Biological Evaluations Of Antioxidative,Anti-glycation,and Anti-Inflammatory Effects Of Pomegranate Extract In Keratinocytes,and The Study Of Paget's Disease

Introduction:Epidermis,the outer layer of skin,is mainly composed of keratinocytes Keratinocytes are constantly exposed to harmful stimuli including extrinsic insults(e.g air pollutants,UV-exposure,and smoking)and intrinsic stress(e.g.endotoxins,oxidation,and glycation).Harmful stimuli lead to elevated level of reactive oxygen species(ROS),glycative stress,and inflammatory response,which further result in the damage of skin integrityPomegranate extract(PE)has been reported on its broad range of bioactivities including antioxidant,anti-glycation,anti-tumor growth,anti-infection,and anti-UV damage effects.It has been reported that a major polyphenol from pomegranate extract,namely,punicalagin(PA),showed skin protective effects against UVA and UVB induced fibroblast damages by reducing ROS and inflammation.PE was also reported on the photoprotective effects in keratinocytes against UVA and UVB mediated inflammation activation through signaling pathways including STAT3,PKB,MAPK or NF-?B.These effects are supported by data from several in vivo studies.A double-blind and placebo-controlled clinical trial revealed that the oral consumption of ellagic acid(EA),another polyphenol from PE,showed photoprotective and anti-hyperpigmentation effects in human skin.Urolithin A(UA),a gut microbial metabolite of PA and EA,has also been studied for photoprotective and skin lightening effects.In addition,PE showed inhibitory effects on the formation of advanced glycation end products(AGEs)and methylglyoxal(MGO;a precursor of AGEs)scavenging capacity.The anti-glycation effects of PE were further investigated by several in vivo studies with high sugar&fat diet mouse model.Furthermore,PE has been reported on the anti-inflammatory effects as it ameliorated poly(I:C)-induced secretion of multiple cytokines in keratinocytes,whilst EA reduced the secretion of IL-1? induced by liposaccharides(LPS)in macrophage cells.Our group has launched a program to systematically examined the chemical composition of PE and has identified over 70 phenolic.Moreover,a standardized PE and its polyphenols including PA,EA,as well as their gut microbial metabolite,UA,have been studied for anti-glycation and anti-neuro-inflammatory effects.However,to date,the protective effects of PE,PA,EA,and UA in human keratinocytes are still unclear Herein,we aim to evaluate the skin protective effects of PE PA,EA,and UA against H2O2-induced oxidative stress,MGO-induced glycative stress,and poly(I:C)-induced inflammatory stress in keratinocytes.In addition,inhibitory effects on cancer cell proliferation in breast carcinoma are studied by analyzing the regulation of a panel of targeted miRNAs.Furthermore,due to that Paget's disease shares certain similar histological features and immunohistological markers with breast cancer,investigated several specific expressed miRNAs in extramammary Paget's disease(EMPD)This work aim to evaluate the antioxidative,anti-glycation,and anti-inflammatory effects of PE PA,EA,and UA in keratinocytes,as well as the miRNAs for EMPD.Results from this study add on growing body of data supporting the utilization of PE for dermatological and/or cosmeceutical applicationsMaterial and Methods:1.Experimental materialsImmortalized human keratinocyte HaCaT cell line was reserved in the biology lab of Providence College.Primary human epidermal keratinocyte from adults(HEKa)cells were purchased from GIBCO(C-005-5C).This study also enrolled 16 EMPD patients,12 eczema or tinea curis patients,as well as 17 healthy volunteers.Clinical information and serum were collected for further investigation.This study was approved by ethics committee of the XX(No.AF-SOP-07-1.0-01)2.ROS ExaminationHaCaT cells were incubated with different concentrations of experimental compounds After that the medium was replaced with DMEM containing 20 ?M DCFDA and H2O2(400?M).The fluorescence signals were read at excitation and emission wavelengths of 485 and 525 nm,respectively3.Viabilities of different experimental compounds/H2O2/MGO on Hacat CellsHaCaT cells were exposed to different concentrations of experimental compounds/H2O2/MGO.Following the pre-determined incubation period,CTG 2.0 was added in a 1:1 ratio with existing media and mixed for 5 minutes on an orbital shaker prior to luminescence measurement.After incubation with different concentrations of experimental compounds/H2O2/MGO and fixation(75%ethanol 15 minutes)crystal violet staining(0.05%w/v)was carried for 10 minutes.After that,cells were washed with PBS for 5 times4.Apoptosis detectionHaCaT cells were exposed to different concentrations of experimental compounds/H2O2/MGO.The cells were harvested and suspended with 500 ?L binding buffer containing 5 ?L FITC-labeled Annexin-V and 5 ?L Propidium iodide(PI).After 15 mins of incubation in the dark,the cells were examined by flow cytometry.Flow cytometry data were analysis with FlowJo5.Detect caspase-3/7,-8,-9HaCaT cells were exposed to different concentrations of experimental compounds for 12h and H2O2 for 24h.Then 100 uL of caspaseGlo reagent were added for the detection of caspase-3/7,-8,-9.After 30 mins incubation,expression levels were detected with using luminescence measurement.6.Detect the integrity of the DNA chainsHaCaT cells were exposed to different concentrations of experimental compounds for 2h and MGO for 24h respectively.After that,cells were detached,and diluted with LMAgarose.Cells were added to the Comet slides,incubated in lysis solution at 4?overnight,immersed in Alkaline Unwinding Solution for 20mins,applied electrophoresis in Alkaline Electrophoresis Solution at 21 volts for 30 minutes,fixed in 75%ethanol for 15 minutes,dried in 37? for 15 minutes and then stained in diluted SYBR GOLD,images were taken with EVOS and analysed via CASP7.DNA oligonucleotide synthesis and LC-MS analysisNine-mer single-stranded DNA oligonucleotides with the sequence 5'-TTTTGTTTT-3' were synthesized by using solid-phase phosphoramidite chemistry.LC-MS analysis was carried out on AB Sciex triple quadrupole-TOF 4600 mass spectrometer.ESI was conducted under a negative ion mode by applying the following parameters.All LC-MS data were analyzed by using AB Sciex Analyst TF software 1.78.Detect the cell adhesion and migration abilityHaCaT cells were exposed to different concentrations of experimental compounds and MGO respectively.CTG 2.0 was used to calculate the cell viability in Oh and 10h after PBS washing in each group.Then the adhesion rate was calculated.Cell migration ability was measured by transwell assay.Cells in upper chamber were stained with Crystal violet and pictures were taken with EVOS.After that stained cells were incubated in 75%ethanol for 15 minutes for decoloration.Then optical density of the decoloration solution in 570 nm was calculated for quantification9.Detect IL-1? secretionHaCaT cells and HEKa cells were treated with different concentrations of experimental compounds and/or Poly(I:C)with lipofectamine.IL-1? in the supernate was quantificated by Elisa10.Detect the transcription of NLRP3,ASC,CASP-1,and pro-IL-1?HEKa cells treated with EA,UA,and Poly(I:C)were detached.Then RNA was collected,and the qPCR was performed using primers of NLRP3,ASC,CASP-1,and pro-IL-1? in each group.?Ct was calculated using automatic threshold11.Detect the mitochondrial ROS and mitochondrial membrane potential levelHEKa cells treated with EA,UA,and Poly(I:C)were detached.Cells were stained with Mito Sox Red or Mito Tracker Red,then analyzed by cytometry.HEKa cells treated with EA,UA,and Poly(I:C).Then cells were stained with Hochester and Mito Sox Red or Mito Tracker Red respectively.Pictures were taken using confocal microscope12.Primary cell culture of EMPDEMPD tumor tissue were transferred quickly to lab to perform primary culture after surgery.Tissues were digested with dispase in 37? water bath for 90mins.After that the dermis tissues were removed and the epidermis tissue was cut into pieces.Then the tissue suspension were centrifuged and the supernatant was removed.The tissue were digested with trypsin in 37? water bath for 90mins,then neutralized with RPMI medium containing 10%FBS.Cells were washed with PBS and filtered with filter net,and then cultured in RPMI medium containing 10%FBS in incubator(37? 5%CO2)13.miRNA array and global normalizationRNA samples from healthy volunteers and EMPD patients were reverse transcribed and pre-amplified.Using Human MicroRNA array card v2.0,plate A,the miRNA expression profiles were detected.Raw cycle threshold(Ct)values were calculated using SDS 2.3 and RQ manager 1.2 software with default baselines and threshold settings.All the Ct values were exported into StatMiner(?)4.2 for global normalization.Heat-map analysis was performed using ?Ct with hierarchical clustering using miRNAs expressed in all samples.14.Detect the expression of differentially expressed miRNAsAccording to the results of miRNA array,differentially expressed miRNAs were selected out and further validated in different groups by qPCR15.Statistical analysisData collected from experiments were analyzed and diagrammed by GraphPad Prism.Groups were compared with one-way analysis of variance(ANOVA)and Newman-Keuls.The statistical significance was announced when p<0.05,p<0.01 or p<0.0001Results:1.Propective effects of PE and polyphenols against H2O2 induced HaCaT cell damage.This work found that PE,PA,and EA ameliorated H2O2 induced ROS production in HaCaT cells;PE,PA,and UA reduced H2O2 induced cytotoxicity of HaCaT cells;PE,PA,and UA inhibited H2O2 induced apoptosis of HaCaT cells;PE,PA,and UA inhibited the Caspase3/7 content of HaCaT cells induced by H2O22.Propective effects of PE and polyphenols against MGO induced HaCaT cell damage.MGO induced cytotoxicity of HaCaT cells without apoptosis.MGO could bind with Guanine and induced HaCaT cells DNA breakage.MGO inhibited the adhesion and migration of HaCaT cells.PE and PA were found with anti-glycation protective effects on MGO induced cell damage3.Propective effects of PE and polyphenols against poly(I:C)induced HaCaT cell damage.Poly(I:C)induced the priming and activation of NLRP3 inflammasome which promoted the secretion of IL-1? in HEKa cells.PE,EA,and UA inhibited poly(I:C)induced secretion of IL-1? in HEKa cells.EA inhibited the priming and activation of NLRP3 inflammasome in HEKa cells4.The miRNA expression profiles of EMPDmiRNA expression profiles of serum EMPD patients were detected in this study Differentially expressed miRNAs were further validated,and found miR-155 overexpressed in EMPD patients compared with healthy volunteers and eczema or tinea cruris patients.Conclusion:1.PE,PA,EA,and UA ameliorated H2O2 induced HaCaT cell damage with antioxidant capacity.2.PE and PA ameliorated H2O2 induced HaCaT cell damage with antiglycation capacity.3.PE,EA,and UA inhibited poly(I:C)induced the secretion of IL-1? in HEKa cells.EA inhibited the poly(I:C)induced priming and activation of NLRP3 inflammasome in HEKa cells.4.Found miR-155 overexpressed in EMPD patients via serum miRNA expression profiles suggesting that miR-155 has the potential to be a serum marker for early diagnosis.