Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction

Abstract

Introduction: Skin aging may occur as a result of increased free radicals in the body. Vitamin E, the major chain-breaking antioxidant, prevents propagation of oxidative stress, especially in biological membranes. In this study, the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing oxidative stress-induced skin aging was evaluated by determining the rate of total collagen synthesis and its gene expression in human skin fibroblasts.

Material and methods: Primary culture of human skin fibroblasts was derived from circumcision foreskin of 9 to 12 year-old boys. Fibroblast cells were divided into 5 different treatment groups: untreated control, hydrogen peroxide (H(2)O(2))-induced oxidative stress (20 µM H(2)O(2) exposure for 2 weeks), TRF treatment, and pre- and post-treatment of TRF to H(2)O(2)-induced oxidative stress.

Results: Our results showed that H(2)O(2)-induced oxidative stress decreased the rate of total collagen synthesis and down-regulated COL I and COL III in skin fibroblasts. Pre-treatment of TRF protected against H(2)O(2)-induced oxidative stress as shown by increase in total collagen synthesis and up-regulation of COL I and COL III (p<0.05) genes. However, similar protective effects against H(2)O(2)-induced oxidative stress were not observed in the post-treated fibroblasts.

Conclusions: Tocotrienol-rich fraction protects against H(2)O(2)-induced oxidative stress in human skin fibroblast culture by modulating the expression of COL I and COL III genes with concomitant increase in the rate of total collagen synthesis. These findings may indicate TRF protection against oxidative stress-induced skin aging.

Keywords: collagen synthesis; gene expression; skin aging; tocotrienol-rich fraction.

Figure 1

Effects of various concentrations of tocotrienol-rich fraction (TRF) on skin fibroblasts after incubation for 24 h at 37°C. Incubation with TRF caused a significant increase in the number of viable cells for skin fibroblasts. The percentage of viable cells was significantly increased at TRF concentrations of 200, 300, 400 and 500 g/ml. Correlation analysis showed a significant correlation between cell viability and concentration of TRF (R²=0.865, p=0.026) ^ap < 0.05 compared to control, data are presented as mean±SD

Figure 2

Comparison of total collagen synthesised in different treatment groups of skin fibroblasts. Total collagen was increased in TRF-treated fibroblasts while H₂O₂-induced oxidative stress caused a reduction in the synthesis of total collagen. Pretreatment of TRF to H₂O₂-induced oxidative stress increased the synthesis of total collagen ^ap < 0.05 compared to untreated control, bp < 0.05 compared to TRF-treated fibroblasts, cp < 0.05 compared to H₂O₂-induced oxidative stress; data are presented as mean±SD

Figure 3

Agarose gel electrophoresis showed that each PCR product appeared as a single band (A) while the melt curve (B) and melt peak (C) analysis showed a single narrow peak of each PCR product without the formation of a primer-dimer structure, indicating that the designed primers and the real-time RT-PCR protocols were specific

Figure 4

Expression of COL I in different treatment groups of skin fibroblasts. COL I was down-regulated in skin fibroblasts with H₂O₂-induced oxidative stress. Pre-treatment of TRF to H₂O₂-induced oxidative stress increased the expression of COL I gene ^ap < 0.05 compared to untreated control, bp < 0.05 compared to TRF-treated fibroblasts, cp < 0.05 compared to H₂O₂-induced oxidative stress, dp < 0.05 compared to TRF pre-treated fibroblasts, data are presented as mean±SD

Figure 5

Expression of COL III in different treatment groups of skin fibroblasts. COL III was downregulated in skin fibroblasts with H₂O₂-induced oxidative stress. Pre-treatment of TRF to H₂O₂-induced oxidative stress increased the expression of COL III gene ^ap < 0.05 compared to untreated control, bp < 0.05 compared to TRF-treated fibroblasts, cp < 0.05 compared to H₂O₂-induced oxidative stress, dp < 0.05 compared to TRF pretreated fibroblasts, data are presented as mean±SD

Figure 6

Expression of COL IV in different treatment groups of skin fibroblasts Data are presented as mean±SD