In this study, we investigated a new functional compound which showed activities like RA without its side effects, such as not inducing IL-1 in keratinocytes. RA and retinoids have been considered among the most beneficial compounds for the improvement of wrinkles [29
]. Many studies have presented functional evidence of these effects by amassing clinical data on skin [30
]. These reports show that RA stimulates cell proliferation in the basal and spinous layers of the skin, leading to thickening. In addition, RA further modulates ECM proteins synthesis and inhibits collagenase production through retinoid receptors and AP-1 activation in vivo
]. Recently, some studies have suggested that RA induced HB-EGF production and EGER signaling [13
]. However in vitro
, meaning in monolayer keratinocytes, the effect of RA has not been clarified and a useful method for screening RA-like compounds has not existed. Therefore, we established an easy screening method to observe morphological changes of differentiated cells under the confluent conditions. Using this method, we investigated new compounds like RA. As a result, we determined that silybin shows cell shape changes similar to RA under a differentiated condition (Fig. A). Silybin treatment does not cause flattened cell shape changes, and show cell shapes like proliferative cells. Furthermore we examined the effect of silybin on the expression of differentiation-associated markers, and showed that silybin reduced the expression level of the terminal differentiation markers transglutaminase1, loricrin and keratin1 (Fig. C). These results demonstrate that both silybin and RA inhibit confluent-induced keratinocyte differentiation.
Silybin has traditionally been used to treat liver disorders [18
]. It has also been known that silybin exerts its effect by it strong anti-oxidant activities [31
]. A number of studies also have shown that silybin suppress UV-induced photocarcinogenesis and cell damage by its antioxidant effects [32
]. Because we used a confluent-induce differentiation condition, it can be assumed that the effect of silybin in this study was independent of silybin’s strong anti-oxidant activities. Furthermore silybin did not bind and activate retinoid receptors such as RARα and RXRα (Fig. ). These data may indicate that silybin shows RA-like cellular morphological changes through other mechanism and signal cascade. Our studies also revealed that RA stimulated IL-1α production in keratinocytes, but silybin did not (Fig. ). This data may support that silybin modulate through the different pathway with RA.
However, our results showed that both RA and silybin stimulated the expression basement membrane proteins, laminin-5 and integrin β4 (Fig. ). In addition our finding, it has been observed that silybin reduces MMPs (matrix metalloproteinase) activation in tumors [34
] and also inhibits UVB- and EGF-induced signaling involving AP-1 and nuclear factor-κB (NF-κB) in JB6 cells [35
]. It has been known that RA also influenced these molecules [6
]. Although there are various difference in cell types and stimulating inducers that used between these studies and our study, these results indicate that silybin and RA show both different and similar effect, and their control mechanism may be partly different. It might be suggested that silybin directly targets downstream of RA signaling pathway or modulate through non-classical routes of RA, such as activation of HB-EGF and unknown mechanism. Although further studies will be necessary to understand the mechanism that silybin affect in this confluent-induced condition, these studies in silybin may be useful tool for particular understanding of RA signaling cascade.
We demonstrated that RA stimulated IL-1α production in keratinocytes, but silybin did not (Fig. ). This difference results in IL-1α induction has led us to conclude that silybin is a more useful and safe compound in keratinocytes and skin, because silybin shows RA-like activities without inducing inflammatory mediators. It seems that silybin does not appear to have a negative RA effect in keratinocytes and skin. Recently it has been shown that silymarin, is composed primarily of silybin, has the ability to protect mice from UVB-induced immunosuppression and that this protective effect is mediated, at least in part, through IL-12 [36
]. This activity which regulates the secretion of cytokines may contribute to the inhibition of IL-1α secretion directly.
We also found that both silybin and RA induced laminin-5 and the laminin receptor integrin β4 (Fig. ). Laminin-5 is a major basement membrane component. Several studies indicate that basement membrane components are affected by photoaging, but that these effects are modulated by RA. In this study, we revealed that laminin-5 protein synthesis was induced by silybin and RA treatment. Another laminin-5 receptor integrin β4, was also induced by silybin and RA treatment. These effects on the production of these two proteins may explain that both silybin and RA induced the similar cell phenotype changes, although RA shows these activities through retinoid receptors while silybin does not. A method that measures the production of these proteins might be useful for the screening of RA-like compounds. In addition, silybin might modulate basement membrane components, and might affect photoaging, inducing an improvement in wrinkling.
In summary, the results presented in this report demonstrate that silybin inhibited confluent-induced keratinocyte differentiation and modulates the production of basement membrane components like RA. However, unlike RA, silybin did not induce inflammatory changes such as the stimulation of interleukin-1α in keratinocytes. These results strongly suggest that silybin might be a potential agent for the prevention and safe treatment of skin aging and wrinkling.