| BackgroundThe skin is not only the largest organ in the human body,but also the body’s first natural protective barrier.It plays an important role for the maintenance of homeostasis and against external harmful substances in the defense.Long-term ultraviolet radiation can cause skin early aging,skin appearance,structure and functional changes,manifested as skin rough,deep wrinkles,pigmentation or depigmentation,precancerous lesions and cancer.Ultraviolet A is a major component of the earth’s surface UV,accounting for about 90%,and penetrating power into the dermis.Human skin fibroblasts is an important part of leather,thus fibroblasts are the main target cells of the role of the UVA.Found in the UVA-induced oxidative damage and apoptosis in vitro and in vivo,on the one hand UVA irradiated skin produce a strong biological activity of the active oxygen species,such as hydroxyl,superoxide anion,singlet oxygen,hydrogen peroxide,on the other hand,it can produce nitric oxide which induced intracellular oxidative damage and apoptosis.NO is an important organism redox signaling molecules and a biological activity of nitrogen.As a unstable biological free radicals it can damage normal cells.UVA irradiation skin can produce strong activity of free radicals.They cause the destruction of proteins and enzymes,oxidative damage to DNA.ROS with nucleic acids may be formed of different types of nucleotide modifications,the most common substance is 8-hydroxy-guanine(8-oxo-dG).Because of the larger number,it usually as an important indicator of oxidative DNA damage.Radicals can start mitochondrial depolarization of the mitochondrial membrane permeability increased,resulting in apoptosis-related proteins and enzymes released into the cell activation of downstream effector,increased apoptosis.There is a close relationship between mitochondria and apoptosis.Studies have shown that a large number of reactive oxygen species generated can lead to changes in mitochondrial membrane permeability.Mitochondrial membrane permeability changes can cause a series of biochemical changes,such as the activation of caspase protease family,causing apoptosis cascade,accelerated apoptosis.Under normal circumstances,Caspase enzyme exists in an inactive zymogen form.Caspase-3 can cut most of the apoptotic cells substrate and to cracking many the apoptosis cascade program protease catalytic reaction protein.So caspase-3 is the apoptosis protease cascade the only way,is also a key enzyme in apoptosis and implementation.There are two major antioxidant system in the body,the enzymatic type and non-enzymatic type.Enzymatic type including superoxide dismutase(SOD),glutathione peroxidase(GPx),glutathione reductase(GR),catalase(CAT).They can be synthesized in the body and clear all kinds of reactive oxygen species.SOD is the main antioxidant enzymes in the tissue cells,the biological effect is the decomposition of superoxide anion,its activity reflects the level of the ability to scavenging oxygen free radicals in tissue cells.When the increased production of intracellular reactive oxygen species,clear decline in reactive oxygen species and decreased antioxidant capacity,they can cause cell damage,cell apoptosis.Explore an efficient and safety,anti-oxidative damage and apoptosis natural products for skin is of great significance.Baicalin is one of the active ingredients in traditional Chinese medicine skullcap,has a wide range of biological effects.Modern pharmacological studies have shown that baicalin has anti-inflammatory,anti-bacterial,anti-virus,free radical scavenging and antioxidant,anti-allergic,anti-tumor,and the protection of the nervous system pharmacological activity.UVA in hunan skin fibroblasts induced oxidative damage and apoptosis intervention has not been reported.ObjectObserve the state of cell growth,cell morphology,oxidizing materials content level of intracellular antioxidant capacity and apoptosis rate changes,to demonstrate baicalin under the UVA irradiation of human skin fibroblasts with light protective effect.On the base of previous experiment,further observation about mitochondrial membrane potential changes and gene oxidative damage products,detected apoptosis related proteins caspase-3 expression levels,to investigate the effect of baicalin by protecting the cells genetic damage and mitochondrial membrane,inhibition of downstream proteins,which play a role in its anti-oxidative damage and apoptosis.Methods1.cell culture Human embryonic skin fibroblast was purchased from the First Affiliated Hospital of Nanjing Medical University.Cells were cultured with DEM medium containing 10%fetal bovine serum.Take 5-10 substituting cells in logarithmic growth phase experiment.2.Ultraviolet radiation According to the experimental design time quantitative ultraviolet radiation,selecting the appropriate concentration of baicalin added to the culture to the point in time with the total dose of radiation 10 J/cm2 UVA.3.Drug concentration screening CCK-8 assay cell proliferation activity,non-cytotoxic drug concentration for subsequent experiments.4.Oxidative stress level detection Light microscope observe cell morphology;fluorescence microscopy and flow cytometry detect intracellular oxidation products content and apoptosis rate;flow cytometry detect cell cycle G1 arrest rate;ultraviolet spectrophotometry to determine cell superoxide dismutase(SOD),malondialdehyde(MDA)and total antioxidant capacity(T-AOC)level.5.Mitochondrial membrane potential change detection Flow cytometry detect mitochondrial membrane potential changes6.Genetic damage detection Immunofluorescence detect photoproducts 8-hydroxy-deoxyguanosine(8-oxo-dG)。7.Apoptosis-related protein detection Western Blot Western blot detection of apoptosis-related proteins caspase-3 expression.Result1.Proliferation of HDFs after baicalin and UVA irradiationCCK-8 confirmed that baicalin for 24 hours,at a concentration of 25μg/ml and below cell activity with the control group show no significant difference(P>0.05).Baicalin in this concentration no significant toxic effects on the cell.In addition to 10J/cm2 UVA irradiated cells.The baicalin treated group with a concentration of 25μg/ml and below cell activity higher than UVA group,the difference was statistically significant(P<0.05).Therefore,25μg/ml,12.5μg/ml,6.25μg/ml three drug-step concentration is chosen to study.2.Changes in reactive oxygen species,superoxide anion and NO content of HDFs after baicalin and UVA irradiationObserved by fluorescence microscopy,the UVA group reactive oxygen species,superoxide anion,NO fluorescence intensity was significantly higher than that in the control group.Baicalin treated groups fluorescence intensity were significantly weakened than UVA group,showing a concentration-dependent.The flow cytometry analysis showed that the fluorescence intensity of the oxidation products of UVA group were significantly higher than that in the control group,showed a significant difference(P<0.05).In reactive oxygen species,baicalin concentration with 25μg/ml and 12.5μg/ml group fluorescence intensity value were significantly decreased compared to UVA irradiation group,the difference was statistically significant(P<0.05);In superoxide anion groups,baicalin concentration with 12.5μg/ml and 6.25μg/ml group fluorescence intensity value were significantly decreased compared to UVA irradiation group,the difference was statistically significant(P<0.05),Compared with the control group,the difiference was not statistically significant;In NO groups NO group,the fluorescence intensity of the drug treatment groups were significantly decreased compared to UVA irradiation group(P<0.05),25μg/ml baicalin group compared with the control group,the difference was not statistically significant(P>0.05).3.Anti-oxidative stress capacity changes of HDFs after baicalin and UVA irradiationSOD,T-AOC vitality in UVA group were significantly lower than the control group(P<0.05),MDA content was significantly higher(P<0.05).SOD,IDA content and T-AOC vitality between drug treated groups and UVA group were compared statistically significant difference(P<0.05)4.Apoptosis rate of HDFs after baicalin and UVA irradiationObserved under the fluorescence microscope,the nucleus in the control group with normal morphology,however most nuclear in UVA irradiation group shows pyknotic,deeply stained.In the baicalin treated groups abnormal nuclear morphology cells decreased,showed some positive correlation with drug concentration.Flow cytometry testing proved the conclusion.5.Changes of mitochondrial membrane potential of HDFs after baicalin and UVA irradiationIn baicalin treated groups membrane potential were significantly lower than theUVA irradiation group,the difference was statistically significant(P<0.05).And with the increase of drug concentration,the fluorescence intensity decreased in a dose-dependent manner,no significant difference(P>0.05)between the drug treatment group(P>0.05),In addition the baicalin group compared with the control group,the difference was not statistically significant(P>0.05).6.DNA photoproduct 8-oxo-dG of HDFs after baicalin and UVA irradiationImmunofluorescence illustrates optical damage marker 8-oxo-dG strongly expressed in the the UVA group of cells.Baicalin group fluorescence intensity was significantly lower than the UVA group,the fluorescence was scarcely observed in the control group.7.Apoptosis-related proteins caspase-3 of HDFs after baicalin and UVA irradiationProcessing by the method described above,UVA group caspase-3 protein expression was significantly higher than that in the control group(P<0.05).After baicalin treatment,the protein expression of UVA group was decreased(P<0.05).In addition,the drug treatment group compared with the control group shows no significant difference(P>0.05).ConclusionThis study is to explore the UVA irradiated human skin fibroblast cell photodamage in the process of oxidative damage and apoptosis.Through the use of immunology,cell biology and molecular biology,observed that baicalin photoprotection UVA irradiation of human skin fibroblasts,and further elaborated in its play of light protective effect mechanism.By screening an effective and non-toxic concentration of baicalin,observed cell morphology,the content of oxidation products,cell cycle and intracellular levels of oxidative stress,DNA damage level,changes in mitochondrial membrane potential and apoptosis-related proteins caspase-3 changes.In summary,this study demonstrated that in UVA irradiation,baicalin has the ability to remove all types of harmful oxidation products,reduce DNA photodamage.Through reducing the mitochondrial membrane depolarization,to improve its permeability,thereby preventing the downstream apoptotic protein caspase-3 activation and expression,to reduce cellular oxidative stress and cell apoptosis. |
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