The Cerebral Protective Effect And Mechanism Of Action Of Vitamin B6 In Rats With Pneumococcal Meningitis

Background and objective:Pneumococcal meningitis is one of the most common central nervous system infections which has a high fatality rate worldwide and a high risk of long-term sequelae in the survivors.Despite effective antimicrobial treatment,about one third of survivors still suffer long-term sequelae such as hearing loss,cerebral palsy,epilepsy,hydrocephalus and cognitive impairment,etc.The average incidence of at least one sequelae in meningitis caused by streptococcus pneumoniae was 24.7%.Therefore,on the basis of giving rational antibiotics to fight infection and using adrenal cortical hormone therapy,to study the pathophysiological mechanism of pneumococcal meningitis and to find new methods to assist in the treatment of pneumococcal meningitis is helpful to improve the prognosis of pneumococcal meningitis.In recent years,studies have confirmed that pneumococcal meningitis causes two different pathological and physiological forms of brain injury,namely hippocampal cell apoptosis and cortical necrosis.Damage to hippocampal structures is associated with learning and memory impairment.The inflammatory mediators of pneumococcal infection can induce immune response and cause tryptophan degeneration through the Kynurenine(KYN)pathway.Kynureninase is one of the most important intermediate metabolites of tryptophan,which can be metabolized into Kynurenic acid(KYNA)through the action of Kynurenine aminotransferase(KAT).KYNA is a broad antagonist of the excitatory amino acid receptor of the central nervous system which is an effective protective factor to avoid excitatory amino acid damage to brain tissue and nervous system and has the anti-convulsion,anti-spasm,protection of the nervous system and other functions.Although KYNA has neuroprotective effect,if the accumulation of KYNA is beyond the normal physiological level in the CNS,it can cause glutamatergic hypofunctioning and then lead to cognitive dysfunction.Other research reports that in pneumococcal meningitis,the KYN pathway may influence the supply of nicotinamide adenine dinucleotide(NAD)in nerve tissue and increase the release of excitatory amino acids such as glutamate.Vitamin B6(vitB6)is a co-factor of two key enzymes,KYN transaminase and Kynureninase.Therefore,in theory,vitamin B6 catalyzes the metabolism of tryptophan and it can reduce the death of nerve cells and play a protective role in brain in bacterial meningitis by reducing the neurotoxic products produced by the Kynurenine pathway by influencing KYN and KYNA and increasing the expression of NAD and ATP.To confirm this hypothesis,we established a model of streptococcus pneumonitis in young rats to study the effects of vitB6 on the clinical symptoms,the expression of KYN?KYNA?NAD?ATP and cytokines TNF-??IL-1??IL-6?IL-10,the expression of Kynurenine 3-monooxygenase(KMO)in brain tissue and the effects on brain histopathology.At the same time,Kynurenine 3-monooxygenase(KMO)inhibitor Ro 61-8048 was applied to observe the effect on the expression of the above indicators and the degree of brain damage in meningitis ratsin in order to further investigate the brain protective effect of vitB6 and its mechanism in bacterial meningitis.Methods:1.Subjects:90 Wistar rats born 21 days old were randomly divided into four groups:36 h group(n = 30),3 d group(n = 20),7 d group(n = 20)and 14 d group(n = 20).Animals in 36 h group(n = 30)were divided into six groups:normal control group,meningitis group,meningitis treated with ceftriaxone group,meningitis treated with ceftriaxone plus vitB6 group,meningitis treated with ceftriaxone plus Ro-61-8048 group and meningitis treated with ceftriaxone,vitB6 plus Ro-61-8048 group(5 animals per group).Animals in 3 d group,7 d group and 14 d group were respectively divided into four groups:normal control group,meningitis group,meningitis treated with ceftriaxone group and meningitis treated with ceftriaxone plus vitB6 group(5 animals per group).The animals in 36h group needed to improve the clinical observation,the determination of KYN,KYNA,NAD,ATP,TNF-??IL-1??IL-6?IL-10,and the detection of KMO expression.Only clinical manifestations were observed in the 3d group and the 7d group.Besides clinical manifestations,Morris water maze behavior test was also required in the 14d group.2.Induction of pneumococcal meningitis in young mice:In addition to the normal control group,the other 70 rats were treated according to the methods of Liu xinjie and Leib.After the animals were anesthetized by intraperitoneal injection of 1%pentobarbital sodium(40mg/kg),Cerebellomedullary cistern puncture was performed,and 10 ?l of saline containing 1×106 of S.pneumoniae(serotype 3)via a 32-gauge needle was injected directly to prepare the meningitis model.Normal control animals were injected with 10 ?l saline.After 18h,the rats were anesthetized and cerebrospinal fluid was taken from the cerebellomedullary cistern.Bacterial culture was conducted in sheep blood AGAR medium to determine the success of the model.3.Drug delivery:At time of infection,animals treated with vitB6 received 360?l of vitB6 intraperitoneally(i.p;600mg/kg).Other animals were injected i.p.with 360 ?l of 0.85%NaCl.Eighteen hours after infection,infection was documented by quantitative culture of 5 uL of CSF and the animals treated with ceftriaxone were treated with ceftriaxone(i.p;100 mg/kg,once daily)and a second dose of vitB6 or 0.85%NaCl was administered.After that,ceftriaxone and vitB6 were administered once a day,and the drug was administered until the end of pretreatment in animal experiments in 36h group,3d group and 7d group,and the drug was discontinued until 7 days after infection in 14d group.At the same time,Ro-61-8048(100mg/kg,sc,59mg/ml soluble in DMSO)was intraperitoneally injected at 18 hours and 24 hours after infection.4.Clinical observation:(1)The clinical manifestations of each group were observed at different time periods and the score was given according to the symptoms.1 point:coma;2 points:does not stand upright after being turned on the back;3 points:stand upright within 30s;4 points:minimal ambulatory activity,stands upright in<5s;5 points:normal.(2)Morris water maze behavior test was conducted on rats in 14d group at day 10 of infection to observe the effect on spatial learning and memory ability.For four days,the average time of each group arriving at the platform per day was calculated.5.Tissue processing:At the end of each group of animal experiments,the animals were anesthetized and executed,and immediately after that,30 milliliters of cold phosphate buffer(PBS)was injected into the left heart and the brain tissue were dissected into the left hemisphere and the right hemisphere.The left brain tissues were quickly put into-80 ? refrigerator in order to be used for ELISA and Western blot method.The right mouse brain was used for pathological observation and immunohistochemistry.After perfusion with 4%paraformaldehyde,it was fixed with 4%paraformaldehyde for 1-3 days and then was given conventional dehydration,waxing and embedding for use.6.Detection method:The concentrations of KYN?KYNA?NAD?TNF-??IL-1??IL-6 and IL-10 in the brain homogenates were measured with a commercial ELISA kit according to the directions provided by the manufacturer.The concentration of ATP in the brain homogenates was determined using a commercially ATP Assay kit and spectrophotometer according to the directions provided by the manufacturer.Western blot was used to detect KMO protein level which was followed by protein extraction,SDS-PAGE electrophoresis,electron transfer membrane,antigen antibody reaction,color rendering and etc.The quantitative analysis of KMO was semi-quantitative using the gel imaging analysis system.The transcription level of KMO gene was determined by reverse transcription polymerase chain reaction(RT-PCR)which was completed by total RNA extraction,synthesis of cDNA,quantitative PCR and result processing.The quantitative expression of KMOmRNA was expressed by amplification coefficient.Method of brain histopathological examination:the wax block was cut into 5 um thick sections and stained with HE.The pathological changes of rat cerebral membranes were observed under microscope.The processs of immunohistochemical staining of KMO in the rat brain included antigen repair,endogenous peroxidase removed,goat serum closed,add one anti and two anti,color,redyeing,dehydration,sealing and etc.7.Statistical analysisSPSS 17.0 was used for all statistical analyses.All results are presented as the mean ± standard deviation(SD).One-way analysis of variance(ANOVA)were performed to compare the differences.A P-value<0.05 was considered statistically significant.Results:1.Rats inoculated with streptococcus pneumoniae were cultured with cerebrospinal fluid,and it was confirmed that the same strain of streptococcus pneumoniae was inoculated with high concentration of bacteria.The cerebrospinal fluid of rats in normal control group showed no bacterial growth.2.Clinical observation:All rats infected with meningitis can show poor spirit,less activity,less food and significantly reduced symptom score.The severe cases can be manifested as cycloplegia,hemiplegia and etc.Treated by Ceftriaxone + vit B6 can improve the clinical manifestations of meningitis in rats more than treated by ceftriaxone alone.In addition,Ro-61-8048 can further aggravate the clinical manifestations of meningitis in rats.3.Morris water maze behavior test:The time of looking for platform of ceftriaxone group was 19.6 ± 2.1 seconds,while that of ceftriaxone + vitB6 group was 14.6 ± 1.1 seconds.T test showed that(P<0.05)which indicated that the time of looking for platform of ceftriaxone + vitB6 group was significantly shorter than that of ceftriaxone group.4.Expression of KYN,KYNA,NAD and ATP:In 36 h group,the level of KYN and KYNA were significantly higher in the animals of the meningitis group compared to the normal control group(P<0.05).Treatment with ceftriaxone and vitB6 moderately decreased the level of KYN and KYNA compared to the animals treated with ceftriaxone alone(P<0.05).Treatment with ceftriaxone plus Ro-61-8048 significantly increased the level of KYN and KYNA compared to the animals treated with ceftriaxone alone(P<0.05).And treatment with ceftriaxone and vitB6 plus Ro-61-8048 significantly increased the level of KYN and KYNA compared to the animals treated with ceftriaxone and vitB6(P<0.05).The change of the level of NAD and ATP is opposite to that of KYN and KYNA.5.Expression of TNF-??IL-1??IL-6 and IL-10:In the 36h group,compared with the normal control group,the inflammatory cytokines TNF-??IL-1??IL-6 and IL-10 in the meningitis group were significantly increased.Ceftriaxone group was lower than meningitis group,and ceftriaxone + vitB6 group was further decreased.There was a significant difference between the groups(P<0.05).However,there was no significant difference between ceftriaxone + Ro-61-8048 group and ceftriaxone group.And there was also no significant difference between ceftriaxone + vitB6+Ro-61-8048 group and ceftriaxone + vitB6 group.6.KMO expression in brain tissue:In the 36h group,compared with the normal control group,KMO expression in brain tissue of meningitis group was significantly increased with significant difference(P<0.05).Compared with ceftriaxone group,KMO expression in brain tissue of ceftriaxone + vitB6 group was significantly decreased(P<0.05).Compared with ceftriaxone group,KMO expression in brain tissue of ceftriaxone + Ro-61-8048 group was significantly decreased(P<0.05).Compared with ceftriaxone + vitB6 group,KMO expression in brain tissue of ceftriaxone + vitB6+ Ro-61-8048 group was significantly decreased(P<0.05).Compared with ceftriaxone + Ro-61-8048 group,KMO expression in brain tissue of ceftriaxone + vitB6+ Ro-61-8048 group was significantly decreased(P<0.05).7.Expression level of KMO mRNA in brain tissue:In the 36h group,compared with the normal control group,KMO mRNA amplification coefficient in brain tissue of meningitis group was significantly increased with significant difference(P<0.05).Compared with ceftriaxone group,KMO mRNA amplification coefficient in brain tissue of ceftriaxone + vitB6 group was significantly decreased(P<0.05).Compared with ceftriaxone group,KMO mRNA amplification coefficient in brain tissue of ceftriaxone + Ro-61-8048 group was significantly decreased(P<0.05).Compared with ceftriaxone + vitB6 group,KMO mRNA amplification coefficient in brain tissue of ceftriaxone + vitB6+ Ro-61-8048 group was significantly decreased(P<0.05).Compared with ceftriaxone + Ro-61-8048 group,KMO mRNA amplification coefficient in brain tissue of ceftriaxone + vitB6+ Ro-61-8048 group was significantly decreased(P<0.05).8.Pathological changes of brain tissue:In the 36h group,The rats infected by S.pneumoniae had the meninges filled with granulocytes,the enlarged subarachnoid space and the significant expansion subarachnoid vessels.Moreover,the degree of the brain damage in the meningitis group was the most serious,and the others were respectively ceftriaxone and Ro-61-8048 group,ceftriaxone group,ceftriaxone and vitB6 plus Ro-61-8048 group and ceftriaxone and vitB6 group in turn.There was not obvious damage in the control group.9.Immunohistochemical expression:KMO immunopositive cells were widely expressed in the meningitis group.The others were respectively ceftriaxone group,ceftriaxone and vitB6 group,ceftriaxone and Ro-61-8048 group and ceftriaxone and vitB6 plus Ro-61-8048 group in turn.KMO was expressed at very low level in the control group.Conclusions:1.The meningitis model was successfully prepared by injecting 1 x 106cfu/ml of S.pneumoniae suspension into the cerebellomedullary cistern.2.Adjuvant treatment with vitB6 in pneumococcal meningitis can improve the clinical manifestations,spatial learning and memory capacity and pathological damage of brain tissue.3.In pneumococcal meningitis,KMO expression increased significantly in protein level,gene transcription level and immunohistochemistry.The application of KMO inhibitor Ro-61-8048 can reduce the expression of KMO,but it increases the degree of meningitis damage which is considered to be related to the excessive increase of KYN and KYNA expression and the reduction of NAD and ATP expression.VitB6 could exert neuroprotective effect by reducing KMO expression which suggests that the KYN pathway involves in the pathophysiological mechanism of pneumococcal meningitis.4.VitB6 can play a role in brain protection in pneumococca meningitis by regulating the levels of KYN metabolites and inflammatory cytokines.