The Role Of Rho/ROCK Signaling Pathway In N-Methyl-D-Aspartic Acid-Induced Destruction Of The Blood-Brain Barrier

The blood brain barrier(BBB)is a selective barrier to protect the central nervous system(CNS)from the influence of peripheral blood circulation.The pathogenesis of most neurological diseases is related to the destruction of BBB.N-methyl-D-aspartic acid(NMDA)is a glutamate analogue,which may cause changes in the permeability of cerebral vascular endothelial cells and destroy neurons by activating N-methyl-D-aspartic acid receptor(NMDAR).The excitotoxicity caused by excessive activation of NMDAR is also closely related to some neurological diseases.Studies indicated that the Rho/ROCK signaling pathway is involved in the regulation of the cytoskeleton and the permeability of the BBB.Our previous research found that the activation of Rho/ROCK induced the down-regulation of tight junction proteins and increase the permeability of the BBB.Other studies have also shown that the Rho/ROCK pathway is closely related to the NMDAR activation in neurons,but whether NMDAR has the same effect on endothelial cells is not clear.This study intends to verify the existence of NMDAR on human brain microvascular endothelium cell(HBMEC),and then explore whether HF(hydroxyfasudil,HF)can improve the permeability of BBB caused by NMDA-induced NMDAR activation.Finally,the protective effect of HF on NMDA induced BBB destruction and cortical neuron damage was verified in vivo.Part one: The role of Rho/ROCK Signaling Pathway in N-methyl-D-aspartatic acid regulating the permeability of human brain microvascular endothelial cellObjective: To explore the role of Rho/ROCK signaling pathway in N-methyl-D-aspartic acid(NMDA)-mediated the increase of permeability in human brain microvascular endothelial cell (HBMEC) and the destructions of the blood brain barrier (BBB).?Methods: In this study,HBMEC were exposed to different concentrations of N-methyl-D-aspartic acid receptor(N-methyl-D-aspartic acid receptor,NMDAR)agonist NMDA and Rho/ROCK signaling pathway inhibitor hydroxyfasudil(HF)for 24 hours.The Cell Counting Kit-8(CCK-8)was used to detect the effects of different drugs on cell viability,and immunofluorescence(IF)was used to detect the expression of NMDAR1 on HBMEC.With the cells treated with NMDA or HF for 24 hours respectively after being pretreated with MK801 or HF for 2 hours,the expression of protein and m RNA of tight junction proteins(occludin and claudin5)and targets myosin phosphatase 1 subunit(MYPT)-1 phosphorylation were evaluated by western blot(WB)and real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)respectively.2’,7’-Dichlorofluorescein yellow diacetate(DCFH-DA)was used as a fluorescent probe to detect the level of reactive oxygen species(ROS)in cells,and cell apoptosis were assayed by flow cytometry.In vitro BBB model,the Millicell-ERS cell resistance meter was used to measure the transendothelial electrical resistance(TEER)and permeability of BBB also evaluated by sodium fluorescein(SF).Meanwhile,the distribution and expression of cytoskeleton F-actin were detected by phalloidin staining.Results:1.NMDAR1 was expressed in HBMEC,and NMDA activated the expression of NMDAR1 in HBMEC while MK801 inhibited this activation.2.The cell viability were significantly reduced by expose with 10mmol/L NMDA for 24h(P<0.05),while 20μmol/L HF treatment for 24 h has no significant effect on the survival rate of HBMEC(P>0.05).Additionally,HF had a protection against the decreased of cell viability induced by NMDA.3.In the NMDA group,the expression levels of occludin and claudin5protein(P<0.01,P<0.001)and m RNA(P<0.01,P<0.01)were significantly decreased,the apoptosis rate and ROS level in the cells were increased(P<0.001).Pretreatment with HF or MK801 can significantly increased the expression of occludin and claduin5(P<0.05/P<0.05,P<0.05/P<0.05)and m RNA(P<0.001/P<0.05,P<0.001/P<0.01),as well as reduced the rate of cell apoptosis and the production of reactive oxygen species in the cell.At the same time,the expression level of p-MYPT1 protein(P<0.01)was increased in the NMDA group,while pretreatment with HF or MK801 down-regulated the protein level(P<0.05,P<0.05).4.The TEER was decreased(P<0.01)and SF permeability was increased(P<0.001)in the NMDA group.While pretreated with HF or MK801 increased TEER(P<0.05,P<0.01),reduced SF permeability(P<0.05,P<0.001)and prevented F-actin remodeling of cytoskeleton.Conclusions:1.NMDAR1 are expressed in HBMEC and activated by NMDA.2.The Rho/ROCK signaling pathway plays an important role in NMDA regulating the permeability of human brain microvascular endothelial cell.Part two: HF prevents blood-brain barrier dysfunction and neuron damage induced by NMDA in miceObjective: To explore whether HF may attenuate NMDA induced-dysfunction of the BBB and neurons in mice.Methods : 24 male C57BL/6 and 24 male Apo E-/-mice with ear tag numbers were randomly divided into 3 groups: control group,NMDA group and HF+ NMDA group with 8 animals in each group.In the control group,100μl0.9% sterile saline was injected into the tail vein after intraperitoneal injection of100μl 0.9% sterile saline for 1 hour.In the NMDA group,100μl NMDA50mg/kg was injected into the tail vein after intraperitoneal injection of 100μl0.9% sterile saline for 1 hour.In the HF+NMDA group,100μl NMDA 50mg/kg was injected into the tail vein after intraperitoneal injection of 100μl HF30mg/kg for 1 hour.The above treatments were applied daily for 3 consecutive days,and the behavior and general conditions of the mice were observed.All mice were sacrificed 24 hours after the last administration,and the mouse brains were extracted.WB and q RT-PCR were performed to detect the expression of occludin and claudin5 proteins and m RNA in the cerebral cortex of mice,respectively.Nissl staining,HE staining and Neu N immunohistochemistry were used to evaluate the damage of cerebral cortex neurons in mice.Results:1.After injection of NMDA into tail vein,stereotypic behaviors such as curling,freezing,or hyperactivity were observed in C57BL/6 mice.Among them,grooming and tail licking/tail biting behaviors were particularly noticeable.After injection of NMDA into tail vein,Apo E-/-mice developed epileptic symptoms such as hyperactivity,front-grab movement,tail-lifting or clonus.However,intraperitoneal injection of HF didn’t significantly reduce the severity of seizures.2.The absence of BBB tight junction destruction and cerebral cortical neuron damage in C57BL/6 mice.However,In Apo E-/-mice,compared with the control group,the expression levels of occludin and claudin5 protein(P<0.01,P<0.01)and m RNA(P<0.05,P<0.05)were significantly decreased in the NMDA group;compared with NMDA group,the expression levels of occludin and claudin5 protein(P<0.05,p <0.05)and m RNA(P<0.05,P<0.05)were significantly up-regulated in the HF+NMDA group.Nissl staining showed that compared with the control group,the cerebral cortex neurons in the NMDA group were arranged disorderly and sparsely,with nuclear pyknosis,and the number of Nissl bodies were decreased(P<0.01).Compared with the NMDA group,the number of cerebral cortex neurons were increased in the HF+NMDA group(P<0.05).HE staining showed that compared with the control group,the NMDA group had fewer neurons and more nucleus pyknosis(P<0.01)in the cerebral cortex.Compared with the NMDA group,the number of neurons in the cerebral cortex were increased and the nucleus pyknosis were decreased in the HF+NMDA group(P<0.05).Neu N immunohistochemistry showed that compared with the control group,the number of positive cells were decreased in the NMDA group(P<0.01).Compared with NMDA group,the number of positive cells were increased in the HF+NMDA group(P<0.05).Conclusion:HF may reverse NMDA induced-dysfuction of BBB and cerebral cortex neurons.