Protective Effect Of Trihexyphenidyl On Blood-brain Barrier In MCAO Rats

Objective: To explore the effect and mechanism of Trihexyphenidyl(THP)on the changes of Occludin on the blood-brain barrier(BBB)in middle cerebral artery occlusion(MCAO)rats.Methods:(1)250g-280 g male SD rats were selected,and the MCAO rat model was established by the modified Longa thread occlusion method;Using the neurological Longa score to judge the degree of neurological deficit,SD rats with Longa score ≥ 2 were selected for subsequent experiments;THP was injected intraperitoneally 30 min before operation,8 h after MCAO,the neurological function of rats was evaluated by the modified Neurological Severity Scores(m NSS)system.Stained with2,3,5-triphenyltetrazolium chloride(TTC)and counted the volume of cerebral infarction;Using immunofluorescence to identify rat brain microvascular endothelial cells(r BMECs);MTT assay detected the cytotoxicity of THP on r BMECs and the effect of THP on the survival rate of r BMECs after Oxygen glucose deprivation(OGD)injury.(2)Fluorescein sodium(Na F)was used to detect the permeability of BBB in MCAO rats;Rhodamine 123(Rh123)was used to detecte the activity of P-glycoprotein(P-gp);The content of reactive oxygen species(ROS)in rat brain tissue was detected by DCFH-DA;r BMECs were co-cultured with astrocytes to establish an in vitro BBB model.In addition,the transendothelial electrical resistance(TEER)was measured every day;The effect of THP on BBB permeability was investigated by measuring the apparent permeability coefficient(Papp)of Na F diffusion from the inner chamber to the outer chamber and from the outer chamber to the inner chamber;The effect of THP on P-gp was investigated by measuring the Papp of Rh123 transport from the inner compartment to the outer compartment and from the outer compartment to the inner compartment;Immunofluorescence staining to detect the effect of OGD injury on astrocytes;ROS content in r BMECs was measured by flow cytometry.(3)The effects of THP on the changes of S1PR2,CRHR1,Erk1/2,p38,c PLA2,MMP-9,Occludin and phosphorylated molecules on cerebral microvessels in MCAO rats were detected by immunofluorescence.(4)The effects of THP and inhibitors on the changes of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2,MMP-9 and Occludin after OGD injury were detected by immunofluorescence and Western blot.Results:(1)The results of the m NSS neurological assessment showed that THP significantly reduced the m NSS score at the dose of 1 mg/kg;Compared with the model group,THP significantly reduced the volume of brain infarction in MCAO rats at the dose of 0.75 mg/kg and 1 mg/kg;The results of factor VIII immunofluorescence staining showed that r BMECs were successfully extracted and isolated;THP had no obvious cytotoxicity to r BMECs;Both NAC and THP increased the survival rate of r BMECs after OGD injury,and the optimal concentration of THP was 1 μM.(2)At doses of 0.75 mg/kg and 1 mg/kg,THP significantly reduced the accumulation of Na F,Rh123 in the ventricle and the level of ROS in brain tissue;The TEER value reached a steady state,which proved that the in vitro BBB model was successfully established;OGD damaged the integrity of the BBB,resulting in a decrease in TEER value,while NAC and THP protected the integrity of the BBB and increased the TEER value;THP decreased the Papp value of Na F from the inner compartment to the outer compartment and from the outer compartment to the inner compartment after OGD injury,reduced the transport of Rh123 from the inner compartment to the outer compartment after OGD injury,and improved the transport of Rh123 from the outer compartment to the inner compartment after OGD injury,indicating that THP reduced the permeability of the BBB and protected the integrity of the BBB;The red fluorescence of astrocytes was enhanced after OGD injury,indicating that astrocytes were activated.OGD injury caused an increase in intracellular ROS levels,and NAC,THP 1 μM,and THP 10 μM pretreatment groups significantly reduced ROS levels after OGD injury.(3)Immunofluorescence results showed that the expression of Occludin was negatively correlated with the activation of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2 and MMP-9.the expression of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2,MMP-9 in the model group were higher than those in sham group,but the expression level of Occludin in the model group was lower than that in the sham group.Compared with the model group,THP inhibited the expression of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2,MMP-9,and promoted the expression of Occludin.The expressions of Erk1/2,p38 and c PLA2 had no significant changes in each group.(4)The effects of THP on the expression of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2,MMP-9 and Occludin on r BMECs after OGD injury were explored by immunofluorescence and Western blot.The results showed that the expression level of Occludin was negatively correlated with the activation of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2 and MMP-9;Compared with the model group,the pretreatment group significantly up-regulated the expression of Occludin.The expression levels of S1PR2,CRHR1,P-Erk1/2,P-p38,P-c PLA2 and MMP-9 were all up-regulated after OGD injury,but this phenomenon was reversed by NAC and THP.By inhibiting the expression of S1PR2,CRHR1,Erk1/2,p38,c PLA2,MMP-9 and phosphorylated molecules,the expression level of Occludin on r BMECs was increased.Conclusions:(1)THP protects MCAO rats by reducing the over-produced ROS in the brain tissue,inhibiting the activation and phosphorylation of S1PR2,CRHR1,Erk1/2,p38,c PLA2,MMP-9,up-regulating the expression of Occludin and reducing the permeability of the BBB.(2)THP increased the expression of Occludin on the BBB by reducing the level of ROS in r BMECs and decreased the permeability of the BBB.The mechanism may be related to the S1PR2/c PLA2/Occludin pathway.