Atentative Study On Functions Of TLRs In The UVB-induced Activation Of NF-κB And Its Significances In The UV-related Skin Diseases

The skin is the biggest apparatus through the whole people body, which is directlyexposed to external environment and easilysuffered damnification itself. It is well-known thatUV can make the skin suffer damnification, and lead to and deteriorate the development of thevarietyof inflammatoryskin diseases. Currentlythe disease rate of skin inflammatorydiseasehas been increasing each year. All the times, it has been a hot research field to studythe skininflammatorydisease, currentlystudies have found that the mechanism of inflammatoryskindisease which is caused byUV mainlyincludes: Oxygen free radicals, DNAmutation, therelease of inflammatory factors,Activation of signaling pathway etc. But the pathogenesiscan not be fullyclarified yet.The epidermis is the outermost layer of the human skin and its playa verycritical role inthe defense of the external environment risk factors.The innate immune system, also known asnon-specific immune system, is the first line of defense to against outside stimulus, takingaveryimportant position inthe body’s immune defense. As an important innate immunemolecule, studies have found that in some UV-related skin diseases such as rosacea, TLRs (TLR2,TLR4) expressionis increased, TLRs maybe involved in UV-induced skininflammation. Andother studies put forward that UVcan induce secretion of TLR2and TLR4ligand, activate TLRs signalingpathway, but theyare onlysome priliminaryhypotheses, it hasnot yet been further confirmed whether TLRs signalingpathwayplayan important role inUV-induced the inflammatoryresponse. Therefore, based on the above analyses, we proposethe followinghypothesis: UV can lead to the hight expressionof TLR2and TRL4inKeratinocytes, and TLRs maybe involved in the UV-induced activation of NF-κB, triggeringthe inflammatoryresponse, and the development of skin inflammatorydisease caused byUVB.Our studywill combine cell experiment, tissue level examination with clinical test toexplore and justifythe hypothesis, it will provide a new idea to elucidate the mechanism ofUVB-induced NF-κB activation and solidate a theoretical basis for the studyon the newstrategyfor the etiologyand the mechanism of inflammatoryskin disease.Research1. Makingsure the effect of UVB irradiation on the expressionof TLR2,TLR4;2. Investigatingthe functions of the expression of TLR2,TLR4in the UVB-inducingNF-κB;3. Testingthe functions ofexpression of TLR2,TLR4and NF-κB in UV-induced theskin injury,4. Makinga clinical studyfor the etiologyof the inflammatoryskin disease (Rosacea)and analysisingits sensitivityto UV.Methods1.Culture the human keratinocytes HaCaT cell in vitro under the different doses of UVBradiation, then the HaCaTcells are divided into fourgroups for the experiment. Respectivelytheyare:①non-irradiated group;②50mJ/cm2UVB irradiated group;③100mJ/cm2UVBirradiated group;④200mJ/cm2UVB irradiated group. UsingWesternblot and the RT-PCR todetect the expression of TLR2,TLR4and NF-κB in HaCaT cell after UVB irradiation;2.Add TLR2and TLR4pathwayinhibitors（OxPAPC）to the cultured HaCaT cell, thenuse Westernblot and the RT-PCR method to detect the change of the expression of NF-κB after100mJ/cm2UVB irradiation; 3.Respectilveyselect20number of rosaceas and20number of polymorphous lighteruptions which have been diagnosised. For normal skin tissue in the irradiatedgroup, usingimmunohistochemistrymethod to detect the expression of TLR2、TLR4and NF-κB;4.Collect and sort out theninety-eight cases of rosacea patients questionnaire,andstatistical analysis is done for all the data, including hot and spicyfood,sun, climate change,psychological factors,drugs, systemic disease, family historyand so on, meanwhile perform aphotosensitivityof UV.Result1.After the HaCaT cells are irradiated bythe different doses with50mJ/cm2UVB,100mJ/cm2UVB,200mJ/cm2UVB, the expression ofTLR2、TLR4and NF-κB in HaCaT cellis increasingand closelydepend on the number of doses;2.NF-κB is activated in the earlier time (1h) after100mJ/cm2UVB irradiation;3.After adding TLR2and TLR4pathwayinhibitors（OxPAPC）, the activation ofNF-κB which is irradiated byUVB is clearlyhold back;4.High expression of TLR2、TLR4appears in the whole epidermis of rosacea andpolymorphous light eruption lesion. To the contrary, the expression of TLR2、TLR4onlyappears in basal epidermis of normal skin, and mainlyappears in normal keratinocyteccytomembrane;5.High expression of NF-κB in the whole epidermis and dermal inflammatorycells ofrosacea and polymorphous light eruption lesion,mainlyappears in the nucleusnand cytoplasmof cells. To the contrary, the expression of NF-κB onlyappears in basal epidermis of normalskin,mainlyappears in the cytoplasm of cells;6.The pathogen of rosacea is related to manyfactors, includinghot and spicyfood,sun,climate change, psychological factors,drugs, systemic disease, familyhistory, and so on, androsacea patients of78percent are proved positive in the photosensitized reactions test.Conclusion1.UVB radiation can increase the expression of TLR2,TLR4in HaCaT cell;2.UVB can activate NF-κB byinducingthe high expression of TLR2and TLR4,furthermore lead to the inflammatoryreaction of backward position,it shows that it is possible that TLR2and TLR4is involved in the UVB-induced activation of NF-κB.3.TLR2, TLR4and NF-κB have higher expression in the UVB-induced inflammatoryskin diseases than normal skin lesions, it shows theyprobablyinfluence the disease process ofUVB-induced inflammatoryskin diseases.4.Manyfactors caused the occurrence of rosacea together, the effect of UV can not beignored in the pathogenesis of rosacea.