Studyonthemechanismunderlyingtheeffectsof Oleuropeinondecreasingthe Bbbpermeabilityinarat Modelof IHC

Part1 Oleuropein decreased the disruption of BBB via inhibitingthe oxidative stress reaction in a rat model of ICHObjectiveThe mNSS score,brain water content(BWC),permeability of the BBB,intensity of oxidative stress reaction,tight junction proteins ZO-1 and Occludin,and expression of ERK,p38,JNK protein were determined to observe if the oleuropein could decrease the blood-brain-barrier(BBB)permeability and secondary brain damages via inhibiting the intensity of oxidative stress in a rat model of intracerebral hemorrhage(ICH).MethodsFourty-eight rats were selected randomly for the present study.The animals were divided randomly into the following groups:sham-operated group,vehicle-treated group(saline treated group),and oleuropein-treated group.An ICH model was established by injection of collagenase Ⅶ into the basal ganglia of the rats’ brain.While the sham-operated group was only received injection of the same amount of saline into the brain.The oleuropein-treated group was subsequently divided into four subgroups with dosages ranged from 20,40,60,80mg/kg(each group included 8 rats).The modified neurological severity score(mNSS)was evaluated at 72 h after the ICH model was established successfully.Twenty minutes after the mNSS was determined,the rats were executed and the brain was removed for measuring the BWC,the BBB permeability,the intensity of oxidative stress reaction.The expression of ZO-1 and Occludin were determined by western blot and RT-PCR.The proteins of the ERK,p38 as well as the JNK were also detected by western blot.ResultsCompared with the sham-operated group,an increase of mNSS,BWC,BBB permeability,intensity of oxidative stress(ROS,MDA)and the upstream proteins such as ERK,p38,JNK in each group was observed,while the indexes of antioxidation(SOD,GSH-Px),ZO-1 and Occludin decreased significantly.Compared with the vehicle-treated group(saline treated),the oleuropein-treated group with a dosage of 20mg/kg showed no significant changes in all outcomes.However,the mNSS,BWC,permeability of BBB,intensity of oxidative stress(ROS,MDA),the upstream relative proteins(ERK,p38,JNK)decreased remarkably,and the indexes of antioxidation(SOD,GSH-Px),the ZO-1 and Occludin were significantly increased in oleuropein-treated groups with a dosage of 40-80mg/kg respectively.A significant difference in all the measurements was noted among all the oleuropein-treated subgroups.Conclusions1 Oleuropein could inhibit the oxidative stress reaction and decrease the disruption of BBB in a rat model of ICH,thereby reducing the secondary brain injury.The therapeutic effect increases as the dosages of oleuropein increase in a certain range.2 In a rat model of ICH,the expression of ERK,p38 and JNK increased,the oxidative stress also increased.The oleuropein could inhibit the oxidative stress reaction,and down regulate the expression of ERK,p38 and JNK.Part2 Possible mechanisms underlying the increase of BBBpermeability induced by oxidative stress reaction in a rat model ofICHObjectiveTo determine the roles of the MAPK(JNK,p38,ERK)pathway in the secondary brain injury and BBB disruption induced by oxidative stress reaction.To observe changes of the mNSS score,the BWC,the BBB permeability,the intensity of oxidative stress reaction,the tight junction proteins ZO-1 and Occludin after administering specific blockade or agonist of MAPK pathway.MethodsNinty-six heathy rats were used in the present study.The animals were divided randomly into the following groups:a sham-operated group,a vehicle-treated group(saline treated),an ERK agonist group,a P38 agonist group,a JNK agonist group,an agonist of the ERK+ p38 group,an agonist of the ERK+p38 +JNK group,an ERK blocker group,a p38 blocker group,a JNK blocker group,a blocker of ERK+p38 group,a blocker of ERK+p38+ JNK group.Each group included 8 rats.An ICH model was established by injection of collagenase Ⅶ into the basal ganglia of the rats’ brain,while the sham-operated group only received injection of the same amount of saline into the brain.The modified neurological severity score(mNSS)in each group was evaluated at 72 h after the ICH model was established successfully.Twenty minutes after determing the mNSS,the animals were executed and the brain was taken out for measuring the BWC,the permeability of BBB,the ZO-1 and Occludin.ResultsCompared with the sham-operated group,the mNSS,the BWC,the BBB permeability in each ICH group increased remarkably,while the expression of ZO-1 and Occludin decreased significantly.Compared with the vehicle-treated group(saline treated),the agonists of the ERK,p38,JNK singnal patway used alone or by combination significantly increased the mNSS,the BWC,BBB permeability,and decreased the expresson of the ZO-1 and Occludin.The combined use of the agonists showed stronger effects than the they were used alone.In all blocker groups,the mNSS score,the BWC,the BBB permeability significantly decreased.The combined use of the blockers decreased the abovementioned indexes remarkably compared with those in the groups with the blockers used alone.Similar results in the changes of the ZO-1 and Occludin were also obtained.Conclusions1.ERK,p38 and JNK blockers could decrease the BBB permeability in the perihematomal brain tissues.The ERK,p38 and JNK agonists displayed contrary effects.3.MAPK(ERK,p38,JNK)pathway plays an important role in the process of BBB destruction caused by oxidative stress reaction in a rat model of ICH.