The Study On The Autophagy And Raman Spectroscopy Of HaCaT Cell Induced By Ultraviolet Radiation

Ultraviolet(UV) radiation from Solar can be divided into UVA(31～400nm), UVB(280-315nm) and UVC(100-280nm) according to the wavelengths. All UVC and about 90% of UVB could be absorbed by ozone, water vapor,oxygen and carbon dioxide when it passes through the atmosphere besides UVA. Therefore, UVA and a small amount of UVB can reach the earth’s surface. With the ozone depletion by global industrialization, the amount of UV reaches the earth increase. It will increase the risk of health effects. Skin that cover the body surfae is the first line of defense against outside injury,95% UV irradiation reaching skin was absorbed by keratinoeytes of skin. Overdose UVB radiation may result in acute and chronic health effect on skin, such as skin erythema, inflammation, skin aging and even increase the likelihood of developing cancer.Previous studies showed that, UVB would damage cell normal functions by at least four ways to affect cell survival conditions, which lead to cell necrosis or apoptosis:1 Direct damage to cell DNA; 2 Activation of sphingomyelinase which can lead to sphingomyelin degradation,result in the increase level of ceramide and their derivatives; 3 Activation of cell surface death receptors such as CD95 etc; 4 Create free radicals and lipid Peroxidation through the cell membrane and mitochondria membrane. The key step of mechanism is energy transfer between UV-Photonen to Molecular cells.There are two types of energy transfer:when UV-Photonen higher than ionization energy it causes damage on atoms and molecules; when lower than ionization energy it can be absorped and initial electronic transition. The main photobiology effect of UV radiation on skin cell is stimulation the molecules by means of selective absorption of ultraviolet spectrum energy by chromosphere, then the stimulated molecules convert energy back to the base state through optical radiation, internal conversion, collision, chemical reaction and energy transfer. The mechanism of DNA damage induced by UV radiation is different, UVB and UVC can be directly absorbed by DNA because their wavelength range are just near the absorption peak of DNA. The most common damages are cyclobutane pyrimidine dimmer, Pyrimidine(6-4) pyrimidine ketone, single adducts of pyrimidine, dimerization base damage; UVA can induce DNA damage by transfer energy to light sensitization agent in cell, while the common damages are base damage, DNA cross-linking, DNA strand break.Raman scattering is the inelastic scattering of a photon, When photons are scattered from an atom or molecule, a small fraction of the scattered photons are scattered by an excitation, with the scattered photons having a frequency different from, and usually lower than, that of the incident photons. It was first observed by the Indian physicist Raman in 1928. Raman spectroscopy was applied to DNA macromolecular conformational study since early 1970s. It could be used to estimate the functional groups or DNA molecular bonds and to determine DNA damage when functional groups and chemical change base on the characteristics of the Raman spectral lines(peak) and location(Raman frequency shift). In our study, we irradiate HaCaT cell with UVA and UVB and conduct raman spectrum analysis HaCaT DNA to explore the mechanism of DNA damage induced by UVA and UVB according to the characters of Raman spectra of the corresponding change of the position and intensity of the spectral and further analysis of the process of energy exchange and function of some molecular structure when damage induced. Resveratrol is an natural stilbene compound extra from rhizome of bullbrier which can decrease malondialdehyde production, aggrandize activities of SOD, GSH-px and CAT, and scavenge free radical. Resveratrol possess the ability to absorpt ultraviolet radiation especially UVB. Resveratrol can interaction with DNA electrostatic attraction and hydrogen bonding through Polyphenol structure. In our experiment, we use raman spectra to study the protection of the secondary structure of DNA before UVB irradiantion pretreatment with resveratrol.Cell autophagy is the degradation of a lysosome dependent pathway widely exists in eukaryotic cells, the damage of intracellular proteins and organelles by autophagy function degradation, degradation products such as amino acids, fatty acids was cells reused again. Lack of growth factors, oxygen free radicals, DNA damage and stress can lead to autophagy. There are three main pathway involved in autophagy according to the pattern of conveyance from cell to lysosome:1 Macroautophagy, the most important autophagy pahtway, occuring from double membrane structure in cell cytoplasm combine with lysosome to generate Autophagy vesicles, mitochondria, endoplasmic reticulum and ribosome are degrade in this way; 2 Microautophagy, occurring by lysosomal membrane invaginat to package around the material and degraded with the help of hydrolytic enzyme; 3 Chaperone-mediated autophagy, a pathway with no lysosomal membrane generated, which means a special base sequence of proteins in the cytoplasm identified by molecular chaperone, material waiting for degradation entrance lysosome by means of combine with lysosome associated membrane protein. Cells that are starving immediately activate autophagy to synthesize new proteins, generate energy and promote sugar dysplasia to maintain the balance of amino acid pool in the cytoplasm. MEFs induced by starvation, the change of LC3-II could be observed. The lifespan of autophagy varies, whereas the half-life time range from 8min to a couple of hours. To investigate the autophagy change of HaCaT cells after stimulated, we cultured HaCaT cell irradiated by wavelength of 305 nm UVB and observed the autophagy changing in the following 5h.PURPOSE1.To study secondary structure damage induced by UVA and UVB in HaCaT cell.2.To Study the protection of resveratrol on the secondary structure in the process of HaCaT cell DNA damage induced by UVB.3.To study the dose-response and time-response effect of autophagy induced by UVB on HaCaT cell.METHOD1. Cell cultureHaCaT cells were inoculated in volume fraction of 10% FBS, 1×105 u/L penicillin, mass concentration of 100 mg/L streptomycin high sugar DMEM medium, At 37℃,5% CO2 incubator in conventional adherent culture.2.UV light sourceUV light was producted by Shanghai Gu village Photoelectric Instrument Factory, and detected by the Shanghai Municipal Bureau. The UV emission peak of 305nm, when the light from the sample at a vertical distance of 40cm. HaCaT cells in a petri dish when grown to 80%, disacard culture medium, PBS wash twice and cover 1.8ml PBS before irradiaon.3.Pretreatment with resveratrolHaCaT cells were cultured in DMEM medium with O.1μmol/ml resveratrol for 6h before UVB irradiation. Disacard culture medium, PBS wash twice and apply UVB irradiation ditto4.Cell genome DNA extractionHaCaT cells were harvested at 0.5th,1.0th,2.0th,3.0th,4.0th, and 5.0% after 82.6 mJ/cm2 UVA or 29.7 mJ/cm2 UVB irradiation, In accordance with the QIAamp Mini DNA Kit protocol, Separately extract HaCaT cells genome DNA for each.5.The laser confocal Raman spectrum measurementUse laser confocal Raman spectrum inverted microscope system to test HaCaT cells DNA. To ensure the equal laser power that exposure to the samples each time, use standard silicon to proofread the wave number axis and laser power before measurement.20 times objective lens, grating with 600 lines/mm,500 microns confocal aperture,600-2000 cm-1 acquisition frequency,1 cm-1 spectral resolution,30 s sample scanning exposure integration time, repeat 10 times.6.acridine orange dyeHaCaT were cultured to 1.0、2.0、3.0、4.0、5.0h after irradiation, disacard culture medium, 1ml PBS wash twice,3ml diluted 100 times of acridine orange dye solution,5% CO2 incubator cultured for 15min, lml PBS wash thrice Fluorescent staining observed under inverted microscope。7.western blotHaCaT were cultured to 1.0、2.0、3.0、4.0、5.0h after irradiation. Total protein was extracted with the full protein extraction kit. The total extracted cellular protein was quantitatived and conducted by SDS-PAGE, transferred to a membrane,5% nonfat dry milk closed, an anti at room temperature incubated for 2h, TBST rinsed, two anti incubated at room temperature for 2h, and the chemiluminescent reaction was conducted and then developped and fixed TBST after rinsing.8.Statistical analysisSPSS 19.0 software was used, Measurement data by the normality test accord with normal distribution or approximate normal distribution were described as the mean ± standard deviation (x±s), Multiple sets of mean comparison using single factor analysis of variance, Multiple comparison using variance together using the LSD method, when the variance is not neat the Dunnett-T3 method. Inspection level of alpha=0.05.Result1.No obvious changes of DNA Raman spectra main line were observed After UVA and UVB irradiate; There were statistically significant differences of guanine hydrogen bonding, electrostatic repulsion of phosphate, adenine base molecular energy and adenine base stacking characteristic frequency spectrum in three groups (P<0.05); In multiple comparisons, there was no statistically significant differences of guanine hydrogen bonding between UVA and control group (P>0.05); There are significant interaction effect between groups and time (P＜0.05)2.No obvious changes of DNA Raman spectra main line were observed After UVB and UVB+ resveratrol irradiate; There were statistically significant differences of guanine hydrogen bonding, electrostatic repulsion of phosphate, adenine base molecular energy and adenine base stacking characteristic frequency spectrum in three groups (P<0.05); In multiple comparisons, there were statistically significant differences of guanine hydrogen bonding in Pair-wise comparison (P<0.01), there were statistically significant differences of electrostatic repulsion of phosphate between UVB and control group (P<0.05), there were no statistically significant differences of adenine base molecular energy bwtween UVB and UVB+ resveratrol group (P>0.05), there were no statistically significant differences of adenine base stacking bwtween control and UVB+resveratrol group (P>0.05); There are significant interaction effect between groups and time (P<0.05)3.HaCaT cells were cultured to 1.0thh、2.0thh、3.0thh、4.0thh、5.0thh after 49.5mJ/cm2 UVB irradiate, autophagy cells increase from 20.15±2.10% at 1.0% to the peak 50.67 ± 7.33% at 3.0%, then decrease to 21.39 ± 3.75% at 5.0% (P< 0.05). HaCaT cells were cultured to 3.0% after 0mJ/cm2(control)、9.9mJ/cm2、 29.7mJ/cm2、49.5mJ/cm2 UVB irradiation, autophagy cells increase from 4.13 ±1.02 % to 50.67 ±7.33%, which is positively related to dose (P<0.05)Conclusion1.UVA and UVB cause HaCaT cell DNA damage by changing on some molecular structure such as guanine hydrogen bonding, electrostatic repulsion of phosphate, adenine base molecular energy and adenine base stacking, UVB contributes more harms effects than UVA.2.Resveratrol can reduce HaCaT cell DNA damage induced by UVB by protecting molecular structure such as guanine hydrogen bonding, electrostatic repulsion of phosphate and adenine base stacking.3.Autophagy in HaCaT cell increase to the peak level at 3.0th after 49.5mJ/cm2 UVB irradiate, autophagy in HaCaT cell is positively related to dose after 9.9～ 49.5mJ/cm2 UVB irradiate.