Potent Effects Of Epigallocatechin-3-gallate Against Oxidative-stress And Inflammation Damage In Human Epidermal Melanocytes Via Attenuation Of Oxidative Stress And And Immune Modulation

Objective①.Damage to melanocytes induced by oxidative stress plays an important role in the pathogenesis of vitiligo.A polyphenol found in green tea,(-)-epigallocatechin-3-gallate(EGCG),exhibits certain antioxidative effects in the treatment of various diseases.The major problem that limits the clinical application is its low bioavailability and stability.Peracetylated EGCG(AcEGCG),a fully acetylated derivative of EGCG,is more stable and bioavailable than EGCG,but the effects of its action on human epidermal melanocytes have not been elucidated.So,the aim of the first part is to compare the protective effects of AcEGCG and EGCG on hydrogen peroxide(H2O2)-induced damage to human melanocytes.②.Vitiligo is an inflammatory skin disorder and activated T cells play an important role in its onset and progression.Epigallocatechin-3-gallate(EGCG),the major chemical constituent of green tea,exhibits remarkable anti-oxidant and anti-inflammatory properties.EGCG was confirmed to directly inhibit protein kinases by working as an ATP analog.However,the underlying mechanism is undetermined.In the second part,we aimed to elucidate the inhibitory effects of EGCG on inflammatory signaling pathways.Methods①.Melanocytes were isolated from human foreskin specimens,and were cultured in Hu16 medium(F12 supplemented with 10%fetal bovine serum(FBS),20 ng/ml bFGF and 20 mg/ml IBMX)and cultured at 37℃ in an incubator with 5%CO2 and 95%humidity.The melanocytes were divided into eight groups:normal control group,H2O2 group,AcEGCG+H2O2 groups(10μM、20μM and 40μM)and EGCG+H2O2 groups(10μM、20μM and 40μM)The cell proliferation was measured with MTS assay.The supernatants were collected after different treatments for measuring LDH level.The reactive oxygen species(ROS)production,the mitochondrial membrane potential(Δψm),apoptosis,caspase-9,caspase-3,and p-p38 and p38 MAPK protein levels in human primary melanocytes were examined to evaluate the protective mechanisms.Statistical analysis was carried out by t test,analysis of variance and Student-Newman-Keul test.②.The molecular docking was performed by using LigandFit module embedded in Discovery Studio.To determine the effect of EGCG binding on JAK2 kinase activity,EGCG and its derivative,peracetylated EGCG(AcEGCG)were tested by Caliper mobility shift assay in vitro.Western blotting was used to determine the effect of EGCG on IFN-y-induced phosphorylation of JAK2 and its downstream STAT1 and STAT3 by.RT-PCR and ELISA assay were performed to examine the effect of EGCG on IFN-y-induced chemoattractant expression in melanocytes.Protein levels of the corresponding receptors including CD 11 a,CXCR3,and CCR2 in human T lymphocytes were measured by Western blotting after treatment by EGCG.The adhesion of human T cells to melanocytes induced by IFN-y were assayed using fluorescence microscope.Results①.Both AcEGCG and EGCG decreased ROS generation,restored lost mitochondrial membrane potential,and reduced H2O2-induced apoptosis in melanocytes.All of these effects were more pronounced with AcEGCG than with EGCG.Furthermore,AcEGCG effectively suppressed H2O2-induced p38 mitogen-activated protein kinase phosphorylation,which has been suggested to contribute to melanocyte damage.②.EGCG directly inhibited the kinase activity of Janus kinase 2(JAK2)In primary cultured human melanocytes,EGCG pre-treatment attenuated IFN-γ-induced phosphorylation of JAK2 and its downstream STAT1 and STAT3 in a dose-dependent manner.We further examined the chemoattractant expression in melanocytes and demonstrated that EGCG significantly inhibited IFN-γ-induced expression of ICAM-1,CXCL10,and MCP-1 in human melanocytes.In addition,EGCG reduced the protein levels of the corresponding receptors including CD11a,CXCR3,and CCR2 in human T lymphocytes.As a consequence,adhesion of human T cells to melanocytes induced by IFN-γ was effectively suppressed by EGCG.Conclusion①.AcEGCG and EGCG exert their effects via the same pathways in human melanocytes.AcEGCG was more effective than EGCG in inducing antioxidation,inhibition of apoptotic signalling and phosphorylation of p38,possibly to its higher stability and higher bioavailability.Taken together,our results indicate that AcEGCG has the potential to act as a therapeutic agent in vitiligo treatment.②.EGCG inhibited the chemoattractant expression in melanocytes and the expression of the corresponding receptors in T lymphocytes via directly targeting JAK2 kinase,leading to the decrease of lymphocyte migration/accumulation in vitiligo lesion as well as the weakening of T cell adhesion to melanocytes.The overall effects will be less activation of auto-reactive T lymphocytes and less destruction of melanocytes.Taken together,our results provided new evidence for the effectiveness of EGCG in vitiligo treatment and supported JAK2 as a molecular target for vitiligo medicine development.