黄芩苷对H_2O_2氧化损伤人黑素细胞的保护机制研究

Protective effects of Baicalin to the human melanocyte cocultured with H_2O_2

目的:探讨黄芩苷对H_2O_2氧化损伤正常人黑素细胞的保护作用及其机制研究。方法:构建H_2O_2氧化损伤黑素细胞模型,通过观察不同浓度的黄芩苷处理H_2O_2氧化损伤正常人黑素细胞,采用倒置显微镜观察黄芩苷对黑素细胞形态的影响;cell counting kit-8(CCK-8)法检测黄芩苷对黑素细胞增殖活力作用;乳酸脱氢酶(lactate dehydrogenase,LDH)法检测黄芩苷对黑素细胞LDH表达的影响,并通过流式细胞仪检测活性氧(ROS)水平,荧光实时定量RT-PCR检测过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶1(peroxidase 1,GPX1)m RNA表达情况,通过ELISA试剂盒检测黄芩苷和H_2O_2处理黑素细胞后分泌的热休克蛋白70(heat shock protein 70,HSP70)。结果:镜下观察黄芩苷给药后细胞较对照组形态明显改变,细胞数增多;黄芩苷作用黑素细胞后增殖率较对照组升高(P<0.05);黄芩苷明显抑制黑素细胞中LDH表达,呈剂量-效应关系(P<0.05)。黄芩苷能显著促进CAT和GPX1m RNA表达,且明显抑制HSP70分泌。结论:黄芩苷对H_2O_2氧化损伤的正常人黑素细胞具有明显保护作用,可为临床白癜风的治疗提供参考。

AIM：To study the mechanism of anti-oxidant and melanocyte protection by Baicalin.METHODS：Different concentration of Baicalin and H2O2 were coeuhured with normal human melano-cyte.Cell proliferation was detected by cell counting kit-8 assay, morphology of the human melanocyte was observed by ordinary inverted microscope,and LDH assay was used to detect cell viability.For mechanism research,DCFH-DA ROS level was assayed by flow cytometry.Induced HSP70 from melanocyte was detected using HSP70 high sensitivity ELISA kit.By RT-PCR,mRNA levels of CAT and GPX1 were dectected.RESULTS：Baicalin has obvious protective effects on melanocytes damage caused by H2O2.Compared with control group,in Baicalin groups,cell shape was changed and increased.In 12.5, 25, 50μmol/L Baicalin groups, the effects of on melanocyte proliferation rate were higher than the control group （P 〈0.05 ）.Baicalin inhibited the expression of LDH in black pigment ceils in a dose-dependent manner,there was statistically significant difference（p〈0.05）.After H2O2 oxidative damage of melanoeytes,Baicalin could sig-nificantly promote the expression of CAT and GPX1 mRNA,and the secretion of HSP70 was significantly increased.CONCLUSION：Baicalin protects human melanocytes from oxidative damage by inhibiting H2O2induced apoptosis.It is referential for the treatment of vitiligo.