The combination of cigarette smoke and solar rays causes effects similar to skin aging in a bilayer skin model

Abstract

Skin aging is a multifactorial process influenced by internal and external factors. The contribution of different environmental factors has been well established individually in the last few years. On the one hand, man is rarely exposed to a single factor, and on the other hand, there is very little knowledge about how these extrinsic factors may interact with each other or even how the skin may react to chronic exposure. This study aimed to evaluate the effect on skin aging of a chronic co-exposure of tissue-engineered skin substitutes to cigarette smoke extract (CSE) and solar simulator light (SSL). Skin substitutes were reconstructed according to the self-assembly method and then exposed to CSE followed by irradiation with SSL simultaneously transmitting UVA1, visible light and infrared. When skin substitutes were chronically exposed to CSE and SSL, a significant decrease in procollagen I synthesis and the inhibition of Smad2 phosphorylation of the TGF-β signaling pathway were observed. A 6.7-fold increase in MMP-1 activity was also observed when CSE was combined with SSL, resulting in a decrease in collagen III and collagen IV protein expression. The secretory profile resulting from the toxic synergy was investigated and several alterations were observed, notably an increase in the quantities of pro-inflammatory cytokines. The results also revealed the activation of the ERK1/2 (3.4-fold) and JNK (3.3-fold) pathways. Taken together, the results showed that a synergy between the two environmental factors could provoke premature skin aging.

Figure 1

Self-assembly method (step 1) and treatment and SSL irradiation procedures (step 2).

Figure 2

(a) UV–visible spectrum of the CSE stock solution and (b) irradiance spectrum of the solar simulator light (SSL) with the CGA-345 long-pass filter.

Figure 3

Effect of cigarette smoke extract (CSE) and SSL irradiation on reconstructed skin substitute morphology. (A) Histological analyses of Masson’s trichrome-stained skin substitutes. Stratum corneum (SC) in dark blue/purple, living epidermis (LE) in violet/pink and dermis (D) in light blue. Objective 10X, scale bar: 100 μm. (B) Fold change in the thickness of the living epidermis and (C) of the dermis. Fold change is defined as the ratio of exposed substitutes’ thickness value to the control (without treatment) thickness value. Two substitutes for each condition were analyzed and confirmed with at least three different cell populations (3 ≤ N ≤ 4, 6 ≤ n ≤ 8). Data are presented as means of the different cell populations ± S.D. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, * p value < 0.05.

Figure 4

Effect of cigarette smoke extract (CSE) and SSL irradiation on procollagen synthesis via the inhibition of the TGF-β/Smad signaling pathway and matrix metalloproteinase (MMP) activity. (A) Procollagen I alpha 1 production assessed by ELISA in cell culture supernatants. Fold change is defined as the ratio of exposed substitutes’ procollagen quantity (in ng/mL) to the control (without treatment). (B) Protein expression of phosphorylated Smad2 in the dermis of reconstructed skin substitutes as determined by western blot. β-actin was used as the loading control. One representative blot is shown. (C) Densitometric quantification of the western blots. (D) MMP-1 activity in cell culture supernatants assessed by a fluorimetric assay (Human Active MMP-1 Fluorokine® E Kit). Analyses were performed with at least three different cell populations (3 ≤ N ≤ 5, 6 ≤ n ≤ 10). Data are presented as means of the different cell populations ± S.D. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001.

Figure 5

Effect of cigarette smoke extract (CSE) and SSL irradiation on dermal components of the extracellular matrix and basement membrane of reconstructed skin substitutes. (A) Dot blot analysis of collagen I, collagen III, collagen IV, and elastin. Dot blots come from the same membrane but were put in a more logical order, hence the black frame around each dot blot. (B) Densitometric quantification of the dot blots. GAPDH was used as loading control. Fold change is defined as the ratio of protein expression in exposed substitutes to that of the control (without treatment). Analyses were confirmed with three different cell populations (N = 3). Data are presented as means of the different cell populations ± S.D. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, * p value < 0.05.

Figure 6

Activation of MAP kinase pathways by cigarette smoke extract (CSE) and SSL irradiation. (a) Levels of phosphorylated p38 MAPK, ERK1/2 and JNK in the epidermis of reconstructed skin substitutes as determined by western blot. β-actin was used as the loading control. One representative blot is shown. (b) Densitometric quantification of the western blots. Analyses were confirmed with three different cell populations (N = 3). Data are presented as means of the different cell populations ± S.D. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, * p value < 0.05, ** p value < 0.01.

Figure 7

Proposed mechanisms of the synergistic effect between cigarette smoke extract (CSE) and SSL irradiation in skin aging.