Effect Of Trihexyphenidyl On The Change Of P-gp In Vitro Blood-brain Barrier After OGD Injury

Objective: To explore the effect and mechanism of trihexyphenidyl(THP)on changes of P-glycoprotein(P-gp)on the blood brain barrier(BBB)after oxygen glucose deprivation(OGD).Methods:(1)Rat brain microvascular endothelial cells(r BMECs)and astrocytes were isolated and cultured in primary cells,and r BMECs were identified by immunofluorescence staining.MTT method was used to detect the effect of OGD at different times on the survival rate of r BMECs.The co-culture system was established by Transwell to simulate the blood-brain barrier in vitro,and the transendothelial electrical resistance(TEER)of the co-culture system was monitored every day,after being stability for subsequent experiments.By measuring the apparent permeability coefficient(Papp)of fluorescein sodium(Na F)transported from the inner chamber to the outer chamber to study the effect of OGD injury r BMECs at different times on the BBB tight junction.Then the Papp values of Rhodamine123(Rh123)in the co-culture system were measured to study the effect of P-gp function after OGD injury.Western Bloting was used to detect the effect of P-gp expression after OGD damaged r BMECs at different times.Based on the above,the damage time of OGD was established.At this damage time,immunofluorescence staining was used to detect the activation of astrocytes after OGD injury.(2)MTT method was used to detect the cytotoxicity of THP on r BMECs and the influence of THP on the survival rate after r BMECs injury,and the morphological changes of the cells were observed under the microscope.Transwell system was used to study the effect of THP on the tight connection of BBB and P-gp function in vitro after r BMECs injury.Western Bloting was used to detect the effect of THP on P-gp expression after r BMECs injury.Then the effect of THP on oxidative stress related indexes ROS,SOD,CAT and GSH after r BMECs injury were detected.(3)Immunofluorescence staining was used to detect the effect of THP on Occludin,NF-?B and P-gp after r BMECs injury.Western Bloting was used to detect expression levels of tight junction-associated proteins after r BMECs injury,for example MMP-9,i NOS,Occludin,and ZO-1.At the same time,the experimental assay was also used to detect the expression levels of P-gp related proteins Akt,ERK1/2,NF-?B and its phosphorylated state molecules were detected.The interaction relationship was studied after the addition of inhibitors.Results:(1)Transwell was used to establish co-culture system between the primary r BMECs and astrocytes.It indicated that the vitro BBB model was constructed successfully.In addition,OGD 4 h could cause damage of r BMECs,destruction of BBB structure,hyperfunction of P-gp and activate astrocytes.(2)THP(0.1 ?M,1 ?M,10 ?M)pretreatment for 2 hours could decrease OGD induced r BMECs damage and repair the tight connections between cells.Also could reduce the expression level of P-gp,and reverse P-gp hyperfunction.THP pretreatment for 2 hours could reduce the ROS content in r BMECs induced by OGD.It could increase the activity of SOD,GSH and CAT enzymes,and it has obvious antioxidant effect.(3)THP could reduce the expression of tight junction related protein MMP-9,i NOS protein and up-regulate the expression of Occludin and ZO-1 protein after r BMECs injury.It could down-regulate the expression level of P-gp in r BMECs by inhibiting the expression of Akt,ERK1/2,NF-?B and its phosphorylated molecules.Conclusions: THP plays an antioxidant role by reducing ROS content in r BMECs after OGD injury,improving antioxidant enzyme activity,and then regulating P-gp changes on the blood-brain barrier in vitro.The mechanism may be related to PI3K/Akt and ERK1/2 signaling pathways.