Fig 1.
Measurement of cell viability and collagen synthesis in NIH 3T3 cells treated with FIR or TGF-β.
(A) Cell viability was measured in NIH 3T3 cells treated with FIR for 30 min and cultured for 12, 24 or 48 h. Cells were treated with TGF-β (20 ng/ml) for 12, 24 or 48 h. (B) The expression levels of TGF-β protein were measured by western blot analysis following treatment with FIR. Cells were treated with FIR for 3, 6 or 12 h. (C) The expression levels of procollagen type 1, p-Smad2 and Smad2/3 proteins were measured by western blot analysis. Cells were treated with FIR for 30 min and cultured for 24 h. Cells were treated with TGF-β (20 ng/ml) for 24 h. TGF-β is a positive control for the TGF-β/Smad axis. (D) Levels of collagen type 1 in the culture medium were measured by ELISA. *p<0.05, FIR or TGF-β versus control. The data are presented as the means ± standard deviation of three independent experiments.
Fig 2.
Measurement of degradation of collagen by MMPs in NIH 3T3 cells treated with UVB and/or FIR.
(A) MMP-1 and MMP-9 expression in UVB-irradiated NIH 3T3 cells. Cells were irradiated with 40, 80 or 120 mJ/cm2 UVB for 48 h. (B) The expression levels of MMP-1 and MMP-9 proteins were measured by western blot analysis following treatment with UVB and/or FIR. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (C) Levels of collagen type 1 in the culture medium were measured by ELISA. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. *p<0.05, UVB versus UVB+FIR. The data are presented as the means ± standard deviation of three independent experiments.
Fig 3.
Measurement of autophagy and the Akt/mTOR signaling pathway in NIH 3T3 cells treated with UVB and/or FIR.
(A) Immunofluorescence staining of LC3 protein in NIH3T3 cells treated with UVB and/or FIR. Representative cell images showing punctate LC3 distribution using a confocal microscope. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (B) Quantitative data calculating the percentage of LC3-positive cells. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. *, p<0.05, versus control. The data are presented as the means ± standard deviation of three independent experiments. (C) The expression levels of autophagic-related proteins were measured by western blot analysis following treatment with UVB and FIR alone or in combination. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (D) The expression levels of Akt/mTOR signaling-associated proteins were measured by western blot analysis. Cells were irradiated with 80 mJ/cm2 UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 6 h.
Fig 4.
Body weight and epidermal thickness in dorsal skin of FIR-treated mice against UVB-induced skin damage.
(A) Measurement of body weight in hairless mice taken once per week. (B) H&E staining and its histogram estimated for epidermal thickness. (C) Quantitative data calculating the epidermal thickness.
Fig 5.
The protein expression and density of collagen fibers in a UVB-exposed hairless mouse model.
(A) Masson’s trichrome staining estimated for relative collagen density. IHC staining of skin tissues was used to determine the expression levels of MMP-9 (B) and LC3 (C). (D) Western blot analysis of protein expression in skin tissues.
Fig 6.
FIR pathways and effects in skin photoaging.
FIR inhibits MMPs and leads to interference with collagen degradation. Furthermore, FIR increases collagen synthesis through the TGF-β/Smad pathway. In addition, FIR-induced autophagy may be mediated by inhibition of the Akt/mTOR signaling pathway. Autophagy can block the epidermal hyperproliferative response to UV and may suppress photoaging.