The Role Of TIR/BB Loop Analogue AS-1 In UVB-induced Acute Light Damage And The Role Of MicroRNA Differential Expression In UVB-induced Chronic Light Damage

Part?The TIR/BB-loop mimetic AS-1 Protects Human HaCaT Keratinocytes and Mouse Skin Against UVB-Mediated inflammation and ApoptosisBACKGROUND;Skin is the largest human organ that is directly exposed to the environment.Ultraviolet(UV)irradiation from the sun is the major environmental cause of skin damage.Acute exposure to UV irradiation causes sunburn,immune suppression,DNA damage,and connective tissue degradation.Accumulated damage,resulting from chronic sun exposure,causes skin cancer and premature skin aging.The incidence of these skin diseases is growing as the population ages.Increased levels of UV irradiation at the Earth's surface,due to depletion of the ozone layer and continuing use of sun-tanning devices for cosmetic purposes,also contribute to rising incidence of actinic damage.UV irradiation causes pathological changes in the skin such as sunburn,skin cancer,and photoaging.These effects are due to both UV-induced DNA damage and altered cellular signaling.UV activates multiple signaling pathways through nongenetic mechanisms,resulting in profound changes in gene expression.This process resembles the response to growth factors and is known as the UV response.Much of the UV response is due to the activation of mitogen-activated protein kinase(MAPK)family members and NF-?B(nuclear factor-?B)pathways.NF-?B induction of numerous cytokines leads to dema and erythema,two components of UV-induced MyD88 is an adaptor protein and is involved in IL-1 receptor/Toll-like receptor(TLR)signals.MyD88 consists of an N-terminal death domain(DD)separated by a short linker from a C-terminal Toll/IL-1 receptor(TIR)domain.Upon ligand stimulation,MyD88 is recruited to the membrane by the interaction of its TIR domain with the analogous domain in the IL-1 receptor or TLR.The MyD88 DD binds to the IL-1 receptor-associated kinase(IRAK)-4,and promotes autophosphorylation of IRAK-1.These events activate both the IkB kinase and the stress-activated mitogen-activated protein kinases such as c-Jun N-terminal kinase and p38.The purpose of this study was to evaluate whether IL-1R/MyD88 pathway is involved in UVB induced damage.We hypothesized that inhibition of the interaction of IL-1R with MyD88 with TIR/BB-Loop mimetic(AS-1)will attenuate UVB induced damage.SPECIFIC AIMS:The present study investigated the possible beneficial effects of AS-1 to inhibit UVB irradiation-induced inflammatory response and apoptosis in vitro and in vivo.MATERIALS AND METHODS:Animals and treatmentMale C57BL/6 mice,6-7 weeks old,were obtained from the Model Animal Research Center(MARC)of Nanjing University(Nanjing,China)and were housed with free access to food and water.Experiments involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health(NIH Publication No.85-23,revised 1996).All aspects of the animal care and experimental protocols were approved by the Nanjing Medical University Committee on Animal Care.The dose of 210 mJ/cm2 was approximately half that required to induce mild oedema with a single exposure.The shaved skin of the mice was exposed to a single dose of 210 mJ/cm2 UVB irradiation.The mice were divided into four groups:(i)unexposed and untreated control;(ii)UVB;(iii)UVB+Vehicle;(iv)UVB+AS-1.Mice that were assigned to the treated group that were received AS-1(50 mg/kg body weight)by intraperitoneal injection immediately 30 min before UV irradiation.AS-1 was prepared by mixing one volume of AS-1 in DMSO with three volumes of saline to give a final AS-lconcentration of 50 mg/kg body weight.Vehicle control was prepared by mixing one volume DMSO with three volumes of saline.The animals were then sacrificed after 24 hours post-UVB exposure and skin biopsies were harvested for immunohistochemical analysis of apoptotic cells.Mice were sacrificed after shaving without any UVB irradiation as controls.Treatment of cellsHuman keratinocyte HaCaT cells were cultured in Dulbecco's Modified Eagle Medium(DMEM,Invitrogen Corporation,USA)supplemented with 10%fetal bovine serum,100 units/ml penicillin/streptomycin and were maintained at 95%humidity in 5%C02 environment at 37?.AS-1(dissolved in DMSO)was used for the treatment of cells.The final concentration of DMSO used was under 0.1%(v/v)for each treatment.The cells(70-80%confluent)were treated with AS-1(100 ?M)for 2 hours in serum-free DMEM after which the media was removed and cells were washed with phosphate-buffered saline(PBS)and then covered with fresh PBS.Then UVB irradiation(30 mJ/cm2)was delivered There were four experimental groups:(?)unexposed and untreated control;(?)UVB;(?)UVB+ Vehicle;(?)UVB+AS-1.RESULTS:1.AS-1 treatment alleviated UVB-mediated decrease in the viability of HaCaT cells.No toxicity of AS-1 were shown to HaCaT cells at all the concentrations of 10?M,50?M,100?M.UVB(30 mJ/cm)irradiation significantly decreased cell viability.However,this UVB-mediated decrease was significantly inhibited by pretreatment of cells with AS-1(25-100?M)in a dose-dependent manner as determined by the CCK-8 assay2.AS-1 inhibited UV irradiation-induced inflammatory cytokines in the culture supernatant of HaCaT cells and mouse skinUVB irradiation induced marked increase of IL-1?.IL-6 and TNF-a compared with sham control,respectively.AS-1 pretreatment suppressed induction of all three inflammatory cytokines compared with the untreated UVB group.Administration of vehicle did not affect UVB-increased levels of inflammatory cytokines in the culture supernatant.3.AS-1 attenuated UVB-irradiation-induced apoptosis in HaCaT cells and mouse skinUsing FACS analyses of annexin V and PI staining,we found that UVB irradiation(30 mJ/cm2)of cells resulted in apoptosis in HaCaT cells.However,AS-1 pretreatment inhibited apoptosis.Vehicle administration did not affect UVB induced apoptosis in HaCaT cells.The same results were found in vivo.Caspase-3 activity was strongly induced in HaCaT cells following exposure to 30 mJ/cm2 UVB,but decreased in cells pretreated with AS-1.The cleaved caspase-3 expression 24 h post-UVB exposure.was significantly increased.They were significantly attenuated in AS-1 pretreatment vehicle group.4.AS-1 administration attenuated the increased associationof IL-1R with MyD88 following UV irradiationUV irradiation significantly increased the association of IL-1R with MyD88 compared with sham control cells.AS-1 administration significantly attenuated the increased interaction of IL-1R with MyD88 induced by UV irradiation.The Administration of vehicle control did not affect the interaction of IL-1R and MyD88 after UV irradiation compared with the untreated UVB group.5?AS-1 decreased NF-?B binding activity and MAPKs phosphorylation in HaCaT cells following UVB exposureUVB irradiation significantly increased NF-?B binding activity and the levels of phospho-p38/p38,and phospho-ERK1/2/ERK1/2 levels,and phospho-JNK/JNK levels,respectively compared with the untreated cells(**p<0.01).In contrast,AS-1 treatment reduced UV irradiation induced increases in NF-?B binding activity,p38,ERK1/2 and JNK phosphorylation compared with the untreated UVB group.There is no significant difference between the vehicle treated UVB group and the untreated UVB group.Immunofluorescence examination showed that UVB irradiation increased NF-?B translocation from the cytoplasm to the nucleus compared with sham control.AS-1 treatment significantly inhibited the UVB induced NF-?B nuclear translocationCONCLUSIONS:We investigated the effects of the TIR/BB loop mimetic(AS-1)on the ultraviolet B(UVB)-induced inflammatory response and apoptosis in human HaCaT keratinocytes and mouse skin.We report here that UVB upregulates the interaction of IL-1R with MyD88 in human HaCaT keratinocytes and mouse skin.We found that pretreatment of cells with AS-1 protected against UVB(30 mJ/cm2 24 hours)-mediated(?)decrease in cell viability(?)induction of apoptosis.(?)increase in Bax;(?)downregulation of Bcl-xL(?)increase in the levels of inflammatory cytokines.(vi)upregulation of the MAPK pathway and the binding activity of NF-?B We also investigated the photoprotective effects of AS-1 in vivo.Male C57BL/6 mice applicated of AS-1 were irradiated with a single dose of UVB(210mJ/cm2),and activation of the MAPK pathway was assessed.AS-1 application significantly decreased UVB-mediated apoptosis and Inflammation.Taken together our results suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in UVB-induced inflammatory response and apoptosisPart?Effect of microRNAs in Chronic Ultraviolet B Irradiation induced skin Photoaging from different siteBackground:Ageing of human skin may result from both the passage of time(intrinsic ageing)and from cumulative exposure to external influences(extrinsic ageing)such as ultraviolet radiation(UVR)which promote wrinkle formation and loss of tissue elasticity.Clinically,severely photoaged skin is characterized by a rough texture,irregular pigmentation,dryness,lack of elasticity,wrinkles,a yellowish color,and teleagiectasia.Notably,the aging is also observed in the skin away from the photodamage area,and the mechanisms are still unclear.MicroRNA has been proved to play a central role in aging.In addition to expression changes in tissues,more recent studies have indicated that miRNAs are detectable and highly stable in plasma,serum,and other biological fluids.And Circulating microRNAs were thought to be mediators among organs and cells.Some researchers even found that microRNAs could be targeted as the target in some diseases.But in photoaging,it is rarely reported and needs more study.Aim:To observe the relationship between the expressions of microRNAs and photoaging in the skin;To study the mechanism of microRNAs involved in photoaging.Methods:C57BL/6 mice were housed according to standard procedures.The backs of all mice were shaved once a week.The animals were divided into a control group that was shaved but not irradiated.The second group was irradiated with UVB light three times per week at a dose of 210 mJ/cm2[?three times minimal erythema dose(MED)]throughout a period of 6 months.UVB irradiation was delivered with a Philips TL 20W/12 lamp(Eindhoven,The Netherlands),which emits radiation at a wavelength of 280-320 nm and with a peak at 313 nm.Irradiation output was monitored with a Waldmann UV-meter(Waldmann,Villigen-Schwenningen,Germany).Microarray was taken for the detection of microRNA;realtime PCR was performed for the expression of the related proteins in aging.Result:1.Microarray showed,17.17%of microRNA had a over 2 fold changes(p<0.05)in UVB irradiated skin compared to control group,13.08%of microRNA had a over 2 fold change(p<0.05)in remote skin from abdominal skin,and 7.27%of microRNA had a over 2 fold change(p<0.05)in serum microRNA compared to control group.2.Compared to control group,there were 13 same microRNAs increased over 2 times in UVB irradiated skin,remote skin and serum,and 2 microRNAs decreased over 2 times.3.P16,P21 and P53 were up-regulated in both UVB irradiated skin and remote skin from abdomen.Conclusions:Photoaging also happened in the remote abdominal skin far away from the irradiated dosal skin.MicroRNA which released from the irradiated skin and exits in the serum may play a central role in the photoaging of abdominal skin.