Shikonin Inhibits IL-6 And IL-17 Production Induced By IL-23 In Peripheral Blood Mononuclear Cells Of Patients With Psoriasis

ObjectiveThe recent discovery of CD4+T cells characterized by secretion of interleukin-17—Th17 cells has been linked to the pathogenesis of psoriasis. Interestingly, recent evidence has suggested that IL-23/Th17 axis plays a provotal role in the pathogenesis of psoriasis. Lithospermum erythrorhizon Sieb. Et Zucc. is a traditional Chinese medicine(TCM), which has cooling blood, activating blood circulation, detoxication, promoting eruptions effects, and used for the treatment of psoriasis by systemic therapy. Shikonin, a fundamental ingredient of Lithospermum erythrorhizon, is a naphthoquinone. Therefore, to be used for the treatment of psoriasis is indispensable. But its exact mechanism remains unknown. To explore the mechanism of shikonin on psoriasis, we investigate the role of shikonin on IL-6 and IL-17 production induced by IL-23 in peripheral blood mononuclear cells of patients with psoriasis.Materials and Methods1.Subjects(1) Psoriasis patient group:twenty psoriasis patients were collected from dermatological institution, The First Affiliated Hospital of China Medical University, including 12 males and 8 females, ages 23～55 years, median 40.9 years.(2) Control group:eleven healthy volunteers, including 5 males,6 females, ages 27～50 years, median 32.1 years.2.MaterialsCapital Materials:rIL-23 was purchased from BD/Pharminen. Human IL-23 ELISA Kit was obtained from R&D. Human IL-17 ELISA Kit was the product of CUSABIO BIOTECH Co.Ltd. Human IL-6 ELISA Kit was obtained from Boster. CCK-8 was purchased from WST. Shikonin was the product of WAKO. CyA was obtained from Merk. RPMI1640 medium and PBS were purchased from Hyclone. Ficoll was obtained from GE-healthcare bio-sciences AB. DMSO was purchased solarbio.3.Methods(1) Reagents:Stock solution of shikonin was prepared in DMSO. The final concentration of DMSO in experimental buffers was <0.5%(V/V). In addition, CyA was dissolved in DMSO. The final DMSO concentration in experimental buffers was <0.5%(V/V).(2) Serum levels of IL-23 were measured by ELISA:Sera were taken from psoriasis patients and healthy volunteers, then analyzed using a human IL-23 ELISA Kit according to the manufacturer instruction.(3) PBMCs:Cells were prepared from Peripheral blood of psoriasis patients. PBMCs isolated from density gradient centrifugation, then washed with PBS and counted. The cells were cultured at 6×108cells/L in RPMI1640 medium.(4) Cell culture:6×108cells/L PBMCs isolated from density gradient centrifugation and were cultured in RPMI1640 medium,obtaining 20% fetal bovine and 1% benzylpenicillin and streptomycin in a 5% CO2 humidified atmosphere at 37℃in the presence or obsence of various stimuli for 72 h.(5) Cytotoxicity assay:Cytotoxicity of shikonin was measured by CCK-8 assay. PBMCs in the presence or obsence of various stimuli for 72 h were harvested and counted, then cultured in 100ul RPMI1640 medium in 96-well plate and 10ul CCK-8 was added to each wells at 37℃for 4h. The absorbance of each well at 450 nm was measured by using an ELISA plate reader. The cell viability(%) of shikonin for PBMCs was calculated:Cell viability(%)=[(As-Ab)/(Ac-Ab)]×100%.(6) Supernatants were harvested and analyzed using human IL-6 and IL-17 ELISA Kits according to the manufacturer instruction.Results1.Serum levels of IL-23A significantly higher level of IL-23 in the sera of psoriasis patients compared with controls (p<0.001).2.IL-23 stumulates production of IL-17PBMCs were cultured with various concentrations of IL-23 (10-100ng/ml), resulted in higher levels of IL-17 production than in the obsence of IL-23 (p<0.001).3.Effect of shikonin on IL-17 production and cell viability(%)PBMCs were cultured with various concentrations of shikonin(0.2-5ug/ml), resulted in higher levels of IL-17 production than medium. In addition, shikonin(2ug/ml) stimulated highest level of IL-17 production. In contrast, shikonin (<7.5ug/ml) decreased IL-17 levels in a dose-dependent manner. PBMCs were cultured with shikonin(>20ug/ml), resulted in lower cell viability(%) than medium in a dose-dependent(p<0.001).4.Effect of shikonin on IL-17 production induced by IL-23 and cell viability(%)PBMCs cultured in the presence or obsence IL-23 (lOng/ml) and increasing concentration of shikonin(<7.5ug/ml) decreased levels of IL-17 production(p<0.001). PBMCs were cultured in the presence IL-23 (10ng/ml) with shikonin(>20ug/ml), resulted in lower cell viability(%) than medium(p<0.001).5.Effect of cyclosporine A(CyA) on IL-17 production induced by IL-23 and cell viability(%)PBMCs cultured in the presence or obsence IL-23 (10ng/ml) and increasing concentration of CyA(<7.5ug/ml) decreased levels of IL-17 production(p<0.001). PBMCs were cultured in the presence IL-23 (10ng/ml) with CyA(≥0ug/ml), resulted in lower cell viability(%) than medium(p<0.001).6.Shikonin inhibits IL-6 and IL-17 production induced by IL-23 and Effect of shikonin on cell viability(%)PBMCs were cultured in the presence IL-23 (10ng/ml) with shikonin (12.5ug/ml), resulted in lower levels of IL-6 and IL-17 production compared with in the presence IL-23(10ng/ml)(p<0.001). PBMCs were cultured in the presence IL-23 (10ng/ml) with shikonin(>20ug/ml), and didn't result in lower cell viability(%) than medium(p>0.05).7.CyA inhibits IL-6 and IL-17 production induced by IL-23 and Effect of shikonin on cell viability(%)PBMCs were cultured in the presence IL-23(10ng/ml) with CyA(12.5ug/ml), resulted in lower levels of IL-6 and IL-17 production compared with in the presence IL-23(10ng/ml)(p<0.001). PBMCs were cultured in the presence IL-23(10ng/ml) with CyA (≥20ug/ml), and didn't result in lower cell viability(%) than medium(p>0.05).ConclusionShikonin could inhibit IL-6 and IL-17 production induced by IL-23 in peripheral blood mononuclear cells of patients with psoriasis.