Experiment 1 Effect And Mechanism Of Poldip2 Mediates The Loss Of Aqp4 Polarity In Mice Brains With Bacterial Meningitis Experiment 2 Resveratrol Protects Cortical Neurons In Infant Rat Brains With Bacterial Meningitis Through MiR-223-3p/NLRP3 Pathway

Objectives: This study aimed to investigate the effect and mechanism of ?-interacting protein 2(Poldip2)mediates the loss of aquaporin-4(AQP4)polarity and this leads to the disruption of blood–brain barrier(BBB)and cerebral edema formation in mouse bacterial meningitis(BM)model.Methods: Mouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern,Small interfering ribonucleic acid(siRNA)targetting Poldip2 was administered by intracerebroventricular(i.c.v)injection at 48 h before BM induction.Recombinant human Poldip2(rh-Poldip2)was administered intranasally at 1 h after BM induction.A specific inhibitor of matrix metalloproteinases(MMPs),UK383367,was administered intravenously at 0.5 h before BM induction.Loeffler scoring method was used to assess the neurobehavioral functions.Dry-wet weight method was used to detect the brain water content.Transmission electron microscopy(TEM)wasused to detect the astrocytic swelling and tight junctions.Evans blue(EB)was used to detect the BBB permeability.Double immunofluorescence was used to detect the expression of AQP4 polarity.Western blot was used to detect the expression level of Poldip2,MMPs,?-dystroglycan(?-DG),zonula occludens-1(ZO-1),Occludin,claudin-5and glial fibrillary acidic protein(GFAP).gelatin zymography assay was used to detect the activity of MMPs.Results: Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model.Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes,alleviated brain edema,preserved the integrity of BBB,and relieved the loss of AQP4 polarity in BM model.Rh-Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein(GFAP)and downregulated the expression of ?-dystroglycan(?-DG),zonula occludens-1(ZO-1),occludin,and claudin-5;whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP,and upregulated ?-DG,ZO-1,occludin,and claudin-5.UK383367 downregulated the expression of GFAP and upregulated the expression of?-DG,ZO-1,occludin,and claudin-5.Conclusions: Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model.Poldip2 mediates the loss of AQP4 polarity via MMPs/?-DG pathway.Poldip2 inhibition relieved the loss of AQP4 polarity,alleviated brain edema and preserved the integrity of BBB.Objectives: To investigate the protective effect of resveratrol on neurons in rat bacterial meningitis(BM)model.Methods: Group B hemolytic Streptococci was injected via the posterior cistern to establish a BM model.Resveratrol was administered intranasally and mi R-223-3p antagomir was administered by intracerebroventricular(i.c.v)injection.HE staining was used to observe the pathological changes of brain tissue.Loeffler scoring method was used to evaluate the neurobehavioral functions.TUNEL staining was used to detect neuronal apoptosis.Immunofluorescence staining was used to detect the expression of IL-1?,IL-18,glial fibrillary acidic protein(GFAP)and ionized calcium-binding adaptor molecule 1(Iba1).Western blot was used to detect the protein expression levels of NLRP3,CC1,IL-1? and IL-18.Real time quantitative PCR was used to detect the expression level of mi R-223-3p.Online software Target Scan was used to search for the mi RNA that might target to NLRP3 m RNA.Results: Compared with sham group,after BM induction,the thickness of meninges in BM model was increased;the neurological score was decreased(P<0.05);the number of TUNEL positive neurons was increased significantly(P<0.05);astrocytes and microglial cells were activated;the fluorescence intensity of IL-1? and IL-18 increased(P<0.05);the expression levels of NLRP3,CC1,IL-1?,IL-18 and mi R-223-3p were increased(P<0.05).Compared with BM group,After treating with resveratrol,the neurological score was increased(P<0.05),the number of TUNEL positive neurons was decreased significantly(P<0.05),the inflammatory reaction of astrocytes and microglial cells were suppressed,the fluorescence intensity of IL-1? and IL-18 was decreased(P<0.05),the expression levels of NLRP3,CC1,IL-1? and IL-18 were decreased(P<0.05),and the expression level of mi R-223-3p was increased in resveratrol treatment group(P<0.05).A nucleotide sequence in the m RNA 3' UTR of NLRP3 might be targeted by mi R-223-3p.In the brain of rat BM model,compared with antagomir control group,the expression level of NLRP3 was increased in mi R-223-3p antagomir treatment group under the condition of treating with resveratrol(P<0.05).Conclusions: Resveratrol may play a protective role on neurons through mi R-223-3P/NLRP3 pathway in BM model of infant rats to reduce the inflammatory death of neurons.