The Anti-Inflammatory Effect Of Gx-50 Via α7 NAChR And TLR4 In Microglia

BackgroundAlzheimer’s disease(AD) is one of the most common dementias and characterized by cognitive deficits and neuron loss. The pathological charecters are the extracellular aggregation of β amyloid and intracellular hyperphosphorylation of tau. Although the neuropathologic hallmarks of AD are well known, these features do not necessarily reflect the fundamental cause of the disease in which many factors and pathways are involved, and complex mechanisms continue to be elucidated. Among the mechanisms, chronic inflammation, which is primarily initiated by over-activated microglia in the brain, accelerates the occurrence and development of AD and has been studied extensively in recent years.Research objectIn our previous studies, a natural compound N- [2-(3,4- dimethoxyphenyl) ethyl]-3-phenyl-acrylamide(gx-50) has been investigated as a novel drug against AD with multiple effects, such as neuroprotection, cognitive improvement and amyloid disaggregation; however, the effects of gx-50 on neuroinflammation has not investigated yet. Here, we designed a series of experiments to investigate whether gx-50 possesses anti-inflammatory effects and the related mechanisms.MethodsWe investigated the effects and mechanisms of gx-50 on inflammation by detecting the cytokines, signaling pathways in Aβ-induced microglia and brains of APP-Tg mice, mainly including α7 nAChR-mediated anti-inflammatory pathways and TLR4-mediated inflammatory pathways.Firstly, the anti-inflammatory effect of gx-50 was investigated by detecting several key pro-inflammatory mediators. In primary microglia, the secretion of TNFα,IL-1β,PGE2 and NO were measured by ELISA and Griess reaction, respectively. And the expression of COX2 and i NOS was measured by Western blot. Together with in vivo expriments, real-time PCR and Western blot were conducted to detect the m RNA and protein of IL-1β, COX2 and i NOS in APP-Tg mice, respectively. In addition, the activation of microglia in brains of APP-Tg mice was detected by immunofluorescence.Then, the effects of gx-50 on the activation of α7 nAChR and α7 nAChR-mediated anti-inflammatory signal pathways were investigated. Gx-50 or Aβ was pre-treated in the BV2 microglial cells in the presence of α-Bungarotoxin-TRITC to detect whether gx-50 could bind to α7nAChR. Real-time PCR and Western blot was conducted to detect the m RNA and protein levels of α7 nAChR. And then, the expression of phosphorylation of JAK2 and STAT3 was measured by Western blot and the location of p-STAT3 in cytoplasm or nucleus was measured by immunofluorescence. Similarly, the expression of phosphorylation of PI3 K and AKT was measured by Western blot. Besides,α-Btx, AG490 or LY294002, the inhibitor of α7 nAChR, JAK2 and PI3 K, was added into the cells respectively to investigate the mechanism of the anti-inflammatory effect of gx-50 by measuring the secretion of IL-1β.Besides, the effects of gx-50 on the activation of TLR4 and TLR4-mediated inflammatory signal pathways were investigated. The levels of TLR4 m RNA and protein were examined by real-time PCR and Western blot. Then, the expression of My D88 and TRAF6 was examined by Western blot. Moreover, the effect of gx-50 on the activation of NF-κB and MAPK signaling pathways in vitro and in vivo was investigated by Western blot and immunofluoresence. The expression of p-IκB and NF-κB p65 was detected by Western blot and the location of NF-κB p65 in cytoplasm or nucleus was measured by immunofluorescence. The expression of p-ERK, p-p38, p-JNK was detected by Western blot as well. Moreover, TLR4 si RNA and TLR4 over-expression plasmid were transfected into BV2 cells to block and over-express TLR4, respectively, followed by the measure of several key factors in TLR4-mediated inflammatory pathway to investigate the relationship between gx-50 and TLR4.ResultsThe anti-inflammatory effect of gx-50 is partly due to the activation of JAK2/STAT3 and PI3K/AKT signal pathways via α7 nAChR as well as the inhibition of TLR4-mediated signal pathway in vitro and in vivo.Gx-50 possessed the anti-inflammatory effects in vitro and in vivo. ELISA and Griess reaction showed that the secretion of TNFα,IL-1β,PGE2 and NO was significantly increased by Aβ, while gx-50 suppressed those increase. Besides, gx-50 inhibited the up-regulation of COX2 and i NOS induced by Aβ. In addition to in vitro results, gx-50 showed anti-inflammatory effect in APP-Tg mice. Microglial activation and the m RNA levels of IL-1β, i NOS, and COX2 were significantly suppressed by gx-50 in APP+-Tg mice.Gx-50 possessed the anti-inflammatory effect by the activation of JAK2/STAT3 and PI3K/AKT signal pathway mediated by α7nAChR. Pre-treatment with gx-50 decreased the red fluorescence degree in present of α-Btx-TRITC,implying that gx-50 could competitive bind to α7 nAChR. Besides, Western blot analyses showed that gx-50 could up-regulate the expression of α7 nAChR in the absent or present of Aβ. These results indicated that gx-50 could bind to α7 nAChR and regulate its expression in microglia. Besides, Western blot analyses showed that the expression of p-JAK2 was significantly increased by gx-50 in the absent or present of Aβ, while reduced by AG490 and α-Btx, the inhibitors of JAK2 and α7nAChR. Immunofluorescence assay showed that gx-50 increased the p-STAT3 in nucleus. Western blot showed the similar result, the expression of p-STAT3 was significantly increased by gx-50 in the absent or present of Aβ, while reduced by AG490 and α-Btx. These results indicated that gx-50 could active JAK2/STAT3 signal pathway via α7nAChR. Similarly, Western blot analyses showed that the expressions of p-PI3 K and p-AKT were significantly increased by gx-50 in the absent or present of Aβ; however, α-Btx and LY294002, the inhibitors of PI3 K significantly suppressed the effect of gx-50, indicating that gx-50 could regulate this signal pathway via α7nAChR. Notably, pre-treatment of α-Btx, AG490 or LY294002 significantly blocked the inhibition effect of gx-50.Gx-50 inhibited TLR4-mediated signal pathway in vitro and in vivo. The expressions of the TLR4- and TLR4-downstream proteins My D88 and TRAF6 were reduced by gx-50 in Aβ-induced primary microglia and in APP+-Tg mice. Besides, Western blot analyses showed that gx-50 significantly inhibited the expression of p-IκB and the translocation of NF-κB p65 from cytoplasm to nucleus induced by Aβ. Immunofluorescence analyses also proved that pre-treatment of gx-50 suppressed the translocation of NF-κB p65 in nucleus induced by Aβ. Similar with NF-κB, Western blot analyses showed that gx-50 significantly inhibited the activations of ERK1/2, p38 in vitro and in vivo. These results showed that gx-50 inhibited the activation of NF-κB and MAPK cascades in vitro and in vivo. Interestingly, silencing of TLR4 greatly reduced Aβ-induced up-expression of IL-1β and TRAF6 by 50% to levels similar to gx-50 inhibition; moreover, overexpression of TLR4 increased the expression of My D88 and TRAF6, which was also significantly reduced by gx-50 by 38% and 15% similar to the exhibition in the presence of Aβ42. These results demonstrated thatIn conclusion, these findings provide strong evidence that gx-50 possesses anti-inflammatory ability in several aspects. First, gx-50 could bind to α7nAChR, leading to the activation of JAK2/STAT3 and PI3K/AKT cascades, which negative regulate several inflammatory cascades. Second, gx-50 displayed directly inhibition on TLR4-mediated inflammatory pathway. Specifically, gx-50 could inhibit Aβ-induced TLR4 activation, leading to the decrease of My D88 and TRAF6, which lead to the inhibition of NF-κB and MAPK and the inhibition of pro-inflammatory mediators.