INHIBITION OF MATRIX METALLOPROTEINASES 235 0.3 0.25 0.2 0.15 0.1 0.05 '• MMP1 [] MMP2 0 10 25 Concentration of 8- Br- cGMP(microM: Figure 3. The effect of 8-Br-cGMP on the production of MMP-1 and MMP-2 by human derreal fibroblasts. *n = 3. p 0.05 vs no treatment. Graillet et al. (13), and they confirmed that NO plays an important role in the paracrine mediation of UV-induced melanogenesis. Figures 4 and 5 show the expression of in- ducible NOS (iNOS) and the production of NO from HDF treated with UV radiation of 30 J/c m2. The mRNA of iNOS was increased by 15% when HDF was treated with UV of 30 J/cm 2 (Figure 4), and the production of NO was increased by 14% (Figure 5), but the results were not significant statistically (p 0.05). Although the increase of iNOS and NO in the UV-irradiated HDF was not meaningful, the source of NO- influencing HDF is not negligible, considering the NO produced by keratinocytes. In a separate experiment, we confirmed that the addition of a culture supernatant of UV-irradiated keratinocytes to the culture medium of HDF also enhanced the produc- tion of MMP-1 and -2 by HDF (data not shown). INHIBITION OF MMP PRODUCTION BY iNOS INHIBITORS To confirm the action of NO on the MMP production by HDF, HDFs were treated with 0.4 0.35 0.3 0.25 0.2 z0.15 0.1 0.05 0 Co ntrol UV30 ( J/c m 2) Figure 4. Production of inducible nitric oxide synthase by UV-irradiated human derreal fibroblasts. The intracellular mRNA of iNOS was determined by using the RT-PCR-ELISA method. *n = 4. p 0.05 vs no UV exposure.

236 JOURNAL OF COSMETIC SCIENCE 1121.001 0 103 I I•1 -1 I-I I•'1 c D Figure 5. Production of nitric oxide by UV-irradiated human dermal fibroblasts. The intracellular NO was measured using NO sensor dye and fluorescence-activated cell sorter. Black line: without UV irradiation. White line: with UV irradiation. A: control. B: 10 J/cm 2. C: 20 J/cm 2. D: 30 J/cm 2. various NOS inhibitors in the presence of UV irradiation (30 J/cm2). AG, NAME, NAL, and BAC are NOS inhibitors that inhibit both the constitutive and inducible NOS. The extract of Scutellaria root is plentiful in various iNOS inhibitors including wogonin, baicalin, and baicalein (22). The enhancement of MMP production by UV irradiation was partially but significantly (p 0.05) blocked by iNOS inhibitors such as AG and BAC, but not by NAME and NAL. Figure 6 shows that the enhanced production of MMP-1 or MMP-2 by UV irradiation was downregulated by the addition of AG and BAC. When 20 microM of BAC was added to the culture media, only 40% of MMP-1 and 42% of MMP-2 were produced, compared to the untreated case. These findings suggest that NO affects MMP-1 and -2 production through the cGMP pathway and is an important signal mediator in regulating the production of MMP by UV-irradiated HDF. CONCLUSIONS The effect of NO and iNOS inhibitors on the production of MMP-1 and -2 by UV- irradiated or non-irradiated HDF was studied. The addition of a NO donor, SNP, to the culture medium of HDF enhanced the production of MMP-1 and -2, while the addition of iNOS inhibitors such as AG and BAC downregulated the production of MMP-1 and -2 by UV-irradiated HDF. Although the production of NO by UV-irradiated HDF was not significant statistically, the influence of NO on HDF might not be negligible, considering the NO produced by UV-irradiated keratinocytes. From these results, we