The Study Of Antioxidants Screening Methods Based On Multiple Cells

Background:With the development of modern industry, the increasingly serious environmentalpollution, a substantial increase compared to the previous daily contact with the typeand frequency of oxidative stress source. Oxidative stress and protection become aresearch hotspot in recent years, especially in the field of health care products andpersonal care products. The a variety of oxidative stress source of the of exogenouswill attack the biological molecules, protein, lipid, or nucleic acid, resulting in thecells of the oxidative damage, directly or indirectly lead to a variety of pathologicalreaction emergence. The the aging of of the oxidative stress caused by by the theultraviolet irradiation and chemical pollutant with the skin, pigmentation, theinflammation and a variety of the the relationship between of the disease has been isinsufficiently substantiated. Many cosmetic companies introduced a variety claim toremove oxide free radicals, anti-aging skin products, but can reach its purportedeffects as yet no standardized evaluation methods. The other hand, the the activity ofraw materials for the obtained from the the plant extracts, chemical synthesis,bio-transcription and and other ways to also need to the establishment of the a set ofscientific and highly efficient screening method. The purpose of this study is toestablish a standardized rapid detection of cellular oxidative damage, antioxidanteffect evaluation for chemicals or cosmetics.The current understanding of the mechanism of the oxidation reaction is not yetentirely clear, but can be considered a complex biological process relates to multi-pathand multi-gene combined effects of oxidative damage. Which the some of the important target cells in the the the process of of the oxidative damage, restrictionenzyme, the intermediates and products is the the key to of the screening pointer andthe markers. And thus can be established a screening methods according to thebiological mechanism of the oxidation reaction the antioxidant compound detection.Antioxidants can be summarized as animal models of law, human evaluation,chemical assays and cell method four have their own characteristics and shortcomings.Especially in the advocate animal welfare, to avoid under the the the background of ofthe unnecessary animal experiments, the establishment of a scientific, efficient, andfast method is a priority to choose. In this study, a variety of cells as experimentalsubjects, analog summer sun type of radiation-induced oxidative damage of the cellmodel, the establishment of a rapid screening method. Use the HaCaT celllines,human skin primal fibroblast cell as the object, detection of the a variety ofbiological of the an oxide injury which was caused by the ultraviolet ray irradiationcells; use the H₂O₂induced Coca-2cells generating oxidation stress, determine thesubstance of the different cell types have the same resistance to oxidation; at the sametime, the use of the skin Episkin model, conducted a preliminary exploration of thewhether the model can be applied to the screening of antioxidants.ObjectiveUsing the in vitro culture cell: HaCaT cell lines, primary skin fibroblast cells,Coca-2cell line and Episkin skin model as objective, detection four types of signs:thecell cycle, cell apoptoic, the ROS level and the SOD level,which was inducedoxidative damage by UV and H₂O₂, the establishment the antioxidant screeningmethods based on a variety of cell lines.Method1.Cell culture: HaCaT cell lines was cultured in MEM medium with10%fetalbovine serum; primary skin fibroblast cells was cultured in DMEM medium with10%fetal bovine serum; the Coca-2cell line was cultured in DMEM medium with12%fetal bovine serum; Episkin skin model was cultured in MEM medium. The cell was seed in T75cell culture flask or diameter65mm petri dish. Episkin was culture in12well plants. The CO2culture incubator condition keep on37℃and5%CO2.2. Test substance:POCI: a plant purified material, the main component of proanthocyanidins;LBPC:a plant purified material, mainly composed of Chinese wolfberrypolysaccharide;FFNT: chemical derivatives;C36D: chemical derivatives3.Oxidant damage model and cell toxiology of test substance: Using the MTTassay to determined the infect of different radiation dose in HaCaT cell lines, primaryskin fibroblast cells and Episkin, and H₂O₂concentration to Coca-2cell line. Thesesteps was to confirm the dose and concentration in the next experiment. Also need todetect the toxicology of the material concentration for the concentration chosen. Theexperiment set the negative control, induced group, VitC group, POCI group, LBPCgroup.4. The screening of antioxidant substance: Respectively, with ultraviolet rays orchemicals inducing cells to produce oxidative stress were added to the screening ofsubstances, by monitoring the type of index prediction screening substanceantioxidant properties. Experiment with the blank control group, model group, theascorbic acid positive control group and the test substance group, HaCaT cells andprimary human skin fibroblasts detected substances; Coca-2cells detected twosubstances4.1.The cell damage induced by oxidative stress studies: Using DCFH-DA, afluorescent probe, to label ROS in order to measure the ROS level in cells. Using thebiochemical method detect the SOD level. PI fluorescein labeling and Annexinv/PIfluorescein di-labeling method to observe the cell cycle and cell apoptosis.Using Vit Cas positive control, to estimate these four index were stable and sensitive.4.2. The effect of material against the oxidative stress: using the four index werementioned above to investigate the index changes after the material join in the cellculture. Examine whether this substance to be measured can antagonistic effects of oxidative stress, and to evaluate the strength of the antioxidant capacity of thematerial. Provide the basis for the antioxidant properties and intensity forecasts5. Statistical method:The result was present as mean±S.D（）. Using the SAS9.0as statics analysis as. Differences between groups were assessed with one-wayANOVA or student’s t-test with SAS9.0, according to data feature. Differences weredetermined to be significant when P<0.05, significant level a=0.05. Use the Modfitsoftware to analysis the cell cycle result.Result1.Established the oxidantive damage model:According to HaCaT cell lines, when the UVA dose achived5J/cm²,corresponding the UVB radiation dose was0.6J/cm²,20%of HaCaT cell lines can beinhibitor.According to primary skin fibroblast cells, when the UVA dose achived8J/cm²,corresponding the UVB radiation dose was1J/cm²,20%of primary skin fibroblastcells can be inhibitor.According to Coca-2cell line, when the H₂O₂concentration was150μmol/L,20%of Coca-2cell line can be inhibitor.In the dose or concentration above, the cell will produce oxidant damage, but willalso keep viability, suitable for further testing.According to Episkin, when the UVA dose were0J/cm²,5J/cm²,15J/cm²,25J/cm²,35J/cm²,45J/cm², corresponding the UVB radiation dose were0J/cm²，1J/cm²，3J/cm²，5J/cm²，7J/cm²，9J/cm²,the cell viability has dose-response relationship.2The cells toxiolog of substance concertration:According to HaCaT cell lines, the Vit C concentration is100μmol/L, POCI is50mg/L, LBPC is400mg/L, FFNT is8mg/L,C36D is90mg/L can inhibitor10%ofcells.According to primary skin fibroblast cells, the Vit C concentration is100μmol/L,POCI is50mg/Ll, LBPC is300mg/L, FFNT is6mg/L,C36D is90mg/L can inhibitor10%of cells.According to Coca-2cell line, the Vit C concentration is100μmol/L, POCI is50 mg/L, LBPC is300mg/L, can inhibitor10%of cells.3Oxidative damage index result.3.1ROS absolute fluorescence intensityShow result in more consistent, an ascorbic acid as a positive control, which iscapable of significantly reducing ADD ROS level of fluorescence intensity of thegroup of substances in each cell. HaCaT cells, ascorbic acid group, POCI group andLBPC group of cells ROS levels than the control group, but low compared with themodel group, the group FFNT group C36D cells ROS levels with the model group, nodifference, and than the control group; primary human fibroblasts after ultravioletradiation, the ROS level is higher than the other two groups of cells, and higher levelsof ROS in the background, ascorbic acid, POCI and LBPC can reduce ROS levelscompared with the model group, group of FFNT and C36D cells ROS levels inHaCaT cells; Caco-2cells, ascorbic acid, POCI and LBPC can reduce ROS levelscompared with the model group.In LBPC group ROS levels is generally higher thanPOCI and ascorbic acid group.3.2SOD levelThe results are displayed in each cell is more consistent. Add the ascorbic acid,POCI and LBPC of substances can help HaCaT cells and primary human fibroblastsincreased intracellular SOD level, so as to achieve the clear ROS antagonisticoxidative damage FFNT and C36D can not rise above two intracellular SOD levels.The bottom of SOD on Caco-2cells, but add substance SOD level trend consistentwith the rest of the cell.3.3Cell apoptoticThree types of cells have shown the impact of oxidative stress on apoptosis. Bothcell lines of the model group, the proportion of early apoptotic cells, primary cells, inthe proportion of late apoptotic than early apoptotic cells surviving cells; the rest ofthe group, an absolute majority apoptosis of cells smaller proportion. Three types ofcells in the added ascorbic acid, POCI and LBPC, can inhibit cell apoptosis; group ofFFNT and C36D group led HaCaT cells and primary human fibroblasts, highproportion of late apoptotic cells, compared with the model a higher proportion ofgroup. Late apoptotic cell proportion FFNT and C36D due inhibit fibroblasts, and therefore primary human fibroblasts in an absolute majority, and HaCaT cells thereare some differences.3.4Cell cycle:Model group, three cell G0/G1arrest, the most obvious of which two skin cellsblock. Caco-2cell model group, other groups with the same kinds of cells in G0/G1phase cells compared to little difference, but the G2phase is significantlyreduced.Ascorbic acid POCI and LBPC group, each of the three cell cycle indicatorswith the control group close to UV-induced HaCaT cells and primary human,; FFNTC36D into the the fiber cell cycle indicators have little effect with the modelgrouplittle difference.Concluds:1. The study based on different stressors mechanisms of injury were establishedoxidative stress model by use UV damage HaCaT cells, fibroblasts and thethree-dimensional skin model, the establishment of a hydrogen peroxide damage theintestinal epithelium oxidative stress model. The results showed that the IC20UV doseand compound exposure dose can induce a variety of cellular oxidative stress modelfor the screening of anti-oxidants.2. In this study, based on the intended use of screening to detect four differentcompounds, results suggest that: UV-induced oxidative stress mode, you can use theHaCaT cells and primary human skin fibroblasts antioxidant screening of substances,four kinds of indicators can be integrated to determine the Antioxidant specialattributes, and only use the ROS level and cycle indicators can also determine theinitial substances have antioxidant properties. In H₂O₂stress mode, produceoxidative stress pathway, suggesting therefore using ROS, SOD levels and apoptosisindex can also be preliminary to determine whether the substance has antioxidantproperties.（Property is determined by the chemical structure, the characteristics of thebiological characteristics of the compound, the property to the characteristics）. 3This study is based screening physical and chemical properties of the differentscreening system for the evaluation of the different experimental substances. Differentcells, skin model can be used for screening. For example, you can use a cell modelsuitable for use in the method of the raw material compound and a solubleformulation into line screening, using the skin model suitable for use in the detectionof the raw materials, formulation and finished screening.4The results suggest that low doses of UV radiation can promote the growth ofHaCaT cells and primary human skin fibroblasts, presumably related to thecompensatory cell proliferation, exactly, but the specific mechanism has yet to beexplored further.