| BackgroundBacterial infections in the central nervous system affect the meninges and brain parenchyma,which is a kind of serious and intractable disease.Bacterial meningitis is a common infectious disease of the central nervous system.Gram-positive Pneumococcal infection is one of the main causes of bacterial meningitis.Streptococcus pneumoniae meningitis is closely related to the intense inflammatory reaction and high mortality rate of the host,and is also the cause of neurological sequelae.Although the use of effective antibiotics and the introduction of prophylactic inoculation have reduced the mortality rate of bacterial meningitis,the related neurological sequelae such as psychomotor disorder,mental retardation and hearing impairment seriously affects the physical and mental health of the surviving children,which brings a great burden to the individual and the society.Innate immunity is the initial stage in which the body' s immune system directly resists the invasion of pathogens.It recognizes the unique conserved structure of pathogens by its own specific pattern recognition receptors and plays an important role in host defense against pathogen invasion.The NLRs family all belong to the cytoplasmic pattern recognition receptor,and three subfamilies have been found so far:nucleotide binding oligomerization domain,NOD-like receptor family pyrin domain ? containing,neuronal apoptosis inhibitory protein.Bacterial peptidoglycan(PGN)be recognized by NOD protein as a component of bacterial cell wall.NOD protein activates NF-? B and participates in innate immune and inflammatory responses to pathogen-associated receptors.Among them,NOD1 recognizes the cytosolic peptides containing meso-DAP on most Gram-negative bacteria and some Gram-positive bacteria PGN.However,NOD2 recognizes muramyl dipeptide(MDP)produced on all bacterial PGN.When NOD2 recognizes the MDP of bacterial cell wall,NOD2 changes in conformation,and activates RIP2 and induces NF-?B translocation to nucleus and transcription of pro-inflammatory gene.Inflammation is then triggered,leading to the production of cytokines,chemokines,and antimicrobial peptides.RIP2 is a serine/threonine protein kinase,which belongs to the RIP family.The N-terminal of RIP2 contains a key serine/threonine kinase domain in the signal transduction pathway.When it was stimulated,176th serine and 474th tyrosine were self-phosphorylated to stabilize the expression of RIP2.Several examples of inflammatory immune regulation abnormalities and related diseases have proved that they originate from the over-activation of NOD2/RIP2.Moreover,the kinase activity of RIP2 is an essential part of the function of NOD2.Several studies have demonstrated that the regulation of RIP2 stability and the self-phosphorylation of 176th serine are essential for cytokine immune response induced by NOD2.Although the role of RIP2 as a NOD binding protein and the theory of ubiquitin have been established,the biological role of RIP2 as a kinase remains to be investigated.It can be a new therapeutic target by regulating the activity of RIP2 kinase to alleviate inflammatory reaction and inflammatory damage.Gefitinib is a ATP competitive kinase inhibitor for EGF-R.It is also used as a first-line treatment for non-small cell lung cancer patients with EGF-R mutations.In vitro kinase assay and tissue culture experiments,it was proved that gefitinib caused RIP2 self-phosphorylation to fail.It indicated that gifitinib inhibited kinase activity completely in vitro,which inhibited the expression of RIP2,thus reducing the stability of RIP2 expression and the production of cytokines stimulated by NOD.However,the same effect has yet to be demonstrated in the studies of diseases in vivo.ObjectiveAn animal model of infective meningitis in young rats was established to explore the effective concentration of bacteria to form bacterial meningitis,and to simulate the response system of the central nervous system stimulated by inflammation during bacterial meningitis caused by Pneumococcal infection.The main objective is to explore the function of immune cells in the central nervous system and to verify that Streptococcus pneumoniae is an effective stimulator for NOD2/RIP2.By detecting the expression of NOD2/RIP2 and NF-?B downstream signaling pathway in mouse nerve cells infected with Streptococcus pneumoniae,the role of RIP2 as a NOD link protein and Ubiquitin theory were analyzed,and the biological role of RIP2 as a kinase was explored to further investigate the effect of RIP2 inhibitor gefitinib on cytokines of bacterial meningitis and its protective effect on brain injury after inflammation,which could help better understand the kinase activity of RIP2 in the NOD2 signaling pathway in the inflammatory response to Streptococcus pneumoniae meningitis,explore the possibility of RIP2 as a therapeutic target protein,and demonstrate that gefitinib may be used as an inhibitor of RIP2 in the treatment of bacterial inflammation caused by Streptococcus pneumoniae meningitis.Methods1.There were 66 C57BL/6 wild male mice,6-8 weeks old,weight 22-28 g.Animals were randomly divided into Control group(n=6)and Experimental group(n=60).The Experimental group was divided into three subgroups according to the different inoculation concentration of bacterial liquid,namely,low(0.5×107cfu/uL),medium(0.5× 108cfu/uL)and high(0.5× 109cfu/uL)concentration groups,20 animals in each subgroup.The clinical manifestations of each group of mice were observed and the scores were recorded according to the prescribed time.Four mice in each group were killed at 0 hours,24 hours,48 hours,72 hours and 120 hours after inoculation,which were used for pathological observation and determination of bacteria culture in brain homogenate.2.The model of streptococcus pneumoniae meningitis was established,the clinical manifestation was observed and the brain homogenate was taken for bacterial culture,and the colony of Streptococcus pneumoniae was counted.HE staining was used to observe the morphological changes of meninges and brain tissues.Localization and expression of P-RIP2 in brain tissue were observed by immunohistochemical technique.Western blot was used to detect the expression changing rules of NOD2 and RIP2 in brain tissue.3.64 C57BL/6 wild male mice were randomly divided into Normal saline group,Streptococcus pneumoniae meningitis group,Gefitinib group and Solvent group,16 mice in each group.The model was established by intracerebroventricular injection of Streptococcus pneumoniae bacterial suspension/normal saline and intraperitoneal injection of gefitinib/solvent.The model was reared in a cage and the clinical manifestations were observed.Animals were sacrificed 72 hours after modeling and brain tissues were taken.Immunohistochemical technique was used to observe the expression of P-RIP2 in brain tissues of each group,and the expression of NOD2/RIP2/P-RIP2/NF-?B in brain tissues were detected by Western blot.Real-time PCR was used to detect the relative expression level of target gene(NOD2 and RIP2)in brain tissues of the mice in each group.4.64 C57BL/6 wild male mice were randomly divided into Normal saline group,Streptococcus pneumoniae meningitis group,Gefitinib group and Solvent group,16 mice in each group.Animals were sacrificed 72 hours after modeling.Enzyme-linked immunosorbent assay(Elisa)was used to detect the concentration changes of IL-6 TNF-? in brain tissues.Morphological changes of meninges and brain tissues were observed by HE staining in brain tissue of mice.Double blind method was used to evaluate the clinical symptoms of mice.Results1.No matter how high or low the inoculation concentration was,all the mice in the experimental group showed the symptoms of brain injury after inoculating bacterial suspensions.All the mice in the high concentration group died 120 hours after inoculation with bacterial suspension.In the Control group,the general condition was good after anaesthesia,the mental and eating conditions were good,the recovery was fast,and there were no symptoms of brain injury.The same strain of Streptococcus pneumoniae was cultured in the brain homogenate of all the experimental groups,and the titer of bacteria was not closely related to the inoculation concentration.After the gross observation of brain tissue,in the Experimental group,the volume of brain tissue enlarged,the blood vessel congested,the gyrus obviously widened and the sulcus was shallower.The brain tissue of the Control group had no such changes.The pathological observation of HE staining showed that no inflammatory cell infiltration was found in the Control group.In the middle and high concentration groups,a little inflammatory cell infiltration appeared in the hippocampus and around the ventricle 24 hours after inoculation of bacterial suspension.Inflammatory changes in subarachnoid space were observed 72 hours after inoculation.HE staining showed the pial meningeal vasodilation and the expansion of the subarachnoid space.The neurons in the inner granular layer were swollen and showed balloon degeneration.Neutrophil infiltration was observed in the hippocampus and around the ventricle.2.The expression of NOD2/RIP2/P-RIP2 protein in cerebral cortex of saline group was relatively lower.The expression of NOD2/RIP2/P-RIP2 protein in Streptococcus pneumoniae meningitis group was significantly increased.In the early stage of infection,that is,within 24 hours after modeling,there was an increase in the rate of infection,and it showed a time-dependent change,and the most significant was at 72nd h,and increased steadily at 120th h.The level of NF-KB protein in cortex increased significantly 72 hours after stimulation by Streptococcus pneumoniae,which was significantly different from that of the model at 0 hour.P-RIP2 immunoreactive cells widely distributed in the cerebral cortex of meningitis mice,and both of the neurons and glial cells were shown to be positive.3.The expression of P-RIP2 immunoreactive cells in cerebral cortex and hippocampus of Streptococcus pneumoniae meningitis group and Solvent group was significantly higher than that in Saline group,while the P-RIP2 immunoreactive cells in Gifitinib group were significantly lower than those in Streptococcus pneumoniae meningitis group.In the Streptococcus pneumoniae meningitis group and Solvent group,the results of Western-blot showed that NOD2,RIP2,P-RIP2 and NF-?B protein in brain tissue of mice were significantly increased.Compared with the saline group,there was a significant difference.However,compared with the Streptococcus pneumoniae meningitis group,gefitinib significantly decreased the expression of P-RIP2 and the difference was statistically significant.Moreover,compared with the Streptococcus pneumoniae meningitis group,the expression of NF-? B protein was significantly lower in Gifitinib group,and there was a significant difference(P<0.05).The expression of NOD2 mRNA in cortex of the mice in Streptococcus pneumoniae meningitis group was significantly higher than that in saline group,and there was a significant difference between the two groups(P<0 05).However,there was no significant difference in the level of NOD2 mRNA between the Gifitinib group and the Streptococcus pneumoniae meningitis group(P>0.05).The expression of RIP2 mRNA in cortex of the mice in saline group was significantly higher than that of Streptococcus pneumoniae meningitis group(P<0.05),and there was a significant difference.There was no significant difference between Streptococcus pneumoniae meningitis group and Gefitinib group(P>0.05).4.The results of ELISA assay showed that the activation of NOD2-RIP2 signaling pathway could increase the secretion of cytokine IL-6,TNF-a in timeliness.That is,within 72 hours after inflammatory stimulation,there was an increasing trend.The level of IL-6 TNF-a in the group of gifitinib was significantly lower than that in the Streptococcus pneumoniae meningitis group and in the solvent group(P<0.05),but still higher than that in the saline group(P<0.05).Observation under the light microscope:In the brain tissue of mice in Streptococcus pneumoniae meningitis group and the solvent group,the pial meningeal vasodilation and the expansion of the subarachnoid space could be observed.The neurons in the inner granular layer were swollen and showed balloon degeneration.Neutrophils infiltration and necrosis were found around the pia mater and their ventricles.Compared with Streptococcus pneumoniae meningitis group,the swelling of brain tissue cells and vacuolar degeneration in gefitinib group were less than those in Streptococcus pneumoniae meningitis group and the solvent group.The tissue structure of cerebral cortex and hippocampus was basically intact,the vascular space was slightly widened,and a little inflammatory cell infiltration was observed.In the saline group,the morphology of the cells was regular and the size was the same.There was no inflammatory reaction or neuronal injury.In Streptococcus pneumoniae meningitis group and solvent group,the symptoms of brain injury such as mental depression,lethargy,rough fur,vomiting,delayed reaction,motor disorder,convulsion,angle arch retraction and other brain injury symptoms were significantly increased compared with those in Gifetini group.In the saline group,the general condition of the mice was better after anaesthesia,the mental and eating conditions were better,and the recovery was faster.None of the above symptoms appeared.Conclusions1.Infectious meningitis animal model can be successfully established by intracerebroventricular injection in C57BL/6 mice.By detecting the neuropathological changes in mice,it is more appropriate to reflect the occurrence and development of human infantile meningitis,thus providing a reliable experimental basis for the study of the mechanism of brain injury.2.Pneumococcal infection significantly increased NOD2,suggesting that NOD2 is involved in the initiation of immune response of bacterial inflammatory host.It is proved that Streptococcus pneumoniae is an effective stimulator of NOD2-RIP2 signaling pathway.That is,intracellular NOD2-RIP2 signaling pathway plays an important role in the intracellular inflammatory response of bacteria and its metabolites.3.Gefitinib can reduce the expression of RIP2 in vivo by inhibiting the phosphorylation of RIP2.That is,RIP2 was inhibited by gefitinib in vivo,suggesting that the inhibition of RIP2 phosphorylation may contribute to the protective effect of gifitinib.4.Under the inhibition of RIP2,the secretion of TNF-a and IL-6 by macrophages decreased significantly,which effectively alleviated the brain injury induced by Streptococcus pneumoniae.SignificanceThis study showed that bacterial meningitis could be induced successfully by injecting bacterial bacteria into the lateral ventricle of mice.When bacterial meningitis occurred,the expression of NOD2-RIP2 in brain tissue was significantly increased,which suggested that NOD2-RIP2-NF-?B/MAPKs signaling pathway is involved in the initiation of immune response of bacterial inflammatory host and promotes the formation and development of inflammation.Gefitinib can inhibit the phosphorylation of RIP2,reduce brain edema and protect neurons from inflammatory damage.The above results suggest that RIP2 may be a feasible pharmacological target for the regulation of inflammation.In the fure treatment of bacterial meningitis,it provides a reference for the prevention and treatment of bacterial meningitis and other central nervous system infectious diseases. |
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