Pilose Antler Protein Relieves UVB-Induced HaCaT Cells and Skin Damage

Abstract

Extended exposure to UVB (280-315 nm) radiation results in oxidative damage and inflammation of the skin. Previous research has demonstrated that pilose antler extracts have strong anti-inflammatory properties and possess antioxidant effects. This study aimed to elucidate the mechanism of pilose antler protein in repairing photodamage caused by UVB radiation in HaCaT cells and ICR mice. Pilose antler protein (PAP) was found to increase the expression of type I collagen and hyaluronic acid in HaCaT cells under UVB irradiation while also inhibiting reactive oxygen species (ROS) production and oxidative stress in vitro. In vivo, the topical application of pilose antler protein effectively attenuated UVB-induced skin damage in ICR mice by reducing interleukin-1β (IL-β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and inhibiting skin inflammation while alleviating UVB-induced oxidative stress. It was shown that pilose antler protein repaired UVB-induced photodamage through the MAPK and TGF-β/Smad pathways.

Keywords: HaCaT cells; UVB; pilose antler; skin damage.

Figure 1

PAP increases UVB-induced HaCaT cell viability and decreases cellular oxidative stress levels (control: without treatment; model: cells + UVB; SP: cells + UVB + sika deer antler proteins; HP: cells + UVB + horse antler proteins). (A) The cell viability of HaCaT cells exposed to UVB with different intensities (n = 6); (B) the cell viability of HaCaT cells exposed to UVB and treated with SPs and HPs. (n = 6); (C) quantitative analysis of reactive oxygen species (ROS) production (n = 3); (D–G) the effects of SPs and HPs on superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content (n = 3). ** p < 0.01 versus the model group, ## p < 0.01 versus the control group.

Figure 2

PAP improves collagen synthesis and barrier function in UVB-induced HaCaT cells. (A,B) The effects of SPs and HPs on type I collagen (ColⅠ) and hyaluronic acid (HA) content (n = 3); (C–H) changes in the expression levels of genes associated with the TGF-β/Smad pathway and skin repair. Real-time fluorescence quantitative PCR showing the mRNA expression of matrix metalloproteinase 1 (MMP1), transformation growth factor-β1 (TGF-β1), type I collagen (ColⅠ), filaggrin (FLG), involucrin (IVL), and aquaporin protein-3 (AQP3) (n = 6); (I,J) representative images of protein blots of MAPK and TGF-β/Smad pathway-related proteins and quantitative analysis of the images (n = 3). * p < 0.05, ** p < 0.01 versus the model group, ## p < 0.01 versus the control group.

Figure 3

PAP improves epidermal thickening, collagen deposition, and inflammation of the skin caused by UVB. The groups were the normal group (BG), UVB model group (MG), 10 mg/mL sika deer antler protein treatment group (SP), 10 mg/mL horse antler protein treatment group (HP), and VC positive treatment group (PG). (A) HE staining of skin tissue (n = 3, scale bar = 100 µm). Black arrows indicate epidermal thickness. The blue dashed line indicates unhealed gaps. Red circles indicate diffuse inflammation. (B) Masson staining of skin tissue (n = 3, scale bar = 100 µm). Orange arrows indicate collagen fiber alignment. The black dotted line indicates decreased collagen fibers. (C) The change in Hydroxyproline (Hyp) levels in skin tissues (n = 3); (D) the change in MMP-1 levels in skin tissues (n = 3); (E–G) expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in serum (n = 3). ** p < 0.01 versus the model group, ## p < 0.01 versus the control group.

Figure 4

SPs and HPs alleviate UVB-induced oxidative stress in ICR mouse skin. (A) The SOD activity in skin tissues (n = 3); (B) the CAT activity in skin tissues; (C) the GSH-Px activity in skin tissues (n = 3); (D) the MDA level in skin tissues (n = 3). ** p < 0.01 versus the model group, ## p < 0.01 versus the control group.

Figure 5

The expression of related genes and proteins in mouse skin tissues (n  =  3). (A) The mRNA level of MMP-1; (B) the mRNA level of tissue inhibitor of metalloproteinase 1 (TIMP-1); (C) the mRNA level of ColⅠ (n = 3); (D) the mRNA level of collagen type III (Col III); (E) the mRNA level of TGF-β1; (F) protein banding; (G) quantitative analysis of TGF-β1 protein; (H) quantitative analysis of p-Smad 2/3 protein. * p < 0.05, ** p < 0.01 versus the model group, ## p < 0.01 versus the control group.

Figure 6

UVB radiation and treatment in mice.