Protective effect of madecassoside on H 2 O 2 -induced oxidative stress and autophagy activation in human melanocytes

// Yuting Ling 1 , Qingli Gong 1 , Xixi Xiong 1 , Li Sun 1 , Wene Zhao 2 , Wenyuan Zhu 1 and Yan Lu 1 1 Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, 210029, P.R. China 2 Department of Analysis and Testing Center, Nanjing Medical University, Nanjing, Jiangsu Province, 210029, P.R. China Correspondence to: Yan Lu, email: luyan6289@163.com Keywords: madecassoside, oxidative stress, autophagy, mitochondria, vitiligo Received: December 28, 2016      Accepted: April 19, 2017      Published: May 07, 2017 ABSTRACT Background: 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. Results: In H2O2-induced oxidative conditions, madecassoside inhibited melanocyte dendrite retraction, improved MMP and reduced the accumulation of [Ca2+]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. Materials and Methods: Normal human melanocytes were treated with 0.01 mM H2O2 and varying 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 ([Ca 2+ ]i) level 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. Conclusions: These results demonstrate the antioxidative effect of madecassoside on human melanocytes subjected to oxidative damage via the activation of autophagy. Moreover, madecassoside could be a promising treatment for vitiligo mainly caused by oxidative stress.