Effects Of Total Glucosides Of Paeony On Expression Of IL-8, ICAM-1and Ki67in Human HaCaT Keratinocytes

ObjectivePsoriasis is the most common dermatological diseases, which is also a kind of chronic, relapsing, inflammatory skin disease, not only the incidence of these diseases, but stubbornly intractable, and is easy to repeat. The specific pathogenesis of this disease remains unclear, therefore becomes a research focus. Psoriasis activates a lot of T lymphocyte, infiltration in pathology. The infiltrating T lymphocytes and keratinocytes play a key role in such interactions in the pathogenesis of inflammatory skin. Because T lymphocytes release large amounts of cytokines. which affect the immune function of keratinocytes; while the skin offer a complex microenvironment for T lymphocyte-mediated immune reactions development. The present study showed that T cell-driven, keratinocytes as target cells of autoimmune inflammation is the main pathogenesis of psoriasis, and the Thl lymphocytes play a key role during the passage. At the same time, IFN-γ is an important Thl lymphocytes secrete cytokines, which is one of the skin inflammation and the regulation of the immune response of the most effective pro-inflammatory cytokines. Also the activation of the keratinocyte cells and keratinocytes can express many chemokines, cytokines and membrane molecules, further cascade of the inflammatory reaction (1). Polymorphonuclear neutrophil aggregation to the skin by the IL-8chemotactic turned to achieve. However, the keratinocytes and inflammatory cell adhesion between ICAM-1is achieved. Therefore, the study of keratinocytes IFN-γ-mediated inflammatory response and its signal transduction mechanisms, that contributes to the Thl dominant skin inflammation reasonable treatment methods, as well as helps to reveal the mechanism of drug treatment of these diseases and efficacy prediction.TGP has multiple autoimmune response pathway inhibitor, anti-inflammatory, anti-viral effects. Therefore, this study made immortalized human keratinocyte cell line (HaCaT cells) as target cells to research TGP on HaCaT cells in IFN-γ-mediated secretion of inflammatory cytokines, signal transduction pathways and diagonalization affected. And it aims to explore TGP on Thl-type inflammatory mode of regulation of HaCaT cells to provide theoretical support in such diseases.MethodsIn cultured HaCaT system, we us experimentally induced IFN-y establish randomly to divide into experimental group (TGP treatment), the blank control group and positive control group (TO group). MTT was used to detect the12h,24h TGP on HaCaT cell viability for the next administration of drug trials to determine the concentration range. Concentrations were added to test drug safety co-culture (96-well cell culture plate, each well0.2mL, three wells per concentration,37℃,5%CO2incubator culture). After24h the cells in the culture supernatant was removed, and then add500U/mL rhIFN-y stimulation of HaCaT cells48h,48h after collecting culture supernatant was measured by ELISA in culture supernatants IL-8, ICAM-1expression. Logarithmic growth phase, cells grew well, were seeded in six-well plates coverslips,12h after adding safe concentrations of the test drug were cultured24h, and then follow the best of each molecule expression duration of action, adding500U/mL rhIFN-y stimulation of HaCaT cells, cells collected at different time points by fluorescence immunohistochemistry positioning Ki-67, ICAM-1expression.Results1、MTT assay TGP on HaCaT cell viability TGP on HaCaT cell proliferation in two-way adjustment, at low concentrations (0.5μg/mL～12.5μg/mL) when HaCaT cell proliferation promoting effect at high concentrations (25μg/mL～125μg/mL) HaCaT cell proliferation when was inhibited.2、Cell supernatants was measured by ELISA IL-8, ICAM-1expression0.5μg/mL～125μg/mLTGP after24h, the supernatant can be reduced in HaCaT cells expressing IL-8, ICAM-1levels (*P<0.05,**P<0.01). And at25μg/mL125μg/mLTGP supernatant concentration range of IL-8inhibited the expression, in the concentration range of0.5μg/mL～25p.g/mLTGP cell supernatants of ICAM-1expression was significantly inhibited.3、Fluorescence immunohistochemical localization ICAM-1, Ki-67expressionDrug concentrations in the TGP when0.5μg/mL～25μg/mL of ICAM-1expression was significantly inhibited. With the decrease in drug concentration TGP, ICAM-1expression was significantly decreased in the nucleus, the nucleus in the cytoplasm of expression did not weaken too obvious, but also weakened. Drug concentration in the TGP0.5μg/mL～25μg/mL fluorescence in the nucleus when Ki67expression was significantly decreased.ConclusionsTGP has a double adjustment function, and suppresses HaCaT cells secrete IL-8, ICAM-1and Ki67expression.