The Effect Of Shikonin On The Proliferation And Apoptosis Of Keratinocytes By Regulating CEBPD Via JAK/STAT3 Pathway

Introduction:Psoriasis represents a chronic inflammatory skin ailment which mainly manifests as hyperkeratosis and parakeratosis of keratinocytes,affecting 2-3%individuals around the world.It is also considered a systemic inflammatory disease.Moreover,the transcription factor STAT3 plays a key role in the development and pathogenetic mechanisms of psoriasis and related conditions.CCAAT/enhancer-binding protein delta?CEBPD?encodes a 269 amino acid protein of the CEBP family,which includes transcription factors modulating cell differentiation,metabolism and immune reactions.Borrelli S et al.by IHC showed that in skin tumors CEBPD is not expressed in basal cell cancer,while most squamous cell cancers show high protein amounts.This indicated CEBPD has an important function in the final steps of keratinocyte differentiation.Shikonin represents an active compound of the oriental traditional plant Lithospermum erythrorhizon,whose antitumor effects have been evaluated for almost three decades.Additionally,shikonin and derivatives display anti-angiogenetic,anti-inflammatory and anti-glycolytic features.Liu L et al.indicated that IFN-?promotes K17 protein overexpression in HaCaT cells via STAT3 activation,with shikonin inhibiting this effect in part by interfering with STAT3.Xu Y et al.demonstrated that shikonin reduces IL-17-associated VEGF overexpression through its blocking effects on JAK2/STAT3 signaling.In a preliminary study,we found that CEBPD expression in psoriasis tissues is lower than that of normal skin tissues.Meanwhile,after stimulation with IL-17 and shikonin,JAK/STAT3 related genes in the HaCaT cell line were screened by the PCR Array technology,and the significant target gene CEBPD was selected.In the present work,the mechanisms of keratinocyte proliferation and apoptosis in response to shikonin in psoriasis were explored.We demonstrated that JAK/STAT3 induction contributes to CEBPD downregulation in psoriasis,by inhibiting or activating STAT3 expression.In addition,shikonin,an effective drug for psoriasis,efficiently inhibited JAK/STAT3activation and increased CEBPD expression,while suppressing proliferation and inducing apoptosis of keratinocytes in psoriasis both in vitro and in vivo.Method:1.Cell cultureThe HaCaT cell line was provided by Kaiji Jiangsu,China,and maintained in RPMI-1640 with 10%fetal bovine serum?FBS?and 1%penicillin-streptomycin at 37?in a humid atmosphere containing 5%CO₂.For JAK/STAT3 activation,recombinant human IL-17A?0,1,10,50,100,200ng/ml?at various concentrations was incubated with cells.For JAK/STAT3 inhibition,Stattic?0,5,10,20,50,100?M?was added at various levels.Shikonin was diluted with DMSO to a stock level of 20 mg/ml and incubated with cells at various concentrations?0,0.5,1,2,4,8,10?M?.Control cells were cultured without additive.2.Lentivirus transfectionHaCaT cell culture was performed in 100mm Petri dishes in medium without antibiotics before lentiviral transfection.To establish STAT3 knockdown HaCaT cells,the cells were infected with packaged lentivirus containing shSTAT3-RNAi?RNAi?or a control scrambled shRNA?NC?.To establish STAT3 overexpression HaCaT cells,the cells were infected with packaged lentivirus containing EGFP-STAT3?LV-STAT3?or a control empty vector?Vector?.All lentiviruses were manufactured by GeneChem,Shanghai,China.HaCaT cells were transfected with Polybrene and Eni.S.Puromycin?1.0?g/ml?was employed for screening cells with successful transfection.Wild type,STAT3-knockout,STAT3 overexpression and empty vector control HaCaT cells cultured in 100mm Petri dishes were observed under an inverted fluorescence microscope.3.Animal modelMale BALB/c aged 6-8 weeks were randomized into 5 groups?n=6?:control?CON?,model?IMQ?,positive drug[Methotrexate at 0.5mg/kg/d?MTX?],low dose shikonin[Shikonin at 5mg/kg/d?SHI5?]and high dose shikonin[Shikonin at 10mg/kg/d?SHI10?]groups.Psoriasis-like inflammatory symptoms were established in mice by topically applying 62.5mg of 5%imiquimod cream to a 3.0cmX2.0cm shaved mouse dorsal epidermal area once daily for ten consecutive days,while control animals were treated with Vaseline.For therapy,drugs were dispensed with edible oil and administered to the corresponding groups by gavage.Meanwhile,the matrix was gavaged to the control and model groups,i.e.edible oil containing 5%DMSO at 1mg/kg/d.Photographs of the lesions were acquired with a digital camera.Upon treatment,the animals were euthanized,and skin tissue samples at lesion sites were obtained.4.Histological and immunohistochemical analysesSkin samples from mice were excised,washed with PBS and fixed in formalin,paraffin embedded and sectioned at 5?m.Then,staining with hematoxylin and eosin?H&E?was performed followed by assessment under a light microscope;epidermal thickness was determined in 6 randomly selected high-power fields for each sample.For immunohistochemical assessment,samples were submitted to incubation with rabbit anti-mouse Phospho-Stat3?Tyr705?,anti-STAT3 and anti-CEBPD antibodies,respectively,overnight at 4oC.Then,EliVision Super Kit was employed for detection.Immune complexes were detected using the DAB kit.A total of 3 randomly selected high-power fields in the slide center were assessed under a light microscope.Mean optical density?MOD?was obtained as integral optic density?IOD?divided by the corresponding area,with Image-Pro Plus 6.0.5.MTS assayThe MTS assay was employed for measuring the viability of wild type and LV-STAT3HaCaT cells,as directed by the manufacturer.Briefly,3000 cells per well?96-well plates?were cultured at 37?in a humid environment containing 5%CO₂.Different concentrations of IL-17A,Stattic or Shikonin were added to cultured cells and incubated for 24,48 and 72 hours,respectively.Then,MTS was added for 3 hours of incubation.Cell viability was then assessed by detecting optical density at 490 nm on a microplate reader.All analyses were performed in three independent triplicate experiments.6.Real-time qPCR assayTotal RNA was extracted from cultured cells with miRNeasy Mini Kit as directed by the manufacturer,and quantitated on a ND1000 spectrophotometer.First strand cDNA synthesis was carried out with 1?g mRNA and GoScript Reverse Transcription Kit based on the manufacturer's instructions.Primers for STAT3,CEBPD and GAPDH were designed using PubMed.The RT2 SYBR Green qPCR Master mix was employed for amplification on a 7900HT Fast Real-Time PCR System.Amplification was performed as follows:95??2 min?;40 cycles at 95??15 s?and 60??1 min?.Melting curves were generated to confirm specificity.The 2^-??Ct??Ct method was employed for data analysis,with GAPDH as a control gene.Three independent triplicate experiments were carried out.7.ImmunoblotTotal protein extraction from cultured cells and mouse skin tissue samples was carried out with RIPA lysis buffer containing protease/phosphatase inhibitors as instructed by the manufacturer.Protein quantitation was performed with a BCA protein assay kit as directed by the manufacturer.Equal amounts of total protein?20 and 30?g for cells and tissue samples,respectively?were resolved by 10%SDS-polyacrylamide gel electrophoresis?SDS-PAGE?and electro-transferred onto PVDF membranes.Upon blocking?5%skim milk or BSA in TBST?for 1h and incubation with respective primary antibodies overnight at 4?,then treated with goat anti-rabbit secondary antibodies for 1h in ambient conditions.Visualization was performed with an ECL kit on a MicroChemi^TMM Chemiluminescent Imaging System.8.Flow cytometryCells?2x105/well?in six-well plates were cultured for 24h.To assess cell cycle distribution,cells were first incubated with serum-free medium for 24h before changing to normal medium containing different doses of shikonin for another 12h of incubation.Then,cells were collected and submitted to fixation with 75%chilled ethanol overnight at 4?and staining with 500?l binding buffer containing 5?l RNase A?5 mg/ml?for 30min at 37?.Next,5?l PI?5 mg/ml?was added for 30 min on ice away from light.To assess apoptosis and senescence,cells were resuspended in 500?l binding buffer and submitted to staining with APC-linked Annexin V?5?l?and PI?5?l;5?g/ml?for 30 min away from light.A BD LSRFortessa was used for analysis,gating 10,000 cells.Cell cycle distribution was assessed with the ModFit software.9.Statistical analysisSPSS v21.0 was employed for all statistical analyses.Group pairs were compared by student's t-test.Results were expressed as the mean±SEM,and the analysis was performed using either a student's t-test when comparing only two groups or one-way ANOVA for greater than two groups.P<0.05 indicated statistical significance.Results:1.JAK/STAT3 activation decreases CEBPD expression in the HaCaT cell line.MTS assay showed that IL-17A treated HaCaT cells had significantly elevated viability in comparison with control cells,with time-and concentration-dependence,while Stattic treated HaCaT cells presented the opposite trend.The result of real-time qPCR showed JAK/STAT3 was time-dependently activated by IL-17A?10ng/ml?,peaking at 6h,while CEBPD was downregulated.After treatment of HaCaT cells with Stattic?2.5?M?,JAK/STAT3 was time-dependently inhibited,while CEBPD was upregulated.Meanwhile,Western blot data showed that JAK/STAT3 was significantly activated by IL-17A?10 ng/ml?and CEBPD expression was slightly increased,while in Stattic?2.5?M?treated HaCaT cells,CEBPD expression exhibited significant upregulation,reflecting JAK/STAT3 signaling inhibition.Then,STAT3 was silenced with packaged lentivirus containing shSTAT3-RNAi?RNAi?,and the cells were treated with IL-17A for 24h.The results of real-time qPCR and immunoblot suggested that STAT3 was markedly downregulated by shSTAT3-RNAi,while CEBPD showed slight upregulation;in addition,no significant upregulation of these proteins was found upon IL-17A administration.These findings jointly indicated that JAK/STAT3 signaling activation played a role in CEBPD downregulation,while JAK/STAT3 inhibition significantly upregulated CEBPD.2.Successful STAT3 overexpression in HaCaT cells by packaged lentivirus.HaCaT cell transfection was carried out as described above.To assess transfection efficiency,cells infected with packaged lentivirus containing EGFP-STAT3?LV-STAT3?or control empty vector?Vector?were examined by fluorescence microscopy.Fluorescent cells were>90%of all cells in all high-power fields.Subsequently,real-time qPCR and immunoblot were employed to quantify STAT3 amounts in HaCaT cells,STAT3 mRNA and protein amounts were markedly higher in the LV-STAT3 group.3.Shikonin significantly suppresses proliferation and enhances apoptosis in HaCaT cells via CEBPD regulation.The MTS assay revealed that shikonin significantly decreased cell proliferation in both wild-type and LV-STAT3 HaCaT cells,in comparison with the control group,time-and concentration-dependently.Flow cytometric analysis further confirmed the changes in cell cycle distribution and apoptosis.Indeed,shikonin remarkably increased the apoptotic rates of both wild-type and LV-STAT3 HaCaT cells,while LV-STAT3 HaCaT cells were more sensitive to shikonin?P<0.05?.In agreement,cell cycle analysis demonstrated that shikonin remarkably arrested the cell cycle at G0/G1.Meanwhile,we assessed the cell cycle and apoptosis-related proteins cyclin E and cleaved-caspase 9 by immunoblot.The results corroborated flow cytometric analysis.Next,we measured CEBPD and STAT3 expression levels.Interestingly,shikonin markedly suppressed the JAK/STAT3signaling pathway and upregulated CEBPD.4.Shikonin alleviates imiquimod-induced mouse skin lesions by upregulating CEBPD.Clearly,IMQ caused psoriasis-like symptoms in the mouse skin.Lavage with shikonin markedly reduced these symptoms which included scaling and epidermal hyperplasia.H&E staining showed that the mouse skin showed severe keratinocyte hyperplasia after IMQ treatment and shikonin reduced epidermal thickness compared with the model group?IMQ?,especially the high dose shikonin group?SHI10?.Further,psoriasis-like lesions were assessed by IHC and Immunoblot.The results showed that JAK/STAT3 signaling was significantly activated in the IMQ group compared with control samples,whereas CEBPD amounts were decreased.Furthermore,shikonin restored the expression levels of STAT3 and CEBPD.Conclusions:1.Shikonin inhibits keratinocyte proliferation and induces apoptosis responsible for psoriasis treatment,mainly through the JAK/STAT3 dependent pathway.2.Activation of JAK/STAT3 downregulates CEBPD in HaCaT cells and IMQ-induced BALB/c mice.3.Shikonin can reverse the effect of JAK/STAT3 activation,suggesting CEBPD as a potential therapeutic target in psoriasis.