The study of Gualou Guizhi Decoction inhibits nerve cell damage induced by microglial activation via TLR3 signaling
OBJECTIVE To explore the neuroprotective molecular mechanism of Gualou Guizhi Decoction(GLGZD)through inhibiting the microglia-mediated inflammatory response via TLR3 signaling pathway.METHODS After GLGZD pre-treatment,BV2 cells were stimulated using LPS to establish inflammatory model,HT22 cells were exposed to microglia-conditioned media of BV2 cells.Cell viability was determined by CCK-8 assay.Cytokines in the cell culture supernatants were detected by ELISA.Expression of TLR3 and TRIF protein was measured by immunofluorescence.The activation of the transcrip-tion factor IRF3 was evaluated by DNA-binding ELISA assay.TLR3 siRNA cell transfection assay was used to verify the target of GLGZD.The levels of the apoptotic proteins Bcl-2 and Bax protein were determined by Western blot.The activation of Caspase-9 was measured by Caspase-9 activity assay.RESULTS GLGZD significantly attenuated the MCM-induced HT22 cell damage,and promoted IFN-β expression and downregulated IL-8 expression in the supernatant of LPS-stimulated BV2 cells.Meanwhile,GLGZD significantly increased the expression levels of the related proteins of TLR3 pathway and IRF3 activation.After knocking down the expression of TLR3,GLGZD could not promote the anti-inflammatory factor IL-10 secretion in BV2.Furthermore,GLGZD significantly increased the expression of Bcl-2 and decreased the expression of Bax,and reduced Caspase-9 activity in HT22 cells exposed to MCM.CONCLUSION GLGZD may ameliorate brain damage through anti-inflammation via activation of theTLR3 signaling pathway following microglial activation,inhibit the neuron apoptosis,subsequently reduce brain injury.
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