Pholiota nameko Polysaccharides Protect against Ultraviolet A-Induced Photoaging by Regulating Matrix Metalloproteinases in Human Dermal Fibroblasts

Abstract

Ultraviolet-A (UVA) exposure is a major cause of skin aging and can induce oxidative damage and accelerate skin wrinkling. Many natural polysaccharides exhibit a UV protective effect. In research on Pholiota nameko polysaccharides (PNPs), a natural macromolecular polysaccharide (4.4-333.487 kDa), studies have shown that PNPs can significantly decrease elastase activity to protect against UVA-induced aging in Hs68 human dermal fibroblasts. Cellular experiments in the present study indicated that PNPs can protect against UVA-induced oxidative damage in Hs68 cells by inhibiting the production of reactive oxygen species. Furthermore, PNPs significantly attenuated UVA-induced cell aging by decreasing the protein expression of matrix metalloproteinase 1, 3, and 9. Pretreatment of Hs68 cells with PNP-40, PNP-60, and PNP-80 before UVA irradiation increased protein expression of tissue inhibitor metalloproteinase 1 by 41%, 42%, and 56% relative to untreated cells. In conclusion, this study demonstrates that PNPs are a natural resource with potentially beneficial effects in protecting against UVA-induced skin aging.

Keywords: Pholiota nameko polysaccharides; aging; human dermal fibroblasts; oxidative damage; ultraviolet-A.

Figure 1

Anti-elastase activity of PNPs (125, 250, and 500 μg/mL) and EGCG (125, 250 and 500 μg/mL). Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD. a–c: Matching lowercase letters between samples at the same concentration indicate significant differences between the samples at that concentration (p < 0.05). A–C: Matching uppercase letters for the same sample at different concentrations indicate significant differences for that sample between different concentrations (p < 0.05).

Figure 2

Cell viability analyzed via MTT assay. Cell viability was assessed using 3-(4,5-dimethylthiazolyl-2) 2,5 diphenyltetrazolium bromide (MTT) assays. Hs68 cells were treated with PNPs for 24 h. Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD. * Significant differences compared to the control group (p < 0.05). a,b: Matching lowercase letters between samples at the same concentration indicate significant differences between the samples at that concentration (p < 0.05). A–C: Matching uppercase letters for the same sample at different concentrations indicate significant differences for that sample between different concentrations (p < 0.05).

Figure 3

Effect of UVA irradiation on cell viability. Cells were irradiated with the indicated single doses of UVA radiation (5–20 J/cm²). Viability was determined via MTT assay at 24 h. Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD. * Significant differences compared to the control group (p < 0.05).

Figure 4

Cell viability of Hs68 cells pretreated with PNPs (62.5, 125, 250, and 500 μg/mL) for 24 h and then subjected to UVA irradiation at 5 J/cm², as determined via MTT assay. Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD. * Significant differences compared to the UVA-induced group (p < 0.05). a–c: Matching lowercase letters between samples at the same concentration indicate significant differences between the samples at that concentration (p < 0.05). A,B: Matching uppercase letters for the same sample at different concentrations indicate significant differences for that sample between different concentrations (p < 0.05).

Figure 5

(a) Radical scavenging activity of PNPs (250 µg/mL) on Hs68 cells against UVA-induced ROS generation (scale bar = 100 µm, magnification: 10 × 10). (b) Quantitative analysis (performed using Image J software) of the radical scavenging effect of PNPs on Hs68 cells against UVA-induced ROS generation. Experiments were independently conducted in triplicate (n = 3). Data are expressed as the mean ± SD. a–c: Matching lowercase symbols indicate significant differences between the different groups (p < 0.05).

Figure 6

Protective effect of PNPs against cellular senescence in Hs68 cells irradiated with UVA. Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD. a–c: Matching lowercase letters indicate significant difference between the different groups (p < 0.05). Experiments were conducted in triplicate (n = 3). Data are expressed as the mean ± SD.

Figure 7

Protective effect of PNPs against UVA irradiation in Hs68 cells. Protein expression of MMP-1, -3, and -9 was analyzed via Western blot. (a) Hs68 cells were pretreated with PNPs (250 µg/mL) and RA (10 µM) for 24 h, followed by UVA-irradiation. Western blotting was performed to examine MMP-1, -3 and -9 expression, with GAPDH used as an internal control. (b–d) MMP-1, -3, and -9 expression after quantification using Image J software. a–e: Matching lowercase letters indicate significant differences between the different groups (p < 0.05). Experiments were conducted in triplicate (n = 3), and the blot shown was the representative result. Data are expressed as the mean ± SD.

Figure 8

1H NMR spectroscopy of PNP-80. a–k: Matching lowercase letters represent hydrogen signals at different positions on the PNP-80 structure.

Figure 9

13C NMR spectroscopy of PNP-80. A–H: Matching capital letters represent carbon signals at different positions on the PNP-80 structure.