| S.Pneumoniae（SP）is one of the main and most common pathogenic bacteria in human bacterial meningitis.Bacterial meningitis is one of serious children infectious diseases of the central nervous system,SP meningitis cases account for more than half of the cases of bacterial meningitis.In recent years,while giving effective antibiotics,bacterial meningitis are still have higher mortality,nearly half of the survivors remaining the cognitive dysfunction,visual damage,hearing damage,epilepsy and other neurological sequelae.Therefore,it is significant to study the immunopathological mechanism of bacterial meningitis and find new means of immune intervention.Bacterial meningitis causes damage to the blood-brain barrier and brain tissue,and the severity of brain tissue damage determines the prognosis of patients with bacterial meningitis.Studies have shown that the inflammatory factors in bacterial meningitis cerebrospinal fluid（CSF）,such as TNF alpha,CRP,IL-6,MCP-1,led to the release of subarachnoid inflammation,and are closely related to the development process of the disease and mortality,suggesting the brain inflammatory mediators in the process of bacterial meningitis immune injury play an important role.Myeloid-related Proteins 8（MRP8）and 14,also known as S100A8 and S100A9,areexpressedbyPolymorphonuclearNeutrophils（PMNs）and monocyte/macrophage,and is closely related to the innate immune function.MRP8 and MRP14 mainly related to the biological characteristics and physiological characteristics of different form dimers,MRP8/14（Calprotectin）,mainly in granulocyte,monocyte,early stage of differentiation and macrophages and microvascular endothelial cells and fibroblasts.In recent years,MRP 8/14 has attracted extensive attention in the field of infectious and inflammatory diseases.Studies show that in arthritis,multiple sclerosis and inflammatory vasculitis and systemic lupus erythematosus（SLE）,abnormal serum MRP8/14 concentration increases,and increased MRP8/14 concentration with the active state of these diseases and other inflammatory parameters,such as CRP and red blood cells sedimentation rate（ESR）.This research through the animal experiments to observe that MRP8/14 increases the inflammation factors:TNF alpha,CRP,IL-6,MCP-1,explore the effects of MRP8/14 in bacterial meningitis inflammation damage,in order to find a new immune intervention targets for bacterial meningitis and provide new train of thought and experimental theory basis.Methods:Part 1.In vivo tests,to detect the effects of MRP8/14 on the change of behavior score、the body weight.48 8 weeks old healthy male BALB/C mice were randomly divided into 4 groups:phosphate buffer saline（PBS group）,MRP8/14 group,Streptococcus pneumonia groups（SP group）,Streptococcus pneumoniae+MRP8/14 protein group（SP+MRP8/14 group）;each group of 12 mice.Anesthesia were induced by intraperitoneal injection of chloral hydrate in mice,and by lateral ventricular injection of40 ul of PBS+5 ul of SP suspension,bacterial meningitis mouse model were established;PBS group mice were injected with 45 ul PBS,and group MRP8/14 with 40 ul MRP8/14(0.5ug?ul-1)+5 ul PBS,SP group mice were injected with 40 ul of PBS+5 ul of Streptococcus pneumoniae suspension,and SP+MRP8/14 group mice with 40 ul MRP8/14(0.5ug?ul-1)+5 ul Streptococcus pneumoniae suspension.Part 2.In lateral ventricle injection of 6h,24 h,48h mice in each group:1)neurological score,2)change,recording of body mass（before and after the injection of mice,we record the mice body weight at different time points and then analyze the difference in weight）,3)The content of brain water was assayed by weighing method.4)HE staining method to observe the brain tissue inflammation,5)Real-time PCR method for detection of relative expression of mRNA of TNF alpha,CRP,IL-6,MCP-1 in brain tissue,6)ELISA method to detect the expression of TNF-alpha,CRP,IL-6,MCP-1 in brain tissue and serum,7)to detect the expression of NF-κB p65 protein in brain tissue with Immunohistochemical method.Results:Part11.Neuroethology evaluation:Both score of MRP8/14 groups compared with PBS groups had no significant difference at 6h、24h、48h after induction of the meningitis 6h（5.0±0）vs（5.0±0）、24h（4.75±0.5）vs（5.0±0）,（P>0.05）;SP groups were relative to the PBS groups decreased significantly at 24h、48h after induction of the meningitis,6h（5.0±0）vs（5.0±0）、the difference has statistically significant at 24h（3.75±0.5）vs（5.0±0）,（P<0.05）;The score of SP+MRP8/14 groups was lower than that of SP groups at 6h、24h、48h after induction of the meningitis 6h（4.94±0.25）vs（5.0±0）、24h（2.5±0.58）vs（3.75±0.5）、48h（1.25±0.96）vs（3.25±0.96）,the differences at 24 h and48h were statistically significant（P<0.05）.2.The changes of body weight:Both of MRP8/14 group compared with PBS group at6h、24h、48 after induction of the meningitis 6h（0.5±0.98）vs（0.52±0.13）g、24h（1.02±0.42）vs（1.26±0.50）g、48h（1.60±0.55）vs（1.29±0.52）g,had no significant difference（P>0.05）;Both of SP groups had an decrease compared to PBS group in body weight at 24h、48h;6h（0.62±0.85）vs（0.52±0.13）g、24h（1.99±0.17）vs（1.26±0.50）g、48h（2.19±0.18）vs（1.29±0.52）g,the difference was statistically significant（P<0.05）;Both of SP+MRP8/14 groups had an decrease compared to SP groups in body weight at 6h、24h、48h;6h（0.65±0.59）vs（0.62±0.85）g、24h（2.62±0.44）vs（1.99±0.17）g、48h（3.30±0.63）vs（2.19±0.18）g,the differences at 24 h and 48 h were statistically significant（P<0.05）.3.Cerebral edema measurement:Both of MRP8/14 group compared with PBS group at6h、24h、48 after induction of the meningitis 6h（77.28±0.46）vs（77.81±0.35）%、24h（76.81±0.89）vs（77.20±0.37）%、48h（76.36±0.69）vs（77.61±0.94）%,had no significant difference（P>0.05）;Both of SP groups had an decrease compared to PBS group in body weight at 24h、48h;6h（78.10±0.86）vs（77.28±0.46）%（P>0.05）、24h（79.16±0.81）vs（76.81±0.89）%、48h（82.28±0.49）vs（76.36±0.69）%,the difference was statistically significant（P<0.05）;Both of SP+MRP8/14 groups had an decrease compared to SP groups in body weight at 24h、48h;24h（81.62±0.83）vs（79.16±0.81）%、48h（87.91±0.92）vs（82.28±0.49）%,the differences at 24 h and 48 h were statistically significant（P<0.05）.4.Brain tissue HE staining:At 6h,SP group compared with PBS group,he subarachnoid space was slightly enlarged,the soft meninges were slightly hyperemic,and the subarachnoid and inflammatory cells were infiltrated.SP+MRP8/14 group compared with SP group of he subarachnoid expansion was obvious,and the vascular congestion of the soft meningioma was more obvious,and the subarachnoid space and inflammatory cells were infiltrated.At 24 h,SP group compared with PBS group,subarachnoid expansion,soft meningeal vascular congestion,meningeal and subarachnoid space and inflammatory cell infiltration;SP+MRP8/14 group compared with SP group of he structural integrity of the arachnoid structure was destroyed,and the vascular congestion of the soft meninges was more obvious,and a large number of inflammatory cells were infiltrated in the meningeal and subarachnoid cavity.48 h,SP relative to the PBS group,subarachnoid significant expansion,pia mater significant vascular congestion,meninges and subarachnoid with inflammatory cells infiltration,SP+MRP8/14 group relative to the SP,arachnoid structural integrity destroyed,subarachnoid open,soft meningeal vascular engorgement more prominent,meninges and subarachnoid inflammatory cell infiltration.5.Brain tissue homogenate Real-time PCR detection of TNF-alpha,CRP,IL-6,MCP-1 mRNA relative expression in bacteria:After injection of 6h,TNF-alpha mRNA relative expression in SP groups compared with PBS groups increased obviously（0.003386±0.0024）vs（0.000849±0.0005）,（P<0.05）;SP+MRP8/14 group compared with SP group increased（0.004229±0.0031）vs（0.003386±0.0024）,（P>0.05）;24h,SP groups compared with PBS groups increased obviously（0.00876±0.0044）vs（0.000847±0.00025）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.0281±0.00806）vs（0.00876±0.0044）,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（0.0135±0.00582）vs（0.000762±0.0002）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.0447±0.03）vs（0.0135±0.00582）,（P<0.05）.After 6h injection,SP group CRP mRNA relative expression quantity is increased than PBS group（0.000995±0.0005）vs（0.000841±0.0005）,（P>0.05）,SP+MRP8/14 group compared with SP group increased（0.001088±0.0008）vs（0.000995±0.0005）,（P>0.05）;24h SP groups compared with PBS groups increased obviously（0.001248±0.0007）vs（0.000577±0.0001）,（P<0.05）,SP+MRP8/14 group compared with SP group increased obviously（0.003416±0.014）vs（0.001248±0.0007）,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（0.002263±0.0008）vs（0.000538±0.0001）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.004476±0.0025）vs（0.002263±0.0008）,（P<0.05）.After injection of 6h,IL-6 mRNA relative expression in SP groups compared with PBS groups increased obviously（0.0063±0.006）vs（0.0003±0.002）,（P<0.05）;SP+MRP8/14 group compared with SP group decreased（0.0055±0.003）vs（0.0063±0.006）,（P>0.05）;24h,SP groups compared with PBS groups increased obviously（0.0124±0.012）vs（0.0006±0.0002）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.0348±0.018）vs（0.0124±0.012）,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（0.0239±0.010）vs（0.0005±0.0002）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.0434±0.020）vs（0.0239±0.010）,（P<0.05）.After 6h injection,SP group MCP-1 mRNA relative expression quantity is increased than PBS group（0.1613±0.1942）vs（0.00695±0.00672）,（P>0.05）,SP+MRP8/14 group compared with SP group increased（0.5132±0.2306）vs（0.1613±0.1942）,（P<0.05）;24h SP groups compared with PBS groups increased obviously（0.2927±0.1291）vs（0.00614±0.0584）,（P<0.05）,SP+MRP8/14 group compared with SP group increased obviously（2.818±1.262）vs（0.2927±0.1291）,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（0.387±0.0841）vs（0.00067±0.00044）,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（0.004476±0.0025）vs（4.721±1.589）vs（0.387±0.0841）,（P<0.05）.6.ELISA detected TNF-alpha,CRP,IL-6,MCP-1 protein content:6.1 ELISA detected the contents of TNF-alpha,CRP,IL-6,MCP-1 in Serum:After 6h of bacterial inoculation,the content of TNF-alpha in SP group was higher than that of PBS group（54.341±5.068）vs（37.379±3.841）pg.ml-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（84.204±7.159）vs（54.341±5.068）pg.ml-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（85.889±1.342）vs（37.183±3.736）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（273.074±31.713）vs（85.889±1.342）pg.ml-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（149.724±10.391）vs（41.859±8.575）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（335.842±20.623）vs（149.724±10.391）pg.ml-1,（P<0.05）.After 6h of bacterial inoculation,the content of CRP in SP group was higher than that of PBS group（16.943±1.932）vs（11.322±1.224）ng.L-1,（P>0.05）;SP+MRP8/14 group was increased compared with SP group（18.712±0.833）vs（16.943±1.932）ng.L-1,（P>0.05）;At 24 h,SP groups compared with PBS groups increased obviously（21.638±2.168）vs（11.880±0.641）ng.L-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（28.093±1.782）vs（21.638±2.168）ng.L-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（26.783±0.591）vs（12.839±2.073）ng.L-1,（P<0.05）,SP+MRP8/14 group compared with SP group increased obviously（30.626±1.774）vs（26.783±0.591）ng.L-1,（P<0.05）.After 6h of bacterial inoculation,the content of serum IL-6 in SP group was higher than that of PBS group（229.024±24.002）vs（62.628±11.571）pg.ml-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（577.111±53.557）vs（229.024±24.002）pg.ml-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（1223.345±103.112）vs（64.044±3.892）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（2326.765±144.669）vs（1223.345±103.112）pg.ml-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（2153.850±182.272）vs（49.309±15.26）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（2895.417±34.544）vs（2153.850±182.272）pg.ml-1,（P<0.05）.After 6h of bacterial inoculation,the content of serum MCP-1 in SP group was higher than that of PBS group（126.990±20.115）vs（67.995±17.733）pg.ml-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（226.832±36.838）vs（126.990±20.115）pg.ml-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（239.921±8.530）vs（52.406±19.259）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（1306.669±102.119）vs（239.921±8.530）pg.ml-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（489.987±27.689）vs（58.294±13.203）pg.ml-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（1513.495±22.769）vs（489.987±27.689）pg.ml-1,（P<0.05）.6.2 ELISA detected the contents of TNF-alpha,CRP,IL-6,MCP-1 in brain tissue homogenate:After 6h of bacterial inoculation,the content of brain tissue TNF-alpha in SP group was higher than that of PBS group（364.884±32.385）vs（300.415±21.272）pg?mg-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（453.451±37.941）vs（364.884±32.385）pg?mg-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（376.230±77.487）vs（287.875±91.347）pg?mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（650.764±145.614）vs（376.230±77.487）pg?mg-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（536.972±47.004）vs（252.222±62.096）pg?mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（1058.782±181.951）vs（536.972±47.004）pg?mg-1,（P<0.05）.After 6h of bacterial inoculation,the content of CRP in brain tissue in SP group was higher than that of PBS group（1.673±0.6411）vs（1.335±0.5090）ng.mg-1,（P>0.05）;SP+MRP8/14 group was increased compared with SP group（2.363±1.8868）vs（1.673±0.6411）ng.mg-1,（P>0.05）;At 24 h,SP groups compared with PBS groups increased obviously（2.908±0.4788）vs（1.455±0.4393）ng.mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（4.854±1.1500）vs（2.908±0.4788）ng.mg-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（7.740±1.248）vs（1.396±0.681）ng.mg-1,（P<0.05）,SP+MRP8/14 group compared with SP group increased obviously（14.293±2.046）vs（7.740±1.248）ng.mg-1,（P<0.05）.After 6h of bacterial inoculation,the content of IL-6 in brain tissue in SP group was higher than that of PBS group（512.102±13.617）vs（397.429±9.998）pg.mg-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（885.784±134.836）vs（512.102±13.617）pg.mg-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（1715.402±142.809）vs（374.470±26.298）pg.mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（1907.884±106.241）vs（1715.402±142.809）pg.mg-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（2142.090±98.117）vs（378.498±34.886）pg.mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（2808.924±171.509）vs（2142.090±98.117）pg.mg-1,（P<0.05）.After 6h of bacterial inoculation,the content of MCP-1 in brain tissue in SP group was higher than that of PBS group（383.366±25.761）vs（240.694±47.047）pg.mg-1,（P<0.05）;SP+MRP8/14 group was increased compared with SP group（579.320±43.008）vs（383.366±25.761）pg.mg-1,（P<0.05）;At 24 h,SP groups compared with PBS groups increased obviously（846.938±137.067）vs（288.277±41.896）pg.mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（1208.930±11.011）vs（846.938±137.067）pg.mg-1,（P<0.05）;48h,SP groups compared with PBS groups increased obviously（1131.959±80.333）vs（256.048±23.459）pg.mg-1,（P<0.05）;SP+MRP8/14 group compared with SP group increased obviously（2156.143±66.382）vs（1131.959±80.333）pg.mg-1,（P<0.05）.7.Brain tissue immunohistochemistry results:four groups of brain cell membrane are visible on NF-κB p65,6 h,24 h,48 h there is a small amount of expression of NF-κB p65 in PBS group and MRP8/14 group;SP group 6h express began to rise（23.54±1.252）vs（0.88±0.835）、24h（38.65±1.959）vs（1.00±0.756）、48h（47.88±1.808）vs（0.88±0.835）,each time point compared with PBS group increased significantly（P<0.05）;SP+MRP8/14 group,the expression of NF-κB trend is consistent with the SP group,but the expression in the SP group at each time point further increase 6h（29.38±2.326）vs（23.54±1.252）、24h（45.88±1.959）vs（38.65±1.959）、48h（67.25±2.765）vs（47.88±1.808）,48 h with statistical significance（P<0.05）.Conclusions:1.This experiment successfully established the mouse SP meningitis model by injecting SP suspension into the lateral ventricle.2.In vitro experiment results confirm,the expression of TNF-α,CRP,IL-6,MCP-1 increased in Streptococcus pneumoniae meningitis model,TNF-α,CRP,IL-6,MCP-1play an role in pathologic injury and immune inflammatory reaction process in the Streptococcus pneumococcal meningitis.3.Myeloid Related Protein 8/14 can promote the disease progression of bacterial meningitis,boost the immune inflammatory reaction of brain tissue,and increase the expression of TNF-α,CRP,IL-6,MCP-1,which indicates that MRP8/14 exacerbates the pathological injury of Streptococcus pneumoniae meningitis,enhanced inflammatory injury of this disease.4.Myeloid related protein 8/14 regulates the immune inflammatory response of SP meningitis through the NF-kappa B signaling pathway,exacerbating the progress of SP meningitis. |
PDF Full Text Request