Photoprotection Of EGCG And Hesperidin On HaCaT Cells Against Oxidative Damage And Apoptosis From UVA Irradiation

BackgroundSkin is the first protective layer against the environment stimulate, such as microorganism infection,physical and chemical materials irritation,to keep the internal environment stable.The numerber of keratinocytes is the richest in the epidermis,accountting for more than 80%.Ultraviolet irradiation of sunlight causes sunburn,photoaging and skin tumors.Long wave ultraviolet(UVA)accounts for 90%in the solar spectrum and penetrates more deeply into human skin.Recent evidence strongly suggested that reactive oxygen species (ROS)in HaCaT cells apoptosis induced by UVA irradiation.ROS, including hydroxide radical(OH-),hydrogen peroxide(H₂O₂),hydroxyl radicals(^·OH),and singlet oxygen(¹O₂),is produced during ultraviolet irradiation.ROS produced by UVA exerts a variety of harmful effects including oxidation of nucleic acids,proteins and membrane lipids, resulting in cell damage.ROS can also induces the depolarizat of mitochondrion,leading to augment membrane permeability and then proteins and enzymes related to apoptosis release to cytoplasm. Antiperoxidases including superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)exist in the cells can scavenge ROS generated by cell activities and lessen cellular oxidation.When ROS increases and antioxidation capacity drops,the cell can be damaged and proced to apoptosis.At present,more and more attention is focused on natural protective agents.Vitamin E,vitamin C and Carotin have been used as antioxidant agents.Traditional Chinese medicines(TCMs):EGCG(the major and most effective component of TP extracted from green tea)and hesperidin(HPD)have wide biological potencies.In the present study we attempted to explore whether EGCG and HPD inhibit the oxidative damage and Apoptosis of HaCaT cells caused by UVA radiation.The experimental results will contribute to the development and application of the natural sunscreens.ObjectiveTo investigate whether EGCG and HPD have toxicity to HaCaT cells;to study the oxidative damage and apoptosis of HaCaT cells caused by UVA radiation;to observe photo-protective efficiency of EGCG and HPD and the possible mechanisms.Materials and methods(1)Cells culture HaCaT cells were cultured in RMPI-1640 medium with 10%fetal bovine serum and cells were plated in 3.5 cm dishs and 96-well plate with an equal number of cells(10⁴～10⁶ cells).(2)Ultraviolet irradiation The time and dosage of UVA irradiation were conducted according to the experiment design.Tested medicines were added into the medium before and after UVA irradiation.(3)Cell viability assay The dose- and time- dependent photodamage was observed by light microscopy;methyl thiazolyl tetrazolium (MTT)assay was used to record cell proliferation and cellular activity.(4)Oxidative damages of HaCaT cells The activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),the total antioxidation capability and the level of maleic dialdehyde(MDA) were detected with colorimetric methods.(5)The mitochondrion membrane potential changes The mitochondrion membrane potential(MP)changes were detected by flow cytometry(FCM).(6)The apoptosis of HaCaT cells The apoptosis rates were detected by TUNEL assay or flow cytometry.(7)Statistical analysis The experimental data were analyzed with SPSS.P values less than 0.05 were considered to be statistically significant.Results:1.Cell viability after 10J/cm² UVA irradiation and photoprotection of EGCG and HPD: The proliferating activity of HaCaT cells was not decreased when HaCaT cells were preincubated with EGCG(under the concentration of 200μg/ml)or HPD(under the concentration of 320μg/ml).After 10J/cm² UVA irradiation,The A value was 0.3426±0.0075 which was significant lower than that of control(0.5437±0.1312).The viability of HaCaT cells preincubated with EGCG or HPD for 24h and then irradiated by 10J/cm² UVA was higher than 10J/cm² UVA irradiation. EGCG and HPD could protect the cellular activity partly and effectively.2.Effects of EGCG and HPD on the activities of SOD,GSH-Px,the total antioxidation capability and MDA in HaCaT cells irradiated by 10J/cm² UVA:After UVA irradiation,the activities of SOD,GSH-Px and total antioxidation capability in HaCaT cells decreased,the content of MDA ascensused(p＜0.05).In EGCG+UVA group,EGCG upregulated the activities of SOD,GSH-Px 31and6.2 times respectively and downregulated the level of MDA by 59%.In HPD+UVA group HPD upregulated the activities of SOD,total antioxidation capability 42 and10 times respectively and downregulated the level of MDA by 65%.3.Effects of EGCG and HPD on the mitochondria membrane potential changes in HaCaT Cells irradiated by 10J/cm² UVA:After UVA irradiation,the mitochondria membrane depolarized,leading to the augment of membrane permeability.EGCG and HPD decreased mitochondria membrane potential changes by 25%and 15%respectively (P＜0.05).EGCG and HPD had no influence on mitochondria membrane potential alone(P＞0.05)。4.Effects of EGCG and HPD on apoptosis changes of HaCaT Cells after 10J/cm² UVA irradiation.By TUNEL method,the apoptosis rate was 0.68%in the blank group and 6.72%in the UVA irradiated group tested by,suggesting that UVA could induce HaCaT Cells to proced to apoptosis.EGCG inhibited UVA-induced apoptosis and the rate was 3.28%(P＜0.05).By Flow cytometry the apoptosis rate was 0.72%in the blank group and 7.86%in the UVA irradiated group,HPD inhibited UVA-induced apoptosis and the rate was 4.34%(P＜0.05).ConclusionsThe viability of HaCaT cells was not decreased when HaCaT cells were preincubated with EGCG(≤200μg/ml)or HPD(≤320μg/ml).EGCG and HPD could protect the cellular viability.After UVA irradiated,the activities of SOD,GSH-Px and the total antioxidation capability in HaCaT cells decreased,the content of MDA and the apoptosis ascensused (p＜0.05).EGCG and HPD could partially recovery these injuries.EGCG and HPD could also decrease mitochondria membrane depolarization caused by UVA.This study confirmed that both EGCG and HPD could relieve the oxidation cell damaged and reduce cellular apoptosis from UVA irradiation by strengthening antioxidation and decreasing oxyradicals.