Pearl extract protects HaCaT cells from UV radiation-induced apoptosis through mitochondrial pathway regulation
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Presented by: Jing Wang
Introduction: Previous studies demonstrated that pearl extract (PE) promotes wound healing and skin whitening. However, whether PE can inhibit ultraviolet (UV)-photodamage in HaCaT cells remains unclear. In this study, an in vitro photoaging cell model was established to observe the effect of PE on UV-induced damage and apoptosis of HaCaT cells. The aim was to provide a reference for future development of natural sunscreen agents.
Method:HaCaT cells were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37℃. The cells were irradiated with 10 J/cm2 UV, while control cells were sham-irradiated by covering with tin foil. Cell viability was assessed by the CCK8 assay. The cell suspensions were collected and assayed for ROS and MDA levels, and GSH-Px and SOD activities using assay kits in accordance with the manufacturer's instructions. Total RNA was isolated from HaCaT cells after treatment using the RNAiso Plus kit according to the manufacturer's guidelines. After 48 h of the indicated treatment, the supernatant of each group of cells in the 6-well plate was collected. TNF-a and IL-10 were detected in accordance with the enzyme-linked immunosorbent assay kit instructions.
Results: PE concentrations of 0.1 and 1 μg/mL were considered as the most effective and safe concentrations. Compared to the control group, superoxide dismutase and glutathione peroxidase activities in the photoaging group were significantly reduced, while malondialdehyde and reactive oxygen species content, along with tumour necrosis factor-alpha (TNF-a) and interleukin (IL)-10 mRNA and protein levels were markedly increased. In contrast, Bcl-2 protein expression was significantly decreased, while caspase-3, caspase-9 and Bax protein expression levels were significantly increased. Compared to the photoaging group, HaCaT cell proliferation was significantly increased in the PE group. Both PE concentrations significantly increased superoxide dismutase and glutathione peroxidase activities in cells, reduced malondialdehyde and reactive oxygen species content, decreased TNF-a and IL-10 mRNA expression in cells, and reduced TNF-a and IL-10 protein levels in the supernatant. Additionally, Bcl-2 protein expression levels were significantly increased, while caspase-3, caspase-9, and Bax protein expression levels were significantly reduced by PE treatment.
Discussion：HaCaT cells reside in the most superficial layer of human skin and are the main target cells of UV radiation [17]. Long-term UV radiation can lead to skin photoaging and even cancer [18]. Apoptosis refers to the gene-regulated process of autonomous and orderly cell death to maintain a stable state in the internal environment [19]. Because of changes in the cellular internal and external environment, as well as stimulation of death signals, this process eliminates aging cells and other cells with potential abnormal growth to maintain a stable state in the cell population [20]. Apoptosis is mainly controlled by three pathways: mitochondrial signalling, death receptor-mediated signalling and endoplasmic reticulum-mediated signalling [21]. Apoptosis is closely related to changes in mitochondrial structure and function.
Conclusions: PE can inhibit UV-induced apoptosis by inhibiting mitochondria-mediated apoptosis and regulating TNF-a and IL-10 expression.
Method:HaCaT cells were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37℃. The cells were irradiated with 10 J/cm2 UV, while control cells were sham-irradiated by covering with tin foil. Cell viability was assessed by the CCK8 assay. The cell suspensions were collected and assayed for ROS and MDA levels, and GSH-Px and SOD activities using assay kits in accordance with the manufacturer's instructions. Total RNA was isolated from HaCaT cells after treatment using the RNAiso Plus kit according to the manufacturer's guidelines. After 48 h of the indicated treatment, the supernatant of each group of cells in the 6-well plate was collected. TNF-a and IL-10 were detected in accordance with the enzyme-linked immunosorbent assay kit instructions.
Results: PE concentrations of 0.1 and 1 μg/mL were considered as the most effective and safe concentrations. Compared to the control group, superoxide dismutase and glutathione peroxidase activities in the photoaging group were significantly reduced, while malondialdehyde and reactive oxygen species content, along with tumour necrosis factor-alpha (TNF-a) and interleukin (IL)-10 mRNA and protein levels were markedly increased. In contrast, Bcl-2 protein expression was significantly decreased, while caspase-3, caspase-9 and Bax protein expression levels were significantly increased. Compared to the photoaging group, HaCaT cell proliferation was significantly increased in the PE group. Both PE concentrations significantly increased superoxide dismutase and glutathione peroxidase activities in cells, reduced malondialdehyde and reactive oxygen species content, decreased TNF-a and IL-10 mRNA expression in cells, and reduced TNF-a and IL-10 protein levels in the supernatant. Additionally, Bcl-2 protein expression levels were significantly increased, while caspase-3, caspase-9, and Bax protein expression levels were significantly reduced by PE treatment.
Discussion：HaCaT cells reside in the most superficial layer of human skin and are the main target cells of UV radiation [17]. Long-term UV radiation can lead to skin photoaging and even cancer [18]. Apoptosis refers to the gene-regulated process of autonomous and orderly cell death to maintain a stable state in the internal environment [19]. Because of changes in the cellular internal and external environment, as well as stimulation of death signals, this process eliminates aging cells and other cells with potential abnormal growth to maintain a stable state in the cell population [20]. Apoptosis is mainly controlled by three pathways: mitochondrial signalling, death receptor-mediated signalling and endoplasmic reticulum-mediated signalling [21]. Apoptosis is closely related to changes in mitochondrial structure and function.
Conclusions: PE can inhibit UV-induced apoptosis by inhibiting mitochondria-mediated apoptosis and regulating TNF-a and IL-10 expression.