Effects Of Photodynamic Therapy Using LED-light With Concomitant Hypocrellin B On Apoptosis Signaling In Keloid Fibroblasts

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Effects of photodynamic therapy using LED-light with concomitant hypocrellin B on activity and apoptosis in keloid fibroblastsBackground:Keloid is a common and refractory disease in dermatology and cosmetic surgery.Keloid fibroblasts(KFB),the most important cells in keloid tissue formation,proliferate abnormally and secrete excessive extracellular matrix,which share certain biological behavior of benign tumor.The current clinical treatments for keloid including local injection of glucocorticoids,compression treatments,external use of silica gel and tretinoin are unsatisfactory.Removal of keloid with surgical resection alone has recurrence rates of 45-100%.Excision with early postoperative radiotherapy is relatively effective than the other treatment options,but possible risk of radiation for children and young adult limits its use in clinical.Therefore,more studies in better understanding the pathogenesis and developing novel therapeutic strategies for keloid are requisite.Hypocrellin,a perylene quinone derivative,was isolated from Hypcrella bambusae Sacc(Hypocreaceae,Ascomycetes).Hypocrellin mainly consists hypocrellin A(HA)and hypocrellin B(HB).HA and HB are similar in structure with a difference in one hydroxyl group.Hypocrellin has the advantages such as high quantum yields of singlet oxygen,strong photogeneration of anion radicals in deoxygenated media,low dark toxicity,quick clearance from normal tissue and availability in pure monomeric form.Hypocrellin has been used in photodynamic therapy as photosensitizer.Photodynamic therapy with HB or HB derivatives was shown to have antitumor activity through inducing tumor cell apoptosis and inhibiting cell viability from several studies.As keloid shares certain feature of tumor with abnormal cell proliferation,we studied the effect of photodynamic therapy by combining HB with yellow LED-light(HB-LED PDT)on apoptosis in KFB cells.The experiment was try to provide the evidence if HB-LED PDT could be a potential clinical therapeutic method for keloid.Objective:To explore the appropriate doses of HB and LED used for KFB treatment in the study and explore the effect of HB-LED on the viability and apoptosis of KFB cells.Methods:1.KFB cells were isolated from keloid tissues.The cells of the third to sixth generation were used in the experiment.Cells were divided into 4 groups:DMEM medium,HB,LED,HB plus LED.2.The keloid fibroblasts were seeded into 96-well plates.After being added HB,cells in each group were radiated by yellow LED-light.The viability of KFB cells were detected by modified MTT.3.The keloid fibroblasts were seeded into 6-well plates.After HB added,cells in each group were radiated by yellow LED-light.The cells were gently trypsinized,washed with PBS.Then,cells were incubated with 5 ?gL/tube Annexin V-FITC for 15 min at 25 ? in the dark.Five ?L/tube PI were added.Samples were analyzed using a flow cytometer.The results were analyzed by BD FACSDiva 7.0.Results:1.1 3J/cm2 of yellow LED-light was a suitable irradiation dose.1.2 IC50(50%inhibiting concentration)of HB was 3.3252×10-7 mol/L in theHB-LED group at 12h after treatment.2.In comparison with negative control group(NC),the results of the MTT assay show that cell activity decreased 50.23%(P<0.001)and 25.77%(P<0.001)at 12h treatment with 0.1 ?mol/L HB plus LED and 0.1 ?mol/L HB alone,respectively.LED along also caused a 16.57%(P<0.001)reduction of cell activity.3.The total apoptosis rate amounted to 57.30%in the HB-LED group.HB alone caused apoptosis in 25.77%of the cells.LED alone also induced apoptosis compared to NC,with a P value of 0.042.Conclusion:1.Photodynamic therapy using yellow LED-light with concomitant hypocrellin B could effectively decrease the activity in keloid fibroblast cells in vitro.2.Photodynamic therapy using yellow LED-light plus hypocrellin B could effectively induced apoptosis of keloid fibroblast cells in vitro.Furthermore,the effects of HB and LED-light are synergistic.Part ? Effects of photodynamic therapy using LED-light with concomitant hypocrellin B on apoptosis signaling in keloid fibroblastsBackground:Apoptosis is an active and programmed cellular death process under certain physiological or pathological conditions,which is an important self-stabilizing mechanism of the body.In recent years,a large number of experimental studies have shown that mitochondrial apoptosis pathway plays an important role in cell survival and death.Furthermore,among the many apoptotic regulatory genes,the BCL-2 family and the caspase family are the most concerned.BCL-2(b-cell lymmpoma/leukemia-2,BCL-2)is an anti-apoptotic gene.BAX gene is the most important apoptotic gene in human body,and the study found that the ratio of BAX/BCL-2 protein is the key factor to induce apoptosis.Caspase-3 is the key protease in cell apoptosis,and because it is at the core of the apoptotic cascade pathway,it is called death protease.As an important second messenger,intracellular calcium ion is involved in regulating cell proliferation,differentiation and apoptosis.In the presence of high sugar,hypoxia and certain drugs,the increase of calcium ion concentration in the cell causes calcium overload,thus promoting cell apoptosis.In order to explore the possible signaling pathway of HB-LED photodynamic therapy inducing the apoptosis of human keloid fibroblasts(HKF),in this experiment,BAX,BCL-2,caspase-3 and calcium ion concentration were detected in HKF cells after HB-LED photodynamic treatment.Objective:To explore whether the mRNA and protein levels of BAX and BCL-2 in KFB cells were influenced by HB-LED PDT and whether intracellular free Ca2+ level and caspase-3 activity arec hanged in the KFB apoptosis upon PDT treatment.Methods:1.Examination of BAX and BCL-2 mRNA by real-time PCR1.1 The keloid fibroblasts were seeded into 6-well plates.After addition of HB,cells in each group were radiated by yellow LED-light.1.2.Total RNA was extracted from KFB cells using the Trizol method.Three ?g of RNA were reverse-transcribed to cDNA1.3.Amplification in a real-time detector after initial denaturation at 95 ? for 8 min,then ran for 45 cycles at 95 ?for 15s,at 59 ? for 20s and at 72 ? for 30s.1.4.The relative expression levels were calculated using the ??Ct method.2.Detection of BAX and BCL-2 protein by intracellular flow immunofluorescence assay2.1.The keloid fibroblasts were seeded into 6-well plates.After addition of HB,cells in each group were radiated by yellow LED-light.2.2.The treated cells in 6-well plates were trypsinized,put into tubes in groups,and centrifuged at 1500 rpm×5min.After discarding the supernatant,samples were washed tw:ice with PBS.2.3.The cells were fixed with 1 mL/tube BioLegend's Fixation Buffer at room temperature in the dark for 15 min and centrifuged.2.4.After discarding the supernatant,the samples were washed once with 1 ml BioLegend's Permeabilization Buffer,and cells were resuspended in 300 ?L Permeabilization Buffer.2.5.Five uL/tube FITC-conjugated anti-BAX and PE-conjugated anti-BCL-2 were added respectively,and samples were incubated at 4? in the dark for 45 min.FITC-conjugated mouse IgGl and PE-conjugated mouse IgG1 were used as isotype controls.2.6.Samples were washed twice with lmL/tube Permeabilization Buffer,resuspended in 500 ?L Permeabilization Buffer,and finally analyzed via flow cytometry with appropriate instrument settings.2.7.The amount of protein was estimated by FlowJo 10.0.7.3.HB-LED PDT increases the level of intracellular free Ca2+3.1.The cells were planted on glass coverslips,then treated with HB-LED PDT.3.2.Fluo-3/AM(5 pmol/L)in Ca2+-free PBS buffer were loaded on cells at 4h after HB-LED PDT treatment and incubated.3.3.After washing twice with PBS,the fluorescence of cells was detected using a confocal laser-scanning microscope at 488 nm for excitation and 530 nm for emission.3.4.Ca2+ levels were presented by mean fluorescence intensity(MFI)of the cells.MFI calculated using the instrument's software(Imaging Software for Microscopy).4.The caspase-3 activity detection by a kit4.1.The keloid fibroblasts were seeded into 6-well plates.After an addition of HB,cells in each group were radiated by yellow LED-light.4.2.Cells were trypsinized,washed with PBS,incubated in lysis buffer on ice for 15 min and centrifuged.4.3.The supernatant were incubated with acetyl-Asp-Glu-Val-Asp p-nitroanilide(Ac-DEVD-pNA)in 96-well plates.4.4.Samples containing yellow p-nitronanilide(p-NA),a lysate from Ac-DEVD-pNA,were measured with a microplate reader at an absorbance of 405 nm.Results:1.At 4 hours after disposal of KFB cells,BAX mRNA in the HB-LED,HB,LED groups increased by 72.67%,23.33%,and 6.61%(P=0.006,<0.05),respectively,in comparison to NC.In contrast,mRNA of BCL-2 in the HB-LED,HB alone and LED alone group decreased by 48.57%,26.77%and 6.73%(P<0.001),respectively.2.Five hours after treatment of KFB cell,BAX protein in the HB-LED group increased by 3.03 times compared to NC.Conversely,the level of BCL-2 protein was downregulated.BCL-2 in the HB-LED group was reduced to 17.50%(P<0.001)of the NC group.Overall,the BAX/BCL-2 ratio increased from 0.1766 in NC to 3.0238 in the HB-LED group.Similar changes occurred in the HB and LED groups.3.By confocal laser microscopy analysis,HB-LED PDT treatment significantly increased intracellular free Ca2+ in KFB cells by 3.77-fold(P<0.001)compared to that in NC cells.4.Data shows that HB-LED significantly increased the activity of caspase-3,which was increased by 5.37 fold compared to NC(P<0.001).HB alone and LED alone raised caspase-3 activity by 2.37 and 1.15 fold(P<0.001,P<0.001),respectively.Conclusion:Hypocrellin B plus yellow LED-light could increase Bax mRNA,but decrease the level of BCL-2 mRNA.These changes also happened in protein level.Hypocrellin B plus yellow LED-light also could upregulate Ca2+ and caspase-3 in KFB cells.Mitochondrial apoptosis pathways are likely to be involved in the apoptosis induced by HB-LED PDT.