Isoegomaketone from Perilla frutescens (L.) Britt Stimulates MAPK/ERK Pathway in Human Keratinocyte to Promote Skin Wound Healing

Abstract

Skin wound healing is essential for recovery from injury, and delayed or impaired wound healing is a severe therapeutic challenge. Keratinocytes, a major component of the epidermis, play crucial roles in reepithelialization during wound healing including cell proliferation. Recent studies have shown that compounds from natural products have candidates for healing skin injury. Isoegomaketone (IK), isolated from leaves of Perilla frutescens var. crispa (Lamiaceae), has various bioactivities. However, the effect of IK on cutaneous wound healing processes has not been studied yet. In this study, we demonstrated that IK exhibits therapeutic wound healing effects using the human keratinocyte cell line HaCaT. Notably, IK promoted cell proliferation and migration in a dose-dependent manner in vitro, and treatment with 10 μM IK upregulated these processes by approximately 1.5-fold after 24 h compared with the control. IK induced the activation of the MAPK/ERK pathway and cell cycle progression to the S and G2/M phases. Thus, this study demonstrates IK as a potential candidate to upregulate wound healing that may provide therapeutic benefits to patients with delayed wound healing.

Figure 1

Effects of IK on cell proliferation in HaCaT cells. (a) Chemical structure of IK. (b) Cell viability of HaCaT cells with various concentrations of IK at 24 h. Cell viability was measured using an Ez-Cytox Kit. The results are presented as the means ± SD of six independent experiments ( ^∗p < 0.05,  ^∗∗p < 0.01, and  ^∗∗∗p < 0.001 versus 0 μM group).

Figure 2

Effects of IK on scratch wound closure of HaCaT cells in vitro. HaCaT cell monolayer was mechanically scratched and incubated with the indicated concentration of IK. HaCaT cell migration was monitored using a microscope at 0 and 24 h after scratch. (a) Images of HaCaT cells treated IK at 0 and 24 h at 10 × magnification. The yellow line shows the border of the wound. (b) Relative wound closure area of HaCaT cells according to IK concentration. The wound area was measured using Image J and calculated as the ratio of the scratch area at 24 h relative to 0 h was calculated. The results are presented as the means ± SD of three independent experiments ( ^∗p < 0.05,  ^∗∗p < 0.01, and  ^∗∗∗p < 0.001 versus 0 μM group).

Figure 3

IK enhances proliferation of keratinocytes through induction of ERK 1/2 pathway. (a) Western blot of HaCaT cells treated with varying concentrations of IK. HaCaT cells were treated with IK (0, 1, 5, and 10 μM) for 24 h. Whole-cell extracts were harvested and analyzed using the antibody against total JNK, ERK1/2, p38, phospho-JNK, ERK1/2, p38, and beta-tubulin was used as the loading control. (b) Viability of HaCaT cells compared with only IK treatments and cotreated with ERK 1/2 inhibitor, PD98059. HaCaT cells were seeded into a 96-well cell culture plate and treated with various concentrations of IK only or with ERK 1/2 inhibitor for 24 h. Cell viability was measured using an Ez-Cytox Kit. The results are presented as the means ± SD of three independent experiments ( ^∗p < 0.05,  ^∗∗p < 0.01, and  ^∗∗∗p < 0.001 versus control group).

Figure 4

Effects of IK on cell cycle progression in HaCaT cells. (a) Cell cycle ratio of HaCaT cells after treatment with IK. (b) A plot of the cell cycle pattern of HaCaT cells under different treatment concentrations of IK. The results are presented as the means ± SD of three independent experiments.