羟基积雪草苷对氧化应激条件下黑素细胞的保护作用及其诱导自噬机制的研究  

Protective effect of madecassoside on H_2O_2-induced oxidative stress and autophagy activation in human melanocyts

作　　者：凌雨婷 龚晴丽 熊喜喜 孙利[1] 丁书红 赵文娥[2] 朱文元[1] 鲁严[1] 

机构地区：[1]南京医科大学第一附属医院皮肤科,江苏南京210029 [2]南京医科大学分析测试中心,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2017年第5期569-574,606,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81171517);江苏省"333"工程项目(BRA2013279)

摘　　要：目的:积雪草是一种传统中医药物,具有很多医学作用,如创伤愈合和抗氧化。中医也发现它可用于白癜风复色和炎症后色素沉着。本研究评估羟基积雪草苷对氧化应激下人黑素细胞作用效果及机制。方法:正常的人黑素细胞予0.01 mmol/L过氧化氢处理,再予不同浓度的羟基积雪草苷(0、10、50、100μg/mL)。评估人黑素细胞树突的回缩速率,流式细胞术及细胞内游离钙浓度([Ca^(2+)]_i)检测线粒体膜电位(MMP),透射电子显微镜(TEM)观察线粒体超微结构,吖啶橙染色检测自噬,蛋白质印迹检测自噬体形成的指标LC3-Ⅱ/LC3-Ⅰ的比值。结果:在过氧化氢引发的氧化应激条件中,羟基积雪草苷在一定浓度下抑制了黑素细胞树突的回缩,提高了膜电位并减少了[Ca^(2+)]_i的积累,减弱了线粒体的损伤,增强了自噬活动。结论:人黑素细胞遭受氧化应激损伤时,羟基积雪草苷可通过自噬激活起到抗氧化作用。因此,羟基积雪草苷可成为氧化应激引起的白癜风的有效治疗手段。Objective： Centella asiatica （L.） Urb. is a traditional Chinese medicine that has many medical applications, including wound healing and anti-oxidation. Some traditional Chinese Medicine doctors have found that it has therapeutic effects for external use in the repigmentation of vitiligo and post-inflammatory hypopigmentation. This study was designed to evaluate the effects of madecassoside, a major bioactive component of C. asiatica, on oxidative stress in human melanocytes and its possible mechanism of action.Methods： Normal human melanocytes were treated with 0.01 mmol/L H2O2 and various concentrations of madecassoside （0, 10, 50, 100 μg/mL）. Subsequently, the retraction velocity of melanocyte dendrites was assessed. Determination of mitochondrial membrane potential （MMP, ΔΨm） was performed by flow cytometry and intracellular calcium （[Ca2＋]i） levels were measured. Alterations of mitochondrial ultrastructure were observed by transmission electron microscopy （TEM）. Acridine orange （AO） staining was used to measure autophagy. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, was analyzed by Western blot.Results： Under H2O2-induced oxidative conditions, madecassoside inhibited melanocyte dendrite retraction, improved MMP and reduced the accumulation of [Ca^2＋]i in a concentration-dependent manner. Observations by TEM showed that madecassoside attenuated the damage of mitochondria in human melanocytes caused by oxidative stress. Furthermore, autophagy activation was demonstrated by AO staining and an increased LC3-Ⅱ/LC3-Ⅰ ratio.Conclusion： These results demonstrate the antioxidative effect of madecassoside on human melanocytes subjected to oxidative damage is activated by autophagy. Moreover, madecassoside could be a promising treatment for vitiligo mainly caused by oxidative stress.

关 键 词：羟基积雪草苷 氧化应激 自噬 线粒体 白癜风 黑素细胞 

分 类 号：R758.41[医药卫生—皮肤病学与性病学]