The effect of long-term cigarette smoking on selected skin barrier proteins and lipids

Abstract

The negative impact of cigarette smoking on the skin includes accelerated aging, pigmentation disorders, and impaired wound healing, but its effect on the skin barrier is not completely understood. Here, we studied the changes in selected epidermal proteins and lipids between smokers (45-66 years, smoking > 10 years, > 10 cigarettes per day) and non-smokers. Volar forearm epidermal and stratum corneum samples, obtained by suction blister and tape stripping, respectively, showed increased thickness in smokers. In the epidermis of smokers, we observed a significant upregulation of filaggrin, loricrin, and a trend of increased involucrin but no differences were found in the case of transglutaminase 1 and kallikrein-related peptidase 7, on the gene and protein levels. No significant changes were observed in the major skin barrier lipids, except for increased cholesterol sulfate in smokers. Liquid chromatography coupled with mass spectrometry revealed shorter acyl chains in ceramides, and an increased proportion of sphingosine and 6-hydroxysphingosine ceramides (with C4 trans-double bond) over dihydrosphingosine and phytosphingosine ceramides in smokers, suggesting altered desaturase 1 activity. Smokers had more ordered lipid chains found by infrared spectroscopy. In conclusion, cigarette smoking perturbs the homeostasis of the barrier proteins and lipids even at a site not directly exposed to smoke.

Figure 1

Evaluation of the selected proteins associated with the skin barrier function in the epidermis of smokers and non-smokers. (a) Gene expression evaluated by qRT-PCR. (b) Protein level evaluated by immunofluorescence staining: representative images of the stained epidermal samples from three smokers and non-smokers (red: protein of interest, blue: cell nuclei stained with DAPI), c quantification of the fluorescence intensity.

Figure 2

Histologic evaluation of the SC and cellular epidermis thickness in smokers and non-smokers. (a) Quantification, (b) representative images of the epidermal samples stained with hematoxylin and eosin from three smokers and non-smokers.

Figure 3

The skin barrier lipids composition and organization in the skin of smokers and non-smokers. (a) Quantification of the main skin barrier lipid groups and their precursors by HPTLC (FFA, free fatty acids; Chol, cholesterol; Cer, ceramide; PL, phospholipids; SM, sphingomyelin; GlcCer, glucosylceramides; CholS, cholesterol sulfate). (b) Percentage changes of ceramides subclasses in smokers compared to nonsmokers (LC–MS²). (c) Ratio of ceramides with double bonds at position 4 of the sphingoid base to ceramides without such double bond (LC–MS²). (d) Conformation of the skin barrier lipids determined by infrared spectroscopy.