extraction solution and 2×SDS-PAGE loading buffer. (B) SDS-PAGE stained with Coomassie brilliant blue R250.Arrow head indicates actin band (MW 43 kDa).

account for the anti-inflammatory effect of CuIIa. However, howCuIIa disrupts actin cytoskeleton needs to be further explored.

Inflammatory responses usually lead to large expansion of activatedimmune cells, which should be eliminated when inflammation hasbeen alleviated, through several negative regulationmechanisms includ-ing induction of apoptotic cell death [31]. LPS is a potent pathogen-associated molecular pattern (PAMP) that can stimulate the activationof innate immune cells via Toll-like receptor 4 (TLR)-4 on cell surface,leading to the expression of several critical pro-inflammatory cytokinessuch as TNF-α and IL-1β [32]. These pro-inflammatory cytokines are re-sponsible for many pathologic conditions associated with acute andchronic inflammation [33]. Although CuIIa did not inhibit TNF-α expres-sion, MAPK phosphorylation and NF-κB activation, it induced significantapoptosis in LPS-activated but not unstimulated RAW 264.7 cells,suggesting that it may suppress inflammation through reducing thenumber of activated inflammatory cells via inducing apoptotic celldeath. This suggested that the anti-inflammatory activity of CuIIa mightbe specific for the activated but not the resting immune cells. Studies in-dicate that apoptosis ofmacrophagesmaybe beneficial to the host duringinnate response. For example, macrophage apoptosis can reduce inflam-matory stress and avoid the establishment of chronic persistent infectionby intercellular pathogens [34]. In a pneumococcal pneumonia mousemodel, alveolar macrophage apoptosis may reduce pulmonary inflam-mation [35]. Therefore, CuIIa-induced apoptosis in LPS-activated cellsmay play a role in its anti-inflammatory activity.

In addition to apoptosis induction, CuIIa also promoted autophagy inLPS-activated cells as revealed by increased LC3-II levels and LC3 punctanumbers, the typical markers of autophagy. Enhancement of autophagy

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may downregulate inflammatory responses during immune responses[36]. Recently, autophagy has been shown to shape inflammation andparticipate in chronic inflammatory diseases and autoimmune diseases[26]. Insufficient autophagy in immune cells may result in necrosis,leading to inflammatory reactions inmacrophages [26,37]. Mechanical-ly, autophagy regulates the activation of inflammasome and reduces theexpression of the pro-inflammatory cytokines IL-1β and IL-18 [38],through yet unknown pathways. Consistent with the notion ofanti-inflammation function of autophagy, the enhancement of autoph-agy by CuIIa may mitigate the inflammatory responses in LPS-activatedmacrophages. On the other hand, CuIIa-induced apoptotic cells could beeliminated through autophagy, thus preventing secondary necrosis ofthe dying cells which may act as a potential pro-inflammation agent.In addition, autophagy has also been shown to play an important rolein combating against infections. In Drosophila, insufficient autophagyincreases the possibility of viral and bacterial infection [39,40]. In linewith this concept, it is presumed that promotion of autophagy byCuIIamay play a role in the treatment of bacillary dysentery and gastro-enteritis, which warrants further investigation.

In conclusion, although CuIIa did not suppress the pro-inflammatorycytokine TNF-α expression,MAPKphosphorylation andNF-κB activationin LPS-stimulated cells, it could effectively induce caspase-3-dependentapoptosis and enhanced autophagy in LPS-activated RAW 264.7 macro-phages as well as disrupted the actin cytoskeleton, which may at leastpartially account for its anti-inflammatory properties. These findingsprovide a novel mechanism of CuIIa in treating inflammation-relateddiseases.

Acknowledgments

This work is supported by grants from the National Natural Sci-ence Foundation of China (No. 81173604) and the Fundamental Re-search Funds for the Central Universities (No. 21612411 and No.21609403).

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