Study Of The Effect Of Heme Oxygenase On UVA-induced Damage In Human Skin Keratinocytes

Background:Ultraviolet A (UVA) radiation can lead to acute inflammation (such as erythema),photoaging, photosensitization, skin diseases, and skin cancer. Heme oxygenase (HO) isan antioxidant enzyme distributed widely in the body, which can protect many tissuesand organs against oxidative damage caused by a variety of stimulus, but its role inresisting UVA irradiation-induced skin cell damage is poorly investigated.Objective:To investigate the oxidative damage effects of different doses of UVA irradiation (100、250、500kJ/m~2) on HaCaT cells; observe the effect of Bach1(BTB and CNC-homology1transcription factor) inhibition on different doses of UVA radiation (250、500kJ/m~2)induced photodamage; and observe the effect of HO deficiency on physiological doseUVA irradiation (250kJ/m~2) induced HaCaT cell injury. To explore whether Bach1andHO can regulate the cell damage caused by UVA irradiation.Methods:①Cells culture: HaCaT cells were cultued in RMPI l640medium containing10%fetal bovine serum; Equal number of cells were plated in6、96-wells or petri plates.②Ultraviolet radiation: According to the experimental design, HaCaT cells weresham or irradiated with different doses of UVA irradiation and pretreated with RNAinterference before exposure.③Cell morphology assay: Cell cytoskeleton were stained with phalloidin andnuclear were stained with DAPI, and observed under fluorescence microscope.④Cell activity assay: MTS assay was used to detect cell proliferation activity.⑤ROS (reactive oxygen species) levels detection: Fluorescent probe DHE wasused to detect cellular ROS levels.⑥Cell DNA damage detection: Single-cell gel electrophoresis technique was usedto detect cell DNA damage.Results:①Different doses of UVAirradiation-induced HaCaT cell injury:Compared with sham group cells, small dose of100kJ/m~2UVA irradiation groupshowed no significant difference in cell morphology, cell activity, cellular ROS levelsand DNA damage; in physiological dose of250kJ/m~2UVA irradiation group, slightshrinkage and a small amount of cell debris was observed, cell viability was significantly decreased, the ROS levels and DNA damage increased significantly, andthe difference was statistically significant (p<0.05); in large dose of500kJ/m~2of UVAirradiated cells, compared with the250kJ/m~2UVA irradiation group, the cell shrinkageand debris is more obvious, cell viability was further reduced, cellular ROS levels andDNA damage increased significantly and the difference was statistically significant(p<0.05).②The influence of Bach1interference on different doses of UVAirradiation-induced HaCaT cell damage:Compared with scrambled group cells, cells treated with2nM and10nM Bach1interfere had no significant difference in cell morphology, activity, ROS levels, andDNA damage. Pretreatment with2nM Bach1interference had no effect on250kJ/m~2and500kJ/m~2UVA induced damage. Pretreatment with10nM Bach1interference canreduce250kJ/m~2and500kJ/m~2UVA irradiation-induced cell damage, Characterized bythe increased cell activity, cell morphology recovery，the reduced ROS production andDNA damage, cmpared with500kJ/m~2UVA irradiated cells,10nM Bach1interferencepretreatment group showed significant difference statistically (p<0.05).③The influence of HO interference on physiological dose of UVAirradiation-induced HaCaT cell damage:Compared with scrambled control group, cells treated with10nM and20nM HOinterfere showed feeble decreased cell growth and cell activity, but no significantchange in cellular ROS levels and DNA damage. Pretreatment with10nM or20nMHO interference can inhibit physiological dose UVA irradiation-induced cell damage,Characterized by the reduced cell activity, serious cell shrinkage，increased ROSproduction and DNA damage.Conclusion:HO may reduce the long-wave ultraviolet (UVA) irradiation-induced HaCaT celldamage, and its protective mechanism may involve the alleviated morphologicaldamage, the increased of cell proliferation activity, the reduction of cellular ROS levelsand DNA damage.