The Role Of Soluble Epoxide Hydrolase Inhibitor In Brain Secondary Damage After Cerebral Hemorrhage In Mice

Objective:Soluble epoxide hydrolase(sEH)is the key enzyme of CYP-EETs signal pathway.TPPU is an inhibitor of sEH.In this paper,sEH is used as the regulatory target to explore the effect and underlying mechanism of TPPU-administration on the secondary injury after cerebral hemorrhage.The aim of this study is to explore the potential of sEHinhibitor in the treatment of cerebral hemorrhage.Methods: Adult male C57BL/6 mice were used to establish the intracerebral hemorrhage(ICH)model by injecting collagenase into basal ganglia.The mice were injected with TPPU per day from the first day after modeling until sacrificed.Immunofluorescence staining,Western blot,TUNEL and Neu N double labeling and RT-PCR were used to detect the expression of sEH and its dynamic changes after ICH and the damage of the blood-brain barrier(BBB),neuron apoptosis,microglia activation and polarization,astrocytes functional and phenotypic changes,peripheral neutrophils,related inflammatory proteins and inflammatory factors,and the expression of related signaling pathway proteins.The method of wet/dry was used to evaluate the water content of brain tissue.Evans blue dye(EB)was injected through the tail vein after ICH to assess the permeability of the BBB.Hematoma volume was measured by hematoxylin staining(HE).The forelimb placement test and modified Neurological Severity Scores(m NSS)were used to evaluate the neurological injury and recovery in mice.Results: ICH model of basal ganglia in mice was successful established.After ICH,the expression of total protein of sEH increased significantly,accompanied by neuron apoptosis,BBB damage,microglia activation,astrocytes activation,peripheral neutrophil infiltration,and the secretion of related inflammatory factors.The administration of TPPU can not only decrease neuron apoptosis,brain edema,the expression of related inflammatory proteins and inflammatory factors,the infiltration of peripheral neutrophils,but also alleviate the damage of BBB,reduce microglia activation and promote its transformation to M2,inhibit astrocytes activation and promote its transformation to A2,and promote the recovery of nerve function.PI3K/Akt/NF-κB signal pathway mediated the protective effect of TPPU on secondary injury of ICH in miceConclusion:sEH-inhibitor TPPU could play an overall neuroprotective role in secondary injury by inhibiting the inflammatory response and promoting the recovery of neurological function after ICH in mice.In conclusion,sEH is one potential therapeutic target for ICH.Part One Protective effect of soluble epoxide hydrolase inhibitor TPPU on secondary injury after intracerebral hemorrhageObjective:To investigate the cellular expression of epoxide hydrolase(sEH)in the brain and the dynamic change in tissues around the hematoma at different time points after ICH.To explore the effects of TPPU on blood-brain barrier damage,neuron death,glia scar formation and neurological function recovery in mice after ICH.Methods: The basal ganglia hemorrhage model(ICH)was performed by stereotactic injection VII collagenase in adult male C57/BL6 mice.The mice were randomly divided into Sham group,Vehicle group and TPPU treatment group.TPPU(0.3 mg/kg)or 1 % DMSO was given to mice intraperitoneally 0.5 hour before modeling,and once a day after modeling until sacrificed.Western blotting(WB)and Immunofluorescence double standard(IF)were used to observe the dynamic alteration of the expression of sEH at different time points after ICH and detect the effect of TPPU,an inhibitor of sEH,on the integrity of blood-brain barrier(BBB),neuron apoptosis,and glial scar formation after ICH injury.To assess the permeability of the BBB,evans blue dye was injected through the tail vein after ICH.Hematoxylin staining(HE)was used to measure the hematoma volume.The modified Neurological Severity Scores(m NSS)and forelimb placement test(FP)were used to assess the recovery of nerve function impairment after ICH in mice.Results: ICH model of basal ganglia in mice was successfully established.TTC staining showed significant hematoma was formed in basal ganglia in Vehicle group.The m NSS behavioral score showed the scores of Vehicle group was higher than the Sham group.FP behavioral score showed that the Vehicle group had lower scores than the Sham group,indicating a loss of neurological function in ICH.Immunofluorescence double labeling showed that the expression of sEH in the basal ganglia of mice was mainly in the synapses,cytoplasm of vascular endothelial cells and astrocytes,and there was also a small amount of colocalization expression with microglia and neuron.The expression level of total protein of sEH increased dramatically after ICH injury.TPPU treatment improved the integrity and permeability of BBB,reduced the water content of brain tissue,eased the hematoma volume,and glial scar formation after ICH.TUNEL and Neu N immunofluorescence double labeling showed that TPPU could reduce the neuron apoptosis and promote the recovery of neural function.Conclusion: sEH were widely expressed in vascular endothelial cells,astrocytes,and a small part of microglia and neuron in healthy animals.However,the expression of sEH in perihematomal tissue increased significantly after ICH.TPPU could play an overall protective role after ICH,alleviate the damage of the BBB,reduce neuron apoptosis,ease glial scar formation and improve nerve function defect.Part Two Effect of soluble epoxide hydrolase inhibitor TPPU on the inflammatory response of perihematomal tissue after ICHObjective: To explore the effects of TPPU,an inhibitor of sEH,on inflammatory cells and inflammatory factors expression after intracerebral hemorrhage in mice.Methods: The ICH model of C57BL/6J mice was established.TPPU was injected intraperitoneally.The mice were randomly divided into Sham group,Vehicle group,TPPU treatment group.TPPU(0.3 mg/kg)or 1 % DMSO was given to mice intraperitoneally once 0.5 hour before modeling,and once a day after modeling until sacrificed.IF and WB were applied to detect the effect of TPPU on the activation of microglia,astrocytes,neutrophil infiltration and the change of expression of matrix metalloproteinase-9(MMP-9)around the hematoma at 1,3 days after operation.Results: IF results demonstrated that the microglia in the perihematomal tissue were activated.The cell morphology changed from branching to amebic.Then the cell body became round,the protuberance became shorter and the number increased considerably.Astrocytes were also significantly activated characterized by hypertrophic processes and intense immunoreactivity of GFAP and their number increased significantly.TPPU treatment significantly decreased the number of microglia activation and astrocytes activation.WB results demonstrated that after TPPU treatment,the protein expression levels of IL-1β and TNF-α dramatically decreased.TPPU treatment can markedly reduce the number of MPO and the expression of MMP-9 in the surrounding tissues of hematoma.Conclusion: The sEH-inhibitor can suppress the inflammatory response,the activation of microglia,astrocytes and the infiltration of neutrophils and decrease expression of inflammatory-related factors and matrix metalloproteinase-9.Part Three Effect of TPPU on phenotypic transformation of microglia and astrocytes after ICH and preliminary exploration of the related mechanismsObjective: To investigate the effect of sEH inhibitor TPPU on the phenotypic transformation of microglia and astrocytes after intracerebral hemorrhage(ICH)in mice and the underlying mechanisms of protective effects.Methods: The ICH model of C57BL/6J mice was established.The mice were randomly divided into Sham group,Vehicle group,TPPU treatment group,and TPPU+LY294002(TPPU+LY).TPPU(0.3 mg/kg)or 1 % DMSO was given to mice intraperitoneally once 0.5 hour before modeling,and once a day after modeling until sacrificed.PI3 K specific inhibitor LY294002 was injected intracerebroventricularly before modeling.IF and WB were used to detect the effect of TPPU on the phenotype transformation of microglia and astrocytes around the hematoma after ICH.Immunofluorescence double labeling,WB,and RT-PCR were used to detect the effects of inhibition of PI3 K pathway on the phenotypic transformation of microglia,astrocytes and neuronal death.Results: The mouse ICH model was successfully established.Around the hematoma area,M1 and M2 cells both increased,and M1 increased more significantly.The expression of A1 and A2 reactive astrocytes was also increased.The expression of phosphorylated PI3 K and Akt decreased while total NF-κB protein increased.After the exogenous sEH-inhibitor TPPU was given,the proportion of M1 microglia decreased while M2 microglia increased significantly.A1 reactive astrocytes decreased and A2 reactive astrocytes increased.The phosphorylation PI3 K and AKT protein obviously increased while the total NF-κB protein decreased significantly.PI3 K inhibitor treatment reversed the effects of TPPU on microglia and astrocytes phenotypic changes and neuronal death.The result was confirmed by IF,WB and RT-PCR.Conclusion: The sEH-inhibitor TPPU could induce the transformation of microglia from M1 to M2 phenotype,the transformation of astrocytes from A1 to A2,reduce the neuronal death which may be mediated through modulating the PI3K/Akt/NF-κB pathway.