The Role Of NOD2 On Streptococcus Pneumoniae Meningitis And The Research Of Its Related Treatment

Streptococcus pneumoniae is the most common pathogen of central nervous system infection.In recent years,high morbidity and mortality have decreased with the improvement of economic and health conditions and the development of health-care.However,the current diagnosis and treatment of clinical outcomes are still unsatisfied,and there are also some neurological sequelae.Therefore,the study of streptococcus pneumoniae meningitis has practical significance.Microglia have been considered as the important contributor to inflammatory immune responses in the brain.As monocytes in the brain,they are thought to have antigen presentation,cytokine secretion and phagocytosis.The study shows that it has the distribution of trans-membrane Toll-like receptors and the cytoplasmic Nod-like receptors.NOD2 protein is one of the important members of NLRs family,which plays an important role in the identification and inflammation induction of pathogenic microorganisms in the central nervous system.NOD2 protein can recognize muramyl dipeptide,which is a pyrolysis product of peptidoglycan existing in the cell wall of Gram-negative and positive bacterial.When NOD2 protein combines with ligand,it induces the release of inflammatory cytokines TNF-? and IL-6 through NF-? B.Although these inflammatory cytokines protect the body from pathogenic microorganisms,the lack of proper regulation leads to excessive production of inflammatory cytokines,which can generate micro-circulation disorders and damage to the normal tissues.It has been proved that the immune tolerance of macrophages mediated by NOD2 can inhibit the overexpression of inflammatory factors and protect the body.Previous researches in our laboratory has confirmed that there are immune tolerance mediated by NOD2 in microglia.Therefore,further study on the pathophysiological mechanism of NOD2 will provide new ideas for clinical treatment.The clinical treatment of streptococcus pneumoniae meningitis is mainly based on the etiology of anti-infection treatment.Simple anti-infective therapy cannot solve the brain tissue immune injury of patients.In vitro studies shows that glial cells were stimulated by lipopolysaccharide and the microglial activation was restricted by glucocorticoids after bacterial exposure.However,many randomized clinical trials have shown that the role of glucocorticoid in the treatment of bacterial meningitis remains unclear.Moreover,no studies have been conducted on the effects of the glucocorticoids on microglial activation in pneumococcal meningitis.RNA interference is the process of silencing the expression of specific gene sequences at the mRNA level by double-stranded RNA(dsRNA)molecules,that is,Sequence-specific post-transcriptional gene silencing.The basic principle of its action is to generate RNA induced silencing complex by combining some 20bp or so double-stranded siRNA(small interferencing RNA)with ribozyme complexes,and the activated RISC combines with the homologous mRNA by base pairing and cleans it.In this way,the mRNAs produced by the transcription of the specific target gene are destroyed,and the function of mRNA is silenced.Since NOD2 has been the focus of the study of central nervous system infections,we intended to research the subject from the following three aspects.1.NOD2 Expression in Streptococcus pneumoniae Meningitis and its Influence on the Blood-Brain BarrierStreptococcus pneumoniae meningitis is one of the most common disorders seen in clinical practice.It is believed that the brain tissue immune injury is caused by the expression of pattern-recognition receptors(PRR)which can further induce the release of other cytokines and inflammatory cascade.The aim of this study is to investigate the expression of Nucleotide-binding oligomerization domain 2(NOD2)and inflammatory factors in rat brain tissues infected with Streptococcus pneumoniae and its influence on the blood brain barrier(BBB)permeability.Rats were given an intracranial injection of Streptococcus pneumoniae to construct the Streptococcus pneumoniae meningitis rat models.The expression change curves of NOD2 and inflammatory factors at different time points(0,12,24,48 h and 7 d)after Streptococcus pneumoniae were evaluated by enzyme-linked immunosorbent assay(ELISA).Western blotting analysis and quantitative real-time polymerase chain reaction(qRT-PCR)were engaged to examine the expression of NOD2.Furthermore,the changing processes of pathological characteristics,nervous system score,cerebral oedema and BBB permeability were observed.Our results showed that NOD2 expression began to increase in the 12 h after Streptococcus pneumoniae infection group,while the remaining inflammatory factors were not obviously increased.Meanwhile,the levels of NOD2,as well as inflammatory factors IL-1?,TNF-a and IL-6 were markedly elevated in 24 h and 48 h infection groups,which were consistent with the increases in BBB permeability and BWC,and the positive expression of NOD2 in the infected rat brain tissues was observed using immunohistochemistry(IHC).This study suggests that NOD2 might be related to the activation of inflammation pathways and the damage to the blood brain barrier.NOD2 and inflammatory factors have played vital roles in the pathogenesis of Streptococcus pneumoniae meningitis.2.Effect of Dexamethasone Adjuvant Therapy on Streptococcus Pneumoniae MeningitisTo study the effect of dexamethasone adjuvant therapy on streptococcus pneumoniae meningitis.Rats were randomly divided into 5 groups,including control group,infection group,ceftriaxone group,early dexamethasone adjuvant therapy group and delayed dexamethasone adjuvant therapy group.Expression of Nucleotide-binding oligomerization domain-containing protein 2,Interleukin-1 beta,Tumor necrosis factor-alpha and Interleukin-6(NOD2,IL-1?,TNF-? and IL-6)in brain tissues was detected using Enzyme-linked immunosorbent assay(ELISA),and histological and morphological changes were observed through Hematoxylin and eosine(HE)staining of brain tissue pathological sections.Neurological scores were rated on day 7 after different treatments in each group,and Morris water maze test were analyzed in all rats.On day 7 after different treatments in each group,compared with the control group,the expression of NOD2,TNF-?,IL-1? and IL-6 in infection group was markedly up-regulated(Ps<0.01),while that in ceftriaxone group and delayed dexamethasone adjuvant therapy group were also up-regulated,and that in early dexamethasone adjuvant therapy group displayed no statistical significance(P>0.01).Compared with ceftriaxone group,the expression in early dexamethason adjuvant group was superior to delayed dexamethasone adjuvant therapy group,along with alleviated pathological changes in rat brain tissues,as well as markedly improved neurological score.Morris Water Maze showed:Compared with infection group,the memory capacity of rats have a slight advantage in three groups of treatment after five days of navigation test,but there was no significant difference among these groups.Early dexamethasone adjuvant therapy can improve the prognosis for streptococcus pneumoniae meningitis on the basis of antibiotic therapy.But neither early dexamethasone adjuvant therapy nor delayed dexamethasone adjuvant therapy has little effect on improving learning and memory function.3.NOD2 siRNA protects against rats brain injury by inhibiting inflammatory responsesIt was suggested by recent research that the Nucleotide-binding oligomerization domain 2(NOD2)gene may serve an important role in meningitis.In the present study,the NOD2 gene was knocked down to evaluate the expressions of NOD2 and inflammatory factors of the Wistar rats and to investigate their pathogenesis to give theoretical basis of treatment.The expression levels of NOD2 in brain tissue sample was identified using quantitative reverse transcription polymerase chain reaction,western blot and immunostaining.Subsequently,the expression levels of the TNF-?,IL-6,and IL-1? levels through enzyme-linked immunosorbent assay(ELISA).Also,Evans blue(EB)staining and water measurement were used to assess BBB permeability.Nervous system score and survival rate were employed to evaluate clinical functions.Results:NOD2 exhibited a higher expression level in the brain tissue samples of Streptococcus pneumonia meningitis.Small interfering(si)NOD2 resulted in a decline in NOD2,TNF-?,IL-6,and IL-1? and inhibited proinflammatory responses by the NF-?B pathway.Brain oedema and BBB leakage were reduced in NOD2-siRNA rats.Moreover,knockdown of NOD2 by small interfering RNA in rats also significantly improved nervous system score and survival rate.In summary,our present study showed that NOD2 small sequence interference exerts a potent regulatory function on the NF-?B pathway in Streptococcus pneumonia meningitis,can attenuate proinflammatory responses,BBB permeability,and brain oedema;and improve survival rate and the prognosis.Based on these findings,we believe that suppressing NOD2 expression may be the new strategy for treating Streptococcus pneumonia infection.