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Photoprotection of EGCG and Hesperidin on HaCaT Cells Against Oxidative Damage and Apoptosis from UVA Irradiation

Author: LiMin
Tutor: LuoDan
School: Nanjing Medical University
Course: Dermatology and Venereology
Keywords: UVA Table epigallocatechin gallate HPD Keratinocytes Oxidative damage Superoxide dismutase Glutathione peroxidase Malondialdehyde Total antioxidant capacity Apoptosis
CLC: R285
Type: Master's thesis
Year: 2008
Downloads: 138
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Abstract

Background: The skin is the body's first natural protective barrier, so that the body against external physical, chemical substances and micro-organisms against maintain body homeostasis. Keratinocytes are the principal components of the epidermal cells, accounting for more than 80% of the epidermal cells, the skin barrier function by keratinocytes completed. Long-term ultraviolet radiation not only causes skin photoaging and skin cancer. Ultraviolet (UV) divided by the wavelength ultraviolet A (UVA, 320 to 400 nm), ultraviolet B (UVB, 290 to 320 nm) and ultraviolet C (UVC, 200 to 290 nm). UVC ozone layer, almost no effect on the human skin; UVB target site is the epidermis, a small part of the superficial dermis, induced erythema, sunburn and skin cancer; ultraviolet A (UVA) accounted for reaching the Earth 90% of the surface UV and penetration can penetrate the epidermis into the dermis, UVA has an important role in UV damage to the skin. , People think UVA ROS lead to oxidative damage of cells and molecules mediated apoptosis induced by UVA study. UV-induced skin to produce ROS hydroxyl (OH ^{- ), superoxide anion (O ₂ ^{- ), singlet oxygen ( ^{1 O ₂) and hydrogen peroxide (H ₂ O ₂). ROS can damage cell proteins and enzyme activity, either directly or indirectly cause oxidative damage to DNA, leading to cell necrosis and apoptosis. Mitochondrial cellular energy power plants. Oxygen free radicals can start mitochondrial depolarization, increase the permeability of the mitochondrial membrane and apoptosis-related proteins and enzymes released into the cytoplasm, and activation of downstream effector induced apoptosis. Under normal circumstances, the memory cells in a series of anti-peroxidase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), they may be cleared of ROS produced within the cell, reduce cell injury oxide. Increased production of intracellular ROS clear decline in the ability of ROS and reduced antioxidant capacity can cause cell damage, cell apoptosis. In recent years, the natural compounds repellant already gained widespread concern. For example, vitamin C, E and beta-carotene have been successfully used in skin care products. The main active ingredient in traditional Chinese medicine extracts of green tea polyphenols (tea polyphenols, TP) epigallocatechin gallate acid resin (epigallocatechingallate EGCG) and hesperidin (hesperidin, HPD) has a wide range of biological effects. In view of the two active ingredients in the the UVA light protective effect is not clear, especially UVA irradiation after generation and removal of reactive oxygen species, and mitochondrial membrane potential protective effect, inhibition of apoptosis has not been reported. The project aims to explore the of EGCG and HPD UVA-induced human keratinocytes (HaCaT cell line) oxidative damage and apoptosis protective effect and its possible mechanism. You can take this provide a theoretical basis for the development and practical application of natural sunscreen and experimental reference data. Objective To observe the EGCG and HPD on cultured HaCaT cells without toxic effects; observed UVA irradiation of HaCaT cells and their mitochondria oxidative damage; observed EGCG and HPD the light protective effect of UVA-irradiated HaCaT cells, to explore its possible mechanism of action . Method 1 Cell culture: containing 10% fetal calf serum RMPI1640 cultured HaCaT cells, quantitative seeded in petri dishes or 96-well plates. Ultraviolet radiation: According to the experimental design time quantitative irradiated cultured cells, respectively, the UVA irradiation before and after drug intervention treatment. Cell injury and cell activity: MTT assay, cell proliferation activity. 4 cells from oxidative damage detection: colorimetric detection UVA irradiation cell supernatant of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, total antioxidant capacity (the total antioxidation capability) and malondialdehyde (MDA) content changes. mitochondrial membrane potential change detection: flow cytometry mitochondrial membrane potential changes. detection of apoptosis: TUNEL method or flow cytometry cells under different treatment conditions apoptosis rate. 7: Statistical analysis using ANOVA analysis, data processing applications SPSS11.0 statistical software, P <0.05 when the difference was statistically significant. The results 1.EGCG and HPD and 10J/cm ^{2 UVA irradiation of HaCaT cells proliferation activity of EGCG concentration 200μg/ml and below, HaCaT cells no significant side effects; HPD concentration of 320μg / ml and below HaCaT cells without significant side effects. A value of (0.3426 ± 0.0075) was significantly less than the control group (0.5437 ± 0.1312) by 10J/cm ^{2 UVA irradiation, UVA significantly inhibited HaCaT cell proliferation activity. HaCaT proliferation activity was significantly higher than the simple 10J/cm ^{2 UVA irradiation group (P <0.05) different concentrations of EGCG and HPD processing. The results showed that EGCG and HPD can significantly reduce the UVA illumination on cell proliferation inhibition. EGCG and HPD and 10J/cm ^{2 UVA irradiation of HaCaT cells SOD, GSH-Px, total antioxidant capacity and the content of MDA. UVA irradiation cell SOD and GSH-Px activity was significantly decreased total antioxidant capacity decreased MDA content increased, the difference was statistically significant (P <0.05). The EGCG treatment the photo therapy group SOD and GSH-Px activity and UVA irradiation alone group, respectively, compared to 31 and 6.2 times higher MDA content was significantly reduced, down 59%. Illuminated HPD treatment group SOD, total antioxidant capacity and UVA irradiation alone group compared significantly higher, respectively 42 times and 10 times higher. MDA decreased by 65%. 3.EGCG and HPD 10J/cm ^{the 2 UVA irradiated HaCaT cells mitochondrial membrane potential. 10J/cm ^{2 UVA irradiation increased mitochondrial membrane permeability, reduced mitochondrial membrane potential depolarization significantly enhanced compared with the control group; EGCG and HPD processing, 10J/cm ^{2 UVA irradiation changes in membrane potential relative separate 10J/cm in ^{2 UVA irradiation group were significantly reduced by 25% and 15%, the difference was statistically significant (P <0.05); EGCG and HPD alone The HaCaT cells processing, the changes in membrane potential with the control group, the difference was not statistically significant (P> 0.05). The impact of EGCG 4.EGCG and HPD 10J/cm ^{2 UVA irradiation of HaCaT cells apoptosis rate: TUNEL method to measure the blank group apoptosis rate was 0.68%, 10J/cm ^{2 UVA light apoptosis was 6.72%, indicating that UVA can induce apoptosis; EGCG treatment after UVA irradiation group positive apoptotic cells was 3.28%, a decrease of approximately 52% (P <0.05 relative UVA irradiation alone group ). HPD: flow cytometry method to measure the blank group apoptosis rate was 0.72%, 10J/cm ^{2 UVA irradiation of apoptotic cells was 7.86%, 4.34% apoptotic cells after treatment with HPD, relative UVA radiation alone group decreased by approximately 45% (P <0.05). Conclusion The results of this study show: EGCG ≤ 200μg/ml concentration, HPD ≤ 320μg/ml HaCaT cells in culture no significant side effects. The EGCG and HPD can significantly reduce 10J/cm ^{2 UVA irradiation inhibitory effects on cell proliferation. The UVA irradiation cell SOD, GSH-Px and total antioxidant capacity decreased, while the EGCG or HPD processing can be partially restored oxidative damage. Plus EGCG and HPD-treated cells can improve UVA irradiation to reduce the mitochondrial membrane potential and to enhance the role of depolarization, thereby reducing the rate of apoptosis. The preliminary results suggested that EGCG and HPD reduce UVA-induced ROS production, reduce apoptosis, which may be related to enhanced HaCaT cells remove free radicals, oxidative damage and protect the role of mitochondria link.}}}}}}}}}}}}}}}

Photoprotection of EGCG and Hesperidin on HaCaT Cells Against Oxidative Damage and Apoptosis from UVA Irradiation

Abstract

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