For centuries, YCJ has been used for its various therapeutic effects, but not for wound healing. We were the first research group to report back in 2006 that ovx rats receiving YCJ at 100 mL/kgBW had significantly better wound healing, including less scarring, brighter skin, and softer hair, as compared to controls [10]. However, these observations were only based on gross morphological evaluation at that time. To take such observations one step further, we conducted the current study aiming at investigating at the microscopic level the changes taking place inside the wound following the intake of rats of YCJ over a seven and 14 days period. Moreover, the phytoestrogenic property of YCJ, as well as the possible role of such property in quicker wound healing was explored.

Our results demonstrated that the intake of YCJ over one week was not enough to produce significant microscopic healing changes in the wound area, as compared to controls. However, such changes were prominent when the intake continued for one more week. This was evident in the accelerated wound healing that was characterized by reduced wound depth and width, increased thickness of the epidermis and dermis, thicker and more abundant collagen fibers and hair follicles, and density of immunostaining against ER-α, and ER-β in the epidermis, dermis, and hypodermis. Such results seem to provide evidence to the observation that YCJ has phytoestrogenic properties, and that the latter could both act as SERM and play a role in the homeostasis of the epidermis, dermis, and hypodermis. This is supported by our recent experiment in which we used gas chromatography-mass spectrometry to confirm that the phytoestrogens of YCJ were sitosterol, stigmasterol, and campesterol [11]. This is also supported by other studies showing a stronger positive in vitro stimulatory effect of a SERM, Raloxifene, on collagen biosynthesis by human skin fibroblasts, as compared to traditional estrogen treatment [12]. This is also supported by the fact that collagen biosynthesis and deposition are an essential aspect of successful wound healing. Fotsis and colleagues reported that Genistein, one of the known isoflavones which have been shown to interact with animal and human estrogen receptors, was able to significantly reduce the degradation of the extracellular matrix in the skin [13]. Pirilä and co-workers investigated the effects of estrogen and a potent matrix metalloproteinase inhibitor, chemically modified non-antimicrobial tetracycline, CMT-8, on wound healing in ovx rats [14]. They observed that estrogen can promote wound healing in ovx rats by normalizing wound bed total collagen structure and content, and by recovering the processing and expression of essential molecules in wound healing, such as laminin-5 gamma2-chain [14]. Pirilä and co-workers also demonstrated in a separate study that estrogen has a beneficial effect on skin wound healing in ovx rats by increasing the collagen content, and by reducing the matrix metalloproteinases (MMP)-mediated collagenolysis [15]. Shuster and co-workers reported a direct relationship between skin collagen and dermal thickness in a study correlating age and sex with skin thickness and collagen density [16]. Azzi and colleagues described specific effects of sex steroids on skin morphology [17].

Both estrogen and SERMs act through binding to homodimeric and heterodimeric ERs. SERMs, in addition, have the potential to selectively induce ER isoforms in a specific target tissue, thus amplifying further the effects on down-stream gene expression. Estrogen acts via ER, and there are currently two known estrogen receptors, denoted ER-α and ER-β. ER-α is expressed in the skin of both humans and rodents, whereas conflicting results have been reported regarding the expression of ER-β in the skin [18–20]. Therefore, we examined in this study the effect of YCJ intake on the expression of ER-α and ER-β in the wounded and normal skin, exploring the possibility that it has phytoestrogenic properties, and that it could act as SERM. Our results demonstrated both ER-α and ER-β were detected in the epidermis, dermis, hypodermis, hair follicles, sebaceous glands, keratinocytes, fat cells, fibroblasts, and panniculus carnosus (skeletal muscles) of the normal and wounded skin. Interestingly, the expression of ER-α and ER-β was significantly the highest in the group which received YCJ following the excision of the ovaries. Such expression was noted in both the nucleus and the cytoplasm. Hart and colleagues reported that the acute effects of estrogen on neuronal signaling were most likely mediated by extra-nuclear estrogen receptors associated with the plasma membrane and/or cytoplasmic organelles [21]. Thornton and co-workers demonstrated that ER-β was strongly expressed in the nucleus, while ER-α was present in diffuse cytoplasmic granules in human dermal papilla cells [22].

Our results showed that the number of hair follicles and their diameter either in the dermis or in the hypodermis of the ovx+YCJ group was the highest and largest, respectively, while the dermis and hypodermis of the ovx+EB group contained the lowest number of follicles and their diameter was the smallest. In contrast, we observed a strong negative correlation between the serum E2 level and the number and size of the hair follicles in the ovx+YCJ and ovx+EB groups. These findings indicate that YCJ had an agonistic effect on hair follicles, while exogenous estrogen (EB) has an antagonist effect. In agreement with our findings, E2 was found to effectively block hair growth in female mice [20]. In humans, E2 was found to be locally produced by the hair follicles [23]. Furthermore, a slower rate of replacement of spontaneous hair loss or plucked hair has been observed in pregnant women, an effect possibly related to the high levels of circulating estrogens [24].

The importance of hair follicles in skin biology does not rest solely with its ability to produce hair. Hair follicles are self-renewing, contain reservoirs of multipotent stem cells that are capable of regenerating the epidermis, and, accordingly, are thought to be actively involved in accelerating wound healing [25, 26]. The present study supports this theory.