CN105030560A - Protection effect of cyanidin-3-glucoside for UVB (ultraviolet B)-induced HaCaT cell injury - Google Patents
Protection effect of cyanidin-3-glucoside for UVB (ultraviolet B)-induced HaCaT cell injury Download PDFInfo
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Abstract
The invention discloses a protection effect of cyanidin-3-glucoside (C3G) for UVB (ultraviolet B)-induced cell injury. The protection effect particularly includes that generation of ROS (reactive oxygen species) of HaCaT cells due to UVB can be reduced by the C3G, accordingly, antioxidant effects can be realized, DNA (deoxyribonucleic acid) can be protected against injury, the survival rate of the HaCaT cells can be increased after the UVB irradiates on the HaCaT cells, and apoptosis reducing effects and the like can be realized. The protection effect has the advantages that the oxidation injury of the selected representative HaCaT cells which are cultivated in vitro is induced by means of UVB irradiation, light injury models of epidermal cells due to ultraviolet rays are imitated, then the C3G is used as a light injury protector, effects and relevant channels of the C3G for relieving UVB injury of the HaCaT cells are evaluated, the protection effect, action mechanisms and the safety of the C3G for light injury of skins are investigated, and accordingly theoretical foundation is laid for clinically effectively preventing and treating light injury of the skins and studying ultraviolet radiation injury protection action mechanisms of natural plant antioxidants.
Description
Technical field
The invention belongs to cosmetic technical field.More specifically, Cy-3-G induces HaCaT cell injury protective action to UVB is related to.
Background technology
Along with atmospheric ozone layer is seriously damaged, ultraviolet (ultraviolet, UV) radiant intensity is irradiated and is strengthened, and living standard improves, outdoor activity, the increasing of sun tan chance, cause people to accept too much ultraviolet radiation, causes light loss evil dermatoses to increase gradually.Ultraviolet (ultraviolet, UV) be irradiated to ground intensity to increase, the ultraviolet causing application on human skin to damage is ultraviolet B radiation (ulrtavioletB mainly, and long wave ultraviolet (ulrtavioletA UVB), UVA), and the former is about 1000 times of the latter to the degree of injury of skin, can causes as the acute injury of sunburn and the chronic cumulative bad of photoaging, skin carcinoma etc. injure skin, potential threat is formed to human health.
UVB induces molecular mechanism and the study on prevention of keratinocyte photic damage, is current one of study hotspot in the world.For a long time, numerous researcheres has done large quantifier elimination in the middle of this, and its molecular mechanism related to is comparatively complicated, and wherein the activation of DNA coup injury, oxidative damage and film surface death receptors is main mechanism.And oxidative damage mechanism to be at present research more and be outbalance.Along with the raising of people's living standard and the growth of the aged; skin aging problem causes the attention of people day by day; therefore; exploitation have high active ingredient sunscreen product, resist ultraviolet radiation, protection skin from photic damage, prevention and delay skin aging, become the focus of current medical research.
Cy-3-G (cyanidin-3-glucoside, C3G) is one of the most common anthocyanidin (anthocyanidin), belongs to the flavonoid class in phenolic compound, molecular formula C
21h
21o
11, it has antiinflammatory, antioxidation and anticancer biological activity as the multi-functional antioxidant of one.Although research has in the past indicated C3G have powerful antiinflammatory, antioxygenic property, at present about the research of the anti-dermal photodamage of C3G, to human epithelial cells, whether there is the effect that alleviates photic damage for C3G and safety there is no correlational study and report.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiency that existing UVB induces horn cell photoprotection, provides a kind of new opplication of Cy-3-G (C3G), the application namely in dermal photodamage protection.Specifically C3G can reduce UVB and causes the ROS of HaCaT cell to generate, and has antioxidation, and DNA can be protected from damage, and anti-apoptotic etc. acts on.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Cy-3-G is preparing the application in sun care preparations and/or medicine.
Cy-3-G repairs the application in cosmetics and/or medicine after preparation is shone.
The application of Cy-3-G after preparing dermal photodamage protective agent and/or photic damage in renovation agent.Particularly, described photic damage refers to the photic damage that UVB causes.
The application of Cy-3-G after preparation HaCaT cell photo-damage protective agent and/or photic damage in renovation agent.Particularly, described HaCaT cell photo-damage refers to the HaCaT cell injury that UVB induces.
In addition, the consumption of above-mentioned Cy-3-G when applying is preferably 80 ~ 200 μm of ol/L.
Cy-3-G is preparing the application in antioxidative cosmetics and/or medicine.Particularly, described oxidation refers to the oxidative damage that UVB causes.More specifically, be the application prevented and treated in preparation in the oxidative damage of the HaCaT cell that UVB causes.Further, UVB is specifically suppressed to cause HaCaT Hemapoiesis ROS.
The application of Cy-3-G in preparation anti-apoptotic preparation.
Survival rate, the ROS growing amount of cell after the present invention adopts the technology for detection UVB such as tetramethyl azo azoles salt colorimetric test (mtt assay), the experiment of fluorescence ROS probe to irradiate respectively, and tentatively probe into the expression of COX-2 and associated protein thereof and the Intervention Mechanism of C3G by protein immunoblotting (Westernblot) technology, inquire into C3G causes human body skin photic damage protective action and safety to UVB.Meanwhile, forming the COX-2 expression that UVB whether also can be suppressed to induce in cell at human epidermal in order to systematically understand C3G, probing into the activation of ROS level and p53 in keratinocyte under UVB damage, analyzing apoptosis-induced with UVB relation between the two; And with " ROS--DNA damages--p53--mitochondrial apoptosis path " serve as theme; probe into Cy-3-G (C3G) to the protection mechanism of dermal photodamage keratinocyte; important evidence is provided, for Prevention and Curation photic damage provides new direction for C3G is applied to control photic damage.
The present invention has following beneficial effect:
The present invention selects the HaCaT cell of In vitro culture to be representative; irradiate with UVB and bring out its oxidative damage; imitate ultraviolet to the photic damage model of epidermis cell; again using C3G as photic damage protective agent; UVB damage effect and the related pathways of immortal human keratinocyte HaCaT cell is alleviated, to inquire into C3G to the protective action of dermal photodamage (especially UVB causes human body skin photic damage) and mechanism of action thereof and safety by evaluate application C3G.Result of study shows, and C3G can reduce UVB and cause the ROS of HaCaT cell to generate, and has antioxidation; And DNA can be protected from damage; the survival rate that UVB irradiates rear HaCaT cell can be improved; reduce apoptosis etc. effect; for effective prevention and therapy dermal photodamage clinically; especially acute photic damage provides new application and thinking, is the mechanism of action based theoretical of research natural plant antioxidant protection ultraviolet radiation damage.
Accompanying drawing explanation
Fig. 1 is the cell survival rate (%) of the HaCaT cell of different UVB dose irradiation.
Fig. 2 is the HaCaT intracellular ROS level that various dose UVB irradiates.
Fig. 3 is the relation of intracellular ROS level and cells survival rate.
Fig. 4 is the impact that UVB irradiates on HaCaT cellular morphology.
Fig. 5 is that the C3G of variable concentrations is to 300mJ/cm
2the impact of the HaCaT cellular morphology that UVB irradiates.
Fig. 6 is variable concentrations C3G irradiates the survival rate of HaCaT cell impact on UVB.
Fig. 7 is the cytoactive impact of HaCaT cell after C3G effect 1h irradiates UVB.
The impact of ROS in Fig. 8 HaCaT cell that to be variable concentrations C3G irradiate 300mJ/cm2UVB.
Fig. 9 is that Westernblot detects variable concentrations C3G to the impact of the HaCaT cell COX-2 that 300mJ/cm2UVB irradiates.
Figure 10 is that Westernblot detects C3G and celecoxib suppresses UVB to induce the phosphorylation of EGFR.
Figure 11 is that Westernblot detects C3G and celecoxib suppresses UVB to induce the phosphorylation of ERK.
Figure 12 is that Westernblot detects C3G and celecoxib suppresses UVB to induce the phosphorylation of JNK.
Figure 13 is that Westernblot detects C3G and celecoxib suppresses UVB to induce the phosphorylation of p38.
Figure 14 is that Westernblot detects C3G and celecoxib suppresses UVB to induce the phosphorylation of Akt.
Figure 15 is the apoptotic impact of HaCaT after C3G irradiates UVB.
Figure 16 is the apoptotic impact of HaCaT after C3G irradiates UVB.
Figure 17 is the impact of HaCaT cell Δ ψ m after C3G irradiates UVB.
Figure 18 is the impact of HaCaT cell Δ ψ m after C3G irradiates UVB.
Figure 19 is the impact that westernblot analyzes that C3G irradiates rear HaCaT DNA Damage on UVB.
Figure 20 is the impact that westernblot analyzes that C3G irradiates rear HaCaT cell p53 phosphorylation on UVB.
Figure 21 is the impact that westernblot analyzes that C3G irradiates rear HaCaT cell death related protein on UVB.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the method and apparatus that the present invention adopts is the art conventional method and equipment.Unless stated otherwise, following examples agents useful for same and material are commercial.
HaCaT cell used is purchased from Wuhan University's preservation center, 37 DEG C, 5%CO
2cultivate in incubator.
The preparation (all solution preparation water is ultra-pure water) of main solution:
(1) C3G: the C3G powder getting 20mg is dissolved in the DMEM of serum-free, is mixed with 4.49 × 105 μ g/ml(and 1mmol/L) storing solution, subpackage is stored in the EP pipe of 1.5ml, keeps in Dark Place at-20 DEG C of refrigerators.Take out in advance during use, experimentally variable concentrations needs, and is diluted to respective concentration with DMEM.
(2) celecoxib: celecoxib DMSO dissolves, is mixed with 281.37 μ g/ml(and 1mmol/L respectively) storing solution, filter after subpackage ,-20 DEG C save backup.
(3) HaCaT cell culture medium (complete medium): DMEM culture medium (high sugared serum-free medium)+10%FBS(hyclone), namely ratio is 9:1; Add in culture medium dual anti-(penicillin+streptomycin), make its final concentration be 100mg/L; Sealed membrane seals, and 4 DEG C save backup.
(4) phosphate buffer (PBS): PBS:KH
2pO
40.20g, Na
2hPO
412H
2o3.56g, NaCl8.00g, KCl0.20g, add ultra-pure water to 1000mL, adjusts pH to 7.2-7.4, autoclaving 4 DEG C preservation.
(5) cells frozen storing liquid: 60%DMEM culture medium, 30% hyclone, 10%DMSO, now with the current before cell cryopreservation.
(6) MTT (5mg/mL): MTT powder 50mg, adds PBS and be dissolved to 10mL, makes MTT concentration be 5mg/mL.0.22 μm of frit sterilizing, subpackage lucifuge 4 DEG C or-20 DEG C of preservations.
(7) fluorescence microplate reader detects ROS reagent D CFH-DA: dilute DCFH-DA with serum-free medium, make final concentration be 10 μm of ol/L.
(8) 0.25% pancreatin: take a certain amount of pancreas enzyme powder, put into the large conical flask of having sterilized, add corresponding PBS by 0.25% concentration, and magnetic agitation until completely dissolved, adjusts between its pH to 7.2-7.4, put into 4 DEG C of refrigerator overnight; Filter sterilizing with 0.22 μm of filter every other day, and point to be filled in 15ml centrifuge tube, perform labelling and be stored in-20 DEG C of refrigerators.
(9) 0.04%EDTA: take disodiumedetate 0.024g, adds in 60mlPBS solution; Solution is placed in 70-80 DEG C of water-bath vibration to dissolving completely; With 0.22 μm of frit sterilizing, subpackage, 4 DEG C of preservations.
In addition, the data of following examples carry out statistical procedures, and all experiments at least in triplicate, all data all represent with mean+SD (x ± s), use SPSS17.0 to carry out statistical analysis, adopt one factor analysis of variance, P < 0.05 is for there being statistical significance.
embodiment 1 Cy-3-G intervenes the research that UVB damages HaCaT cell ROS/COX-2 path
One, experimental technique
1, HaCaT cell culture
(1) cell recovery
1) from-80 DEG C of refrigerators, take out the cryopreservation tube that HaCaT cell is housed, drop in 37 DEG C of warm water immediately, and shake makes it be heated evenly frequently, melts as early as possible;
2) when cryopreserving liquid dissolves completely, take out cryopreservation tube, at aseptic middle sucking-off cell suspension, be injected in 9ml cell culture medium, evenly, 1000rpm × 5min is centrifugal in piping and druming gently;
3) abandoning supernatant, adds 6ml culture medium, and piping and druming discrete cellular, moves in 10mm culture dish, be placed in 5%CO
2, cultivate in 37 DEG C of incubators.
(2) cell culture
1) Growth of Cells is in containing in the DMEM culture medium of 10%FBS, is placed in containing 5%CO
2, cultivate in 37 DEG C of incubators; Within 2 days, change liquid to go down to posterity 1 time, trophophase cell of taking the logarithm during experiment;
2) passage: when cell covers with culture dish, discard culture fluid, PBS rinses once, adds the trypsin 3ml of 0.25%, gently wave and culture bottle, make Digestive system flow through cell surface in culture bottle; Place 2min in incubator, observe under inverted microscope, find that intercellular substance increases, the culture medium added immediately containing serum stops digestion; Being divided by single cell suspension under digestion is filled in new culture dish, continues to cultivate.
(3) cell cryopreservation
1) choose the HaCaT cell being in exponential phase, use 0.25% trypsin digestion and cell, the centrifugal 1000rpm × 5min of cell suspension;
2) abandoning supernatant, adds cells frozen storing liquid, resuspended mixing cell;
3) add above-mentioned cell suspension 1 ~ 1.5ml in every cryopreservation tube, screw lid, and on cryopreservation tube, write freeze-stored cell, cryopreserved human and frozen time exactly;
4) cryopreservation tube is placed in-20 DEG C of refrigerators, after cryopreserving liquid solidifies, turns and deposit in-80 DEG C of refrigerators.
2, the photic damage model of HaCaT cell is set up in UVB radiation
Time when about cell fusion to 80%, carry out UVB radiation.NB-UVB fluorescent tube (Philip, wavelength 311nm, radiant intensity 0.42mw/cm2) is placed in operation in super-clean bench: carry out UVB radiation after sucking culture fluid, adds the appropriate culture medium containing serum immediately and be placed in incubator continuation cultivation after radiation terminates.Radiation dose is respectively 100,200,300,400mJ/cm2; Be respectively 0h, 3h, 6h, 12h, 24h, the 48h after radiation detection time.
3, MTT experiment detects cell survival
(1) various dose UVB irradiates the Survival Effects of HaCaT cell
1) HaCaT cell culture is to logarithmic (log) phase, collecting cell; Establish negative control group, positive controls and various dose processed group in 96 orifice plates, negative control group adds complete medium, and positive controls and processed group adjustment concentration of cell suspension, with 7.5 × 10
4/ ml, 200 μ L cell suspension are inoculated in every hole, often organize and all establish 6 experimental ports; The aseptic PBS of edge hole fills, 5%CO
2, hatch for 37 DEG C;
2) after cultivation 24h cell grows to monolayer, siphon away culture medium, the UVB that processed group gives 4min, 8min, 12min, 16min respectively irradiates; Positive controls gives false photograph;
3) after irradiating, every hole adds 200 μ L complete mediums continuation cultivations.After irradiation when 0h, 3h, 6h, 12h, 24h, 48h, siphon away culture medium, every hole adds 50ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h;
4) stop cultivating, carefully suck culture fluid in hole, inversion is blotted.Every hole adds 100 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value OD value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm place;
5) survival rate of each group of UVB radiation is calculated:
Cell survival rate (%)=(OD
processed group-OD
negative control group)/(OD
positive controls-OD
negative control group) × 100%
(2) C3G is on the impact of the HaCaT cell survival that UVB irradiates
1) HaCaT cell culture is to logarithmic (log) phase, collecting cell; Establish negative control group, positive controls and various dose processed group in 96 orifice plates, negative control group adds complete medium, and positive controls and processed group adjustment concentration of cell suspension, with 7.5 × 10
4/ ml, 200 μ L are inoculated in every hole, often organize and all establish 6 experimental ports; The aseptic PBS of edge hole fills, 5%CO
2, hatch for 37 DEG C;
2) after cultivation 24h cell grows to monolayer, siphon away culture medium, give UVB and irradiate 12min;
3), after irradiating, processed group gives 80 μm of ol/L respectively, the C3G100 μ L of 160 μm of ol/L and 200 μm ol/L hatches 1h; Matched group gives complete medium and hatches 1h;
4) suck after 1h and hatch liquid, every hole adds complete medium respectively and continues to cultivate 5h, 11h, 23h, 47h;
5) UVB postradiation 6,12,24,48h, siphon away culture medium, every hole adds 50ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues to cultivate 4h;
6) stop cultivating, carefully suck culture fluid in hole, inversion is blotted.Every hole adds 100 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value OD value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm place;
7) cell survival rate of each group is calculated:
Cell survival rate (%)=(OD
processed group-OD
negative control group)/(OD
positive controls-OD
negative control group) × 100%
4, the intracellular ROS level of DCF fluoremetry HaCaT
(1) the HaCaT intracellular ROS level of various dose UVB irradiation
1) DCFH-DA preparation: dilute DCFH-DA with serum-free medium, make final concentration be 10 μm of ol/L;
2) logarithmic (log) phase HaCaT cell suspension is inoculated into 96 orifice plates with the density of 1.5 × 104/mL, every hole 200 μ L cell suspension;
3), after cultivating 24h, old culture fluid is discarded.Add the DCFH-DA that 50 μ L have diluted, in 37 DEG C of cell culture incubators, hatch 30min.With serum-free cell culture medium washed cell 3 times, do not enter intracellular DCFH-DA fully to remove;
4) UVB giving 0min, 4min, 8min, 12min, 16min respectively irradiates.After irradiating, every hole adds 100 μ L complete mediums;
5), after cultivating 30min, detect with fluorescence microplate reader, 488nm is as excitation wavelength, and 568nm is emission wavelength;
(2) C3G irradiates the impact of HaCaT intracellular ROS level on UVB
1) DCFH-DA preparation: dilute DCFH-DA with serum-free medium, make final concentration be 10 μm of ol/L;
2) logarithmic (log) phase HaCaT cell suspension is inoculated into 96 orifice plates with the density of 1.5 × 104/mL, every hole 200 μ L cell suspension;
3), after cultivating 24h, old culture fluid is discarded.Add the DCFH-DA that 50 μ L have diluted, in 37 DEG C of cell culture incubators, hatch 30min.With serum-free cell culture medium washed cell 3 times, do not enter intracellular DCFH-DA fully to remove;
4), after irradiating 12min with UVB, cell is divided into 7 groups, gives the C3G of complete medium and 10,20,40,80,160,200 μm of ol/L respectively;
5), after cultivating 30min, detect with fluorescence microplate reader, 488nm is as excitation wavelength, and 568nm is emission wavelength.
5, Westernblot detects COX-2 and the correlative protein expression of HaCaT cell
(1) total protein extraction
1) by HaCaT cell culture to logarithmic (log) phase, with concentration of cell suspension for 1 × 10
6individual/1mL is inoculated in 10mm culture dish and cultivates, and every ware 8mL, is placed in 37 DEG C, 5%CO
2adherent growth in incubator;
2), after cultivating 24h, old culture fluid is discarded; If negative control group, positive controls and various dose processed group, the UVB that positive controls and processed group give 12min irradiates, and negative control group gives false photograph; Irradiation post treatment component does not give 80 μm of ol/L, the C3G3mL of 160 μm of ol/L and 200 μm ol/L hatches 1h; Matched group gives complete medium and hatches; Suck after 1h and hatch liquid, each group all adds complete medium and continues to cultivate 11h;
3) get each group of cell suspension, the centrifugal 5min of 400 × g, remove supernatant;
4) often organize and first add 10 μ L lysates (RIPA and PMSF mixes with 100:1), cooled on ice 5min after vortex cell lysis 1min, again add 10 μ L lysates every 20min, continue vortex cracking;
5), after 1h, 4 DEG C 10, the centrifugal 15min of 000 × g, moves on in 1.5mLEP pipe by supernatant; Be placed in-80 DEG C of Refrigerator stores.
(2) determination of protein concentration
1) quantity per sample, according to BCA method detection kit preparation BCA working solution (BCA reagent A: BCA reagent B is 50:1), fully mixes;
2) 7 0.2mL specification PCR pipe are taken out, and numbering 1-7,2-7 pipe adds the ultra-pure water of 5 μ L;
3) standard substance (2mg/mL) sucking-off 10 μ L is joined in No. 1 pipe, takes out in 5 μ L to 2 pipes, make 2 by above method successively, 1,0.5,0.25,0.125,0.0625mg/mL;
4) standard substance and the sample of the variant concentration of 2 μ L is added in 96 orifice plates;
5) each hole adds 200 μ LBCA working solutions, places 30min for 37 DEG C;
6) 570nm place measures OD value, calculates protein concentration according to standard curve;
7) by each applied sample amount 40 μ g subpackage protein sample.Protein solution is mixed by 5:1 with 5 × sampleloadingBuffer.
(3) SDS electrophoresis
(4) transferring film
(5) immuning hybridization
1) primary antibodie diluted primary antibodie is to debita spissitudo (1:1000-1:2000), 4 DEG C of overnight incubation;
2) suck primary antibodie Incubating Solution, box will be hatched and be placed on decolorization swinging table, and wash film with TBST, 5min × 3
Secondary;
3) resist to debita spissitudo (1:2500-1:5000) containing 5% defatted milk powder dilution two, incubated at room temperature 1h;
4) discard two anti-Incubating Solutions, will hatch box and be placed on decolorization swinging table, TBST washes film, 5min × 3
Secondary.
(6) develop the color: adopt the colour developing of ECL chemical luminescence for liquid in dark place, after development terminates, room temperature dries film, after scanner scanning, by ImageJ software analysis result.
Two, experimental result
1, the foundation of UVB oxidative damage HaCaT cell model
(1) survival condition of the HaCaT cell of various dose UVB irradiation
Cell survival rate (Cellviability) is used to assess UVB(100 ~ 400mJ/cm2) impact of irradiation on HaCaT cell viability measurement, as shown in figure 1 and table 1.
The analysis of mtt assay determination data shows, 100mJ/cm
2the cell survival rate of group is without significant change, and other irradiation groups are all with the prolongation of incubation time after irradiating, and cell survival takes the lead in the decline (P<0.05) occurred in various degree, and all after cultivating 12h, reaches minimum.
UVB exposure rate (400mJ/cm
2) higher, HaCaT cell survival rate lower (77.59%), fall is larger, in certain dose relationship.
Table 1 various dose UVB irradiates the impact (%) on HaCaT cell survival rate
* P< 0.05 is relative to the matched group of incubation time
(2) the HaCaT intracellular ROS level of various dose UVB irradiation
Fluorescence microplate reader testing result shows, and 200mJ/cm2UVB group has small size rising, but does not have significant difference compared with matched group; 300 ~ 500mJ/cm2UVB irradiates the ROS remarkable rising compared with matched group produced HaCaT cell, increases gradually, (P<0.05) (as shown in Figure 2) in dose-dependent relationship along with dosage increases ROS generation.
(3) the intensity screening of UVB oxidative damage HaCaT cell model
Do linear regression analysis by above-mentioned ROS level and cells survival rate result, can think there is negative linear correlation (P < 0.05) between the two, namely ROS level is higher, cells survival rate lower (as shown in Figure 3).300mJ/cm2UVB all shows in two experiments has statistical significance to the impact of HaCaT cell, can be considered that UVB causes the minimum dose of oxidative damage.Therefore we select 300mJ/cm2UVB to set up UVB oxidative damage HaCaT cell model.
2, the change of HaCaT morphocytology
Normal HaCaT cellular morphology: observe under inverted microscope, visible HaCaT cell attachment growth, in epithelial cell type.Its form is flat polygon, and there are circular kernel in central authorities.Cell is closely linked to be monolayer each other, is membranaceous expansion during growth.Go down to posterity adherent after 10 ~ 12h and growth is stable, can exponential phase be entered after the 24 ~ 48h that goes down to posterity.
(1) morphological change after HaCaT cell UVB pre-irradiation
The HaCaT cell of Normal group is typical Epithelial feature, high caryoplasm ratio, and cell arrangement is tight, and profile knows that refractive power is good; UVB group cell connects loose, and refractivity comparatively matched group is poor, part cell death, de-wall (as shown in Figure 4).
(2) C3G HaCaT morphological changes of cell that UVB is irradiated
The HaCaT cell rounding that UVB irradiates, shrinkage, suspension cell increases, and iuntercellular is separated from one another, and ordered state is upset.The cell adding C3G group is subject to repair in various degree, show as cell comparatively matched group arrangement closely, swelling degree is low, and profile is clear, and refractive power is good, cell debris reduces, and cell injury degree and C3G dosage are negative correlation (as shown in Figure 5).
3, C3G is on the impact of the HaCaT cell survival that UVB irradiates
(1) as shown in table 2 and Fig. 6,80 μm of ol/LC3G 12h after UVB irradiates improve cell survival rate, after irradiation 6,24h cell survival rate still a little higher than matched group.160 μm of ol/LC3G improve cell survival rate in 12 hours after irradiation.200 μm of ol/LC3G 6h after UVB irradiates just increase cell survival rate.Illustrate that C3G is improved the protective effect of survival rate to the HaCaT cell that UVB irradiates, and concentration is higher, the protective effect persistent period is longer, and protective capability is stronger.
In addition, the survival rate of C3G group reduces in time, and matched group is in 24h comparatively 12h rising.Illustrate that HaCaT cell has self-repairing capability in 24h after UVB irradiates, cell survival rate raises comparatively before, but still lower than C3G group.Prove that C3G has the effect effectively improving cell self-regeneration efficiency.
Table 2300mJ/cm
2the survival rate (%) of UVB irradiation and the rear HaCaT cell of C3G process
* P< 0.05 is relative to C3G matched group
(2) 300mJ/cm
2after dosage UVB irradiates HaCaT cell, variable concentrations C3G acts on cell 1h, then after continuation cultivation 12h on the impact of cytoactive as shown in Figure 7.
As seen from Figure 7, compared with normal cell controls group (NC), cell is subject to 300mJ/cm
2after dosage UVB irradiates, then after continuing to cultivate 12h, the survival rate of cell significantly declines (P<0.01).But when adding each group of C3G effect 1h, the survival rate of cell raises all to some extent.Compared with UVB irradiation group, three groups of UVB+80,160 and 200 μm of ol/LC3G can significantly improve the survival rate (P<0.01) of cell, so C3G effect 1h also can increase by the survival rate of UVB damaging cells.
4, C3G is on the impact of the HaCaT intracellular ROS level that UVB irradiates
As seen from Figure 8, along with the increase of C3G concentration, the scavenging action of C3G to the ROS that UVB damage produces strengthens gradually, and has dose dependent.Wherein, 80 ~ 200 μm of ol/LC3G act on the HaCaT cell of UVB irradiation, the ROS remarkable decline compared with matched group produced in cell, and increase with dosage and reduce the more (P<0.05).
5, C3G irradiates the impact of HaCaT cell COX-2 and correlative protein expression on UVB
According to above-mentioned cell by according to after to found that UVB irradiates the activity of cell after HaCat cell 12h minimum, therefore we infer that the damage that now cell is subject to is the most serious.For obtaining the data of COX-2 and correlative protein expression, we select to irradiate HaCat cell with 300mJ/cm2UVB, are then 80,160 or 200 μm of ol/LC3G or 80 μm of ol/L celecoxib process 1h with concentration, and in irradiation 12h collecting cell afterwards.Extract albumen, survey protein concentration, Westernblot observes the change of the signal intensitys such as COX-2, p-EGFR, p-p38.
(1) C3G suppresses the COX-2 of UVB induction to express
Westernblot result display UVB can increase COX-2 in HaCaT cell to express, and 80 ~ 200 μm of ol/LC3G and celecoxib all can reduce COX-2, but and have no dose dependent (as shown in Figure 9).80 μm of ol/LC3G and 80 μm of ol/L celecoxibs suppress COX-2 to express degree difference not statistically significant.
(2) C3G suppresses the EGFR of UVB induction to activate
As shown in Figure 10, UVB irradiates the phosphorylation obviously increasing EGFR in cell to result, and the p-EGFR that C3G and celecoxib all can suppress UVB to induce activates, but and has no dose dependent.In addition, 80 μm of ol/LC3G reduce the degree of EGFR phosphorylation obviously compared with 80 μm of ol/L celecoxibs strong (P<0.05).
(3) C3G suppresses HaCaT cell ERK, p38, JNK and the Akt phosphorylation of UVB induction
1) C3G is on the impact of the HaCaT cell ERK phosphorylation that UVB induces
Westernblot result shows: after UVB irradiates, in cell, p-ERK obviously increases, and the p-ERK that C3G and celecoxib all can reduce UVB induction activates, but and has no dose dependent.In addition, 80 μm of ol/LC3G compare not statistically significant (as shown in figure 11) with 80 μm of ol/L celecoxibs.
2) C3G is on the impact of the HaCaT cell JNK phosphorylation that UVB induces
As shown in figure 12, after UVB irradiates, in HaCaT cell, JNK phosphorylation increases, and the p-JNK that C3G and celecoxib all can suppress UVB to induce activates, but dose dependent not statistically significant.In addition, 80 μm of ol/LC3G reduce the degree of JNK phosphorylation compared with 80 μm of ol/L celecoxibs weak (P<0.05).
3) C3G is on the impact of the HaCaT cell p38 phosphorylation that UVB induces
As shown in figure 13, UVB irradiates the phosphorylation obviously increasing p38 in cell, and the p-p38 that C3G can suppress UVB to induce activates, but and has no dose dependent.80 μm of ol/L celecoxibs to postradiation p38 phosphorylation without obvious effect.
4) C3G is on the impact of the HaCaT cell Akt phosphorylation that UVB induces
As shown in figure 14, after UVB irradiates, in HaCaT cell, the phosphorylation of Akt increases, and the p-Akt that C3G and celecoxib all can suppress UVB to induce activates, and in dose dependent (P<0.05), 200 μm of ol/LC3G rejection ability are the strongest.In addition, 80 μm of ol/LC3G reduce the degree of Akt phosphorylation compared with 80 μm of ol/L celecoxibs weak (P<0.05).
In sum, the technology for detection C3G such as research and utilization mtt assay, the experiment of fluorescence ROS probe, Westernblot of the present embodiment, on the impact of the cell survival rate of UVB oxidative damage HaCaT cell model, ROS level, COX-2 and correlative protein expression thereof, draws to draw a conclusion:
(1) UVB irradiation HaCaT cell impels ROS generation to increase, and cause cell oxidative damage, cell-proliferation activity is suppressed.
(2) UVB increases by inducing the generation of ROS, activates EFGR, thus promotes the phosphorylation of ERK, p38, JNK and Akt, regulate the expression of COX-2, infers that UVB causes HaCaT cell injury by ROS/COX-2 path.
(3) C3G can reduce UVB and causes the ROS of HaCaT cell to generate, and the proliferation activity of Cell protection, alleviates oxidative damage.
(4) C3G is by reducing ROS level, intervenes the ROS/COX-2 path that UVB damages HaCaT cell, thus minimizing COX-2 expresses.
the apoptosis impact of HaCaT cell after embodiment 2C3G irradiates UVB
1, apoptosis detects---the two dye method flow cytomery of AnnexinV-FITC/PI
The two dye method of AnnexinV-FITC/PI detects apoptotic principle: at apoptotic commitment, be positioned at the Phosphatidylserine (phosphalidylserine inside cell membrane, PS) can migrate to outside cell membrane, and AnnexinV is as cardiolipin binding protein, PS is had to the adhesion of height, therefore the fluorescence intensity of AnnexinV mark fluorescent element (FITC) can reflect the cell of early apoptosis; Meanwhile, because the PS of non-viable non-apoptotic cell also can be exposed to outside cell membrane, and to PI high dye, so be combined PI to refuse the cell that dye method just can distinguish apoptosis (early apoptosis) and downright bad (late apoptic).On flow cytometry analysis scatterplot; the double-negative cell (right lower quadrant) representing normal activity cell (left lower quadrant) the AnnexinV positive, PI negative representative generation early apoptosis of AnnexinV, PI, the two positive cell (right upper quadrant) representing late apoptic or necrosis of AnnexinV, PI.
(1) process and collecting cell: get the HaCaT cell dissociation cultivated and be in exponential phase in culture bottle, with the cell density kind of 250,000/ml in six orifice plates, every hole 2ml, is placed in incubator and cultivates 24h post processing cell.Give 300mJ/cm2UVB according to grouping by four holes to irradiate, non-irradiated hole aluminium-foil paper blocks.After having irradiated in three holes the C3G(80 of the variable concentrations that add-backs at once prepare, 160,200 μm of ol/L), matched group add-back be complete medium.After C3G effect 1h, the C3G of C3G group is gained complete medium, continue to hatch common 12h, collecting cell carries out dyeing parallel type cell instrument and detects.
(2) dyeing and flow cytomery: collecting cell, comprises the cell swum in culture medium; Afterwards, EDTA need be washed away, the resuspended sample presentation of 200 μ lPBS.By resuspended cell centrifugation, 1500 turns of 5min, abandon supernatant.Adding 200 μ lBindingBuffer mixes resuspended, then adds 5 μ lAnnexinV-FITC, and room temperature lucifuge hatches 10min.Centrifugal with 1500 turns of 5min again, after abandon supernatant.Adding 200 μ lBindingBuffer mixes resuspended, then adds 5 μ lPI, directly goes up machine.By BDFACSDivaV4.1.2 software analysis data.
2, experimental result
After the UVB irradiation HaCaT cell of 300mJ/cm2, then add the C3G effect of variable concentrations, on apoptotic impact by flow cytomery result as shown in figs.
From Figure 15 and 16, compared with Normal group, through 300mJ/cm
2uVB irradiate after the apoptosis rate of cell obviously raises, the ratio of competent cell obviously declines (P<0.01).And after adding C3G effect; the C3G of three concentration all can reduce apoptosis; the cell of apoptosis drops to 26.14%, 25.38%, 21.40% respectively than from 58.01%, although the apoptosis rate after C3G protection is higher than Normal group cell, but has not had obvious significant difference.And the cell of late apoptic/necrosis remains at low levels always, and there is no significant change.
the impact of HaCaT mitochondrial membrane potential in anoxic after embodiment 3C3G irradiates UVB
1, mitochondrial membrane potential (Δ ψ m) detects---and JC-1 staining for flow cytometry detects
The principle of JC-1 detection line mitochondrial membrane potential change: JC-1 is a kind of cation lipid fluorescent dye, has monomer and polymer two kinds of existences, exists, exist when high concentration with multimeric forms when low concentration with monomeric form.In normal cell, because mitochondrial membrane potential (Δ ψ m) has polarity, the polarity that JC-1 relies on Δ ψ m is taken in rapidly in mitochondrion, and in mitochondrion, form polymer because its concentration raises, polymer utilizing emitted light is red fluorescence, can be arrived by the redness of flow cytometer (FL-2) Air conduct measurement; When apoptosis, mitochondrial membrane potential is depolarized, and causes JC-1 to discharge in mitochondrion, and its concentration is reduced, therefore exists with the form of monomer and send green fluorescence.The red green change of root Ju fluorescence just reflects the change of mitochondrial membrane potential.The green fluorescence percentage ratio of JC-1 monomer represents the cell percentages of surveyed Δ ψ m reduction.
Dyeing and flow cytomery: collecting cell, comprises the cell swum in culture medium; Afterwards, the resuspended sample presentation of 200 μ lPBS.By resuspended cell centrifugation, 1500 turns of 5min, abandon supernatant.Add 200 μ lJC-1 dye liquor mixing cell dyeings, room temperature lucifuge hatches 15min, upper machine.By BDFACSDivaV4.1.2 software analysis data.
2, experimental result
The destruction of mitochondrial membrane potential (Δ ψ m), be considered to one of event the earliest in apoptotic cascade course of reaction, before it occurs in nucleus apoptosis feature (Chromatin condensation, DNA break) appearance, once mitochondrial membrane potential collapse, then apoptosis is irreversible.300mJ/cm
2uVB irradiate HaCaT cell after, then add the C3G effect of variable concentrations, the impact changed mitochondrial membrane potential is by flow cytomery result as shown in FIG. 17 and 18.
From Figure 17 and 18, compared with Normal group, through 300mJ/cm
2uVB irradiate after the Δ ψ m of cell decrease (show as green fluorescence and be increased to 5.6% from 2.73%) (P<0.05).And after adding C3G effect, the C3G of three concentration all can make Δ ψ m raise to some extent.
the impact of HaCaT DNA Damage after embodiment 4C3G irradiates UVB
1, ATM and ATR, is the main kinases of core element sensor, can be convened, stimulate activation by DNA damage, finally cause cell cycle arrest or apoptosis, so ATM, ATR are used as the mark substance of cell generation DNA damage usually.
The present embodiment analyzes the protein level detecting be activated ATM, ATR that phosphorylation occurs by westernblot, reflect the level of DNA Damage, analyzes the impact of C3G on HaCaT DNA Damage after UVB irradiation.
2, the protein expression band of phosphorylation ATM, ATR as shown in Figure 19 A; Carry out strip analysis with QuantityOne software, result as shown in Figure 19 B.Result shows, 300mJ/cm
2uVB irradiate after cause obvious DNA damage, show the rise of ATM, ATR phosphorylation level.And after the C3G effect adding variable concentrations, all serve protection DNA from the effect of damage, show the minimizing of phosphorylation ATM, ATR.
the impact of HaCaT cell p53 and apoptosis-related protein after embodiment 5C3G irradiates UVB
1, westernblot analyzes C3G irradiates rear HaCaT cell p53 phosphorylation impact on UVB
P53, as an important nuclear factor in cell, is by being activated, phosphorylation occurs just to play regulating action, so illustrate cell injury or protection mechanism by the level of detection Intracellular phosphorylation p53.
The protein expression band of phosphorylation p53 as shown in FIG. 20 A; Carry out strip analysis with QuantityOne software, result as shown in fig. 20b.
As seen from Figure 20, the level of cell phosphorylation p53 is in normal state very low, but through 300mJ/cm
2uVB irradiate after the p53 showed increased of phosphorylation, and after the C3G effect adding variable concentrations, all downwards of generation p53 phosphorylation level.
2, C3G irradiates the impact of apoptosis-related protein in rear HaCaT cell on UVB
In order to confirm that Bcl-2 family protein take part in the apoptosis of UVB induction, analyze the level of pro apoptotic protein Bax in Bcl-2 family protein and anti-apoptotic proteins Bcl-2 with westernblot, and the cracking situation of downstream effect albumen caspase-3.
Above-mentioned protein expression band as illustrated in fig. 21; Carry out strip analysis with QuantityOne software, result as illustrated in fig. 21b.Result shows, and UVB significantly increases the expression of pro apoptotic protein Bax after irradiating, and decreases the expression of anti-apoptotic proteins Bcl-2, and too increases the cracking of caspase-3.And after the C3G effect adding variable concentrations, all can reverse above-mentioned effect, the cracking of the up-regulated of the down-regulated expression of Bax, Bcl-2, caspase-3 is reduced.
In sum, we can draw to draw a conclusion:
(1) UVB irradiates the survival rate decreasing HaCaT cell, makes HaCaT apoptosis.
(2) UVB cell death inducing,, by the excessive generation of ROS, induction, DNA damage occurs, activate p53 to express, p53 acts on Bcl-2 family protein on mitochondrial membrane, mitochondrial membrane potential is changed, discharge a series of cytokine, finally cause activating caspases and active cell apoptosis cascade reaction.
(3) C3G has the natural plants chemical substance compared with strong anti-oxidative activity, can improve the survival rate of the postradiation HaCaT cell of UVB, and HaCaT apoptosis is reduced.
(4) C3G suppresses the apoptosis caused by UVB, by removing excessive ROS, reducing DNA damage, reduce the activation of p53, the permeability regulating and controlling the expression of Bcl-2 family protein on mitochondrial membrane, recover mitochondrial membrane potential, reduce mitochondrial membrane, the release of apoptosis relevant cell factor is reduced, thus reduce the cracking activation of caspases, the final generation suppressing apoptotic response caused by UVB.
Claims (10)
1. Cy-3-G is preparing the application in sun care preparations and/or medicine.
2. Cy-3-G repairs the application in cosmetics and/or medicine after preparation is shone.
3. the application of Cy-3-G after preparing dermal photodamage protective agent and/or photic damage in renovation agent.
4. apply according to claim 3, it is characterized in that, described photic damage refers to the photic damage that UVB causes.
5. the application of Cy-3-G after preparation HaCaT cell photo-damage protective agent and/or photic damage in renovation agent.
6. apply according to claim 5, it is characterized in that, described HaCaT cell photo-damage refers to the HaCaT cell injury that UVB induces.
7. according to the arbitrary described application of claim 1 ~ 6, it is characterized in that, the consumption of described Cy-3-G is 80 ~ 200 μm of ol/L.
8. Cy-3-G is preparing the application in antioxidative cosmetics and/or medicine.
9. apply according to claim 8, it is characterized in that, described oxidation refers to the oxidative damage that UVB causes.
10. the application of Cy-3-G in preparation anti-apoptotic preparation.
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