Regulation Of GX50 On The Nuclear Transcription Fator-κB Pathway Associated With The Aging Of The Skin

Backgroud At present, studies have shown that nicotine can activateα7 nicotinacetylcholine receptor to suppress nuclear transcription fator-κB signaling pathway, thusprevent Alzheimer's disease[1]. But nicotine is addictive and re-using nicotine increaseheart rate and raise blood pressure and reduce appetite. Large doses of nicotine can causenausea and vomiting, and be fatal in severe. Since the main effect of nicotineα7 nicotinacetylcholine receptor site, the researchers of the laboratory of Professor Wei Dongqingdesigned and screened a compound called GX50 using bioinformatics tools and methodsaccording toα7 nicotin acetylcholine receptor role for nicotine. GX50 happens to be anatural component in wild pepper with the use of security. In the previous study in ourlaboratory we find GX50 has a regulatory role of the nuclear transcription fator-κBsignaling pathway on macrophages, neural cells and endothelial cells, so we apply to theskin in order to more fully explore its effect and prepare further development of thecompound . Here we provide evidence for widespread involvement of nucleartranscription fator-κB in mammalian aging, and demonstrate a reversal of many featuresof aging upon acute blockade of nuclear transcription fator-κB in aged skin[2]. It isidentified nuclear transcription fator-κB as a candidate activator of aging-relatedtranscriptional changes in multiple human and mouse tissues. Genetic blockade ofnuclear transcription fator-κB in the skin of chronologically aged mice reverse the globalgene expression program and tissue characteristics to those of young mice[3].Objective Detection of the pole of GX50 in the skin and the path of nuclear transcriptionfator-κB , to prepare for the clinical applying of the compound .Methods In order to explore the GX50's role in skin aging, we mainly research on bothtissue level and cell level. At the tissular level , we apply drugs on the back skin of theaged mouse with mixture of GX50 and dimethyl sulfoxide and glycerine on the left andonly mixture of dimethyl sulfoxide and glycerine on the right as control for two weeksand then cut both sides of the skin and produce frozen sections. First , we make thehemoatoxylin and eosin staining to detect the changes of the two sides. In order to furtherillustrate the difference between both sides of the skin, we make theimmunohistochemistry of collagen protein expression in the skin of the frozen sections .To find out whether the target of GX50 in the process of reversing the old skin tissues tothe young skin is the nuclear transcription fator-κB signaling pathway, we carried out EMSA (electrophoretic mobility shift assay) experiment to test the nuclear transcriptionfator-κB DNA-binding activity.We carry out Western Blot to detect the plasma protein of skin tissue according to thestandard of phosphorylation degree of IκB which restrain the activity of nucleartranscription fator-κB.At the cellular level, we conduct the primary culture of mice epidemis and identify purityby immunofluorescence staining choosing anti-PCK (Mouse Anti-Pan Cytokeratin) asthe epidemal cell's surface markers. We treat the cells with GX50 and LPS and blankcontrol and extract membrane protein, nucleoprotein and plasma protein and measuretheir concentration by ultraviolet specrophotometer. It is found that GX50 can effectivelyactivateα7 nicotin acetylcholine receptor through drug design method.We detect theability of GX50 combine toα7 nicotin acetylcholine receptor of epidemis membranewith the application of Octet RED biological interactions analyzer.We carried out EMSAexperiment to test the nuclear transcription fator-κB activity of the epidemal cells. Wecarry out Western Blot to detect the phosphorylation degree of IκB.Results (1)The results of the hemoatoxylin and eosin staining show that epidermis isflatter on the left than the right and the size and the shape of the basal cells is moreregular and extracellular matrix arranged closely;Dermal has more follicle and moredense and more melanocytes and less melanin deposition on the left than the right;Borders of Epidermal and dermal are more obviously and the connection beween theepidermis and the dermis is more firmly on the left than the right.(2) Immunohistochemistry of collagen protein expression in the skin of the frozensections show that the density of collagen protein on the left is higher than that on theright(P＜0.05).(3) The results of EMSA of the skin tissues show that the volume of the nucleartranscription fator-κB shuffling into the nuclear in the GX50-treated skin sample was lessthan the control group(P＜0.05). The result suggested that GX50 had inhibitory effecton the activation of nuclear transcription fator-κB to some extent.(4) The results we obtain from the experiment is that the control group got a faint p-IκBbands while the GX50-treated skin sample had none in the condition of adding moresample in the experimental group than the control. Though internal reference correction,the results could serve as a reference, that GX50 might play its role by restraining thephosphorylation of IκB and thus prevent the activation of nuclear transcription fator-κBwhich may weaken the regulation of downstream target genes.(5) The trend of the combine curve indicate thatα7 nicotin acetylcholine receptor might be the target of the regulation of GX50 on the skin.(6) Lag belt gray analysis of EMSA of mice epidemis show that the volume of thenuclear transcription fator-κB shuffling into the nuclear in the GX50-treated group is lessthan the control groups(P＜0.05)and the results indicate that GX50 reduce the activityof nuclear transcription fator-κB.(7) The results of Western Blot of mice epidemis show that the volume of IκB inGX50-treated group is higher than that of control group while p-IκB is the opposite to theIκB(P＜0.05). These indicate that GX50 restrain the phosphorylation of IκB.Conclusion To conclude, GX50 can reverse the aging of the skin by regulate the nucleartranscription fator-κB pathway when it arrive atα7 nicotin acetylcholine receptor.