UV Radiation-induced DNA Damage And The Protective Effect Of Resveratrol In Keratinocyte

According to the wavelengths, ultraviolet (UV) radiation from sun can be divided into UVA(320～400nm), UVB(280～320nm) and UVC(100～280nm).The ozone layer at the height of 10km is like a blanket blocking the majority of UVA、 UVB and 100%UVC. Since 1974, scientists have found the ozone layer destructed, so far the ozone hole appears in antarctic and arctic. Due to the destruction of the ozone layer, the amount of UV reaching the earth increases. The epidermis is the outermost layer as a natural barrier in the human body. It is the organ that direct contact with the outside world protecting the body from the bad environment. Ultraviolet radiation is one of important physical factor while excessive ultraviolet radiation may cause inflammation, erythema, immunosuppression and even skin cancer. As early as 1997, Osaka University proved that ultraviolet can induce skin cancer. In the past few decades, the incidence of skin cancer is increasing dramaticly in European and America. According to the statistics, every 1% reduction in ozone content, the risk of skin cancer will increase by 2%～3%.UV-induced DNA damage and gene mutation result in skin cancer. DNA damage will affect cell growth, development and proliferation. Moreover it even can lead to gene mutation, cancer and death. UV can induce DNA damage in a direct or indirect way corresponding to different wavebands. For example, UVA mainly induce DNA damage indirectly. UVB and UVC can be absorbed by DNA base for in the UV absorption peak to make DNA damage directly. Besides UVB is also proved to induce DNA damage indirectly by producing excess free radicals. HaCaT is a model to study UV-induced damage. Therefore in this study we selected HaCaT cells to study DNA damage caused by different wavebands and different dose of UV irradiationAs to the protection for DNA damage, antioxidant has always been a research hot spot except for UV fabric physical blockers and chemical absorbent. However, the existing antioxdants are not ideal for their unstable characteristic and not all-powerful. Resveratrol (RSV) is a natural polyphenol structurally similar to diethylstilbestrol and estradiol. It is known for its anti-inflammatory, anti-tumor, cardioprotective, neuroprotective. Moreover, RSV is an important antioxidant. So in this experiment comet assay was employed to detect the RSV protection of DNA damage induced by UV and explore the protection mechanism preliminarily.PURPOSETo study DNA damage induced by UV and the protection of resveratrol in HaCaT cell, and to explore the protection mechanism of resveratrol preliminarily.METHOD1. Cell cultureHuman keratinocyte cell line, HaCaT cells, were maintained in DMEM supplemented with10% FBS and 100 U/ml penicillin and 100 μg/ml streptomycin.in a humidified atmosphere with CO2(5%, v/v) at 37℃.2. UV irradiationCell will be irradiated when grown to 80%-90%. Prior to UV exposure, cells were washed twice with phosphate buffered saline (PBS) and covered with a thin layer of PBS. In parallel, non-irradiated cells were treated similarly and kept in the dark in an incubator.3. Comet assay detecting DNA damage induced by UV radiationThe cells were exposed to different dose of UVA, UVB or UVC. Irradiated and non-irradiated control cells were harvested and made into suspension. The slides were immersed in ice-cold alkaline lysis solution, unwinding, electrophoresis, neutralization, dyeing and rinsing. The coded slides were examined under a fluorescence micro-scope. And then images were analysed by CASP software.4. Comet assay detecting of resveratrol function in DNA damage induced by UV radiationDifferent concentrations of resveratrol were added to the culture medium for 12h. Then cells were irradiated with 30mJ/cm2UVB or UVC. The method was described as above. Irradiated and non-irradiated control cells were harvested and made into suspension.The slides were immersed in ice-cold alkaline lysis solution, unwinding, electrophoresis, neutralization, dyeing and rinsing. The coded slides were examined under a fluorescence micro-scope. And then images were analysed by CASP software.5. Comet assay detecting the effect by resveratrol and SIRT inhibitor pretreatment in DNA damage induced by UV radiationHaCaT cells were pretreatment with 25μmol/L resveratrol or 5mmol/L SIRT inhibitor for 16h before 30mJ/cm2 UVB irradiation.The irradiation method was described above. Irradiated and non-irradiated control cells were harvested and made into suspension.Then comet assay was done immediately.And the images were analysed by CASP software.6. Western blot (WB) testing the expression of SIRT6 after different dose of UVB irradiationHaCaT were harvested after different dose of UVB 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 PVDF membrane, blocked incubated primary antibody for 2h at room temperature, TBST rinsed, incubated second antibody for 2h and the chemiluminescent reaction was conducted and then developped and fixed TBST after rinsing.7. Comet assay detecting DNA damage induced by UV radiation after transfection with siSIRT6.After transfection for 24h, cells were irradiated by UVB. And the exposure dose was 30mJ/cm2. Then cells were harvested and made into suspension. The comet assay was done immediately.8. qPCR and Western blot detecting the effect by resveratrol and SIRT inhibitor pretreatment in DNA damage induced by UV radiationHaCaT cell were pretreated with resveratrol and harvested after irradiation immediately. Total RNA was extracted by trizol. We amplified SIRT6 and the products were identified by agarose gel electrophoresis. qPCR was employed to test SIRT6 mRNA expression. While total protein was extracted with the full protein extraction kit. The method of WB was described as above.9. 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 α=0.05.Result1. UVA irradiation had no significant effects on HaCaT cells.However, TailDNA%、TailLength、CometLength、TailMoment and OliveTailMoment showed both UVB and UVC induced DNA damage in a dose-dependent manner. UVC was more harmful than UVB at the same dose.2. No obvious DNA damage was observed by comet assay after resveratrol pretreatment compared with control;that is to say:HaCaT cell pretreated with resveratrol alone do not cause DNA damage.3. DNA damage induced by UVB descreased significantly after treated with 0.1μmol/L resveratrol examined under a fluorescence microscope directly or evaluating by TailDNA%、TailLength、ComeLength、TailMoment and OliveTailMoment indirectly.4. DNA damage induced by UVC descreased significantly after treated with 0.1μmol/L and 10μmol/L resveratrol examined under a fluorescence microscope directly or evaluating by TailDNA%、TailLength、ComeLength、TailMoment and OliveTailMoment indirectly.5. TailDNA%、TailLength、ComeLength、TailMoment and OliveTailMoment showed that pretreatment with 25μmol/L resveratrol groups had significant effects in descreasing DNA damage induced by UVB. However, when pretreated with 5mmol/L SIRT inhibitor, UVB induced DNA damage showed more serious examined under a fluorescence micro-scope directly or evaluating by TailDNA%、TailLength、 ComeLength、TailMoment and OliveTailMoment.6. Western blot revealed that the expression of SIRT6 was induced by UVB in a dose-dependent manner.7. The images of comet assay showed that siSIRT6+UVB group had longer tail than siNC+UVB group. TailDNA%、TailLength、ComeLength、TailMoment and OliveTailMoment had a significant increase in siSIRT6+UVB group.8. The mRNA and protein expression levels of SIRT6 were up-regulated after UV irradiation stress tested by q-PCR and western blot assay. Moreover, the two genes were also induced by different concentrations of resveratrol treatment alone or combined with UVB irradiation. And the expression of SIRT6 level was more obvious in the resveratrol and UVB combinded groups compared with resveratrol treatment alone.Conclusion1. In 0-90 mJ/cm2, UVA irradiation had no significant effects on HaCaT cells tested by comet assay. Both UVB and UVC induced DNA damage in a dose-dependent manner. UVC was more harmful than UVB at the same dose.2. Reservatrol protection mechanism may be related to SIRT6.