Protective Effect of Spirulina-Derived C-Phycocyanin against Ultraviolet B-Induced Damage in HaCaT Cells

Abstract

Background and objectives: Reactive oxygen species (ROS) overwhelm the antioxidant defense system, induce oxidative stress, and increase matrix metalloproteinase (MMP) expression, resulting in skin aging. Thus, preventing ultraviolet B (UVB)-induced skin damage can attenuate skin aging. Spirulina (a biomass of cyanobacteria, also called blue-green algae) is comprised of prokaryotes, whereas microalgae are eukaryotes and are rich in phycocyanin, a powerful antioxidant. Materials and Methods: Here, we investigated the photoprotective effects of spirulina-derived C-phycocyanin (C-PC) against UVB radiation using keratinocytes (HaCaT cells). Results: UVB radiation increased MMP-1 and MMP-9 expression but decreased involucrin, filaggrin, and loricrin expression. C-PC showed no toxicity at concentrations of 5-80 μg/mL in terms of HaCaT cell viability. UVB-irradiated HaCaT cells had a 50.8% survival rate, which increased to 80.3% with C-PC treatment. MMP expression increased with UVB treatment, whereas MMP-1 and MMP-9 concentrations decreased with C-PC treatment. UVB reduced involucrin, filaggrin, and loricrin expression in HaCaT cells, but 80 μg/mL C-PC increased their expression by >25%. In the UVB radiation group, dichlorofluorescin diacetate fluorescence intensity in HaCaT cells increased by 81.6% compared with that in the control group, whereas ROS production was reduced by 51.2% and 55.1% upon treatment with 40 and 80 μg/mL C-PC, respectively. Conclusions: C-PC might reduce or prevent skin aging by reducing UVB irradiation-induced skin wrinkles and free radicals.

Keywords: C-phycocyanin; ROS; UVB; filaggrin; involucrin; loricrin; matrix metalloproteinase.

Figure 1

Chemical structure of C-phycocyanin.

Figure 2

Cell viability of human keratinocytes after ultraviolet B (UVB) exposure. (a) HaCaT cell viability after pretreatment with various concentrations of C-phycocyanin (C-PC); (b) toxicity of UVB according to the dosage; (c) viability of cells treated with C-PC, followed by UVB irradiation. Data are presented as percentages of the control group. ### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. UVB/vehicle group.

Figure 3

Effects of C-phycocyanin (C-PC) on matrix metalloproteinase (MMP)-1 and MMP-9 secretion by ultraviolet B (UVB)-induced HaCaT cells. Cells were treated with C-PC before UVB irradiation. The levels of secretion of (a) MMP-1 and (b) MMP-9 were measured in the culture medium of UVB-irradiated HaCaT cells. ### p < 0.001 vs. control group. ** p < 0.01 and *** p < 0.001 vs. UVB/vehicle group.

Figure 4

Effects C-phycocyanin (C-PC) on (a) western blot band (b) involucrin, (c) filaggrin, and (d) loricrin levels in ultraviolet B (UVB)-induced HaCaT cells, as assessed using Western blot analysis following treatment C-PC (20, 40, 80 μg/mL) These results are representative of three experiments performed in triplicate. Band intensities were quantified, and data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. UVB/vehicle group.

Figure 4

Effects C-phycocyanin (C-PC) on (a) western blot band (b) involucrin, (c) filaggrin, and (d) loricrin levels in ultraviolet B (UVB)-induced HaCaT cells, as assessed using Western blot analysis following treatment C-PC (20, 40, 80 μg/mL) These results are representative of three experiments performed in triplicate. Band intensities were quantified, and data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. UVB/vehicle group.

Figure 5

Effects of C-phycocyanin (C-PC) on ultraviolet B (UVB)-induced intracellular reactive oxygen species (ROS) levels. (a) HaCaT cells were seeded in 96-well plates and incubated with 2′,7′-dichlorofluorescin diacetate (DCFDA) dye prior to UVB exposure. (b) ROS levels were measured at 6 h post-UVB radiation along with treatment with the indicated concentrations of C-PC. Scale bars, 200 µm. Data are presented as the mean ± SD of three independent experiments. ### p < 0.001 vs. control group. * p < 0.01 and *** p < 0.001 vs. UVB/vehicle group.