The Functional Research On GSK3β And PIN Genes Related To The Anti-inflammatory And Immuno-regulatory Actions Of EsA

Esculentoside A (EsA) is a kind of saponin isolated from the Chinese herb Phytolacca esculenta. It has been showed that EsA possessed comprehensive pharmacological effects during the last twenty years. Previous experiments demonstrate that it has strong anti-inflammatory and immunoregulatory effects. Moreover, ESA significantly diminishs macrophage production of tumour necrosis factor (TNF) , platelet activating factor(PAF), interleukin-1( IL-1),interleukin-6(IL-6),prostaglandin E2(PGE2)and nitric oxide(NO), and inhibits lymphocyte release of interleukin-2(IL-2) , interferon-γ(IFN-γ),which play important roles in the inflammatory and immunological response. EsA has been shown to inhibit phagocytic activity in mouse macrophages and has the positive curative effect on autoimmunity in the mouse model. With analysis of cDNA microarry, it is found that EsA regulates the expression of a group of genes including glycogen synthase kinase 3 beta (GSK3β), protein inhibitor of nitric oxide synthase (PIN ), suggesting GSK3βand PIN may be related to the anti-inflammatory and immunoregulatory effects of EsA.Glycogen synthase kinase-3βis a ubiquitously expressed constitutively active serine/threonine kinase that phosphorylates cellular substrates and thereby regulates a wide variety of cellular functions, including development, metabolism, gene transcription, protein translation, cytoskeletal organization, cell cycle regulation, and apoptosis. The recent discoveries reveal that GSK3 promotes the production of inflammatory molecules and cell migration, which make GSK3 a powerful regulator of inflammation. The involvement of GSK3 in inflammation highlights its contributions to the pathologies of several prevalent diseases which involve inflammation, including mood disorders, Alzheimer's disease, diabetes, and cancer. The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS). It binds to neuronal nNOS, thereby inhibiting nNOS enzymatic activity and decreasing the production of NO. PIN is also identified to be the same as light chain 8 (LC8), wihich regulates the many biological processes. The actions of their anti-inflammation and immunoregulation, however, are not well clarified.To gain a better understanding of the effects of GSK3βand PIN and possible mechanisms of ESA on anti-inflammatory and immuno-regulatory effects, this study were designed to study the regulatory functions of inflammatory and immune responses of GSK3βand PIN in a murine macrophage RAW264.7 cell line transfected with the eukaryotic expression vector of pcDNA3.1-GSK3βand pcDNA3.1-PIN.The results are showed as follow:1. To establish murine macrophage RAW264.7 stably overexpressing GSK3βand PIN, GSK3βand PIN cDNA were cloned, verified by sequence analysis, and constructed into an eukaryotic expression vector of pcDNA3.1. RAW264.7 were subsequently transfected with pcDNA3.1-GSK3βand pcDNA3.1-PIN using lipofectamine2000 and selected with G418. Western blot confirmed the overexpression of GSK3βin the stably transfectants.2. Phagocytosis was assessed by neutral red analysis. RAW264.7 cells stably overexpressing GSK3βand PIN resulted in reduced phagocytosis compared with that of pcDNA3.1 control.3. Nitric oxide (NO) activities in the supernatant of over expressing GSK3βand PIN RAW264.7 cells in response to LPS (1μg/mL) were determined by Griess method. RAW264.7 cells stably overexpressing GSK3βand PIN significantly suppressed NO production.4.Tumor necrosis factor (TNF) activities in the supernatant of overexpressing GSK3βand PIN RAW264.7 cells in response to LPS were determined by crystal violet staining assay. RAW264.7 cells stably overexpressing GSK3βand PIN promoted LPS-stimulated TNF release .5. Western blot assay was used to assess the synthesis of IL-1 and IL-6 protein by LPS(1μg/mL)-stimulated RAW 264.7 cells stably overexpressing GSK3βand PIN . The overexpression cells markedly promoted LPS-stimulated IL-6 production, but did not changed IL-1 production.In conclusion , the murine macrophages RAW264.7 stably overexpressing GSK3βand PIN significantly suppressed the phagocytosis and LPS-stimulated NO production, whereas selectively promoted LPS-stimulated TNF and IL-6 productions.These results indicated GSK3βand PIN exerted the regulatory functions of inflammatory and immune response in macrophages. Based on the observed actions of GSK3βand PIN and our previous results that EsA up-regulated the expression of GSK3βand PIN genes, it may speculate that ESA exerts strong anti-inflammatory and immunoregulatory effects through the GSK3βand PIN.