Resveratrol Inhibits Secretion of Interleukin 8 by Regulation of Autophagic Flux

Hong-Ying Chen,Xu Chen,Li Li,*,Heng Gu,*

1Jiangsu Key Laboratory of Molecular Biology for Skin Disease and STIs,2Department of Physical Therapy,Hospital for Skin Diseases(Institute of Dermatology),Chinese Academy of Medical Science and Peking Union Medical College,Nanjing,Jiangsu 210042,China.

Abstract Objective:The plant polyphenol resveratrol(3,4′,5-trihydroxystilbene)(RSV)has been proposed for use because of its protective effect on ultraviolet(UV)-induced skin disorders.In UVB-induced skin damage,cell autophagy and apoptosis have been approved to prevent the damage and to contribute to the cytoprotective role of RSV;however,the detailed mechanism remains unknown.So,we conducted this study to investigate the cytoprotective effects of RSV on UVB-irradiated human epidermal keratinocytes(HEKs)and its undergoing mechanisms.

Keywords:ultraviolet,resveratrol,autophagy,IL-8,keratinocyte

Introduction

Ultraviolet B(UVB)causes skin damage by inducing secretion of various cytokines,such as interleukin(IL)-8,1-2 which is a well documented pro-inflammatory cytokine and an important chemoattractant and activator for neutrophil and many other cell types in response to pathologic stresses.3Multiple mechanisms involving autophagy and apoptosis were reported to participate in protecting skin against UVB-induced damage.4-5However,the relation between IL-8 and autophagy or apoptosis remains uncertain.

The polyphenol resveratrol(3,4′,5-trihydroxystilbene)(RSV)originates from various natural products and the protective effect of topical RSV against UV radiationinduced skin damage has also been revealed in vivo and in vitro.6-8Our previous study demonstrated that RSV decreased cell death by increasing autophagy.5The potential use of RSV in skin flap transplantation was proposed because RSV showed a protective role in skin flap survival,and this role was thought to be related to enhancement of autophagy.9The induction of autophagy by RSV has been considered to be one of the mechanisms that prevents cell apoptosistoprotect cells.10Theregulatory effect of RSV on cytokines in response to UV radiation is another important mechanism related to its cytoprotective role,and the NF-κB signaling pathways are thought to contribute to this role.11However,the underlying mechanism by which RSV decreases the expression of cytokines in UV-irradiated cells remains unclear.

We conducted this study to investigate if autophagy and apoptosis is involved in the RSV-mediated decrease of inflammatory cytokine secretion by UVB-irradiated human epidermal keratinocytes(HEKs),and through which pathway or relative molecular.

Materials and methods

Reagents and antibodies

The compounds used in this study were RSV,chloroquine,and 3-methyladenine(3-MA)from Sigma-Aldrich(St.Louis,MO,USA),as well as procaspase activating compound 1(PAC-1),apoptosis activator 2(AA2),and LY294002 from Selleck Chemicals(Houston,TX,USA).The antibodies used were anti-c-Jun(#9165),anti-c-Fos(#4384),anti-phospho-NF-κB p65 Ser536(#3033),anti-NF-κB p65(#8242),anti-IκBα(#4814),anti-LC3A/B(#12741),anti-GAPDH(#5174),anti-histone H3(#4499),and anti-β-actin(#8457)(Cell Signaling Technology,Danvers,MA,USA).

Cell culture and grouping

HEKs were obtained from neonatal foreskin.The cells were cultured in defined keratinocyte serum-free media(Gibco,Invitrogen,Thermo Fisher,Carlsbad,CA,USA)and incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide.

UVB-challenged HEKs cells,which were treated with autophagy inhibitors LY294002 and 3-MA combined with or without RSV,were divided into groups of UVB,UVB+RSV,UVB+LY294002,UVB+LY294002+RSV,UVB+3-MA,and UVB+3-MA+RSV,to evaluate the contribution of autophagy to the RSV-mediated suppression of IL-8 secretion.Cells treated with PAC-1 or AA2 were divided into groups of UVB,UVB+RSV,UVB+PAC-1,UVB+PAC-1+RSV,UVB+AA2,and UVB+AA2+RSV,to evaluate the contribution of apoptosis to RSV-mediated suppression of IL-8 secretion.

UVB radiation

UV light with wavelength of 290 to 320nm(peak,310nm)was delivered by a UVB irradiation apparatus with a UVB lamp(UVB Broadband PL-S 9W/12;Philips,Amsterdam,Netherlands).Cells at 80%to 90%confluence were used and irradiated with a total exposure dose of 50mJ/cm2one time.

Cytokine array assay

Cells were cultured for 12,24,and 48hours after radiation.The cytokines in the culture supernatants were analyzed using a human cytokine array(ARY005;R&D Systems,Minneapolis,MN,USA)according to the manufacturer’s instructions.Briefly,the collected cell culture supernatant samples were added to membranes and incubated overnight at 4°C.The captured target proteins on the membrane were detected with biotinylated detection antibodies and visualized using a chemiluminescence imaging method.The secretion profiles included the cytokines Chemokine(C-C Motif)Ligand 1(CCL1/I-309),human macrophage chemoattractant protein-1(CCL2/MCP-1),macrophage inflammatory protein 1(MIP-1)α/β(MIP-1α/β),regulated upon activation normal T cell expressed and secreted factor(RANTES/CCL5),CD40 ligand,complement component C5/C5a,growth-related oncogeneα(GROα/CXCL1),interferon-inducible protein-10(IP10/CXCL10),Chemokine(C-X-C motif)Ligand 11 Protein(CXCL11),CXCL12,granulocyte-colony stimulating factor(G-CSF),granulocyte-macrophage colony stimulating factor(GM-CSF),intercellular cell adhesion molecule-1(ICAM-1),interferonγ(IFN-γ),IL-1α(IL-1F1),IL-1β(IL-1F2),IL-1ra(IL-1F3),IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12 p70,IL-13,IL-16,IL-17A,IL-17E,IL-18,IL-21,IL-27,IL-32α,memory initialization file(MIF),serpin E1,tumor necrosis factor alpha(TNFα),soluble triggering receptor expressed on myelocytes-1(sTREM-1).The cytokines were quantified by assessing the pixel density in each spot as measured by Quantity One software(Bio-Rad Laboratories,Hercules,CA,USA).

ELISA

The IL-8 protein level in the culture media was determined by a human IL-8 ELISA kit(BD Biosciences,Franklin Lakes,NJ,USA)according to the manufacturer’s instructions.

RNA interference

The transfection of small interfering RNA(siRNA)into HEKs was performed using Lipofectamine 2000(Invitrogen,Thermo Fisher)according to the manufacturer’s instructions.In brief,the cells were plated in 24-well plates and transfected with 100nmol/L ATG5 siRNA.After 48 hours of transfection,the cells were treated with 50mJ/cm2UVB exposure followed by RSV or not.Cells treated with siATG5 or RSV were divided into groups of UVB,UVB+RSV,UVB+siATG5,and UVB+siATG5+RSV,to evaluate the contribution of autophagy to RSV-mediated suppression of IL-8 secretion.The ATG5 siRNA sequences were as follows:5′-GUG AUG AUU CAU GGA AUU GTT-3′(sense)and 5′-CAA UUC CAU GAA UCA GCA CTT-3′(antisense).

Western blot assay

Cells were lysed in RIPA lysis buffer(Beyotime Biotechnology,Haimen,Jiangsu,China)containing protease and phosphatase inhibitor cocktail(Roche Applied Science,Penzberg,Germany).The protein concentration was measured with a BCA assay(Beyotime Biotechnology).Equal amounts of protein lysates were loaded on 4% to 15%MiniPROTEANTGprecastpolyacrylamidegels(Bio-Rad Laboratories)followed by transfer onto polyvinylidene fluoride membranes(Bio-Rad Laboratories).After blocking in 3% bovine serum albumin,the membranes were incubated with the indicated primary antibodies(1:1,000)and secondary antibodies(1:2,000).To visualize the protein bands,an Immun-Star WesternC Chemiluminescence Kit(170-5070;Bio-Rad Laboratories)was used through a chemiluminescence imaging method.The intensities of the protein bands were quantified with Quantity One software(Bio-Rad Laboratories).

Flow cytometry

Cell apoptosis was analyzed by flow cytometry using a fluorescein isothiocyanate(FITC)Annexin V Apoptosis Detection Kit I and propidium iodide(PI)/RNase Staining Buffer(both from BD Biosciences)according to the manufacturer’s instructions.Briefly,after treatment,the cells were collected by 0.25% trypsin without ethylenediaminetetraacetic acid.The cells were stained with 5μ L Annexin V-FITC and 5μL PI for 20 minutes in the dark at 37°C and then resuspended in 0.5mL for detection using BD FACS Verse.FlowJo software(FlowJo,LLC,Ashland,OR,USA)was used for data analyses.

Statistical analyses

The data of cytokine assay were analyzed using Student ttest using SPSS software(SPSS inc,Chicago,IL,USA),mean pixeldensity of IL-8 after 24and48hourswere comparedto 0 hour(control).Other data were analyzed by ANOVA using SPSS software.Multiple comparison between groups were performed using Tukey test with GraphPad Prism software.Independent experiments were performed at least three times.Significance was defined as P＜0.05.

Results

RSV inhibited UVB-induced secretion of IL-8 in HEKs

Thirty-six cytokines released by HEKs listed in the“Material and methods”were analyzed using ELISA assay at 12,24,and 48hours after 50mJ/cm2UVB exposure.The release of IL-8 and GROαsignuificantly changed after UVB radiation.In particular,the release of IL-8 started to rise at 12hours(0 vs.12hours:[12,834.94±1974.77]vs.[13,595.61±6096.11],P＞0.05)after UVB radiation and peaked after 24hours(0 vs.24hours:[12,834.94±1974.77]vs.[23,890.45±11,862.61],P＜0.05).There was no significant difference in the mean pixel density of IL-8 between 24 and 48hours(24 vs.48hours:[23,890.45±11,862.61]vs.[27,226.57±10,999.89],P＞0.05).In contrast to IL-8,the secretion of GROαdecreased at 12hours and increased from 24 to 48hours after UVB exposure(Fig.1A).And then,we tested the intervention effect of RSV on IL-8 secretion of cells exposed to UVB.The results showed that RSV increased the secretion of IL-8(CON vs.RSV:[23.77pg/mL±3.46pg/mL]vs.[148.09 pg/mL±19.72pg/mL],P＜0.001)and significantly decreased the secretion of IL-8 induced by UVB radiation(UVB vs.UVB+RSV:[1454.05pg/mL±52.95pg/mL]vs.[553.68pg/mL±206.03pg/mL],P＜0.001)(Fig.1B).

RSV inhibited UVB-induced c-Fos signaling

The western blot results showed that RSV did not affect the expression of c-Jun,c-Fos,or NF-κB/p65 or the phosphorylation of NF-κB/p65 in HEKs.UVB radiation increased the expression of c-Fos(CON vs.UVB:[0.063±0.014]vs.[0.068±0.011],P＜0.05)and significantly decreased the expression of c-Jun(CON vs.UVB:[0.025±0.002]vs.[0.015±0.004],P＜0.001),but did not affect the expression or phosphorylation of NF-κB/p65.RSV treatment inhibited the levels of c-Jun(UVB vs.UVB+RSV:[0.008±0.003]vs.[0.003±0.002],P＜0.05)and c-Fos(UVB vs.UVB+RSV:[0.103±0.009]vs.[0.048±0.015],P＜0.01)in UVB-irradiated cells(Fig.2A).NF-κB is sequestered in the cytoplasm and bound by members of the IκB family of inhibitor proteins,and degradation of IκB leads to translocation of NF-κB to the nucleus and its activation.12Next,the expression of NF-κB/p65 in the nucleus and IκBαin the cytosol was detected.The results showed that RSV did not affect the level of NF-κB/p65 in the nucleus but slightly inhibited IκBαin the cytosol regardless of whether HEKs were exposed to UVB,suggesting that RSV did not affect the translocation of NF-κB/p65 to the nucleus(Fig.2B).

Figure 1.Effects of RSV on UVB-induced IL-8 secretion.(A)HEKs were exposed to UVB and incubated for 0,12,24,and 48hours.Chemokines in the culture supernatants were measured.The mean pixel density of IL-8 was calculated.(B)HEKs were exposed to UVB and then treated with or without 100μmol/L RSV for 36hours.The level of IL-8 protein in the cell culture supernatants was determined by ELISA.The data are shown as mean±standard deviation of three independent experiments.***P＜0.001,**P＜0.01,*P＜0.05.Representative figures are shown.HEKs:human epidermal keratinocytes;IL-8:interleukin 8;RSV:resveratrol(3,4′,5-trihydroxystilbene);UVB:ultraviolet B.

RSV downregulated apoptosis and upregulated autophagy levels of UVB-challenged HEKs

We evaluated the ability of two apoptosis activators,PAC-1 and AA2,on prevention of apoptosis inhibited by RSV.The flow cytometry results showed that PAC-1 and AA2 increased the RSV-inhibited apoptosis of UVB-irradiated HEKs(UVB+RSV vs.UVB+PAC-1+RSV:[19.56%±0.62%]vs.[94.33%±0.15%],P＜0.001;UVB+RSV vs.UVB+AA2+RSV:[19.56%±0.62%]vs.[94.97%±1.91%],P＜0.001)(Fig.3A).Microtubule-associated protein 1 light chain 3(LC3)is a widely used autophagy marker.LC3-I,the proteolytically processed form of LC3,is converted into the conjugated form LC3-II with the induction of autophagy.In the present study,we used chloroquine(an inhibitor of the degradation function of lysosomes)and two autophagy inhibitors,3-MA and LY294002,and the LC3-II/GAPDH ratio was calculated to measure the autophagic flux by western blot.In HEKs,3-MA and LY294002 slightly decreased LC3-II accumulation with no significant difference(UVB+CQ vs.UVB+CQ+3-MA vs.UVB+CQ+LY294002:[0.97±0.03]vs.[0.89±0.06]vs.[0.47±0.17]),and the RSV-induced LC3-II accumulation was decreased by both 3-MA and LY294002 in UVB-irradiated cells(UVB+CQ+RSV vs.UVB+CQ+3-MA+RSV vs.UVB+CQ+LY294002+RSV:[1.15±0.03]vs.[0.77±0.13]vs.[0.67±0.13],P＜0.01)(Fig.3B).These results suggest that PAC-1 and AA2 reversed the RSV-mediated inhibition of apoptosis and 3-MA and LY294002 reduced the RSVinduced autophagic flux in HEKs exposed to UVB.

Figure 2.Effect of RSV on AP-1 and NF-κB signaling in HEKs exposed to UVB.HEKs were treated with or without 100μmol/L RSV for 12h after UVB radiation or control.(A)The total protein levels or phosphorylation of c-Jun,c-Fos,and NF-κB/p65 were determined by western blot.GAPDH served as the loading control.The data are shown as mean±standard deviation of three independent experiments.***P＜0.001,**P＜0.01,*P＜0.05.(B)The expression of NF-κB/p65 in the nucleus and IκBαin the cytoplasm was evaluated by western blot.Histone H3 and β-actin served as the loading controls.HEKs:human epidermal keratinocytes;RSV:resveratrol(3,4′,5-trihydroxystilbene);UVB:ultraviolet B.

Figure 3.Effects of RSV on apoptosis and autophagy in UVB-challenged HEKs.(A)HEKs were treated with 100μmol/L RSV,10μmol/L PAC-1,or 20μmol/L AA2 for 12hours after UVB radiation.Cell apoptosis was analyzed by flow cytometry.(B)HEKs were treated with or without 100μmol/L RSV,10μmol/L LY294002,or 5μmol/L 3-MA for 12hours in the presence of 50μmol/L chloroquine after UVB radiation.The protein levels of LC3A/B were determined by western blot,and GAPDH served as the loading control.The LC3-II/GAPDH ratio was calculated.The data are shown as mean±standard deviation of three independent experiments.***P＜0.001,**P＜0.01,*P＜0.05.HEKs:human epidermal keratinocytes;RSV:resveratrol(3,4′,5-trihydroxystilbene);PAC-1:procaspase activating compound 1;AA2:apoptosis activator 2;3-MA:3-methyladenine;UVB:ultraviolet B.

Autophagy may mediate the inhibitory effect of RSV on IL-8 release by UVB-challenged HEKs

UVB-challenged HEKs were treated with autophagy inhibitors or apoptosis activators in the presence or absence of RSV to evaluate the contribution of autophagy and apoptosis to RSV-mediated suppression of IL-8 secretion.First,two autophagy inhibitors,LY294002 and 3-MA were used to suppress the induction effect of RSV on autophagy.The results showed that RSV treatment decreased IL-8 secretion by 3.5 times compared with UVB radiation alone(UVB vs.UVB+RSV:[2,817.75 pg/mL±227.46pg/mL]vs.[796.40pg/mL±28.99pg/mL],P＜0.01).LY294002 and 3-MA treatment did not exert obvious effects on IL-8 secretion;however,RSV treatment decreased the secretion of IL-8 by 2.0 after LY294002(UVB+LY294002 vs.UVB+LY294002+RSV:[3,283.00 pg/mL±444.05pg/mL]vs.[1,608.58pg/mL±128.42pg/mL],P＜0.05)and 1.6 times after 3-MA treatment(UVB+3-MA vs.UVB+3-MA+RSV:[2,941.88pg/mL±103.80 pg/mL]vs.[1,867.51pg/mL±153.84pg/mL],P＜0.01),respectively,and these values were lower than those observed after RSV treatment alone.These findings suggest that the two autophagy inhibitors attenuated the RSV-mediated inhibition of IL-8 protein secretion by UVBirradiated HEKs(Fig.4A).In HEKs with ATG5 knocked down to inhibit autophagy,RSV slightly increased the secretion of IL-8 with no significant difference,RSVinhibited IL-8 secretion was prevented by siATG5(UVB+siATG5 vs.UVB+siATG5+RSV:[2,530.11pg/mL±685.34pg/mL]vs.[3,011.42pg/mL±435.69pg/mL],P＞0.05)(Fig.4B).Next,in UVB-challenged HEKs,apoptosis activators PAC-1 and AA2 did not exert obvious effects on IL-8 secretion,whereas the two apoptosis activators could enhance the RSV-mediated decrease in IL-8 secretion because RSV decreased IL-8 secretion by 4.9 and 16.0 times after PAC-1(UVB+PAC-1 vs.UVB+PAC-1+RSV:[3325.16pg/mL±347.58pg/mL]vs.[684.12pg/mL±53.70pg/mL],P＜0.001)and AA2 treatment(UVB+AA2 vs.UVB+AA2+RSV:[2,925.20pg/mL±54.04pg/mL]vs.[182.73pg/mL±149.11pg/mL],P＜0.001),respectively.These results indicate that the two apoptosis activators did not affect the RSV-mediated inhibition of IL-8 protein secretion by UVB-irradiated HEKs(Fig.4C).

Figure 4.LY294002,3-MA,and ATG5 siRNA prevent the RSV-mediated inhibition of UVB-induced IL-8 secretion.(A)HEKs were treated with or without 100μmol/L RSV,10μmol/L LY294002,or 5μmol/L 3-MA.(B)HEKs were treated with or without 10μmol/L PAC-1 or 20μmol/L AA2.(C)HEKs were transfected with ATG5 siRNA for 36hours after UVB exposure.The IL-8 protein levels in the cell culture supernatants were measured by ELISA.The data are shown as mean±standard deviation of three independent experiments;***P＜0.001,**P＜0.01,*P＜0.05.HEKs:human epidermal keratinocytes;IL-8:interleukin 8;RSV:resveratrol(3,4′,5-trihydroxystilbene);PAC-1:procaspase activating compound 1;AA2:apoptosis activator 2;3-MA:3-methyladenine;UVB:ultraviolet B.

Discussion

In our study,we found that 50mJ/cm2UVB-induced IL-8 secretion in HEKs,and 100μmol/L RSV inhibited UVBinduced secretion of IL-8.RSV inhibited the levels of c-Fos in UVB-irradiated HEKs.RSV regulated autophagy flux to influence IL-8 expression in UVB-irradiated HEKs.

In the begining,we detected 36 cytokines released by HEKs,and among these cytokines,IL-8 and GROαwere changed after UVB exposure.After UVB exposure,the secretion of IL-8 began to increase at 12hours,whereas the secretion of GROαfirst obviously decreased and then increased from 24hours.The secretion of GROαremained at a relatively low level and was lower at 48hours after exposure than at baseline.Thus,we determined the release of IL-8 to evaluate the influence of RSV on UVB-induced inflammation.

RSV can induce IL-8 gene transcription and expression via NF-κB.13-14In this study,we found that RSV-induced IL-8 expression but did not exert an effect on NF-κB signaling,either on its phosphorylation or nuclear translocation.Although RSV decreased the expression of IκBαin the cytoplasm,NF-κB signaling was not activated.Inhibition of NF-κB by RSV is not thought to be cell type-specific(in T,epithelial,and glioma cells),but NF-κB does not seem to be activated in keratinocytes.15In addition to NF-κB,AP-1 is another signaling cascade transcription factor revealed to induce IL-8 gene transcription.16In this study,UVB decreased the protein levels of c-Jun and increased the protein levels of c-Fos,suggesting that UVB exposure activated AP-1 signaling.Moreover,RSV decreased the protein level of c-Fos in UVB-irradiated HEKs;thus,we hypothesized that RSV probably induced IL-8 secretion by c-Fos signaling in HEKs.

Our study revealed that RSV has a potential protective role because it reduced IL-8 secretion by HEKs exposed to UVB radiation.However,Pastore et al.17reported that RSV enhanced the UV-induced response,including IL-8 expression,NF-κB activation and nuclear translocation,which differs from our results.Notably,a much lower dose of UVB(0.1mJ/cm2)was used in their study.In our previous study,LC3 or related proteins in the mechanistic target of rapamycin signaling pathway were detected after exposure to different doses of UVB(1.5-50mJ/cm2),and we found that only under the condition of 50mJ/cm2UVB did the proteins change.18RSV protects normal skin keratinocytes against UV damage while augmenting UV damage to tumor cells.7,19Thus,we infer that RSV tends to eliminate damaged cells exposed to mild UV levels while rescuing cells exposed to relatively high doses.

In our previous study,we demonstrated that RSVinduced UVB-inhibited autophagic flux.18In the present study,we demonstrated the contribution of autophagy to the RSV-mediated inhibition of IL-8 in the context of UVB radiation.Traditionally,inflammation and autophagy are considered two distinct pathways,and very few reports on the role of autophagy in modulating inflammation have been published in the last decade.Qiang et al.20reported that the ATG7 and ATG5 genes regulate UV-induced inflammation.In our study,inhibiting autophagy by ATG5 knockdown or autophagy inhibitors attenuated the RSVmediated decrease in IL-8.SIRT1 was recently concluded to act as a switch between autophagy and inflammation,and high doses of RSV decreased the release of IL-1βvia upregulation of SIRT1 to promote autophagy.21In addition to autophagy,apoptosis is another critical mechanism regulated by RSV that influences the fates of cells.22,23However,in this study,the inhibition of apoptosis by RSV in UVB-challenged HEKs was not attenuated by PAC-1 and AA2,suggesting that apoptosis does not contribute to the RSV-mediated inhibition of IL-8.

In summary,our study demonstrated the contribution of RSV-induced autophagy to IL-8 inhibition in UVBchallenged keratinocytes,indicating the molecular mechanisms underlying the photoprotective effect of RSV.Actually,studies have indicated the possibility of using RSV as a therapeutic agent for psoriasis and atopic dermatitis.24The limitations of this study were as follows:firstly,only 36 inflammatory cytokines were evaluated,more inflammatory cytokines should be investigated in the future;secondly,we did not evaluate other inflammatory signaling pathways like JAK/STAT;thirdly,we could not confirm that RSV induce IL-8 expression through c-Fos signaling,and we plan to up-regulate c-Fos expression in UVB-challenged HEKs and evaluate IL-8 secretion by ELISA;finally,the detailed mechanism by which RSV regulates autophagic flux to influence IL-8 expression was not revealed in this study and is worth further investigation.

Source of funding

This study was supported by National Natural Science Foundation of China(No.81703153),CAMS Innovation Fund for Medical Sciences(No.2016-I2M-1-005),and the NanjingIncubation Program for National Clinical Research Center(No.2019060001).