Abstract:

In this study, normal human skin cells extracted from an aged donor were used to establish the in vitro senescent skin cell model. The bioenergetics effect was evaluated by measuring cellular ATP content using CellTiter-Glo® Reagent. Microscopic inspection and the semi-quantitative analysis methods of mitochondrial networks have been used to evaluate the mitochondria assembled into flexible networks in metabolically active cells. In the meantime, immuno-cytochemistry staining was used to futher understand the mechanism of mitochondria network formation, and the expression of Mitofusin-1 was investigated. With this model, the efficacy of Dragon’s Blood Complex （DBC）, a botanical complex composed of four active ingredients, was evaluated with normal human dermal fibroblasts and epidermal keratinocytes about its biofunctions on ATP production and mitochondrial integrity including morphology of mitochondria, aboundance of mitochondrial networks, and the expression of Mitofusin-1. The results show that the ATP level in senescent fibroblasts and keratinocytes is significantly increased by Dragon’s Blood Complex. The relative ATP content of fibroblasts is 117.7%±4.3% compared with the control group （P<0.01） when treated with 0.25% DBC. When treated with 0.5%, 0.25%, and 0.1% DBC, the relative ATP content of keratinocytes, compared with the control group, are 149.8%±9.0%, 147.5%±6.4%, and 134.0%±8.3%, respectively （P<0.001）. By incubation with 0.25% DBC, the count of mitochondrial network in senescent keratinocytes is significantly promoted （P<0.05）. And in the DBC-treated fibroblasts, the mitochondria networks are less fragmented and more extensively branched in morphology. Besides, Mitofusin-1 is more widely and abundantly expressed in the DBC-treated fibroblasts. This might partly explain the promotion effect of DBC on mitochondria networks. In summary, DBC shows anti-aging effects on fibroblasts and keratinocytes by promoting the cellular ATP and mitochondria. The evaluation method used in this study can also be applied to the screening of more active ingredients targeting the functions relevant with mitochondrial aging and bioenergetics.

Key words: skin aging, Dragon’s Blood Complex, mitochondrion, mitochondrial network, Mitofusin-1