The Investigation Of Mechanisms And Influential Factors Of Tempol Against UVB-induced Skin Photoaging

Ultraviolet is an electromagnetic wave.Its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length,200nm to 290nm is called UVC,290nm to 320nm is called UVB and 320nm to 400 nm is called UVA.UVC is the most harmful to skin.but can be almost completerly absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA.and UVB is the main reason.In recent years,skin diseases are increasing which results from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction.Exposure to ultraviolet extensively can cause skin photodamaging,cutaneous carcinoma and etc.Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet,especially in the field of skin photodamaging induced by UVB which has become a hot research field in nowadaysIn order to reduce skin damage caused by ultraviolet,anti-oxidant application has akeady become utility preference to protect the skin and to treat skin diseases. These researches are carrying on.Many studies have demonstrated that more oxygen free radicals are produced in human and animal skin after radiation with ultraviolet. Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radicals in vivo can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed,which causes injury of DNA,proteins and fat,destruction of cells structures and biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can prevent from skin photodamage caused by ultraviolet,such as tocophero,antiscorbic acid,β-carotene,catalase, SOD and Tretinoin etc.They have good effect on animals and cultrured cell system, but the effect on human is unclear.Some substances require a high concentration to become effective,however to maintain the concentration is harmful to the health.Nitroxide is a common biophysics agent.Compared with SOD.it has a small molecular weight,good stability,permeability and innocuity.Many studies have shown that nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.The study was divided into two parts,HaCaT cell and KM hairless mice were used as in vitro and in vivo models respectively.Tempol was used as protectant to establish the models against skin photoaging.We investigated the mechanisms of Tempol against skin photoaging by analyzed the indexes of the models,so that to provide laboratory data for prevention and treatment of photoaging diseases. PART ONEThe mechanisms of Tempol's protective effects on photodamaging in HaCat cells under UVB radiationBackgroundUltraviolet is divided into UVA.UVB and UVC.Only UVA and UVB can pass through atmosphere to the Earth.The wave length of UVA is 320 to 400 nm and UVB is 280 to 320 nm.Epidermis is outer layer of the skin.which is the first natural barrier to protect organism from physical,chemical as well as microorganism damage and maintain internal environment stable.This function is performed by keratinocytes, which account for 90%of epidermal cells.Keratinocytes are not only protective cells but also participate in biological processes(e.g.immunity,inflammation, proliferation,tumor transformation and etc.).Some studies showed that 95% ultraviolet are absorbed by keratinocytes.International investigations have already separately established model of keratinocytes oxidation damage caused by UVA, UVB.and conducts some researches,which mainly concentrates on immunosuppression,DNA injury,apoptosis and inflammation.It was showed that UVB is the main reason,which causes non-melanocyte skin cancer.More than one million people were diagnosed with skin cancer every year in the USA.One patient dies of the skin malignant tumor each hour.It was estimated that there will be 40,000,000 skin cancer patients in the U.S.A.at 2060.So far,although we don't have the statistical data.but skin diseases caused by ultraviolet have attracted much people high attention,particularly skin photoaging caused by UVB,which becomes a hot research field now.In order to reduce skin damage caused by ultraviolet,anti-oxidant application has already become utility preference to protect the skin and to treat skin diseases. These researches are carrying on,Stewart MS and Cameron GS have discovered that application of the Vitamin C or E may suppress keratinocytes damage after UVB radiation.Many researchers have discovered that after application of anti-scorbutic acid or hydroxyacetic acid may suppress apoptosis related transcription factor expression about apoptosis after UVB radiation.Valeria M et al.have discovered that the anti-oxidant and Vitamin C may suppress JNK expression of keratinocytes,which suppresses AP-1 expression,and suppresses the keratinocytes apoptosis.These substances have good effect against ultraviolet on animals and cultured cell system. but the effect on human is unclear.Some substances require a high concentration to become effective,but to maintain the concentration is harmful to the health.Nitroxide is a common biophysics agent.Samuni A.found thatit is similar to SOD disproportionate superoxide anion in 1988.so its effects against oxidative damage were attracted attention.Compared with SOD,it has a smaller molecular weight. better stability,permeability and innocuity.Nitroxide was widely applied in the reaserch of bacterium,cultured myocardial cells,isolated heart perfusion and animals in vivo.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide,which has a protective effect on skin photoaging caused by ultraviolet in our recent studies.HaCaT cell belongs to keratinocytes,which is one kind of transformed but non-carcinogenic cell and is evolved from abdomen epidermis.HaCaT cell retains all epidermis differentiation ability,which is similar to the normal cell.It is similar to the normal cell on bionomics.Therefore we have established cell damage model with HaCaT cells radiated by 30mJ/cm² UVB to imitate the keratinocytes photodamage. Take Tempol as the protecting agent,we investigated the possible mechanism and the influencing factors of Tempol against UVB radiation on HaCaT cells damage.The study was unique in domestic and foreign,and it has provided a brand-new mentality for Tempol and other anti-oxidants and has laid the rationale for the possible mechanism.ObjectiveTo establish a phtodamage model of HaCaT cells with UVB radiation and study the mechanisms of Tempol on HaCaT cells against UVB and the influencial factors.Materials and methods1.Phtodamage model establishment1.1 HaCaT cells cultureHuman keratinocytes cell line HaCaT cells were cultured with DMEM supplemented with 10%fetal calf serum(FBS)at 37℃in a humidified incubator containing 5%CO₂. 1.2 UV lightThe wave length of UVB ranges from 290 to 320 nm and the peak is 297 nm. Each lamp box contians four lamps and each lamp's power is 40w.Put the lamps into lamp box and radiated HaCaT cells.2.Experiment of Tempol's protective effects on photodamaging2.1 HaCaT cells groupsHaCaT cells were divided into seven groups,which were marked group A,B,C,D,E,FandG.Group A was control group,which was not applied Tempol and without radiation.Group B was UVB radiation control,which was not applied Tempol but radiated with ultraviolet.Group C was supplied with 0.5mM Tempol.Group D was supplied with 1mM Tempol.Group E was supplied with 2mM Tempol.Group F was supplied with 4mM Tempol.Group G was supplied with 8mM Tempol.2.2 Cell culture and Dose of radiationWhen the cytomixis was to 80%,they were taken out from cell incubator and Tempol was added aseptically.Medium was added to control group and radiation control group,the volume is the same in these groups at least.Put human keratinocytes cell line HaCaT cells to be cultured in a humidified incubator containing 5%CO₂ at 37℃for one hour.Except for control group,other six groups were radiated with UVB;the dose was 30mJ/cm² and the vertical dimension from the light to the cells was 15cm.2.3 Measurement of cell proliferation in HaCaT cellsHaCaT cells were incubated for 18 hours and add MTT to culture for 4 hours. Then add dimethyl sulfoxide was added to dissolve the crystal.Measure the extinction value with enzyme immunity instrumentation on 490 nm.2.4 Measurement of apoptosis in HaCaT cells HaCaT cells After HaCaT cells were incubated for i8 hours,digested,centrifugated and adjusted to 1×10⁶/ml.Added Annexin V-FITC and PI,analyzed with FCM and measured the rate of apoptosis in HaCaT cells.2.5 Measurement ofFoxO3a mRNA expression in HaCaT cellsHaCaT cells were incubated with UVB for 18 hours.The total RNA was extracted from HaCaT cells.The cDNA was obtained by reverse transcription, and FoxO3a mRNA expression was measured with RT-PCR.Results1.The effect of Tempol on the HaCaT cell proliferation The OD scores were elevated in group A,C,D,E and F significantly.The differences between group A,C,D,E,F and group B,G were significant(p＜0.01). The score in group A and C was higher than that of other groups,and the differences between group A and C was no significant(p＞0.05).The OD scores in group D,E and F decreased one by one.The differences between group B and G were no significant(p＞0.05).2.The effect of Tempol on HaCaT cell apoptosisThe apoptosis rates were elevated in group B,C,D,E,F and G significantly.The differences between group B,C,D,E,F,G and group A were significant (P＜0.01).The apoptosis rate in group B was the highest.The apoptosis rate was decreasing as the concentration of Tempol increased.The differences between eachgroup were significant(p＜0.01).3.The effect of Tempol on FoxO3a mRNA expressions in HaCaT cellFoxO3a mRNA expression was increased in group B,C,D,E,F and G significantly compared with group A(p＜0.01).FoxO3a mRNA expression in group B was the highest.FoxO3a mRNA expression was increasing as the concentration of Tempol increased in other groups.The differences between each group were significant(p＜0.01).ConclusionAt a certain range of concentration(0.5mM,1mM,2mM,4mM,8mM), Tempol could have a protective effect on photodamaging caused by radiation with UVB.The study showed that Tempol may increase HaCaT cell proliferation. inhibit HaCaT cell apoptosis and inhibit over-expression of FoxO3a in HaCaT cell. Its protective effect of Tempol was decreased as the concentration increased.Tempol at concent ration of 0.5mM had the st rongest protective effect. PART TWOThe investigation of mechanisms and influential factors of Tempol against UVB- induced skin photoaging in hairless miceBackgroundIn recent years,skin diseases are increasing which result from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction. Exposure to ultraviolet can cause skin photoaging,cutaneous carcinoma and etc. Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet.Ultraviolet is an electromagnetic wave,and its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length.200nm to 290nm called UVC,290nm to 320nm called UVB and 320nm to 400 nm called UVA.UVC is the most harmful to skin.but can be almost completely absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA,of which UVB is the main reason.Many studies have demonstrated that skin photodamage by ultraviolet related with oxygen free radicals.EPR equipment has detected more oxygen free radicals in human and animal skin after radiation with ultraviolet.Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radical can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed, which causes injury of DNA,proteins and fat,cells structures destruction and disorder of biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can protect skin from photodamage caused by ultraviolet,such as SOD,tocophero,antiscorbic acid,β-carotene,catalase and Tretinoin etc.They have good effect on animals and cultured cell systems,but the effect on human is unclear.Some substances require a high concentration to come effective,however to maintain the concentration is harmful to the health. Nitroxide is a common biophysics agent.Samuni A.found that it is similar to SOD disproportionate superoxide anion in 1988.Then its effects against oxidative damage was attracted more attention.It has a smaller molecular weight,better stability,permeability and mnocuity compared with SOD.Nitroxide was widely applied in the research of bacterium,cultured myocardial cells,isolated heart perfusion and animals.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.KM hairless mice were selected as objects against skin photoaging to establish a model,and Tempol as protectant.We analyzed the skin surface,the changes of collagen and elastic fibers in the dermis,the malondialdehyde(MDA)and hydroxyproline(HYP)content,and mtp53 expression.These indexes were analyzed; we investigated the mechanisms of the protective effect of Tempol against skin photoaging and effects of time on the protection,in order to provide laboratory data for photoaging disease therapy.ObjectiveThe study was designed to investigate the changes of skin surface condition,collagen fibers and elastic fibers in the dermis,MDA and HYP content in the skin,fibroblast and collagen fiber ultrastructure,mtp53 expression in hairless mice induced by UVB radiation after application with Tempol in different time-spans.The effect of time on Tempol against photoaging was observed.Materials and methods1:Animal models establishment1.1 AnimalsSeventy-two KM hairless mice(6-8 weeks old;20-25 grams of body weight: half male and half female)were used in the study.They were kindly supplied by Beijing University and were fed on the same condition.1.2 UV lightThe wave length of UVB ranges was from 290 to 320 nm and the peak was 297 nm.Each light lamp power was 40w.Put the lamps into lamp box and the height of radiation was 30cm. 2.Experiment of Tempol against photoaging2.1 Animal groupsSeventy-two hairless mice were divided into six groups randomly and each group had twelve mice.Group A was control group.The skin was applied TDW without radiation.Group B was UVB radiation control.The skin was applied TDW but radiated with ultraviolet.Group C was applied 0.36%Tempol and radiated after an hour.Group D was applied 0.36%Tempol and radiated after two hours.Group E was applied 0.36%Tempol and radiated after four hours.Group F was applied 0.36%Tempol and radiated after eight hours.2.2 Dose of radiationExcept for the control group,other five groups were radiated with UVB every other day.The radiation dose was 0.111J/cm² every time.The total time was 14 weeks and the total dose was 5.45J/cm².2.3 Measurement of surface condition changes on hairless mice skin0.36%Tempol or TDW was applied on dorsal skin(2×3cm² areas)of hairless mice and the study according to above experimental design,and then the surface changes were scored every other week.The total time was 14 weeks.2.4 Measurement of collagen and elastic fibers changes in hairless mice skinAfter the last radiation,the mice were killed and shin tissue was cut off the tissue (about 0.5cm² areas),then the cut sheets were fixated,dehydrated,embedded and stained,the content and histological changes of collagen and elastic fibers in the dermis were observed.2.5 Measurement of fibroblast and collagen fiber ultrastructure changes in hairless mice skinThe skin tissues were cut from the dorsal of the mice(2 or 3 pieces from each mouse and each was about 0.1cm³ area),then were fixated,dehydrated,embedded and located.The samples were cut to extra thin sections(50nm)and stained by uranyl acetate and lead citrate.Ultrastructure changes of fibroblast and collagen fibers were observed by transmission electron microscope.2.6 Measurement of MDA and HYP content changes in hairless mice skin2.6.1 The homogenate preparation and measurement of MDA content The skin was drilled down with corneal trephine,and then it was weighed without subcutaneous fat to prepare 10%homogenizer.Put 0.1ml 10%homogenizer and then added No.1,2,3 reagents misce bene.The mixture was warmed for 40 minutes in 95℃water,centrifugated 10 minutes at 3500-4000r/min.The supernatant was taken,and was shaded selection with 532nm spectrophotometer.2.6.2 Measurement of HYP contentThe skin was drilled down with corneal trephine,and then about 30 to 100 grams tissues(wet weight)was put into test tube.Added 1ml water to digest,warmed the mixture for 20 minutes.Adjusted PH to 6.0 to 6.8 and added distilled water to 10ml. Put active carbon into 3ml solution to misce bene.Centrifugated the mixture for 10 minutes at 3500r/mm and taken 1ml the clear supernatant and added No.1,2,3 agents to misce bene.The mixture was warmed for 60 minutes in 60℃water,centrifugated for 10 minutes in 3500r/min.The supernatant was taken and shaded selection with 532nm spectrophotometer.2.7 Measurement ofmtp53 expression in hairless mice skinThe materials were cut from the dorsal skin of the mice and the expression of mtp53 was measured with immunohistochemical method.The results were analyzed by America Leica DMI4000B Image Processing System.Results1.The skin surface changes on hairless miceThe scores were elevated in group B,C,E and F significantly.The differences between group B,C,E,F and group A,D was significant(P＜0.01).The score in group B and F was the highest;the score in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).2.The changes of collagen and elastic fibers in hairless mice skinThe collagen and elastic fibers in the dermis arranged in bunchy regularly without marked rupture in group A and D.Epidermis in group B,C,E and F was hyperkeratinization.Papillar layer was flatted.The collagen and elastic fibers in the dermis of hairless mice became thicker,fragmented and disarranged. 3.The ultrastryctue changes of fibroblast and collagen fibers in hairless mice skinThe fibroblast and collagen fibers were normal in group A and D.We could observe karyopycnosis,the collagen and elastic fibers disarranged,the boundary among fibers unclear and some fibers was dissolved in group B,C,E and F.4.The changes of MDA and HYP content in hairless mice skinThe MDA contentThe MDA content was significant increased in group B,C,E and F.The differences between group B,C,E,F and group A,D was significant(P＜0.01). The MDA content in group B and F was the highest;the MDA content in group C and E was lower than that of group B and F.The differences between group A and D. group B and F was no significant(p＞0.05).The HYP contentThe HYP content was significant decreased in group B,C,E and F.The differences between group B,C,E,F and group A,D was significant(P＜0.01). The HYP content in group B and F was the lowest;the HYP content in group C and E was higher than that of group B and F.The differences between group A and D,group B and F,group C and E was considered no significant(p＞0.05).5.The changes of mtp53 expression in hairless mice skinMtp53 expression measurement was increased significant in group B,C,E and F.The differences between group B,C,E,F and group A,D was significant (P＜0.01).The mtp53 expression in group B and F was the highest;the mtp53 expression in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).6.The affection of time on the effect of Tempol against photoagingThe results showed that 0.36%Tempol had determinate protection to skin photoaging of hairless mice caused by radiation with UVB for 1 to 4 hours after external application,and the protection effect would be the best in 2 hours after external application,no effect after 8 hours.Conclusions External application with 0.36%Tempol had protection to skin photoaging of hairless mice caused by radiation with UVB,and its protection were affected by time. The effective time-spans are from 1 to 4 hours after external application,and the best time-span is around 2 hours after medication.Its mechanisms may have a relationship to inhibiting degradation and degeneration of the collagen fibers and elastic fibers. inhibiting apoptosis of fibroblasts,inhibiting lipid peroxidation and inhibiting gene mutation of p53 in dermis.