The Role Of NHE1 In Early Brain Injury After Experimental Subarachnoid Hemorrhage And Its Mechanism

Part Ⅰ:The expression and interaction of NHE1 and CHP1 in the brain of rats after subarachnoid hemorrhageObjectiveTo investigate the expression of transmembrane transport glycoprotein NHE1 and calcineurin B homologous protein CHP1 in brain tissue after subarachnoid hemorrhage(SAH)and the relationship with early brain injury(EBI).The changes of NHE1 and CHP1 interaction after subarachnoid hemorrhage were also observed.Methods1、Animals groups:72 healthy adult male Sprgue-Dawley(SD)rats were randomly divided into 6 groups of 12 rats in each,a sham group and 5 SAH groups arranged by time:2,12,24,48 and 72 hours after SAH.2、Animal model preparation:A rat SAH model was established by injection of fresh arterial nonheparinized blood into the prechiasm.According to the experimental grouping,the rats were sacrificed at various time points after modeling,the brain was perfused,and the cerebral tissue samples were taken for analysis.3、Detection methods and indicators:Western-blot(WB)and immunofluorescence were used to detect the changes of NHE1 and CHP1 protein levels in brain tissue at diferent time points after SAH,and co-immunoprecipitation(Co-IP)was applied to detect the changes of interaction between NHE1 and CHP1 in brain tissue after SAH.Results1、The western-blot analysis showed that the expression of NHE1 and CHP1 in brain tissue increased 2 hours after subarachnoid hemorrhage in rats and peaked at 24 hours after SAH(P<0.05).2、The Co-IP results showed that the NHE1 and CHP1 interaction was significantly enhanced 24 hours after subarachnoid hemorrhage in rats compared with the sham group(P<0.05).3、The double immunofluorescence staining results demonstrated that the expression trends and interactions of NHE1 and CHP1 in rat brain tissue were similar to those of Western-blot analysis.Conclusion1、NHE1 and CHP1 may participate in the pathological process of early brain injury after subarachnoid hemorrhage through enhanced interaction.2、The next most appropriate time point for studying the role of NHE1 and CHP1 in early brain injury after subarachnoid hemorrhage may be 24 hours after SAH.Pare Ⅱ Experimental study of the effect of intervention in the regulation of NHE1 on EBI after SAH and its possible mechanismObjectiveTo investigate whether the regulation of NHE1 has an intervention effect on early brain injury after SAH in rats and its possible mechanism.Methods1、Animal groups:144 healthy adult male SD rats were randomly divided into 8 groups:sham group,SAH group,SAH+HOE642 vehicle group,SAH+HOE642 group,SAH+SiNHE1 control group,SAH+SiNHEl group,SAH+ rhNHE1 control group,SAH+rhNHE1 group,18 rats in each group.2、Animal model preparation:SAH model was established by injecting autologous blood into the prechiasmatic cistern.3、Drug administration:In the SAH+HOE642 group,the HOE642 is injected intravenously in rats with a dose of 15 mg/kg at 20 min before induction of SAH.The SAH+HOE642 vehicle group was intravenously infused with an equal amount of sterile saline 20 minutes before the rat model induction.In the SAH+SiNHE1 control group and the SAH+SiNHE1 group,100 pmol/μl of NHE1 siRNA or ControlsiRNA was injected into the intracerebroventricularly under the guidance of a stereotactic apparatus at 24 hours before the model establishment.In the SAH+ rhNHEl control group and the SAH+rhNHEl group,15 μl of transfected rhNHEl or Vector plasmid was injected into the intracerebroventricularly under the guidance of a stereotactic instrument 24 hours before the rat model established.4、Detection methods and indicators:Each group of rats was evaluated for their behavioral ability by a behavioral score table 24 hours after SAH,and then the rats were sacrificed.For 6 rats,the total coronal sections containing temporal base tissues is obtained for immunofluorescence,FJB,Nissl,and TUNEL stainings.The underlying temporal base brain tissues of other 6 rats are collected and used in western blot analysis,immunoprecipitation analysis,ROS assay,and blood brain barrier(BBB)permeability.The last 6 rats in each group are sacrificed for brain edema test.Results1、Compared with the Sham group:the expression of NHE1 in brain tissue was significantly increased(P<0.05),and the interaction between NHE1 and CHP1 was significantly enhanced(P<0.05);neurobehavioral damage was aggravated 24h after SAH(P<0.05);brain edema and brain tissue albumin content increased significantly(P<0.05);inflammatory factors TNF-α,IL-1β levels increased significantly(P<0.05);ROS and LDH increased significantly(P<0.05);TUNEL and FJB positive cells increased significantly(P<0.05);Niss body is reduced(P<0.05).2、Compared with the Sham group:after inhibition of NHE1 by HOE642 or small interfering RNA,the expression of NHE1 in brain tissue and its interaction with CHP1 were significantly decreased(P<0.05);rats neurobehavioral damage relief(P<0.05);the cerebral edema and brain tissue albumin content decreased(P<0.05);the inflammatory factors TNF-α,IL-1β levels decreased significantly(P<0.05);ROS and LDH reduced significantly(P<0.05);TUNEL and FJB positive cells decreased significantly(P<0.05);Niss body increased(P<0.05).3、After intervention with human recombinant NHE1,the opposite effects were obserbved:the expression of NHE1 in brain tissue and its interaction with CHP1 were significantly increased(P<0.05);rats neurobehavioral damage aggravated(P<0.05);the cerebral edema and brain tissue albumin content raised(P<0.05);the inflammatory factors TNF-α,IL-1β levels increased significantly(P<0.05);ROS and LDH raised significantly(P<0.05);TUNEL and FJB positive cells increased significantly(P<0.05);Niss body decreased(P<0.05)..4、Immunofluorescence staining results also showed that compared with SAH control group,inhibition of NHE1 with HOE642 or NHE1 siRNA intervention reduced the expression of NHE1 in brain tissue(p<0.05).After intervention with rhNHE1,The expression of NHE1 in neurons was significantly increased(p<0.05).Conclusion1、After SAH,NHE1 in brain tissue and neurons promotes brain damage by enhancing interaction with CHP1,and participates in the pathological process of early brain injury after SAH.2、HOE642 inhibits NHE1 or siNHE1 intervention has a protective effect on early brain injury after SAH.3、Human recombination NHE1 aggravated early brain damage after SAH.4、NHE1 may be a new target for the treatment of SAH.Part Ⅲ:NHE1 participates in brain injury after subarachnoid hemorrhage by promoting neuronal apoptosis in vitroObjectiveTo investigate the role of NHE1 in neuronal apoptosis after SAH in vitroMethods1、SAH in vitro model preparation and grouping:the primary cultured neurons treated with OxyHb are used to mimic SAH in vitro.Primary cortical neurons are divided into 4 groups as below:control group,OxyHb group,OxyHb+Vehicle group and OxyHb+HOE642 group.2、Administration method:The final concentration of HOE642 was adjusted to 1μM.Cultured neurons were pretreated with HOE642 for 2 hours at 37 ℃,then thoroughly rinsed with PBS,and fresh medium was added and incubated with OxyHb(5 μM)for an additional 24 hours at 37℃.3、Detection index:Western blot(WB)method was used to detect the NHE1 protein levels in cultured neurons and the interaction changes after OxyHb and HOE642 treatment between NHE1 and CHP1 was detected by Co-Immunoprecipitation(Co-IP).Neuronal apoptosis was detected by Annexin V and PI staining.Result1、Compared with the Control group:after treatment with OxyHb for 24 hours,the expression of NHE1 in neurons was significantly increased(P<0.05),the interaction between NHE1 and CHP1 was enhanced,and neuronal apoptosis was significantly increased(P<0.05).2、Compared with OxyHb group:after inhibition of NHE1 by HOE642,the expression of NHE1 in neurons was significantly decreased(P<0.05),the interaction between NHE1 and CHP1 was weakened,and neuronal apoptosis was significantly decreased(P<0.05).Conclusion1、In vitro,treated the cultured neurons with OxyHb,the expression of NHE1 was increased,and neuronal apoptosis was promoted by enhancing the interaction with CHP1.2、Inhibition of NHE1 with HOE642 has a protective effect on neuronal apoptosis in SAH vitro model.