Puerarin Reduces Oxidative Damage and Photoaging Caused by UVA Radiation in Human Fibroblasts by Regulating Nrf2 and MAPK Signaling Pathways

Abstract

Fibroblasts account for more than 95% of dermal cells maintaining dermal structure and function. However, UVA penetrates the dermis and causes oxidative stress that damages the dermis and accelerates skin aging. Puerarin, the main active ingredient of Puerariae lobata, has been demonstrated to withstand oxidative stress caused by a variety of factors. However, there are limited findings on whether puerarin protects fibroblasts from UVA-induced oxidative stress damage. The effects of puerarin on human skin fibroblasts (HSF) under UVA-induced oxidative stress were investigated in this study. It is found that puerarin upregulates antioxidant enzymes' mRNA expression level and their content through modulating the KEAP1-Nrf2/ARE signaling pathway, thus improving cell antioxidant capacity and successfully eliminating UVA-induced reactive oxygen species (ROS) and lipid oxidation product malondialdehyde (MDA). Additionally, puerarin blocks the overexpression of human extracellular signal-regulated kinase (ERK), human c-Jun amino-terminal kinase (JNK), and P38, which downregulates matrix metalloproteinase 1 (MMP-1) expression and increases type I collagen (COL-1) expression. Moreover, preliminary research on mouse skin suggests that puerarin can hydrate, moisturize, and increase the antioxidant capacity of skin tissue. These findings suggest that puerarin can protect the skin against photoaging.

Keywords: anti-photoaging; antioxidant; oxidative stress; puerarin.

Figure 1

Effects of puerarin on UVA-induced cytotoxicity in human skin fibroblasts (HSF): (a) Toxicity of different concentrations of puerarin on HSF; (b) Toxicity of different doses of UVA on HSF; (c) Preventative effects of puerarin on cytotoxicity in UVA-irradiated HSF. ^## p < 0.01, ^### p < 0.001 as compared to the control group; *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 2

Effects of puerarin on cellular antioxidant indexes: (a) Effects of puerarin on total antioxidant capacity of HSF with UVA-induced oxidative damage. (b) Effects of puerarin on reactive oxygen species (ROS) production induced by UVA stimulation. (c) Effects of puerarin on lipid oxidation product malondialdehyde (MDA) content in HSF with UVA-induced oxidative damage. ^## p < 0.01, ^### p < 0.001 as compared to the control group; * p < 0.05, *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 3

Effects of puerarin on UVA-induced Nrf2 signaling in HSF: (a,b) RT-PCR analysis of KEAP1 and NRF2 expression levels; (c,d) ELISA analysis of KEAP1 proteins and Nrf2 proteins in nucleus and cytosol. ^# p < 0.05, ^### p < 0.001 as compared to the control group; * p < 0.05, *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 4

Effects of puerarin on UVA-induced antioxidase in HSF: (d–f) RT-PCR analysis of puerarin on UVA-induced SOD, CAT, and GSH-Px expression in HSF; (a–c) ELISA analysis of puerarin on UVA-induced SOD, CAT, and GSH-Px; (j–l) RT-PCR analysis of puerarin on UVA-induced HO-1, NQO1, and GCLC expression in HSF; (g–i) ELISA analysis of puerarin on UVA-induced HO-1, NQO1, and GCLC. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 as compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 5

Effects of puerarin on UVA-induced mitogen-activated protein kinase (MAPK) signaling in HSF: (a–c) RT-PCR analysis of human extracellular signal-regulated kinase (ERK), human c-Jun amino-terminal kinase (JNK), and P38 mRNA expression levels after treatment and UVA irradiation of samples. ^# p < 0.05, ^## p < 0.01 as compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 6

Effects of puerarin on UVA-induced skin aging markers in HSF: (a,b) Effects of puerarin on MMP-1 and COL-1 expression of HSF with UVA-induced oxidative damage; (c,d) Effects of puerarin on MMP-1 and COL-1 content of HSF with UVA-induced oxidative damage. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 as compared to the control group; *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 7

Effects of puerarin on UVA irradiation in mouse skin: (a) Results of moisture content (MMV) measurement value in mice skin; (b) Results of transepidermal water loss (TEWL) in mice skin; (c) Effects of puerarin on antioxidant capacity in mice after UVA irradiation; (d) Effects of puerarin on CAT activity in mice after UVA irradiation. ^## p < 0.01, ^### p < 0.001 as compared to the control group; ** p < 0.01, *** p < 0.001 as compared to the damage model group; Values do not have a common mark (^#, *) when p > 0.05, (n = 3).

Figure 8

Effects of puerarin on mouse skin H&E staining: (a) Blank group mice; (b) Blank group skin H&E staining (10×); (c) Blank group skin H&E staining (20×); (d) Model group mice; (e) Model group skin H&E staining (10×); (f) Model group skin H&E staining (20×); (g) Positive control group mice; (h) Positive control group skin H&E staining (10×); (i) Positive control group skin H&E staining (20×); (j) Puerarin group mice; (k) Puerarin group skin H&E staining (10×); (l) Puerarin group skin H&E staining (20×).