Paeoniflorin Upregulates β-Defensin-2 Expression in HumanBronchial Epithelial Cell Through the p38 MAPK, ERK,and NF-κB Signaling Pathways

Yuying Gan,1 Xuefan Cui,1,2 Ting Ma,1 Yanliang Liu,1 Amin Li,1 and Mao Huang1

Abstract—Paeoniflorin (PF) is one of the principal components of peony, a plant widely used in tradi-tional Chinese medicine for its anti-inflammatory and immunomodulatory effects. Human β-defensin-2(hBD-2) is an antimicrobial peptide that acts as the first line of defense against bacterial, viral, and fungalinfections. This study aims to determine whether or not PF can regulate the expression of hBD-2 and itspossible molecular mechanism in human bronchial epithelial cells (HBECs). Real-time quantitativereverse transcription PCR showed that PF can enhance the mRNA expression level of hBD-2 in a co-ncentration- and time-dependent manner in HBECs. Further studies demonstrated that the mRNA andprotein expression levels of hBD-2 were attenuated by the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, the extracellular signal-regulated kinase (ERK) inhibitor PD98059, andthe nuclear factor kappa B (NF-κB) inhibitor (pyrrolidine dithiocarbamate (PDTC)). The phosphoryla-tion of p38 MAPK, ERK, and c-Jun N-terminal kinase was detected by Western blot analysis, and theNF-κB translocation of 16HBECs after PF treatment was analyzed by immunofluorescence. These re-sults support that PF upregulates hBD-2 expression in HBECs through the p38 MAPK, ERK, and NF-κB signaling pathways. These findings provide a new pharmacological mechanism of PF for the treat-ment of microbial infections by strengthening epithelial antimicrobial barriers.

KEYWORDS: paeoniflorin; human β-defensin-2; p38 MAPK; ERK; NF-κB; 16HBEC.

INTRODUCTION

The airway epithelium helps prevent the invasion ofmicrobial pathogens. It makes up the mechanical barrierand mediates the initial host immunological response.Defensins are small, cationic, and highly disulfide-bondedantimicrobial peptides. According to the spatial distribu-tion of cysteine residues and the pattern of disulfide brid-ges, the defensin family is subdivided into α-, β-, and θ-defensins [1, 2]. Human β-defensin-2 (hBD-2) was firstdiscovered from human psoriatic skin in 1997. hBD-2 ismostly expressed by epithelial cells, especially those in therespiratory tract and skin. Aside from its direct, antibiotic-like activity against bacterial, viral, and fungal infections,

hBD-2 serves as a chemoattractant for immature dendriticcells, memory T cells, and mast cells and stimulates theproduction of proinflammatory cytokines and chemokines;therefore, hBD-2 has been regarded important in bothinnate and adaptive immune responses to microbial inva-sion [3–7]. hBD-2 was the first inducible human antimi-crobial protein discovered. hBD-2 is induced by variousproinflammatory agents, such as interleukin (IL)-1β andtumor necrosis factor (TNF)-α, or by contact with bacteria,viruses, and fungi [8–11]. The mitogen-activated proteinkinase (MAPK) pathway, which includes stress-activatedp38 MAPK, extracellular signal-regulated kinase (ERK),and c-Jun N-terminal kinase (JNK), is assumed crucial tothe induction of hBD-2 expression. Both the 5″ flankingregion and intron of the hBD-2 gene contain several bind-ing sites for nuclear factor kappa B (NF-κB); NF-κB is alsoinvolved in hBD-2 expression [8, 12–14].

Paeoniflorin (PF), a monoterpene glycoside, is themain active constituent in the roots of Paeonia lactiflora,a plant that has been used in traditional Chinese medicine

Yuying Gan is the first author.

1 Department of Respiratory Medicine, The First Affiliated Hospital ofNanjing Medical University, 210029 Nanjing, China

2 To whom correspondence should be addressed at Department ofRespiratory Medicine, The First Affiliated Hospital of Nanjing MedicalUniversity, 210029 Nanjing, China. E-mail: xuefancui@163.com

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Inflammation (# 2014)DOI: 10.1007/s10753-014-9872-7

for more than 1,200 years to alleviate many inflammatorydiseases. PF possesses multiple pharmacological activities,including antioxidant, anti-inflammatory, anticancer, andimmunomodulatory effects [15, 16]. PF can protect miceagainst lethal lipopolysaccharide (LPS) challenge and canimprove survival in experimental sepsis by regulating thebalance between inflammatory and anti-inflammatory cy-tokines [17, 18]. However, the mechanisms behind theprotective effects of PF against sepsis remain unknown.Hence, we hypothesized that PF resists microbial infectionby enhancing the expression of endogenous hBD-2 tostrengthen innate and adaptive immunities. In this study,we investigated the effect of PF on hBD-2 expression inhuman bronchial epithelial cells (HBECs) and then exam-ined its possible molecular mechanism.

MATERIALS AND METHODS

Cell Culture and Reagents

The immortalized HBEC line 16HBEC was culturedin RPMI 1640 (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C under a 5 %CO2 humidified air atmosphere. PF, with a purity of >98 %as determined by HPLC, was dissolved in sterilized salinewater, LPS, SB203580, PD98059, SP600125, or pyrrol-idine dithiocarbamate (PDTC) and then in dimethyl sulf-oxide. These reagents were purchased from Sigma. Thecell line 16HBEC was purchased from the Type CultureCollection of the Chinese Academy of Sciences, Shanghai,China. Antibodies against phospho-p38 (p-p38), p38,phospho-ERK (p-ERK), ERK, phospho-JNK (p-JNK),JNK, NF-κB p65, and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) were obtained from Cell SignalingTechnology (MA, USA). BD OptEIA hBD-2 Elisa Kit IIwas purchased from BD Biosciences.

Cell Viability Assay

Cell Counting Kit (CCK)-8 detection kit (Dojindo,Japan) was used to evaluate cell viability for the determi-nation of noncytotoxic concentrations. Cells were seededat a concentration of 5×103 cells per well in 96-well plates.All experiments were conducted in triplicate. Afterreaching approximately 70 % confluence, the cells weremixed with different concentrations (μM) of PF. At 48 hafter treatment, the CCK-8 solution was applied at 10 μLper well and then incubated at 37 °C for 2 h. The

absorbance values of all wells were then measured at450 nm in a microplate reader (Bio-Rad, USA).

PF Stimulation and RNA Isolation from 16HBECs

Cells at 70% confluence were stimulated for differenttimes (0, 1, 6, 12, 24, and 48 h) and concentrations (0, 6.25,12.5, 25, 50, and 100μM) of PF and were then dissolved insterilized saline water. The LPS (5 μg/ml)group was usedas the positive control. For the inhibition of MAPK andNF-κB signaling, the cells were incubated with 25 μMSB203580, 25 μMPD98059, 30 μMSP600125, or 50 μMPDTC; dissolved in dimethyl sulfoxide; and then added0.5 h prior to stimulation. For RNA isolation, 16HBECswere washed with 0.01 mM phosphate-buffered saline(PBS) to clean the medium. TRIzol (0.5 mL) per 1×10616HBECs was then added, and the solution was collectedin 1.5-mL Eppendorf tubes. After adding 0.1 mL chloro-form, the aqueous phase was isolated using phase-locktubes (Eppendorf, Hamburg, Germany). RNAwas precip-itated with 0.4 mL of isopropyl alcohol and then washedtwice with 100 % ethanol. The concentration and purity ofRNA were measured at 260/280 nm using a NanoDropspectrophotometer.

Expressions of hBD-2 mRNA by Real-timeQuantitative Reverse Transcription PCR

The reverse transcription (RT) reaction was per-formed using the PrimeScript™ RT Master Mix (Takara)in a final volume of 20 μL containing 1 μg total RNA,4 μL 5× PrimeScript RT Master Mix (Perfect Real Time),and 20 μL RNase-free water. The RT reaction was per-formed at 37 °C for 15 min and then terminated by heatingat 85 °C for 5 s. Real-time quantitative PCR using SYBR®Green 1 was performed with StepOnePlus™ real-timePCR system. The hBD-2 primers were forward 5′-GACTCAGCTCCTGGTGAAGCTC-3′ and reverse 5′-CACCAAAAACACCTGGAAGAGG-3′. The GAPDHprimers were forward 5′-CGGAGTCAACGG ATTTGGTCGTAT-3′ and reverse 5′-AGCCTTCTCCATG GTGGTGAAGAC-3′. Real-time quantitative PCR was alsoperformed in a 20-μL reaction mixture prepared with areal-time PCR Master Mix kit containing a diluted com-plementary DNA solution, 10 μMof each primer, 10 μL ofSYBR® Premix Ex Taq (Tli RNaseH Plus; 2×), 0.4 μL ofROX Reference Dye (50×), and RNase-free water underthe following conditions: 1 cycle at 95 °C for 30 s, follow-ed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Allexperiments were conducted three times. Human GAPDH

Gan, Cui, Ma, Liu, Li, and Huang

was measured as the internal control. Data analyses wereperformed according to the 2-ΔΔCT method.

Measurement of hBD-2 Secretion by ELISA

Cell supernatants were recovered by centrifugationand assayed for the hBD-2 detection by BD OptEIAhBD-2 Elisa Kit II (BD Biosciences) according to themanufacturer’s instructions to quantify hBD-2 releasedbefore and after adding inhibitors.

Western Blot Analysis

After treatment with 100 μM PF, the cells were ana-lyzed by immunoblotting for different time periods (0, 15,30, 60, 120, and 180 min). The treated cells were washedand scraped into cold PBS. The cell pellets were resus-pended in lysis buffer and centrifuged to yield whole-celllysates. The protein (30 μg) for each sample was separatedby SDS-polyacrylamide gels with electrophoresis (Bio-Rad, Hercules, CA, USA), and the gel was transferred topolyvinylidene fluoride membranes. The membranes wereblocked with 5 % nonfat dried milk for 2 h and thenincubated overnight at 4 °C with the corresponding prima-ry antibodies, such as p-ERK (1:2,000), ERK (1:1,000), p-JNK (1:2,000), JNK (1:1,000), p-p38 MAPK (1:1,000),p38 MAPK (1:1,000), and GAPDH (1:2,500). After wash-ing, the membranes were incubated with the appropriatesecondary antibody conjugated with horseradish peroxi-dase and then developed in electrochemiluminescentWestern detection reagents (Bio-Rad, Hercules, CA,USA). The results are representative of three independentexperiments.

NF-κB Immunofluorescence

Immunofluorescence staining was conducted to de-tect NF-κB p65 subunit nuclear translocation. The cellsgrown on glass cover slips were fixed with 4 % paraformfor 10 min at room temperature (RT). After washing thricewith PBS, they were permeabilized with 0.2 % Triton X-100 for 5 min, rinsed again with PBS, and then blockedwith 4 % goat serum for 1 h. The cover slips were incu-bated overnight at 4 °C in a primary antibody solution(rabbit anti-NF-κB p65), washed and incubated with asecondary antibody (fluorescein isothiocyanate-conjugatedgoat anti-rabbit IgG) for 1 h at RT, stained with 5 ng/mL4′,6-diamidino-2-phenylindole to visualize the nucleus for5 min, and then washed repeatedly. The cover slips weremounted on microscope slides, and fluorescence was visu-alized under a microscope.

Statistical Analysis

Normally distributed data are presented as means andstandard errors of the means from at least three independentexperiments. Group differences were calculated by one-wayANOVA, with p<0.05 as the level of significance.

Fig. 1. Effect of paeoniflorin (PF) on 16HBEC viability. 16HBECs weretreated with PF at various concentrations (0, 10, 30, 100, and 300 μM) for48 h. Cell viability was analyzed by a CCK-8 assay. Data are shown asmeans ± standard deviation in five parallel experiments. *p<0.05 denotesthe comparison with the control without PF. The data are representative ofthree independent experiments.

Fig. 2. Real-time quantitative RT-PCR assay for hBD-2 mRNA stimulat-ed with paeoniflorin (PF). Cells were incubated with 0, 6.25, 12.5, 25, 50,and 100 μM PF for 12 h. The expression of hBD-2 was quantified byTaqMan real-time RT-PCR. GAPDH mRNA was used as an internalcontrol. Data are the means ± standard error of the mean (SEM) of sevenseparate experiments. The mRNA expression of hBD-2 is significantlyhigher at 50 and 100 μM PF than the control without PF. The LPS(5 μg/ml) group was used as the positive control (*p<0.05). The dataare representative of three independent experiments.

Paeoniflorin Upregulates β-Defensin-2 Expression

RESULTS

Cytotoxicity of PF on 16HBECs

We used CCK-8 detection kit to detect the cytotoxic-ity of PF by treating 16HBECs with PF at various concen-trations (0, 10, 30, 100, and 300 μM) for 48 h. The viabilityof 16HBECs was not significantly affected by 100 μM PFcompared with that of the control (Fig. 1). This resultsuggests that treatment with PF at the concentration rangeof 0 to 100 μM for 48 h elicits no cytotoxicity on16HBECs. Thus, this concentration range was applied insubsequent experiments.

PF Induces hBD-2 Gene Expression in HBECs. To initial-ly assess the time and dose dependency of PF-inducedhBD-2 gene expression, 16HBECs were treated withPF, and the induction of the hBD-2 gene wasassayed. Total RNA was isolated from the cells,and the expression level of the hBD-2 gene wasanalyzed by real-time RT-PCR. The mRNA expres-sion level of hBD-2 increased after PF treatment ina dose- and time-dependent manner. Considering

Fig. 3. Real-time quantitative RT-PCR analysis on the mRNA expressionof hBD-2 in 16HBECs after 100 μM paeoniflorin (PF) stimulation for d-ifferent times (0, 1, 6, 12, 24, and 48 h). Data are shown as means ± SEMof six separate experiments. The expression of hBD-2 mRNA is signifi-cantly higher at 12 and 24 h than the control at 0 h (*p<0.05). The data arerepresentative of three independent experiments.

Fig. 4. 16HBECs were treated with 100 μM paeoniflorin (PF) for the indicated times. At each time, whole-cell lysates were prepared and used for p-ERK,ERK, p-p38MAPK, p38MAPK, p-JNK, JNK, or GAPDHwestern with respective antibodies. Data are shown as means ± SEMof six separate experiments.The treatment with 100 μM PF triggered the phosphorylation (activation) of ERK, p38 MAPK, and JNK (*p<0.05). The data are representative of threeindependent experiments.

Gan, Cui, Ma, Liu, Li, and Huang

that the stimulation with 100 μM of PF at 12 hshowed the strongest response (Figs. 2 and 3), weselected this time–dose relationship for subsequentexperiments.

Role of MAPKs on PF-Induced hBD-2 Expression. Thefamily of MAPKs regulates hBD-2 mRNA expression byvarious inflammatory stimuli. Therefore, we determinedthe effect of PF on the activation of ERK, p38 MAPK,and JNK in 16HBECs. As shown in Fig. 4, the treatmentwith 100 μMPF triggered the phosphorylation (activation)of ERK, p38 MAPK, and JNK. The maximal activation ofERK and JNK occurred at 15 min, and that of p38 MAPKoccurred at 120 min, following the treatment with PF. PFhad almost no effect on nonphosphorylated protein kinase,indicating that PF induced the activation of preexistingERK, p38 MAPK, and JNK. We then tested the functionsof these signaling proteins in PF-induced hBD-2 mRNAand protein expression. Pretreatment with PD98059 and

SB203580 effectively inhibited PF-induced hBD-2 mRNAand protein expression, whereas pretreatment withSP600125 did not show inhibition (Figs. 5 and 6). Theseresults suggest that p38 MAPK and ERK are involved inPF-induced hBD-2 mRNA and protein expression. Thechemical inhibitors used in these experiments neither re-duced the viability of the 16HBECs nor induced the mor-phological signs of cytotoxicity (data not shown).

Function of NF-κB in PF-Induced hBD-2 Expression. NF-κB is involved in hBD-2 transcriptional activation byextracellular stimuli. We determined the effect of PF onNF-κB activation to test whether or not NF-κB mediatesPF-induced hBD-2mRNA expression in 16HBECs. In thisstudy, the activation of NF-κB was assessed by the degreeof nuclear translocation of p65 NF-κB. The marked nucle-ar translocation of p65 NF-κB occurred at 15 to 30 minafter treatment of 16HBECs with 100 μM PF (Fig. 7).Using PDTC, an inhibitor of NF-κB, we determined thefunction of NF-κB in PF-induced hBD-2 mRNA and pro-tein expression in 16HBECs (Figs. 5 and 6). These results

Fig. 5. Real-time RT-PCR assay for the upregulated expression of hBD-2mRNA treated with inhibitors for intracellular pathways. The cells weretreated with inhibitors for intracellular pathways in a series of experiments.16HBECs were preincubated with SB203580 (an inhibitor of p38 mito-gen-activated protein kinase), PD98059 (an inhibitor of ERK), PDTC (aninhibitor of nuclear factor kappa B), and SP600125 (an inhibitor of JNK)for 30 min and then treated with 100 μM paeoniflorin (PF) for 12 h. S-B203580 (an inhibitor of p38 mitogen-activated protein kinase), PD98059(an inhibitor of ERK), and PDTC (an inhibitor of nuclear factor kappa B)completely inhibited the upregulated expression of hBD-2 by PF, whereasSP600125 (an inhibitor of JNK) did not show inhibition (*p<0.05, Stu-dent’s t test). The data are representative of three independent experiments.

Fig. 6. Competitive ELISA for the quantification of hBD-2 secretion bycultured 16HBECs after paeoniflorin (PF) and inhibitor treatments. 16H-BECs were preincubated with SB203580, PD98059, PDTC, and S-P600125 for 30 min and then treated with 100 μM PF for 12 h. Serial d-ilutions of synthetic mature hBD-2 peptide were used to generate a stan-dard curve, and the hBD-2 peptide concentration of each experimental g-roup was considered. In accordance with the RT-PCR result, SB203580,PD98059, and PDTC completely inhibited the secretion of hBD-2 by PF,whereas SP600125 did not show inhibition (*p<0.05, Student’s t test).The data are representative of three independent experiments.

Paeoniflorin Upregulates β-Defensin-2 Expression

suggest that NF-κB mediates PF-induced hBD-2 expres-sion probably by upregulating hBD-2 transcription.

DISCUSSION

This study first demonstrated that PF stimulates theupregulated mRNA expression of hBD-2 in a concentra-tion- and time-dependent manner in HBECs. This findingis in contrast to that of Zhou et al. [19], who reported thatPF reduces the expression of hBD-2 in the colonic mucosaof mice with oxazolone-induced colitis. The discrep-ancy between these results may be related to differentPF doses, organ types, or in vivo and in vitro actionsof PF. hBD-2 is induced by various proinflammatoryagents or LPS. A previous study indicated that IL-17is the most potent cytokine to induce hBD-2 messagein the primary human tracheobronchial epithelial cells[20]. hBD-2, with its strong bactericidal activityagainst both Gram-negative and Gram-positive

bacteria, fungi, and enveloped viruses, has an impor-tant function in the respiratory airway defense system[1, 21]. The precise mechanism underlying the anti-microbial activity of human defensins remains un-clear. A previous finding revealed that the cationiccharacteristic and amphipathic nature of antimicrobialpeptides allow binding to and direct interaction withthe lipid bilayer of cell membrane, leading to theleakage of the internal aqueous contents of cells [22].

We further investigated the signaling mechanisms ofhBD-2 gene upregulation following PF stimulation in16HBECs. NF-κB and MAPKs are crucial pathways inthe induction of hBD-2. MAPKs are a family of key pro-teins involved in the regulation of gene expression in re-sponse to the extracellular stimulation signals and activationof a wide range of cell responses, including cell prolifera-tion, differentiation, and apoptosis [23]. MAPKs includestress-activated p38 MAPK, ERK, and JNK. SeveralMAPKs are involved in the expression of hBD-2 by variousstimuli. Madi et al. [24] reported that Pseudomonasfluorescens can induce hBD-2 production in Caco-2/TC7cells via p38 and ERK–MAPK-dependent pathways. Li et

Fig. 7. Zeiss AxioSkop fluorescence microscope (×40 objective) performed on 16HBECs shows cytoplasmic and nuclear localization of the NF-κB p65subunit. Co-staining with DAPI shows green staining of the nuclei, confirming the nuclear localization of the NF-κB p65 subunit in 16HBECs at 15 to 30minfollowing paeoniflorin treatment. The data are representative of three independent experiments.

Gan, Cui, Ma, Liu, Li, and Huang

al. [13] reported that lipopeptide LP01 from normal com-mensal Staphylococcus epidermidis increases antimicrobialpeptide hBD-2 expression via the activation of p38 MAPK.Ju et al. [10] showed that the pretreatment of B cells with aJNK inhibitor suppresses HIV-1 Tat-induced hBD-2 expres-sion. Jang et al. [25] reported that IL-1β induces HBD-2mRNA expression in A549 cells through p38 MAPK andJNK, but not ERK. In our study, PF increased the levels ofphosphorylated ERK, p38 MAPK, and JNK in 16HBEC.We examined the function of the MAPK signaling pathwayin the induction of hBD-2 expression with ERK1/2, p38,JNK, and ERK kinase inhibitors. Pretreatment withPD98059 and SB203580 effectively inhibited PF-inducedhBD-2 mRNA and protein expression, whereas SP600125did not show inhibition. This result suggests that PFupregulates hBD-2 expression in HBECs through the p38MAPK and ERK signaling pathways, and the JNK is notnecessary. Thus, the induction of hBD-2 release appears tobe determined by different signalingmolecules and seems tobe dependent on stimulus and tissue. Meanwhile, NF-κB, acritical transcription factor for the expression of many cyto-kines, is normally retained in the cytoplasm in an inactiveform through being associated with an inhibitor of κB (IκB)protein. Following activation, IκB protein breaks down andliberates NF-κB to enter the nucleus, where it binds to thepromoter regions of target genes and eventually induces theexpressions of cytokines [26]. Given that the proximalpromoter of the hBD-2 gene contains several binding sitesfor NF-κB, several studies have shown that NF-κB is es-sential in hBD-2 regulation. In the present study, the sup-pression of NF-κB activity attenuated PF-induced hBD-2expression. We also observed the NF-κB translocation of16HBECs after PF treatment.

Some studies have shown that the anti-inflammatoryaction of PF may be related to its regulation of an imbal-anced cytokine production. Wang et al. [27] demonstratedthat the anti-inflammatory effect of PF in a murine modelof allergic contact dermatitis might be associated with thedecline in IL-2, IL-4, and IL-17 levels and with increasingIL-10 level.

Similarly, Nam et al. [28] suggested that PF possessesa neuroprotective activity by reducing the production ofNO, TNF-α, and IL-1β from the primary microglial cellsfor neuroprotection. Zhang et al. [29] reported that the pre-and co-administration of PF can significantly reduce theseverity of colitis and downregulate several inflammatoryparameters in the colon. These parameters include theactivity of myeloperoxidase, the levels of TNF-α and IL-6, and the mRNA expression of proinflammatory media-tors (MCP-1, Cox2, IFN-γ, TNF-α, IL-6, and IL-17) in

dextran sulfate sodium-induced colitis. Yan et al. [30] firstreported the induction of heat shock proteins (HSPs) by PFin cultured mammalian cells. Although the molecularmechanisms underlying the pharmacological functions ofPF remain unclear, these activities might be ascribed totheir positive effect on the induction of molecular chaper-ones. Asai et al. [31] reported that the intraperitonealadministration of PF clearly induces Hsp70 in mousestomach and that PF has a protective effect on HCl- andethanol-triggered gastric mucosal injury. Hence, PF mightbe used clinically as an HSP inducer for the prevention andtreatment of diseases associated with protein conformationand of other pathological states, such as stress ulcers andirritant- or ischemia-induced injuries.

In summary, this study is the first to demonstrate thatPF significantly upregulates hBD-2 expression in HBECsthrough the p38 MAPK, ERK, and NF-κB signaling path-ways. These results suggest that PF is a promising drugcandidate for the treatment of microbial infections bystrengthening the innate antimicrobial response. In vivostudies are needed to confirm the in vitro effects of PF onthe inducible production of human defensins.

ACKNOWLEDGEMENTS

The authors are grateful to the editor, the associateeditor, and reviewer. This research was supported in part byJiangsu Provincal Special Program of Medical Science(BL2012012).

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Gan, Cui, Ma, Liu, Li, and Huang