JAK/STAT Signaling Pathway Mediates β-amyloid Protein-induced TNF-α Action Of Microglia In Vitro

The deposition of the P-amyloid protein (AP) induces the chronic inflammatory reaction caused by the activation of glial cells in the brain, which may be the most important pathological mechanism of Alzheimer disease pathogenesis. The activation of microglia plays a vital roal in the inflammation of AD. A(3 is a form of peptide chain cut by unusual mechanism in essence belonging to advanced glycation(AGEs). The oligomers induce the inflammation of microglia by receptor of advanced glycation(RAGE) which high expressed on the microglial cell's surface. And tumor necrosis factor-a(TNF-a) is the major inflammatory factor released by microglia.Objective:To explore the inflammatory response of microglia induced byβ-amyloid (AP) oligomers through the receptor for advanced glycation end product (RAGE).To further analyze the relationship between Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway and tumor necrosis factor-α(TNF-α) released by microglia induced by Aβoligomers.Methods:1. Primary microglial cells cultured in vitro, purified and identified.2. Purified microglia was inoculated into 96-well plates by the same density, divided into six groups.①Blank group;②DMSO group;③:Aβoligomers stimulated microglia for 24h;④:After microglia was treated by 1:50 anti-RAGE for 2 h, then stimulated with 1μmol/L Aβoligomers for 24 h;⑤:After microglia was treated by 10μmol/L AG490 for 2h, then stimulated by 1 u mol/L Aβoligomers for 24 h;⑥:After microglia was treated by 1:50 anti-RAGE for 1h, then stimulated by 10μmol/L AG490 for 1h,and then stimulated by 1μmol/L Aβoligomers for 24 h.We observed the microglia's form which treated by Aβoligomers under the inverted phase contrast microscope.3. We can observe the form of the microglia treated by Aβoligomers under inverted phase contrast microscope.4. We detected levels of TNF-αin cell's supernatant liquid by enzyme-linked immunosorbent assay(ELISA).Results:1. Aβoligomers maked microglia inflammated. Microglia was amoeba-like under inverted phase contrast microscope. The expression of TNF-αwas obviously increased after the treatment of oligomer,compared with blank group. Dimethyl sulfoxide (DMSO) used for the dissolution have no effection on the inflammation.2. Aβoligomer induced the activation of JAK/STAT signal transduction pathway in microglia. AG490,the drug inhibited JAK/STAT pathway,and anti-RAGE IgG despectively inhibited the produce of TNF-αobviously. AG490 and anti-RAGE IgG also can inhibite the activation of microglia together.Conclusions: Aβoligomer can induce the activation and inflammatory reaction of microglia,and mediate the releasion of TNF-αby JAK/STAT signaling pathway through RAGE. But dimethyl sulfoxide with lower concentration cann't cause inflammatory reaction.Significance: This study further confirmed the inflammatory pathogenesis of alzheimer's disease and the importance of microglia in the inflammatory reaction. The research stated that JAK/STAT was the significant signal transduction pathway in the inflammatory reaction in microglia which induced by AP oligomer.Innovation: The study which used microglia as carrier and made RAGE and Aβoligomer as starting point approached the initiating agent of activation of microglia in alzheimer's disease,the mechanism of inflammatory reaction and intervention study.To look for definited inflammatory damage mechanism and new interventional target this study provided scientific evidence.