The Protective Effect And Mechanism Of Brain-derived Neurotrophic Factor In Pneumococcal Meningitis

Objective:Streptococcus pneumoniae meningitis is one of the most common infectious diseases of the central nervous system(CNS)in children.Despite the continuous use of prophylactic vaccines and effective antibiotics,it still has high mortality and disability rate.Therefore,it is of great significance to further study the pathogenesis of pneumococcal meningitis and find new adjuvant treatment.Our previous studies found that brain-derived neurotrophic factor(BDNF)can not only promote nerve repair but also have anti-inflammatory function in bacterial meningitis.Based on these results,the effects of endogenous Bdnf deficiency on inflammatory response and brain injury of pneumococcal meningitis were investigated by using cortical and hippocampal conditional BDNF knockout mice,and the effect and mechanism of exogenous BDNF on macrophage/microglia polarization in inflammatory response were investigated by cellular and animal experiments.Methods:Part ? We used Cre-lox P system to construct cortical and hippocampal conditional Bdnf gene knockout mice(Cre+/FF)and their little-mate controls(Cre-/FF).The genotypes of mice were identified by agarose gel electrophoresis,and the effect of gene knockout was determined by Real-time PCR and immunofluorescence.The mouse model of pneumococcal meningitis was established,and the survival rate,disease score and body weight of the mice were recorded.The pathological condition of brain tissues was observed by HE staining,and the necrosis and apoptosis of neurons were studied by Nissl and TUNEL staining.The proliferation and activation of macrophage/microglia were detected by immunofluorescence staining.The expression of inflammatory cytokines in brain homogenates was detected by Real-time PCR.The expression of inflammation-related signaling molecules TLR2,My D88,NOD2,phosphorylated p38 MAPK,and phosphorylated p65 NF-?B were detected by Realtime PCR and Western blot.Part ? Rat microglial cell line HAPI and primary microglia were pretreated with BDNF for 24 h,and then stimulated by LPS for 6 h.The expression of proinflammatory mediators and M2 markers was detected by Real-time PCR,and the secretion of NO in cell supernatant was detected by Griess assay.Microglia were treated with BDNF alone,and the expression of M2 marker arginase1 was detected by Western blot.Microglia were pretreated with BDNF for 24 h,and then stimulated by LPS for 6h.The expression of STAT3 phosphorylated protein was detected by Western blot,and then STAT3 pathway was blocked by inhibitor,and the expression of M1 marker i NOS was detected by Western blot.Microglia were stimulated with BDNF to observe the effect on its receptor Trk B phosphorylation,and then Trk B receptor and its downstream signal pathways were blocked with inhibitors,and the expression of STAT3 phosphorylated protein and i NOS were detected by Western blot.Part ? SD rats were pretreated with exogenous BDNF prior to intracisternal infection with live Streptococcus pneumoniae and the brains were harvested at 24 h after inoculation.The expression of inflammatory mediators in brain tissues was detected by Milliplex? MAP assay,the expression of M2 macrophage/microglia markers was detected by Real-time PCR,and the expression of i NOS and arginase1 on macrophages/microglia was detected by immunofluorescence staining.Results:Part ? 24 h after Streptococcus pneumoniae infection,the weight loss,clinical manifestation,inflammatory cell infiltration,neuronal injury and hippocampal apoptosis in Cre+/FF meningitis group were more significant than those in Cre-/FF meningitis group.The expression of pro-inflammatory cytokines in Cre+/FF meningitis group was higher than that in Cre-/FF meningitis group.Meanwhile,the expression of TLR2,My D88,NOD2,phosphorylated p38 MAPK and phosphorylated p65 NF-?B in cortex and hippocampus of Cre+/FF meningitis group was significantly higher than those of Cre-/FF meningitis group.Part ? BDNF pretreatment could significantly down-regulate LPS-induced the expression of pro-inflammatory mediators and up-regulate the expression of M2 microglia markers.BDNF pretreatment further up-regulated the phosphorylated STAT3 protein expression induced by LPS,and the expression of i NOS was significantly upregulated after inhibition of STAT3 activation.Microglia were treated with different concentrations of BDNF,and the phosphorylated Trk B protein increased in a dosedependent manner.Cells were pretreated with Trk B,MEK,PI3 K or PLC inhibitors then stimulated by BDNF and LPS,the phosphorylated expression of STAT3 was downregulated and the expression of i NOS was up-regulated in Trk B receptor inhibitor group and PI3 K inhibitor group compared with BDNF control group.In addition,after pretreatment with Trk B receptor inhibitor,the expression of BDNF-induced phosphorylated Akt was significantly down-regulated.Part ? Intracerebroventricular administration of BDNF could significantly downregulate the expression of pro-inflammatory factors in rats with Streptococcus pneumoniae meningitis.The result of immunofluorescence showed that compared with PBS meningitis group,the expression of M1 marker i NOS of macrophage/microglia in BDNF meningitis group was down-regulated.On the other hand,BDNF pretreatment significantly up-regulated the expression of M2 markers in meningitis rats.Compared with PBS meningitis group,the expression of M2 macrophage/microglial marker arginase1 in BDNF meningitis group was up-regulated.Conclusion:(1)Cortical and hippocampal conditional Bdnf gene knockout aggravated the severity of brain injury in Streptococcus pneumoniae meningitis,promoted brain inflammation,increased neuronal necrosis and apoptosis associated with meningitis.These may be associated with TLR2/NOD2-related signal pathways in brain tissues.(2)BDNF significantly reduced LPS-induced the expression of pro-inflammatory mediators and increased the expression of anti-inflammatory mediators by promoting the transformation of microglia phenotype from M1 to M2.This effect was mainly mediated by the Trk B receptor on the microglia membrane through Akt/STAT3 signal pathway.(3)In Streptococcus pneumoniae meningitis,BDNF could effectively inhibit the inflammation of macrophage/microglia,induced the transformation of macrophage/microglia to M2 phenotype,reduced the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors.