DISCUSSION

TA is widely considered as one of the treatment options for melasma. In 1979, its role in melasma was first demonstrated (Sadako, 1979). Afterwards, many clinical studies with oral TA treatment showed favorable responses in melasma. Other forms of TA administration for melasma also have been investigated. Weekly intradermal injections of TA (Lee et al., 2006), ionotophoresis of TA using chemical enhancer and constant electric current (Todo and Sugibayashi, 2012), and topical TA in liposome formulation was also introduced (Manosroi et al., 2002). Until now, only two clinical studies using topical TA has been reported (Kanechorn Na Ayuthaya et al., 2012; Na et al., 2013). However, the histopathologic analysis was not available in the study of Kanechom Na Ayuthaya et al. and the concurrent use of oral and topical TA in the study of Na et al. could not demonstrate the independent effect of topical TA in melasma. In our study, we investigated the independent effect of topical TA treatment on melasma with histopathologic analysis. Several findings were noted and discussed below.

First, with clinical assessment, time-sequential improvement of MSS and mMASI were noted, especially significant during the first 8 week. However, the additional improvement was minimal after 8 week. Consistent with the change of MSS and mMASI score, subjective satisfaction score showed the similar tendency during 12 weeks. With chromameter measurement, the significant improvement of the value of L* and a* in lesional skin was performed using oral TA, the results were relatively comparable to our results. It is explained that TA has a limitation of binding capacity in that TA reversibly blocks lysine binding sites on plasminogen molecules (Tse and Hui, 2013).

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Contrary to our result, TA rather increased pigmentation of the perilesional skin from 0 week to 4 week, and to 8 week in previous report (Na et al., 2013). They suggested the darkening of perilesional skin is affected by seasonal changes, from spring to summer (Na et al., 2013). However, in our study, the L* and a* value indicated that the perilesional normal skin after treatment became brighter than those of baseline status, which showed a similar pattern to those of lesional skin. Considering our result, topical TA might effect on perilesional normal skin as well as lesional skin of melasma. Also, the aggravated erythema at 12 weeks on TA-treated site was reported previously (Kanechorn Na Ayuthaya et al., 2012), however, our result showed no worsening in erythema, although no further improvement was shown in the erythema index after 4 week. No other significant adverse effects, such as irritation were reported except mild dryness in some subjects.

Secondly, with histological analysis, we focused on three main factors i.e., pigmentation, vascularity, and mast cell. Consistent with our clinical assessment and previous report, the epidermal pigmentation significantly reduced in both lesional and perilesional normal skin. It has been known that TA could suppress the process of angiogenesis as well as induce the melanogenesis in melanocytes (Kal et al., 2004; Na et al., 2013). It was reported that TA also inhibits the neovascularization induced by basic fibroblast growth factor (bFGF) (Kal et al., 2004). The positive correlation between the induction of pigmentation and increase of dermal blood vessels in lesional skin of melasma was suggested (Kim et al., 2007), and the significant decrease of vessel numbers as well as the reduction of pigmentation during TA treatment was also reported (Na et al., 2013). In this study, the decreased CD31-stained blood vessel in dermis was observed in lesional and perilesional normal skin after topical TA treatment, though not statistically significant. In terms of mast cells, which were more prominent in the elastotic areas of melasma skin (Hernandez-Barrera et al., 2008), significantly decreased number of mast cells was observed after 8-week use of oral and topical TA (Na et al., 2013). However, there was little difference in the number of c-kit-stained mast cell after topical TA treatment in our study. Taken together, topical TA treatment could reduce the epidermal pigmentation, although their effect on dermal environment showed limited, compared with oral TA.

Thirdly, our data showed the significant decrease of ET-1 expression after treatment in epidermis of both lesional and perilesional normal skin. It has been reported that paracrine

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linkages among keratinocytes, fibroblasts, and melanocytes play important roles in regulating epidermal pigmentation. In response to various stimuli, human keratinocytes secrete various cytokines that serve as mitogens or melanogens for human melanocytes, including ET-1, α-MSH, and SCF (Imokawa et al., 1992; Morita et al., 1994). Among them, ET-1 can trigger melanogenesis via the MITF-regulated GPNMB pathway and plays an indispensable role in epidermal pigmentation in hyperpigmentary disorders (Zhang et al., 2013). We demonstrated the significant decrease of ET-1 expression in the epidermis of both lesional and perilesional normal skin. Also, the expression of SCF, one of the cytokines secreted by human keratinocytes and human fibroblasts (Imokawa et al., 1998), was significantly decreased after treatment in the epidermis of lesional skin. However, there was no significant difference in the expression of α-MSH after treatment in both lesional and perilesional normal skin. Therefore, our data is speculated that both ET-1 and SCF might play an important role in action mechanism of TA on melasma. TA is known to suppress the production of prostaglandins, and subsequent UV-induced melanogenesis through the suppression of the epidermal plasmin activity. Lesser free arachidonic acids and depleted production of prostaglandins result in the reduction of tyrosinase activity in melanocytes (Maeda and Naganuma, 1998; Maeda and Tomita, 2007). However, there was no significant difference in the expression of EP2 after treatment in both lesional and perilesional normal skin. Also, human melanocytes may respond to angiogenic factors because normal human melanocytes express functional VEGF receptors (Kim et al., 2005), and the expression of VEGF was significantly increased in melasma (Kim et al., 2007). In contrary, the expression of VEGF after treatment showed minimal difference. It is suggested that dermal structures were not affected by topical TA possibly because of their low penetration into the dermis.

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