Protective Effect of Amaranthus cruentus L. Seed Oil on UVA-Radiation-Induced Apoptosis in Human Skin Fibroblasts

Abstract

Since the exposure of fibroblasts to prolonged UVA radiation induces oxidative stress and apoptosis, there is a need for effective skin protection compounds with cytoprotective and antioxidant properties. One of their sources is Amaranthus cruentus L. seed oil (AmO), which is rich in unsaturated fatty acids, squalene, vitamin E derivatives and phytosterols. The aim of this study was to evaluate whether AmO evokes a protective effect on the apoptosis induced by UVA radiation in human skin fibroblasts. UVA radiation at an applied dose of 10 J/cm² caused a significant reduction in the survival of human skin fibroblasts and directed them into the apoptosis pathway. Increased expression of p53, caspase-3, caspase-9 and PARP proteins in UVA-treated fibroblasts suggests the intrinsic mechanism of apoptosis. Application of the oil at 0.1% and 0.15% concentrations to UVA-treated cells decreased the expression of these proteins, which was accompanied by increased cell survival. Similarly, the UVA-dependent decrease in the expression of p-Akt and mTOR proteins was restored under the effect of the studied oil. The molecular mechanism of this phenomenon was related to the stimulation of antioxidant processes through the activation of Nrf2. This suggests that AmO stimulated the antioxidant system in fibroblasts, preventing the effects of UVA-induced oxidative stress, which may lead to pharmaceutical and cosmetological applications as a sun-protective substance.

Keywords: Amaranthus cruentus L. seed oil; UVA radiation; antioxidant; apoptosis; cosmetology; dermal fibroblasts; oxidative stress; pharmacy; sun-protective substance.

Figure 1

(A) The effects of different doses of UVA radiation on human skin fibroblasts’ cell viability. The mean ± standard error (SEM) values from the experiments performed in triplicates. * Statistically significant difference at p < 0.05 compared with the control. (B) The effects of different doses of AmO in human skin fibroblasts’ cell viability. The mean ± standard error (SEM) values from experiments performed in triplicates. * Statistically significant differences at p < 0.05 compared with the control.

Figure 1

(A) The effects of different doses of UVA radiation on human skin fibroblasts’ cell viability. The mean ± standard error (SEM) values from the experiments performed in triplicates. * Statistically significant difference at p < 0.05 compared with the control. (B) The effects of different doses of AmO in human skin fibroblasts’ cell viability. The mean ± standard error (SEM) values from experiments performed in triplicates. * Statistically significant differences at p < 0.05 compared with the control.

Figure 2

Fluorescence analysis of ROS generation in UVA irradiated fibroblasts treated with AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Red fluorescence intensity represents the amount of generated ROS. The images were obtained at a 20× magnification.

Figure 3

Cell viability in fibroblasts irradiated with UVA and treated with AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. The mean ± standard error (SEM) values from the experiments performed in triplicates. * Statistically significant differences at p < 0.05 compared with the control.

Figure 4

Cytometric assay of apoptosis in UVA-irradiated fibroblasts treated with AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. The cells were stained with fluorescent dyes using Propidium Iodide and Annexin V.

Figure 5

Cytometric assay of reduced thiols level in UVA-irradiated fibroblasts treated with AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. The cells were stained with fluorescent dyes Propidium Iodide and VB-48^TM.

Figure 6

Immunofluorescence staining of p53 expression in fibroblasts irradiated by UVA in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents p53 expression. The images were obtained at a 20× magnification.

Figure 7

Immunofluorescence staining of caspase-3 expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents caspase-3 expression. The images were obtained at 20× magnification.

Figure 8

Western blot analysis (A) and densitometry results via ImageJ^® (B) for caspase-9 expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. The mean values of 3 pooled cell homogenate extracts from 3 independent experiments are presented.

Figure 9

Immunofluorescence staining of p-Akt protein expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents p-Akt protein expression. The images were obtained at 20× magnification.

Figure 10

Immunofluorescence staining of mTOR protein expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents mTOR protein expression. The images were obtained at 20× magnification.

Figure 11

DNA biosynthesis in the UVA irradiated fibroblasts in the presence of AmO at concentrations 0.05%, 0.1% and 0.15% vs. the control. The mean ± standard error (SEM) values from the experiments performed in triplicates. * Statistically significant differences at p < 0.05 compared with the control.

Figure 12

Immunofluorescence staining of PARP protein expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents PARP protein expression. The images were obtained at 20× magnification.

Figure 13

Immunofluorescence staining of Nrf2 protein expression in UVA-irradiated fibroblasts in the presence of AmO at concentrations of 0.05%, 0.1% and 0.15% vs. the control. Blue staining indicates the nuclei and red staining represents Nrf2 protein expression. The images were obtained at 20× magnification.