The Effects And Mechanisms Of TRIF Signaling Pathway In Primary Astrocytes Release Of Inflammatory Cytokines

Astrocytes(Astroeyte, AS) is the most widely distributed in the central nervous system cells that have complex and diverse structures and functions in the secretion of nutrients, regulate all aspects of neuronal microenvironment are playing an important function, involved in a variety of pathological processes of neurological disorders.Astrocytes play an important role in regulating immune function in brain by producting inflammatory cytokines.Toll-like receptors(Toll-like receptors, TLR) are involved in non-specific immunity(innate immunity) of an important class of protein molecules, is also connected to the non-specific immunity and specific immune.In the central nervous system, TLRs mainly expressed in microglia and macrophages, but there are also expressed in astrocytes.Effect of astrocytes may be associated with inflammation related to TLRs.ObjectiveThis study in primary cultured astrocytes by inducing stimulation of astrocyte activation, observe the changes of cytokines in astrocytes, and to observe the change in touch with TRIF signaling pathway, further investigate the effect of TRIF signaling pathway to astrocyte inflammatory response.Methods1.The primary cultured astrocytes: 1-2 days using newborn suckling mice C57,prepared to take primary cortical astrocytes, passaged three times, a few cells grow when astrocytes passage on 24 well plates slide with a time of 24 h, the cells were completely adherent paraformaldehyde, were rabbit anti-GFAP polyclonal antibody immunocytochemistry cell culture, while taking advantage of CD11 b antibody microglia, with TLR3 and TRIF Antibodies were detected to observe. Astrocyte count purity higher than 95% when used in subsequent experiments.2.The rats were divided into four groups: astrocytes conventional training, without any treatment;(2) LPS treatment groups::(1) a control group treated with lipopolysaccharide(LPS)(100ng / ml) cells 24 hours using quantitative PCR was observed iNOS, TNF-α IL-6, expression, IL-1β in;(3) IL-4 treatment groups:(IL-4)(20ng / ml) was treated with interleukin-4 cells 24 hours, fluorescence quantitative PCRwas observed ARG1, CD206, IL-10, IFN-β, TNF-α, IL-6 mRNA expression changes;(4)Poly(I: C) treatment groups: with Poly(I: C)(25μg / ml) cells were treated for 24 hours, fluorescence quantitative PCR was observed IL-6, iNOS, TNF-α, IL-1β, ARG1,CD206, IL-10, IFN-β, CD68 mRNA expression changes.3.The expression of inflammatory factors after knock down TRIF: the use of siRNA-transfected primary cultured astrocytes, get TRIF decreased expression of specific astrocytes, respectively, with LPS, IL-4 treatment knock down TRIF and did not knock down TRIF astrocytes. Before and after knocking down TRIF secreted inflammatory cytokine mRNA expression levels change.Results1.Immunohistochemical results showed that after 3 passages, primary marker GFAP positive astrocytes cultured purity count up to 95%, CD11 b detection basic no positive mark, can eliminate the influence of microglia. TRIF and TLR3 antibody test,some cells labeled positive.2.Q-PCR results showed that the use of LPS treatment,the inflammation-promoting factor IL-6, mRNA expression of iNOS, TNF-α and other rises. After stimulation with IL-4, inflammation inhibitors ARG1, mRNA expression of CD206, IL-10, etc. rises;pro-inflammatory cytokines TNF-α mRNA expression of IL-6 decreased. With Poly:after stimulation(I:C),proinflammatory cytokines IL-6, iNOS, TNF-α, mRNA expression of IL-1β, and the anti-inflammatory factor ARG1, CD206, IL-10, IFN-β,CD68 mRNA expression were increased.3.Q-PCR results showed that, siRNA transfection knock down TRIF primary cultured astrocytes after 24 hours in the stimulation of IL-4, visible Arg1 and mRNA CD206 expression in knockout after TRIF significantly reduced, TNF-α mRNA and IL-6 expression after knockout(siTRIF + IL-4 group) compared siNC + IL-4 group increased; after siRNA transfection of primary cultured astrocytes for 24 hours use LPS stimulation, IL-6, the expression of TNF-α, iNOS, IL-1β knockout after LPS treatment group(siTRIF + LPS group) compared siNC + LPS group were significantly decreased.Conclusion1.The use of birth 1-2 days C57 suckling mice after three passages, to cultivate purity of greater than 95% of primary astrocytes, and can eliminate the influence ofmicroglia and neurons, and TLR3 and TRIF in astrocytes are also highly expressed.2.Use of inducers stimulate primary astrocytes, the expression of various inflammatory factors changed significantly, confirming primary astrocytes cells can be highly induced activation.3.After knocking down the primary signal TRIF astrocytes, changes of the inflammatory factors may be increased or decreased significantly, confirmed TRIF signaling pathway plays an important role in the activation of astrocytes.