The Mechanisms Of Post-ischemic Neuroinflammation Regualted By Gualou-Guizhi Decoction Based On TLR3/4 Signaling Pathway

ObjectiveTo investigate the inhibitory effect of Gualou-Guizhi decoction(GLGZD) on the neuroinflammation following ischemic stroke through Toll-like receptor 3/4 signaling pathway and its underlying mechanisms in vivo and vitro.Methods1. Sprague-Dawley rat(n=90) were randomly devived into three groups:Sham-operated group, MCAO model group, GLGZD treatment group. The ischemic models were established by 2h left middle cerebral artery occlusion (MCAO) followed by reperfusion. GLGZD was administered daily for 7 days to the rat at the concentration of of 1.16g/ml. The sham group only isolated artery, without ligation and insertion. Cerebral infarct volume was detected by TTC staining. Neurological deficits were evaluated and the modified Ashworth scale was also applied per each group. The changes of individual paw parameters were assessed by using CatWalk gait analysis. All animals were sacrificed after treatment, cortical tissue and blood were collected. Inflammatory mediators in plasma obtained from collected blood were determined using ELISA. RT-PCR and Western-blotting/Immunohistochemistry assaywere performed to analyze gene and protein expression associated with neuroinflammation through TLR3/4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity involved in inflammatory pathway.2. In vitro model was utilized in which LPS-induced BV2(a microglia cell line) models were treated with GLGZD. The cell viability was evaluated by MTT assay to identify the concentrations of GLGZD and/or LPS, various concentrations had no cytotoxic action on the BV-2 cells. The levels of nitrite and cytokines from culture supernatant related to inflammation were examined by Griess reaction and ELISA in LPS-activated microglia. The mRNA and protein were extracted after GLGZD treatment. The mRNA expression of inflammation related gene were detected by RT-PCR/qRT-PCR, the protein expression levels of TLR3/4 pathway were assessed by Western Blot. The involving transcription factors, including NFκB and IRF3, were determined by immunofluorescence and DNA binding ELISA. To investigate the effect of GLGZD on PC 12 neurons induced by conditioned medium obtained from LPS-activated microglial cells, PC 12 cell viability was evaluated by MTT assay. In addition, PC 12 morphological changes was detected with immunofluorescence.Result1. MCAO followed by reperfusion could result in severe nerve effects and cerebral infarction. Treatment of the rats with GLGZD could reduce cerebral infarct volume and improve the nerve defects. Additionally, the activation and infiltration of inflammatory cells was signicantly reduced by GLGZD treatment.2. GLGZD significantly inhibits the MCAO-induced pro-inflammatory mediators and enhances anti-inflammatory mediators at mRNA and protein levels. In addition, GLGZD could increase the related protein expression involving NFκB signaling pathway and the activation of NFκB, up regulated the levels expression of protein in IRF3 signaling pathway and IRF-3 activity.in ischemic cerebral tissue after MCAO.3. The excessive production of inflammatory mediators in LPS-stimulated microglia treated with GLGZD were decreased and the anti-inflammatory factors increased significantly. Meanwhile, GLGZD significantly suppressed the gene/protein levels and NFκB activity of pro-inflammation and enhanced the expression of gene/protein and IRF3 activity associated with anti-inflammatory signaling.4. Cell viability of PC 12 cells stimulated with microgial conditioned mediums was increased by GLGZD treatment. The damage changes of PC 12 cell morphology was also ameliorated by GLGZD.ConclusionThese findings suggest that GLGZD, a promising agent for the treatment of speciticity following stroke, may be exert its anti-inflammatory properties through TLR3/4 signaling, contributing to neuroprotection.