The Effects Of Myeloid-Associated Protein MRP8/14 On Brain Injury Caused By Streptococcus Pneumoniae Meningitis In Mice

Objectives:Bacterial meningitis(BM)refers to the inflammatory diseases of the brain parenchyma,capsule or blood vessels caused by various bacteria invading the central nervous system,of which Streptococcus pneumoniae meningitis is more common.In the process of bacterial meningitis disease,blood-brain barrier disruption and brain injury often occur,leading to necrosis and apoptosis of brain neurons.S100A8/A9,also known as Myeloid-related protein-8/14(MRP8/14),calprotectin,is a member of the S100 protein family and is a protein associated with the movement inhibitory factor.Migrating inhibitory factor-related protein(MRP)heterodimers consisting of type 8 and type 14 were originally found in neutrophils as immunogenic proteins belonging to the S100 family of calcium-binding proteins.EF-hand structure is an important functional domain of proteins that can bind to Ca2+ to change the conformation of the protein and expose the site of interaction with the target protein,thereby exerting corresponding biological functions.S100A8/A9 expression has organization and the cells are characteristically expressed mainly in myeloid cells and are most abundant in neutrophils,accounting for 50%of the cytosol soluble components of neutrophils,and are also expressed constitutively in monocytes.MRP8/14 plays a role as a chemotactic target in inflammation and can participate in inflammatory reactions,regulate cell growth and differentiation,inhibit growth,and induce apoptosis.The incidence of brain neuron apoptosis was higher in bacterial meningitis.It was shown that apoptosis-mediated brain injury is an important factor for brain injury in the course of bacterial meningitis,while high concentrations of MRP8/14 have pro-apoptotic effects.Therefore,whether it is used as a target to inhibit brain neuronal apoptosis may be one of the important ways to prevent bacterial meningitis brain injury.At the same time,whether it can be used as a new therapeutic target,so as to inhibit the Streptococcus pneumoniae infection,the brain tissue damage caused by inflammation and neuronal apoptosis is our concern.This study was first designed to establish a mouse model of meningitis induced by Streptococcus pneumoniae(Streptococcus pneumoniae,SP).In order to explore the effect of MRP8/14 on brain damage caused by bacterial meningitis in mice and to understand its role in the progression of bacterial meningitis,SP mouse behavior,brain edema,brain tissue homogenate IgG,albumin levels,and the expression of brain damage markers NSE and caspase3 were studied.The study was supposed to provide new ideas and experimental evidence for bacterial meningitis therapies.Methods:1.To study the effect of MRP8/14 on neurobehavioral changes and body weight changes in SP meningitis mice,48 healthy male BALB/C mice(aged from 1.5 to 2 months)were randomly divided into 4 groups:Phosphoric acid Buffered saline solution control group(PBS group),MRP8/14 protein group(MRP8/14 group),Streptococcus pneumoniae group(SP group),Streptococcus pneumoniae+MRP8/14 protein group(SP+MRP8/14 group).After anesthesia was induced by intraperitoneal injection,a bacterial bacterial meningitis model was established by injecting 40 ul phosphate buffered saline(PBS)+5 ul Streptococcus pneumoniae suspension into the lateral ventricle;45 ul phosphate buffered saline(PBS)was injected into the PBS group;MRP8/14 groups were injected with 40 ul of MRP8/14 protein and 5 ul phosphate buffer saline solution(PBS).In SP group,40 ul of phosphate buffered saline(PBS)and 5 ul of Streptococcus pneumoniae suspension were injected,and 40 ul of MRP8/14 protein was injected into SP+MRP8/14 groups.5ul of Streptococcus pneumoniae suspension.2.The lateral ventricle injection was performed at 6h,24h,48h for each group of mice:1)neurobehavioral score;2)record changes in body weight(before and after injection at different time points before and after the injection of the weight of mice,before and after analysis(Changes in body weight difference);3)Changes in brain edema were recorded(weights of brain tissue of mice at different time points were recorded,and changes in brain tissue weight and ratio before and after analysis).3.To analyze the effect of MRP8/14 on blood-brain barrier and brain injury in SP meningitis mice.To determine the levels of IgG and Albumin in brain homogenate by ELISA method,and detect the expression of NSE and caspase3 protein in brain injury by immunohistochemistry.Animal models and grouping with experimental methods were shown as follows:IgG,Albumin levels in brain homogenates were determined by enzyme-linked immunosorbent assay(ELISA)at 6 h,24 h,and 48 h after injection into the lateral ventricle,respectively.The expression of NSE and caspase3 protein from brain tissue was detected.4.To investigate the effect of MRP8/14 on neuronal necrosis(Nissl staining)in the brain of SP meningitis mice and evaluate the brain injury.The animal model and grouping with the experimental methods:The necrosis of the nerve cells in the mouse brain tissue was detected at 6h,24h,and 48h after injection into the lateral ventricle.The number of Nissl-positive cells and necrosis-positive cells in the mouse brain tissue at different time points was observed by microscopy.Results:Part 1:Establishment of SP meningitis model in mice1.Effect of MRP8/14 on clinical symptoms of SP meningitis in mice.(1)Neurobehavioral score:Recorded at 6h.24h,and 48h after injection of each lateral ventricle in each group Bed score.The clinical scores of MRP8/14 and PBS groups were compared:6h(5.0±0)vs(5.0±0),24h(4.75±0.5)vs(5.0±0),48h(5.0±0)vs(5.0±0).There was no significant difference between the two groups(P>0.05).In the SP group,the clinical scores at 6h,24h,and 48h were significantly lower than those in the PBS group:6h(4.75±0.5)vs(5.0±0),24h(3.5±0.577)vs(5.0±0),48h(3.25±0.957)vs(5.0±0).The differences between the two groups were statistically significant(P<0.05).The clinical scores of the SP+MRP8/14 group at 6h,24h,and 48h were all significantly lower than those of the SP group(P<0.05):6h(4.5±0.577)vs(4.75±0.5),24h(2.5±0.577)vs(3.5±0.577),48h(1±0.816)vs(3.25±0.957).(2)Changes in body weight:Recorded before and 6h,24h,and 48 h after injection in each group of mice.The amount of change.Changes in body weight at 6h,24h and 48h in MRP8/14 and PBS mice:6h(2.237±0.407)vs(2.152±0.403),24h(4.458±1.784)vs(5.338±2.049),48h(7.032±2.600)vs(5.683±2.480),there was no significant difference in body weight between the two groups(P>0.05).Body weights of the SP group at 6 h,24 h,and 48 h after bacterial inoculation decreased compared with the PBS group:6h(2.749±0.309)vs(2.152±0.403),24h(9.125 ±0.365)vs(5.338±2.049),48h(10.036±0.905)vs(5.683±2.480),the differences were statistically significant(P<0.05).The weight of rats after inoculation of bacterial and protein suspensions was significantly lower than that of the SP group(P<0.05):6h(2.92±0.357)vs(2.749±0.309),24h(11.336±1.454)vs(9.125±0.365)、48h(13.803±2.303)vs(10.036±0.905).2.The effect of MRP8/14 on brain water content in SP meningitis mice.Brain tissue water content(%)=(wet weight-dry weight)/wet weight × 100%.At 6h,24h,and 48h after infection with Streptococcus pneumoniae,the water content of brain tissue in mice showed an increasing trend.There was no significant difference in clinical scores between MRP8/14 group and PBS group 6h(76.859±0.308)vs(77.076±0.756)%,24h(77.149±0.928)vs(76.047±0.744)%,48h(78.785±0.779)vs(76.998±0.832)%±The water content of brain tissue in the SP group at 6h,24h,and 48h was significantly higher than that of the PBS group(P<0.05):6h(78.055±0.774)vs(77.076±0.756)%,24h(79.154±1.235)vs(76.047±0.744)%,48h(82.87±0.626)vs(76.998±0.832)%.The ratio of brain tissue water content in SP+MRP8/14 group was significantly higher(P<0.05)than the SP group at 6h(78.909±1.195)vs(78.055±0.774)%,24h(81.179±1.053)vs(79.154±1.235)%,48h(88.019±1.036)vs(82.87±0.626)%.Part 2:The Effects of myeloid-associated protein MRP8/14 on brain injury caused by Streptococcus pneumoniae meningitis in mice.1.The effects of MRP8/14 on neuronal loss in cerebral neurons in SP Meningitis mice.Nissl staining results:There were positive staining cells in the brain tissue of the four groups.In the brain tissue,Nissl-positive neurons in the MRP8/14 group had no significant difference(P>0.05)from the PBS group:6h(112±0.816)vs(113.25±2.362),24h(109.5 ±1.290)vs(117±3.464),48h(105.5 ±2.886)vs(115.25±2.986).SP group vs.PBS group:6h(106±2.943)vs(113.25±2.362),24h(94±1.825)vs(117±3.464),48h(85.25±1.707)vs(115.25±2.986),and the difference was statistically significant(P<0.05).Compared with SP group,the value in SP+MRP8/14 group significantly reduced(P<0.05)at 6h(91.75±4.031)vs(106±2.943),24h(80.75±5.560)vs(94±1.825)and 48h(74.25±3.685)vs(85.25±1.707).2.The effect of MRP8/14 on IgG and Albumin of blood brain barrier markers in brain of SP meningitis mice.The IgG levels in the mouse brain increased at 6 h5 24 h,and 48 h after infection with S.pneumoniae.No significant difference in IgG expression between the MRP8/14 group and the PBS group was observed for 6h(56.010±14.336)vs(59.594±22.975),24h(58.905±9.057)vs(71.025±21.050),48h(49.972±7.450)vs(53.992±12.488).In PBS group,the difference was statistically significant at 6h(218.548±50.900)vs(59.594±22.975),24h(659.693±223.473)vs(71.025±21.050),48h(1224.340±266.477)vs(53.992±12.488)(P<0.01).The expression of IgG in brain tissue of mice in SP+MRP8/14 group was significantly higher than that in SP group at 6h(288.155±67.407)vs(218.548±50.900),24h(1461.726±26.531)vs(659.693±223.473),48h(2680.163±307.636)vs(1224.340±266.477),the difference was statistically significant(P<0.05).At 6h,24h and 48h after infection with streptococcus pneumoniae,Albumin in the brain tissue of mice showed an increasing trend.The experiment found that there was no significant difference in expression of Albumin between the MRP8/14 group and the PBS group mice at 6h(10.208±3.567)vs(9.708±4.769),24h(8.933±1.589)vs(13.109±2.672),48h(10.265±1.898)vs(14.098±2.889).Compared with SP group,the expression of Albumin in SP group significantly increased(P<0.01)at 6h(14.215±3.660)vs(9.708±4.769),24h(18.021 ±1.292)vs(13.109±2.672),48h(27.334±2.736)vs(14.098±2.889).Compared with SP group,the value in SP+MRP8/14 was also increased(P<0.05)at 6h(19.440±4.677)vs(14.215±3.660),24 h(25.322±3.153)vs(18.021±1.292),48h(38.214±4.255)vs(27.334±2.736).3.The effect of MRP8/14 on NSE and caspase3 in brain lesions in brain of SP meningitis mice.Brain tissue immunohistochemical analysis:(1)Expression of NSE protein in brain tissue:In brain tissue,NSE positive cells were expressed in each group of mice,and intracellular matrix had tan particles deposited under light microscope.We found that the number of NSE-positive cells in the MRP8/14 group was not significantly different from that in the PBS group after bacterial injection:6h(100±4.209)vs(100.63±3.543),24 h(100.25±3.240)vs(100.38±2.774),48h(99.75±5.994)vs(99.5±2.563),the difference was not statistically significant(P>0.05).The number of NSE positive cells in SP group was significantly reduced by compared with PBS group.The differences were statistically significant 6h(96.38±1.685)vs(100.63±3.543),24h(86.25±2.252)vs(100.38±2.774),48h(77.25±2.915)vs(99.5±2.563)(P<0.05).we found that the number of NSE positive cells in the SP+MRP8/14 group was less than that in the SP group(88.25±2.915)vs(96.38±1.685),(68.38±2.446)vs(86.25±2.252),(56±4.690)vs(77.25±2.915)at the three time points of 6h,24h,and 48h.The difference was statistically significant(P<0.05).(2)Expression of caspase3 protein in brain tissue:In brain tissue,caspase3 positive cells were expressed in all groups of mice.In the MRP8/14 group and PBS group,caspase3 immunohistochemical reaction was positive at 6h,24h,and 48h,but the number of positive cells was not significantly different at 6h(5.43±0.850)vs(6.25 10.957),24h(6.75±2.062)vs(6.25±1.893),48h(5.75±0.957)vs(8±2.160)(P>0.05).The expression of caspase3 in SP group was significantly higher than that of PBS group(P<0.05).The data were showed:6h(16.38±0.955)vs(6.25±0.957),24h(21.7512.754)vs(6.25±1.893),48h(42.5±2.380)vs(8±2.160).The expression of caspase3 in SP+MRP8/14 group was significantly higher(P<0.05)than that in SP group at 6h(21.29±2.525)vs(16.38±0.955),24h(36.5±3.512)vs(21.75±2.754),and 48h(63.25±1.259)vs(42.5±2.380).Conclusions:1.A mouse SP meningitis model was induced by injecting with Streptococcus pneumoniae suspension via the lateral ventricle injection route.In this model,S.pneumoniae infection resulted in the derease of neurobehavioral score and body weight,and the increase of brain edema.2.S.pneumoniae infection increased the expression of blood-brain barrier markers IgG and Albumin in brain homogenates,but decreased the expression of the brain injury maiker NSE protein and the number of Nissl-positive cells in brain samples,as well as upregulated caspase3 protein expression.The data indIcated that brain damage was induced by the establishment of the SP meningitis model in mice.3.Myeloid-associated protein MRP8/14 exacerbated the clinical symptoms of SP meningitis,and promoted cell loss in brain tissues(the number of Nissl positive neurons decreased significantly).It also further increased the expression of blood-brain barrier markers IgG and Albumin in brain homogenates,and the caspase3 expression,but reduced NSE protein expression,indicating that MRP 8/14 aggravated SP brain damage caused by meningitis.