| Objective:In this study,we established a model of SPM mice,and studied the effect of MRP8/14 protein on the expression of NLRP3/NLRC4 signaling pathway,so as to provide new experimental ideas and basis for the mechanism of SPM immune inflammation.Methods:1.The standard strain ATCC-6303 of SP-? was resuscitated overnight in 37? water and inoculated on blood agar medium.The SP suspension with a concentration of 4.5×108 CFU/ml was prepared for infection.2.In vivo tests,48 healthy male BALB/C mice aged about 6 to 8 weeks were randomly divided into 4 groups,12 mice in each group:phosphate buffer saline(PBS)group,MRP8/14 group,SP group and SP+MRP8/14 group.The lateral ventricles was located,the model of mice with SPM was established by injecting 40?l PBS and 5?l SP suspension(4.5×108 CFU/ml).The PBS group was injected with 45?l PBS,the MRP8/14 group with 40?l MRP8/14(0.5?g/?l)and 5?l PBS,the SP group with 40?l PBS and 5?l SP suspension,and the SP+MRP8/14 group with 40?l MRP8/14(0.5?g/?l)and 5?l SP suspension.3.After lateral ventricular injection,mice in each group were randomly divided into 3 groups and killed at 6h,24h and 48h.Behavioral changes and reduction of body weight were observed and recorded.The mice in each group were killed after anesthesia,half of the brain tissues were taken and HE staining was used to observe the changes of brain inflammation.The renaining brain tissue was prepared into brain homogenate specimens,after extracting total RNA,and reverse transcription,Real-time PCR was used to detect the relative expression of NLRP3,NLRC4,IL-1? and Gasdermin D mRNA in brain homogenate.Results:1.Neurobehavioral score(score(s)):Both scores of mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.The scores of mice in SP group compared with PBS group,were significantly decreased at 24h and 48h,the difference was statistically significant(P<0.05).Both scores of mice in SP+MRP 8/14 group compared with SP group,were significantly decreased at 24h and 48h,the difference was statistically significant(P<0.05).2.Reduction of body weight(gram,g):Both the reduction of body weight of mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.The reduction of body weight of mice in SP group compared with PBS group,was significantly decreased at 24h and 48h,the difference was statistically significant(P<0.05).The reduction of body weight of mice in SP+MRP8/14 group compared with SP group,was significantly decreased at 24h and 48h,the difference was statistically significant(P<0.05).3.Brain tissue HE staining:MRP8/14 group compared with PBS group,the meningeal structure of mice was normal at 6h,24h and 48h,and there were no hyperemia and inflammatory cells.SP group compared with PBS group,at 6h SP group soft meninges were slightly hyperemic,the subarachnoid space was slightly enlarged,at 24h SP group soft meningeal vascular congestion,subarachnoid expansion,and meningeal and subarachnoid space inflammatory cell infiltrated,at 48h SP group pia mater significant vascular congestion,subarachnoid significant expansion,meninges and subarachnoid with inflammatory cells infiltration.SP+MRP8/14 group compared with SP group,at 6h SP+MRP8/14 group soft meninges were significant hyperemic,the subarachnoid space was slightly enlarged with inflammatory cells infiltration,at 24h SP+MRP8/14 group soft meningeal vascular congestion and inflammatory cell infiltrated were more obvious,the structural integrity of the arachnoid structure was destroyed,at 48h SP+MRP8/14 group pia mater congestion is more obvious,the arachnoid structure is destroyed,and a large number of inflammatory cells infiltrate.4.Real-time PCR was used to detect the relative expression of NLRP3,NLRC4,IL-1?and Gasdermin D mRNA in brain homogenate:(1)Relative expression of NLRP3:Both mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.Both the relative expression of NLRP3 mRNA in SP group at 6h,24h and 48h was higher than PBS group,there was no significant difference(P>0.05).Both the relative expression of NLRP3 mRNA in SP+MRP8/14 group at 6h,24h and 48h was higher than SP group,the difference was statistically significant(P<0.05).(2)Relative expression of NLRC4:Both mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.The relative expression of NLRC4 mRNA in SP group at 6h,24h and 48h was higher than PBS group,the difference of 24h and 48h was statistical significant(P<0.05).The relative expression of NLRC4 mRNA in SP+MRP8/14 group at 6h,24h and 48h was higher than SP group,the difference was statistically significant(P<0.05).(3)Relative expression of IL-1?:Both mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.Both the relative expression of IL-1? mRNA in SP group at 6h,24h and 48h was higher than PBS group,the difference was statistically significant(P<0.05).The relative expression of IL-1? mRNA in SP+MRP8/14 group at 6h,24h and 48h was higher than SP group,the difference was statistically significant(P<0.05).(4)Relative expression of Gasdermin D:Both mice in MRP8/14 group compared with PBS group had no statistically significant difference(P>0.05)at 6h,24h,48h.Both the relative expression of Gasdermin D mRNA in SP group at 6h,24h and 48h was higher than PBS group,the difference was statistically significant(P<0.05).Both the relative expression of Gasdermin D mRNA in SP+MRP8/14 group at 6h,24h and 48h was higher than SP group,the difference was statistically significant(P<0.05).Conclusions:1.In this experiment,SP suspension was injected into lateral ventricle,the model of SPM in mice was successfully established.2.In SPM mice,the expression of inflammatory body NLRP3/NLRC4,downstream pro-inflammatory factor IL-1? and porin Gasdermin D was upregulated,which suggests that NLRP3/NLRC4 signaling may be involved in the inflammatory response of SPM.3.In SPM mice,MRP8/14 furtrther upregulated the relative expression of NLRP3/NLRC4,IL-1? and Gasdermin D mRNA,boost the immune inflammatory reaction of brain tissue,which suggests that MRP8/14 promotes the inflammatory process of SPM. |
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