The Role Of TLR4-mediated PTEN/PI3K/AKT/NF-κB Signaling Pathway In Neuroinflammation In Rat Hippocampal Neurons

Objective primary culturing rat hippocampal neurons,and using LPS(lipopolysaccharide), which is the natural ligand of toll-like receptor 4(TLR4), and other different drugs to activate or block TLR4 signaling pathway, to investigate a possible role for a TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in neuroinflammation in rat hippocampal neurons. And to reveal that the activation of this signaling pathway involved in neuroinflammation and to provide more experimental basis in the role of TLR4- mediated neuroinflammation.Methods Rat hippocampal neurons were cultured in vitro and the purity was identified by immunofluorescence assay. Cultured neurons were treated with lipopolysaccharide(LPS), a TLR4 ligand, or pre-treated with TLR4 antibody to block TLR4 signaling. Neurons were also treated with dipotassium bisperoxo(pyridine-2-carboxyl) oxovanadate [bp V(pic)] and pyrrolidine dithiocarbamate(PDTC), selective inhibitors of PTEN and NF-κB, respectively, in the presence of LPS. The levels of PTEN, AKT and phspho protern PTEN, AKT were tested by Western blot. The nuclear translocation of nuclear factor-κB(NF-κB) in hippocampal neurons was tested by double immunofluorescence assay. The expression of m RNA of TNF-α and IL-1β were tested by real-time quantitative PCR(RT-q PCR). The content of tumor necrosis factors alpha(TNF-α) and interleukin-1β(IL-1β) were tested by Enzyme-linked immunosorbent assay(ELISA).Results After being primary cultured for seven days,the purity of rat hippocampal neurons might be above 95%. The protein expression of AKT did not change after stimulation with LPS in different doses for different time. The protein expression of PTEN did not change after stimulation with LPS in different doses for 8 h. However, it decreased after LPS-stimulated for 24 h and increased for 48 h in dose-dependent, with the greatest decreased or increase observed at a concentration of 1μg/ml(P<0.01, P<0.05). The m RNA expression of AKT unchanged with a concentration of 1μg/ml LPS stimulation of hippocampal neurons at different times, however, the m RNA expression of PTEN began to increase at 12 h after LPS stimulation(P<0.01, P<0.05), The levels of PTEN, AKT, and their phosphorylated forms were evaluated, along with the nuclear localization of NF-κB, to assess pathway activation. The induction of a neuroinflammatory response was determined by measuring TNF-α and IL-1β m RNA and protein levels. The results indicated that while LPS stimulation had no effect on PTEN and AKT expression, the levels of phosphorylated PTEN(p PTEN) decreased, while phosphorylated AKT(p AKT) levels increased(P<0.01, P<0.05). Moreover, LPS treatment induced the nuclear translocation of NF-κB, and increased the expression and secretion of TNF-α and IL-1β. Blocking TLR4 increased the levels of p PTEN and decreased the levels of p AKT, while pre-treatment with bp V(pic) led to a reduction in levels of p PTEN and p AKT(P<0.01, P<0.05). Furthermore, treatment with TLR4 antibody, bp V(pic), and PDTC decreased LPS-induced nuclear translocation of NF-κB, and resulted in a downregulation of TNF-α and IL-1β expression(P<0.01, P<0.05).Conclusion1. The gene and protein expression of AKT and PTEN did not change significantly in the early of inflammatory response induced by LPS stimulation. In the interim and late of inflammatory response, the gene and protein expression of AKT did not change significantly; the protein of PTEN decreased in the interim of inflammatory response,casing reflectively to the increase of gene expression and with the increase of inflammation, the protein and gene expression were both increased.2. At earlier of LPS stimulation, AKT and PTEN play the role through their phosphorylated or dephosphorylated form in rat hippocampal neurons. And TLR4 antibody and PTEN inhibitor can reduced by the effects of LPS stimulation on AKT and PTEN.3. LPS treatment induced the nuclear translocation of NF-κB, and increased the expression and secretion of TNF-α and IL-1β. Treatment with TLR4 antibody, bp V(pic), and PDTC decreased LPS-induced nuclear translocation of NF-κB, and resulted in a downregulation of TNF-α and IL-1β expression.4. Taken together, these results provide evidence for a TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in rat hippocampal neurons, which is associated with the activation of a neuroinflammatory response.