| Vitiligo is an acquired and progressive disorder manifested by circumscribed depigmented patches on the skin. It affects approximately 0.1 to 2.0% of the world population[1]. The etiology is still unknown but several hypotheses (including neural, radical, autoimmune,self-destruction and inherent defect theory) have been proposed to explain the selective destruction of melanocytes. Recent studies revealed: there were accumulation of hydrogen peroxide (H2O2) in vitiligo patients and resulted in melanocyte morphology and function [2-5]. Dell, etc. [6] adopted the combined antioxidants and NB-UVB treatments, got good results. These showed that an imbalance of oxidative stress existed in vitiligo patients, and H2O2-mediated oxidative injure played a key role in vitiligo.Objective:To investigate the possible protective effect of Quercetin, Resreratol, Extract of Ginkgo Biloba leaf and Vitmin C on the immortalized mouse melanocyte (B10Br) through the establishment of oxidative stress in vitro model and their impact on the biological activity of melanocytes. To find an adjunctive therapeutic approach in vitiligo. Methods:Cell viability was measured by using MTT assay, H2O2–induced apoptosis rate and cell morphology changes were observed by flow cytometry and microscope, the effect of quercetin on tyrosinase activity and melanin synthesis were measued by DOPA oxidation assay and NaOH-assay.Results:1 Establishment of hydrogen peroxide-induced oxidative stress model of melanocyte in vitroThe activity of melanocytes decreased significantly after treated by different concentrations of hydrogen peroxide for 24 hours, and showed a concentration-dependent manner. The cell activity decreased to about (53.88±3.00) % (P <0.01), when treated with 200μM hydrogen peroxide. The activity of melanocytes decreased significantly after treated with 200μM hydrogen peroxide for different hours and showed a time-dependent manner. The cell activity decreased to about (54.53±2.23) % (P <0.01), when treated for 24 hours. Flow cytometry: Early and late the percentage of apoptotic cells was significantly increased. Microscopic pictures showed: dendrite shortened or disappeared, the number of adherent cells reduced, suspended cells gradually increased, cell debris appeared, showed a dose/time dependent manner.2 The effect of Chinese monomer on the melanocytesThe activity of melanocytes was measured after treated by different concentrations of antioxidants for 24 hours. Selection of the activity of melanocytes smaller (P> 0.05) concentration of the antioxidants to continue the following tests. 3 The protective of Chinese monomer on the melanocytes against hydrogen peroxide-induced oxidative stressCell viabilities were measured when B10Br cells were pretreated with 33.33μM Quercetin, 0.33μM Resreratol, 16.67μM Extract of Ginkgo Biloba leaf for 24h prior to exposure to 200μMH2O2 for 24h. Cell activity reversed to (94.22±3.36) % (P <0.01); (77.42±7.48) %; (P <0.05) and (76.57±2.99) % (P <0.01), respectively. However, cell acitivity pretreated by vitamin C didn't increase, only (59.93±2.16) % (P> 0.05). Flow cytometry: Early and late the percentage of apoptotic cells was significantly decreased after pretreated with antioxidants. Microscopic pictures showed: dendrite didn't retract apparently, the number of adherent cells increased. But, dendrite shortened significantly when pretreated by Vitamin C.4 The biological effects of Chinese monomer on melanocytes The tyrosinase activity and melanin content of melanocytes decreased after treated with 200μM hydrogen peroxide. However, they increased significantly after pretreated with antioxidants. Querccetin showed more significantly.Conclusions:Hydrogen peroxide decreased melanocytes activity by induced cells apoptosis, and showed in a concentration/time-dependent manner. Quercetin, resveratrol, and Ginkgo biloba extract can increase the activity of cells by inhibiting their apoptosis.Moreover, Quercetin, resveratrol, and Ginkgo biloba extract can increased tyrosinase activity and melanin content of melanocytes.
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