Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression

Abstract

Exposure to ultraviolet (UV) light can cause skin photoaging, which is associated with upregulation of matrix metalloproteinases (MMPs) and downregulation of collagen synthesis. It has been reported that MMPs, especially MMP-1, MMP-3 and MMP-9, decrease the elasticity of the dermis by degrading collagen. In this study, we assessed the effects of Pinus densiflora extract (PDE) on photoaging and investigated its mechanism of action in human skin fibroblast (Hs68) cells after UVB exposure using real-time polymerase chain reaction, Western blot analysis, and enzymatic activity assays. PDE exhibited an antioxidant activity and inhibited elastase activities in vitro. We also found that PDE inhibited UVB-induced cytotoxicity, MMP-1 production and expression of MMP-1, -3 and -9 mRNA in Hs68 cells. In addition, PDE decreased UVB-induced MMP-2 activity and MMP-2 mRNA expression. Moreover, PDE prevented the decrease of type I procollagen mediated by exposure to UVB irradiation, an effect that is linked to the upregulation and downregulation of Smad3 and Smad7, respectively. Another effect of UV irradiation is to stimulate activator protein 1 (AP-1) activity via overexpression of c-Jun/c-Fos, which, in turn, upregulates MMP-1, -3, and -9. In this study, we found that PDE suppressed UV-induced c-Jun and c-Fos mRNA expression. Taken together, these results demonstrate that PDE regulates UVB-induced expression of MMPs and type I procollagen synthesis by inhibiting AP-1 activity and restoring impaired Smad signaling, suggesting that PDE may be useful as an effective anti-photoaging agent.

Keywords: Anti-oxidant; Matrix metalloproteinases; Photoaging; Pinus densiflora; TGF-β/Smad; Type I procollagen; UV irradiation.

Fig. 1

Antioxidant effect and elastase inhibition of Pinus densiflora extracts (PDE). (A) DPPH radical scavenging activity of PDE. (B) The inhibition (%) of porcine elastase activity of PDE. Ursolic acid was used as positive control. (n = 3; significant difference versus CTRL: ^* <0.01, ^*** <0.0001) Ascorbic: Ascorbic acid, U.A.: Ursolic acid).

Fig. 2

Effects of PDE on Hs68 cell viability. (A) Hs68 cells were treated with various concentrations of PDE for 24 h and cytotoxicity was determined by MTT assay. (B) Cell viability of Hs68 cells exposed to various UVB irradiation intensities prior to 24 h incubation. (C) Protective effects of PDE against UVB-induced cell death. Hs68 cells were treated with different doses of PDE for 24 h and then the cells were exposed to UVB (100 mJ/cm²) irradiation. After UVB exposure, fresh media was added. At 24 h after UVB irradiation, percent cell viability was assessed by MTT assay. Results are expressed as the mean ± SD (% control) of three independent experiments. (^*P < 0.05, ^**P < 0.005 and ^***P < 0.0001 versus UVB-treated control. Values determined by one-way analysis of variance and subsequently applying Tukey's test; ^#P < 0.01 versus control group by the student's t-test)

Fig. 3

Effect of PDE on MMP-1 production, MMPs mRNA expression and type 1 pro-collagen protein in UVB-induced Hs68 cells. Hs68 cells were treated with various concentrations of PDE for 24 h and then exposed to UVB irradiation (100 mJ/cm²). At 24 h after UVB exposure, (A) release of MMP-1 into the culture media was determined by ELISA and (B–D) the expression of MMP-1, -3, -9 mRNA was measured by real-time PCR. β-Actin was used as an internal control for real-time PCR. (E) The expression of MMP-1 and type 1 pro-collagen (Pro-COL) protein was determined by Western blotting. Data are shown as the mean ± SD of three independent experiments (^#P < 0.05 and ^###P < 0.001 versus control group by student's t-test; ^***P < 0.0005 versus UVB-treated control by one-way analysis of variance and subsequently applying Tukey's test).

Fig. 4

Effect of PDE on UV-induced expression of MMP-2 in human fibroblasts. (A and B) Zymography assay showing the effect of PDE on MMP-2 activity in the culture medium of human fibroblasts. (C) Quantitative MMP-2 mRNA expression was analyzed by real-time PCR. The data represent mean ± SD (^#P < 0.05 and ^##P < 0.01 versus control group by student's t-test; ^**P < 0.005 and ^***P < 0.001 versus UVB-treated control by one-way analysis of variance and subsequently applying Tukey's test).

Fig. 5

Effect of PDE on expression of c-Jun, c-Fos, Smad3, and Smad7. Quantitative measures for (A) c-Fos mRNA expression, (B) c-Jun mRNA expression, (C) Smad3 mRNA expression and (D) Smad7 mRNA expression in Hs68 cells irradiated with UVB were determined by real-time PCR. The data represent mean ± SD (^#P < 0.05 and ^###P < 0.005 versus control group by student's t-test; ^*P < 0.05, ^**P < 0.005 and ^***P < 0.0001 versus UVB-treated control by one-way analysis of variance and subsequently applying Tukey's test).