IN VIVO PHOTOCHEMOPROTECTIVE EFFECT OF QUERCETIN INCORPORATED IN W/O MICROEMULSION BY INHIBITION OF METALLOPROTEINASES ACTIVITY

TF12

Fabiana Testa Moura de Carvalho Vicentini (1), Maria Vitória Lopes Badra Bentley (1), Yiru Xu (2), Gary Fisher (2), Maria José Vieira Fonseca (1)

(1) Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil(2) Department of Dermatology, University of Michigan, Ann Arbor, USA. 

Introduction: It has been demonstrated that ultraviolet exposure leads to sustained elevation of matrix metalloproteinases (MMPs), a family of proteolytic enzymes which contribute to skin photoaging and in skin carcinoma to the spreading of metastatic cells by specifically degrading skin collagen and elastin. Molecular mechanisms underlying the induction of MMPs after exposure to UV irradiation have been proposed. It has been suggested that UV irradiation activates growth factor receptors, which induce the activation of protein kinase cascades, like the MAPK cascade. The activation would be followed by an increase in the expression of the c-Jun and c-Fos and formation the AP-1 complex, a MMPs transcription regulator. Topical treatment with antioxidants may interrupt the activation of MAP kinase pathways, and thus inhibit UV-induced MMP expression in human skin. Objectives: Therefore, firstly the present work investigated the in vivo effectiveness of w/o microemulsion containing quercetin (ME+Q), a well-known antioxidant, in inhibiting the proteinase secretion/activity increase induced by UVB irradiation in hairless mice skin. Then, finally the quercetin photoprotective mechanism of action against UV induction of MMPs was evaluated. Methods: To evaluate the in vivo topical effectiveness, ME+Q was applied on the dorsal skin of hairless mice 1 h before, immediately before and after exposure to UVB irradiation (2.87 J/cm²). Then its effectiveness was evaluated by the measurement of its capability to decrease the cutaneous proteinase secretion/activity induced by UVB irradiation using SDS-PAGE zymography. The investigation of quercetin photoprotective mechanism of action against the secretion/activity of UV-induced MMPs was done using primary human keratinocytes as an in vitro model and by evaluation of quercetin effect against UV-induced (100 mJ/cm²) activation of MAP kinases (ERK, JNK, and p38), using Western blot analysis, and against UV (50 mJ/cm²)  induction of c-Fos, c-Jun, MMPs-1, 3 and 9 mRNA levels, using real-time RT-PCR analysis. Results: SDS-PAGE zymography detected inhibition of proteinase secretion/activity increase induced by UVB irradiation when ME+Q was applied. Furthermore, supporting these results, the control dosage of total proteins in the skin detected no significant statistical difference among the samples. On the other hand, the pretreatment of primary human keratinocytes with methanolic solution of quercetin (20 µg/mL) had no blocking effect on UV activation of all three MAP kinases evaluated and consequently c-Jun and c-Fos mRNA upregulation was not prevented. Furthermore, this flavonoid did not inhibit the increase in mRNA levels of MMPs-1, 3 and 9 induced by UV irradiation. Conclusion: Based on the current observations, it seems that in vivo photoprotective effects of quercetin incorporated in w/o ME are mediated mainly by inhibition of MMPs activity once no effect of this flavonoid was observed against the UV-induced AP-1 activation and consequently increase in MMPs expression.