Shikonin Inhibits VEGF, IL-6 And IL-23 Production In IL-17-stimulated HaCaT Cells

ObjectivePsoriasis is a chronic autoimmune disease characterized by a strong genetic basis, infiltration of the dermis and epidermis by activated T cells and the hyperproliferation and abnormal differentiation of keratinocytes (KCs). The typical lesions of psoriasis are erythemas and scales. Nowdays the pathogenesis of psoriasis is regard as a kind of T cells mediated immune response resulting in epidermal KCs hyperproliferation and differentiation. The cytokines expressed by KCs take part in the memory and activation of T cells. The cytokines secreted by T cells and KCs may compose a complex cytokine network and lead to specific immune pathological damage of psoriasis. Recent studies consider that Th17 cells are a newly discovered CD4+ helper T-cell subpopulation which can secrete interleukin-17 (IL-17A). TGF-β, IL-1β, IL-6, IL-21 and IL-23 involved in Th17 cells differentiation. IL-17 can stimulate KCs to produce cytokines including vascular endothelial growth factor (VEGF), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-a) and chemokine (C-X-C motif) ligand 10 (CXCL10). The cytokines may induce and aggravate the psoriasis.Lithospermum erythrorhizon (LE) belonging to Boraginaceae perennial has long been used in traditional Chinese herbal medicine. The roots of LE have anti-inflammatory, anti-tumor, hepatoprotective effect, immune regulation, contraception, sterilization and other anti-virus effect. Lithospermum aqueous extract, ethanolic extract and ether extract have the same effect. Lithospermum erythrorhizon (LE) is considered as the main drug in the treatment of psoriasis. The main components of Lithospermum erythrorhizon (LE) are fat-soluble pigments of naphthalene quinones including shikonin and its derivatives and a water-soluble components including Lithospermum polysaccharide that accounts for about 2%. In this study, we observed the serum levels of IL-17 with psoriasis patients and investigated whether IL-17 could stimulate the production of cytokines on HaCaT cells alone. We also investigated whether shikonin could inhibited the proinflamation effects of IL-17 acting on HaCaT cells. The purpose of this study was to explore the effect of cytokines in the pathogenesis of psoriasis and the mechanism of shikonin treatment for psoriasis.Subjects and MethodsSubjects and Main Materials1. Subjects(1) Psoriasis patients The patients diagnosed with psoriasis clinically have a total of 23 (17 male and 6 female) at Dpartment of Dermatology, No.1 Hospital of China Medical University. The age distribution of patients is 23-74 years (mean 49±15 years). All patients without history of other autoimmune diseases.(2) Normal control group The voluntary donors whose number is 16 have no history of psoriasis and other autoimmune diseases. The sex and age of donors match with the group of psoriasis patients.All patients gave their informed consent to participate in the study. According to PASI (psoriasis area and severity index, psoriasis area and severity index), the patients with psoriasis were divided into mild, moderate and severe skin lesions patients.(3) HaCaT cell HaCaT cell was kindly provided by Benjamin Franklin Medical Center, Free University of Berlin, German.2. Main Materials(1) Main Reagents Recombinant human IL-17 was purchased from peprotech (USA). Shikonin was obtained from Wako (Japan) and cyclosporin A (CsA) from Merk company (Germany). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and antibioticantimycotice was purchased from Hyclone (USA). Trypsinase was purchased from GIBICO (USA). DMSO was obtained from AMRESCO (USA). Recombinant human IL-17 ELISA kit was purchased from Rapidbio (USA). Recombinant human IL-23 ELISA kit was obtained from R&D Systems (USA).Recombinant human VEGF and recombinant human IL-6 ELISA kit were purchased from Wuhan Boster (China). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo, Kumamoto (Japan).(2) Experimental instruments CO2 incubator, superclean bench, microscope, low speed centrifuge, table top high speed centrifuge, low temperature refrigeration,-80℃deep freezer refrigerator,-20℃freezer refrigerator,4℃refrigerator, computer pressure steam sterilizer, electric constant temperature water tank, microplate reader.Methods1. Experimental cells were divided into the stimulated group by IL-17 and the drug group depending on the different treatments administered HaCaT cells (1×105 cells per ml) were seeded on 12-well plate for 3 days in the different treatment conditions. Each group was established three holes and cultured in 6 times repeatedly.①The stimulated group by IL-17 were divided into four groups:control group (without agent, only DMEM for 24h), IL-17 group (IL-17 80ng/ml and DMEM for 24h), IL-17 group (IL-17 80ng/ml and DMEM for 36h), IL-17 group (IL-17 80 ng/ml and DMEM for 48h).②The drug groups were divided into four groups:control group (without agent, only DMEM for 24h), shikonin and IL-17 group (shikonin 50umol/L, IL-17 80ng/ml and DMEM for 48h), CsA and IL-17 group (CsA 10umol/L, IL-17 80ng/ml and DMEM for 48h), IL-17 group (IL-17 80 ng/ml and DMEM for 48h).2. The serum levels of IL-17 with psoriasis patients and the levels VEGF, IL-6, IL-23 in different treatment groups were detected by ELISA.3. The viability of HaCaT cells in the drug group was detected by CCK-8.ResultsThe serum levels of IL-17 with psoriasis patients were significantly higher than the healthy controls (18040.23±8045.54 v.s 2954.86±1433.74) and the difference was significant (P<0.05). According to PASI score, the serum mean relative expression levels of IL-17 in the patients of mild, moderate and severe skin lesions were 3466.37±1294.77,7928.59±1143.94,20319.74±6260.72 separately and the difference was significant (P<0.001).In the stimulation of IL-17 (80ng/ml) on HaCaT cells for Oh 24h,36h,48h the mean relative expression levels of VEGF were 340.76±57.80,333.54±56.58,510.11±107.62,791.70±55.85 respectively; the mean relative expression levels of IL-6 were 107.50±23.80,144.4844.26,210.48±30.02,220.37±35.74 and the mean relative expression levels of IL-23 were 96.49±4.35,62.37±8.50,121.35±13.10,161.91±12.77 separately. The expression of VEGF, IL-6, IL-23 in different time stimulation groups were higher than the control group and the differences were significant statistically (P <0.001).In the treatment with shikonin(50umol/L), CsA(10umol/L) and IL-17(80ng/ml) on HaCaT cells for 48h, the mean relative expression levels of VEGF were 328.45±35.97, 511.22±50.29,490.47±33.15,798.42±99.52 separately in the control group, 50umol/L shikonin+IL-17 group, lOumol/L CsA+IL-17 group and IL-17 stimulation group. The mean relative expression levels of IL-6 were 107.95±20.04,133.88±33.85,135.57±26.41,220.59±34.66 in the above-mentioned group. The mean relative expression levels of IL-23 were 100.07±10.25,119.16±16.47,129.28±13.36, 178.63±27.01 respectively. The expression of VEGF, IL-6, IL-23 in different treatment groups of drug on HaCaT cells were lower than the stimulated group by IL-17 and the differences were significant statistically (P<0.001).The cell survival rate of shikonin group was 101% and the CsA group also was 101%. The OD values of cell viability were 1.115±0.011,1.125±0.142,1.125±0.085, 1.126±0.129 separately in the control group,50umol/L shikonin+IL-17 group, lOumol/L CsA+IL-17 group and IL-17 stimulation group. The cell viability of different drug treatment groups have no significant difference (P>0.05). Conclusions1. It demonstrated that the serum levels of IL-17 of psoriasis patients were significantly higher than the healthy controls.2. IL-17 is proinflammatory cytokine, which can induce the secretion of proinflammatory cytokines (VEGF, IL-6, IL-23) in HaCaT cells.3. Shikonin markedly inhibits the proinflammatory effect of IL-17 on HaCaT cells.