Effects Of MRP8/14 On The Expression Of HMGB1、AIF、GFAP And P-p38 MAPK In Streptococcal Meningitis Mice

Streptococcus pneumoniae(SP)is a gram-positive bacterium that can cause bacterial meningitis(BM).Streptococcus pneumoniae meningitis(SPM)can produce the severe sequelae and complications in central nervous system diseases.Although regular antimicrobial treatment,streptococcus pneumoniae meningitis still has a high incidence,disability rate and fatality rate.About 50%of survivors left neurological sequelae,most of them showed mental retardation,eonvulsion,hearing loss and so on.Therefore,the further understanding of the immune and pathological mechanism of BM is of great significance to the evaluation of its efficacy and outcome.High mobility group box-1 protein(HMGB1)is a highly conserved nuclear protein widely distributed in mammalian cells.Some studies have found that HMGB1 is highly expressed in cerebrospinal fluid of children with bacterial meningitis.Apoptosis inducing factor(AIF)is a kind of conserved flavin protein,which exists in the inner and outer membrane of mitochondria.Under the stimulation of apoptosis signal,AIF can induce nuclear chromatin condensation and caspase-independent DNA fragmentation.lt plays an important role in infectious brain injury.Glial fibrillary acidic protein(GFAP)is a specific cytoskeleton protein of astrocytes,which is elevated in brain trauma and neurodegenerative lesions.lt is a characteristic marker of mature and reactive astrocytes.lt was found that the expression of GFAP was significantly increased in purulent meningitis,which was of great value in the diagnosis of early suppurative meningitis.MRPS/14 is one of the important damage-associated molecular patters.MRP8 protein and MRP 14 protein often form heterodimer MRP8/14 protein in calcium dependent manner to play an important role.In recent years,the research of MRP8/14 in infectious diseases has attracted wide attention.For example,in multiple sclerosis,arthritis,inflammatory vasculitis and inflammatory bowel disease,serum MRP8/14 levels are significantly increased.But the role of MRP8/14 in central nervous system infectious diseases is poorly understood.In SPM,it has been found that neutrophil derived MRP 14 induces inflammation after bacterial clearance.However,the roles of heterodimer MRP8/14 protein in the pathophysiological process of streptococcus pneumoniae meningitis and its mechanism are of clinical significance.Therefore,on the basis of previous work,we further observed and analyzed the effect of MRP8/14 on brain injury markers(HMGB1,AIF,GFAP)of bacterial meningitis,and explored the effect of MRP8/14 on brain injury in mice with bacterial meningitis.Studying its role in the progression of bacterial meningitis in order to provide a new idea and experimental theory basis for the immune intervention of bacterial meningitis.Methods:Part 1.To study the effects of MRP8/14 on neurobehavioral changes,body mass changes.1.Groups:48 strong male BALB/C mice aged about 2 months were randomly divided into 4 groups:PBS control groups,MRP8/14 protein groups,SP groups,SP+MRP8/14 groups;There were 12 mice in per group;After induced anaesthesia by intraperitoneal injections mouse model of SPM was established by intraventricular injection of 40ul PBS combined with 5ul SP suspension.hnjection of 45ul PBS into PBS groups;40ul MRP8/14 protein combined with 5ul PBS were injected into mice of MRP8/14 groups;40ul PBS combined with 5ul SP suspension were injected into mice of SP groups;40ul MRP8/14 combined with 5ul SP suspension were injected into mice of SP+MRP8/14 groups.2.At 6h,24h,48h after intracerebroventrieular injection,each group of mice was given:1)Neurobehavioral score,2)Change of body mass recorded.Part 2.To study the effects of MRP8/14 on the pathological changes of brain tissue in SPM mice.Animal modeling and grouping are the same as the first part of the experiment.At 6h,24h,48h after intracerebroventricular injection,each group of mice was given:1)Brain water content changes,2)Evaluation the damage of neurons in brain tissue by Nissl staining.Part 3.To study the effects of MRP8/14 on the relative expression of HMGB1、AIF、GFAP and p-p38 MAPK in the brain of SPM mice.Animal modeling and grouping are the same as the first part of the experiment.At 6h,24h,48h after intracerebroventricular injection,the relative expressions of HMGB1、IF、GFAP were detected by real-time PCR method in mice;The expression of p-p38 MAPK protein in the brain tissue of mice was detected by immunohistochemical method.Results:Part1.The effects of MRP8/14 on clinical manifestation of SPM mice1.Neurobehavioral score:The clinical manifestations of mice at 6h,24h,48h were evaluated by Loeffie’s five-point neurobehavioral scoring system.We found that there were no significant difference in clinical score(P>0.05)between MRP8/14 groups and PBS groups at each time point;There were no significant difference in clinical scores between SP groups and PBS groups at 6h(P>0.05);At 24h,48h,SP groups’s clinical scores were lower than that of PBS groups(P=0.000);There were no significant difference in clinical scores between SP＋MRP8/14 groups and SP groups at 6h(P>0.05);At 24h,48h,the clinical scores of SP+MRP8/14 groups were significantly lower than that of SP groups(P=0.000).2.The Body mass changes(g):The body mass of mice before and after injection was detected at each time point in each group.lt was found that there were no significant differences in body mass between MRP8/14 groups and PBS groups at each time point(P>0.05);There were no significant differences in body mass between SP groups and PBS groups、between SP+MRP8/14 groups and SP groups at 6h(P>0.05);At 24h,48h,the body mass of SP groups were lower than that of PBS groups(P<0.05);At 24h,48h,the body mass of SP+MRP8/14 groups were significantly lower than that of SP groups(P<0.05).Part 2.The effects of MRP8/14 on the pathological changes of brain tissue in SPM mice1.Brain water content changes(%):The weight of brain tissue of mice before and after drying at each time point in each group was recorded.lt was found that there were no significant differences in brain water content between MRP8/14 groups and PBS groups at each time point(P>0.05);There were no significant differences in brain water content between SP groups and PBS groups.between SP+MRP8/14 groups and SP groups at 6h(P>0.05);At 24h,48h,the brain water content of SP groups were significantly higher than that of PBS groups(P＝0.000);At 24h,48h,the brain water content of SP+MRP8/14 groups were significantly higher than that of SP groups(P=0.000).2.Brain Nissl staining:At 6h,24h,48h after intracerebroventricular injection,it was found that there was no significant difference in the number of neurons between MRP8/14 groups and PBS groups at each time point(P>0.05);The number of neurons in SP groups was lower than that in PBS groups at different time points(P<0.05);The number of neurons in SP＋MRP8/14 groups was lower than that in SP groups at different time points(P<0.05).Part 3.The effects of MRP8/14 on the expression of HMGB1、AIF、GFAP and p-p38 MAPK in brain tissue of SPM mice1.Detection of relative expression of HMGB1 mRNA in brain tissue by real-time PCR:At 6h,24h,48h after intracerebroventricular injection,there were no significant differences in the relative expression of HMGB1 mRNA between MRP8/14 groups and PBS groups at each time point(P>0.05);The relative expression of HMGB1 mRNA in SP groups was higher than that in PBS groups at different time points(P=0.000);The relative expression of HMGB1 mRNA in SP+MRP8/14 groups was higher than that in SP groups at different time Points(P=0.000).2.Detection of relative expression of AIF mRNA in brain tissue by real-time PCR:At 6h,24h,48h after intracerebroventricular injection,there were no significant differences in the relative expression of AIF mRNA between MRP8/14 groups and PBS groups at each time point(,P>0.05);The relative expression of AIF mRNA in SP groups was higher than that in PBS groups at different time points(P＝0.000);The relative expression of AIF mRNA in SP+MRP8/14 groups was higher than that in SP groups at different time points(P＝0.000).3.Detection of relative expression of GFAP mRNA in brain tissue by real-time PCR:At 6h,24h,48h after intracerebroventricular injection,there were no significant differences in the relative expression of GFAP mRNA between MRP8/14 groups and PBS groups at each time point(P>0.05);The relative expression of GFAP mRNA in SP groups was higher than that in PBS groups at different time points(P<0.01);The relative expression of GFAP mRNA in SP+MRP8/14 groups was higher than that in SP groups at different time points(P<0.01).4.Detection of p-p38 MAPK protein expression by immunohistochenical method:p-p38 MAPK positive cells are visible in cerebral cortex of four groups.At 6h,24h,48h after intracerebroventricular injection,it was found that there were no significant differences in the expression of p-p38 MAPK protein between MRP8/14 groups and PBS groups at each time point(P>0.05);The expression of p-p38 MAPK protein in SP groups was higher than that in PBS groups at different time points(P=0.000);The expression of p-p38 MAPK protein in SP+MRP8/14 groups was higher than that in SP groups at different time points(P＝0.000).Conclusions:1.In this experiment,SP suspension was injected into lateral ventricle,and the SPM model of mice was successfully constructed.2.MRP8/14 aggravated the clinical symptoms of SPM,reduced neurobehavioral score,lost body weight,increased brain water content and reduced number of Nissl-positive neurons.At the same time,the relative expressions of brain injury markers(HMGB1,AIF,GFAP)mRNA were increased in SPM model,indicating that MRP8/14 aggravated experimental SPM brain injury and promoted the mRNA expressions of markers of brain injury.3.MRP8/14 significantly up-regulated p-p38 MAPK protein in the brain of SPM mice,suggesting that MRP8/14 can aggravate the progression of SPM in mice,and the mechanism of brain injury may be related to the activation of p38 MAPK signaling pathway.