Protective Effects of Euphrasia officinalis Extract against Ultraviolet B-Induced Photoaging in Normal Human Dermal Fibroblasts

Abstract

Ultraviolet (UV) radiation induces skin photoaging, which is associated with the elevation of matrix metalloproteinases (MMPs) and the impairment of collagen. The Euphrasia species play a well-known role in the treatment of certain eye disorders through their anti-oxidative and anti-inflammatory activities. However, their protective activity toward UVB-induced damage remains unclear. In the present study, we investigated the protective effect of Euphrasia officinalis (95% ethanol extract) on UVB-irradiated photoaging in normal human dermal fibroblasts (NHDFs). Our results show that Euphrasia officinalis extract exhibited obvious reactive oxygen species (ROS) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, enhanced NHDF cell migration, and reduced UVB-induced apoptosis. The UVB-induced increases in MMP-1 and MMP-3 and decrease in type I procollagen were ameliorated by Euphrasia officinalis treatment, which worked by suppressing the mitogen-activated protein kinase (MAPK) and nuclear transcription factor activator protein 1 (AP-1) signaling pathways. Taken together, our data strongly suggest that Euphrasia officinalis ethanol extract could reduce UVB-induced photoaging by alleviating oxidative stress, proinflammatory activity, and cell apoptosis.

Keywords: Euphrasia officinalis; apoptosis; matrix metalloproteinases; photoaging; type I procollagen; ultraviolet B.

Figure 1

Effect of E. officinalis on cell viability and ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging ability. (A) Cell viability after 72 h with or without ultraviolet B (UVB) (144 mJ/cm²) and E. officinalis (1 µg/mL, 10 µg/mL, or 50 μg/mL); (B) ABTS radical scavenging ability of E. officinalis. Arbutin was used as a positive control. Values are means ± standard deviation (SD). * and # indicate significant differences between the non-irradiated control and the UVB-irradiated control, respectively. * p < 0.05 versus the non-irradiated control.

Figure 2

Effect of E. officinalis on the generation of reactive oxygen species (ROS) in UVB-irradiated normal human dermal fibroblasts (NHDFs). (A) ROS levels in NHDFs were determined after 24 h of UVB radiation with and without E. officinalis treatment. The number of cells is plotted versus the dichlorofluorescein fluorescence detected by the FL-2 channel; (B) The relative ROS generated by NHDFs is shown. Values are means ± SD. *** p < 0.001 versus the non-irradiated control. # p < 0.05 and ## p < 0.01 versus the UVB-irradiated control.

Figure 3

Effect of E. officinalis on NHDF cell migration as determined by a wound-healing assay. (A) NHDF cells cultured for 72 h were fixed and stained with crystal violet. The dotted lines represent the wound boundary of Control. Magnification scale: 100×. Scale bar: 200 µm; (B) Graphical presentation of wound-healing assay. Values are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the untreated control.

Figure 4

Anti-apoptotic effect of E. officinalis on UVB-irradiated NHDFs. (A) Apoptotic cells were detected by Hoechst 33258 staining. Cells that underwent apoptosis are indicated by arrows. Magnification scale: 400×. Scale bar: 10 µm; (B) Apoptotic rate was calculated as a percentage of apoptotic cells over the total number of cells numbers in five randomly selected areas. Values are means ± SD. *** p < 0.001 versus the non-irradiated control. # p < 0.05, and ## p < 0.01, and ### p < 0.001 versus the UVB-irradiated control.

Figure 5

Effect of E. officinalis on the secretion of the matrix metalloproteinases (MMPs) and type I procollagen. The production of (A) MMP-1, (B) MMP-3, (C) interleukin-6 (IL-6), (D) transforming growth factor-β1 (TGF-β1), and (E) type I procollagen in non- and UVB-irradiated NHDFs. Values are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the non-irradiated control. # p < 0.05 and ## p < 0.01 versus the UVB-irradiated control.

Figure 5

Effect of E. officinalis on the secretion of the matrix metalloproteinases (MMPs) and type I procollagen. The production of (A) MMP-1, (B) MMP-3, (C) interleukin-6 (IL-6), (D) transforming growth factor-β1 (TGF-β1), and (E) type I procollagen in non- and UVB-irradiated NHDFs. Values are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the non-irradiated control. # p < 0.05 and ## p < 0.01 versus the UVB-irradiated control.

Figure 6

Effect of E. officinalis on skin aging–related mRNA expression. (A) mRNA expression of MMP-1 and type I procollagen in UVB-irradiated NHDFs. The mRNA levels of (B) MMP-1 and (C) type I procollagen were quantified and normalized to the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) value. Densitometry data are expressed as the percentage relative to the level in the non-irradiated control and shown as the mean ± SD. * p < 0.05 and ** p < 0.01 versus the non-irradiated control. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the UVB-irradiated control.

Figure 7

Effect of E. officinalis on activator protein 1 (AP-1) signaling proteins. (A) The protein levels of p-c-Jun and p-c-Fos in UVB-irradiated NHDFs were measured by Western blot analysis. The signal intensities for (B) p-c-Jun and (C) p-c-Fos were quantified and normalized to the corresponding β-actin value. Densitometry data are expressed as the percentage relative to the non-irradiated control and shown as the mean ± SD. ** p < 0.01 and *** p < 0.001 versus the non-irradiated control. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the UVB-irradiated control.

Figure 8

Effect of E. officinalis on mitogen-activated protein kinase (MAPK) signaling–related proteins. (A) The protein levels of the phosphorylation of c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated kinase (p-ERK), and p38 (p-p38) in UVB-irradiated NHDFs were measured by Western blot analysis. The band intensities for (B) p-JNK, (C) p-ERK, and (D) p-p38 were quantified and normalized to the corresponding β-actin value. Densitometry data are expressed as the percentage relative to the non-irradiated control and shown as the mean ± SD. * p < 0.05 and ** p < 0.01 versus the non-irradiated control. # p < 0.05 and ## p < 0.01 versus the UVB-irradiated control.