Luteolin Prevents UVB-Induced Skin Photoaging Damage by Modulating SIRT3/ROS/MAPK Signaling: An in vitro and in vivo Studies

Abstract

The aim of this study was to investigate the role of luteolin in the mechanism of ultraviolet radiation B (UVB)-induced photoaging. An in vivo photoaging model was established using UVB irradiation of bare skin on the back of rats, and an in vitro photoaging model was established using UVB irradiation of human dermal fibroblasts (HDF). Skin damage was observed using hematoxylin-eosin (HE) and Masson staining, skin and cellular reactive oxygen species (ROS) levels were detected by DHE and DCF fluorescent probes, mitochondrial membrane potential was detected by JC-1 staining, and protein expressions were detected by immunofluorescence and Western Blot. Results from animal experiments showed that luteolin reduced UVB-induced erythema and wrinkle formation. Results from cellular assays showed that luteolin inhibited UVB-induced decrease in cell viability. In addition, in vitro and in vivo experiments showed that luteolin reduced oxidative stress levels, decreased activation of matrix metalloproteinases (MMPs) and increased collagen expression. Continued cellular experiments using 3-TYP, an inhibitor of Sirtuin 3 (SIRT3), revealed a loss of cellular protection by luteolin and a decrease in collagen, suggesting that luteolin acts by targeting and promoting SIRT3. luteolin is involved in the protection of skin cells against UVB radiation-induced ageing via the SIRT3/ROS/mitogen-activated protein kinases (MAPK) axis and it may be a promising therapeutic agent for the prevention of UVB photoaging.

Keywords: MAPK; MMPs; ROS; UVB photoaging; luteolin.

FIGURE 1

Luteolin improves the condition of the skin on the back of rats damaged by UVB irradiation. (A) Changes in skin appearance, Masson and HE sections of rats in each experimental group. (B) The content of ROS in the skin of rats in each experimental group, red fluorescence indicated ROS levels. (C) The relative epidermal thickness of skin in each group. (D) JC-1 staining results of skin in each group, green represents monomers and red represents aggregate. (E) The red/green fluorescence ratio of skin in each group, the lower the ratio the lower the membrane mitochondrial potential. (F) Representative image of immunofluorescence staining for MMP-1 and Collagen I. (G) The relative fluorescent intensity of MMP-1 of skin in each group. (H) The relative fluorescent intensity of Collagen I of skin in each group. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 2

Luteolin protects the skin from UVB-induced oxidative damage. (A) The contents of MDA in skin tissues. (B) The contents of SOD in skin tissues. (C) The contents of LDH in skin tissues. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 3

Luteolin inhibits UVB-induced activation of MMPs and p38 MAPK in vivo. (A) Representative immunoblots of SIRT3, p-P38, p-JNK, c-Jun, MMP-1, MMP-3, TGF-β, Smad3 and Collagen I protein expressions in skin tissue. (B) Optical density histograms of SIRT3/Tubulin, p-P38/Tubulin, p-JNK/Tubulin, c-Jun/Tubulin, MMP-1/Tubulin, MMP-1/Tubulin, MMP-3/Tubulin, TGF-β/Tubulin, Smad3/Tubulin and Collagen I/Tubulin. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 4

Luteolin improves biochemical parameters on HSF cells exposed to UV radiation. (A) Cell viability at different concentrations of luteolin. (B–D), The contents of MDA, SOD and LDH in skin tissues. (E) luteolin decreased the content of SAβ-gal of cells under UVB irradiation. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 5

Luteolin increases intracellular Collagen I expression and reduces ROS accumulation. (A) luteolin reduced intracellular ROS accumulation. (B) Representative image of immunofluorescence staining for MMP-1 and Collagen I. (C) luteolin reduced intracellular levels of MMP-1. (D) luteolin increased intracellular levels of Collagen I. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 6

Luteolin inhibits UVB-induced activation of MMPs and p38 MAPK in vitro. (A) Representative immunoblots of SIRT3, p-P38 MAPK, p-JNK, c-Jun, MMP-1, MMP-3, TGF-β, Smad3 and Collagen I protein expressions in cell. (B) Optical density histograms of SIRT3/Tubulin, p-P38/Tubulin, p-JNK/Tubulin, c-Jun/Tubulin, MMP-1/Tubulin, MMP-3/Tubulin, TGF-β/Tubulin, Smad3/Tubulin and Collagen I/Tubulin. # indicates significant difference between the model group and the blank group (#p < 0.05; ##p < 0.01); * indicates significant difference between the administered group and the model group (*p < 0.05; **p < 0.01).

FIGURE 7

3-TPY counteracts Luteolin’s protection of cells from UVB-induced damage. (A) 3-TPY inhibited the inhibitory effect of luteolin on intracellular ROS accumulation. (B) Representative image of immunofluorescence staining for MMP-1 and Collagen I. (C) 3-TPY inhibited the inhibitory effect of luteolin on intracellular MMP-1 expression. (D) 3-TPY inhibited the promotion of luteolin on intracellular Collagen I expression. (E) Representative immunoblots of c-Jun, MMP-1, Smad3 and Collagen I protein expressions in cell. (F) Optical density histograms of c-Jun/Tubulin, MMP-1/Tubulin, MMP-1/Tubulin, Smad3/Tubulin and Collagen I/Tubulin. * indicates a significant difference between the two groups (*p < 0.05; **p < 0.01).