| Objective：Ischemic cerebrovascular disease (ICVD) is one of high mortality and morbiditydisease. Ischemia-reperfusion injury is a major pathological process of ICVD. Thedisruption of blood-brain barrier induced by oxidative stress during the reperfusionresults in the increase of permeabitily and is associated with the second injury ofneurons. Therefore, antioxidative stress has been proposed as a potential target forprotecting the BBB thereby alleviation of ischemia-reperfusion damage. Previousstudies of our lab have been proved that the ethyl acetate extraction of Gastrodia elatahas protective function on cerebral ischemia and eight phenolic compounds (1#-8#)have been isolated from this fraction. To screen active compounds of the phenoliccompounds (1#-8#) those of protecting BBB, bEnd.3cells, a mouse cerebral capillaryendothelial cell line, and astrocytes were used as blood-brain barrier model forresearch, and the related mechanism were determined.Methods:1. H2O2-induced oxidative injury of the blood brain barrier: The BBB is mainlyconsisted of vascular endothelial cells and astrocytes. As an well-accepted method,H2O2-induced oxidative injury was employed to induce the BBB damage. bEnd.3cells or astrocytes were exposed to a different concentration of H2O2(0.125mM,0.25mM,0.5mM,0.75mM) for4h or exposed to0.5mM H2O2for1h,2h,4h,6h todetect the reasonable condition for oxidative injury by LDH assay.2. Screening the active compounds protection of the BBB: The protective effectof phenolic compounds1#-8#on blood-brain barrier disrupture induced by H2O2willbe detected by LDH assay to screen the active compounds. 3. The effect and mechanism of active phenolic compounds on vascularendothelial cells3.1The effect of active phenolic compounds on bEnd.3cell proliferation wasdetected byAlamar blue.3.2The expression of tight junction protein including occludin, claudin-1andclaudin-5in bEnd3cells were determined after treatment with phenolic compoundsby Western blot and immunofluorescence; The Nrf2/ARE pathway including itsresponse proteins were detected by western blot to study its protective mechanism onblood-brain barrier.4. The effect and mechanism of active phenolic compounds on astrocytes4.1The protective effect of phenolic compounds on astrocytes injury induced byH2O2was detected by LDH assay.4.2The Nrf2and its downstream proteins BNDF, HO-1and NQO1expression inboth normal astrocytes and H2O2-induced damaged astrocytes were detected bywestern blot to investigate the protective mechanism of the active phenoliccompounds in astrocyte.Results:1. Phenolic compounds from ethyl acetate extraction of Gastrodia elata Blumeprotect the endothelial cells of blood-brain barrier1.1Compared with the control, bEnd3cells exposed to different concentration ofH2O2(0.25mM,0.5mM,0.75mM) for4h and exposed to0.5mM H2O2for6h resultedin a significantly increasing of the LDH leakage (P<0.01). The reasonable conditionof H2O2induced damage in bEnd.3cells is set up as exposure the cells to0.5mMH2O2for4h.1.2Pre-treatment with different concentrations (15μg/ml,25μg/ml,50μg/ml,100μg/ml) of phenolic compounds1#,2#,4#,7#had no effect on the LDH leakage ofbEnd.3cells damage induced by H2O2.The LDH leakage in H2O2-induced damage ofbEnd.3cells were markedly reduced by pre-treatment of different concentrations(12.5μg/ml,25μg/ml,50μg/ml) of phenolic compounds3#,5#,6#and different concentrations of8#(5μg/ml,10μg/ml,20μg/ml)(P<0.01).1.3Phenolic compounds3#,5#can promote the proliferation of bEnd.3,6#,8#didn’t show any effect on the proliferation.1.4Compared with control, the expression of claudin-1, claudin-5, occludin,Nrf2, HO-1in bEnd.3cells by phenolic compound5#treatment were increasedsignificantly (P<0.05). There is no significant effect on the expression of claudin-5,occludin, Nrf2, HO-1, NQO1in bEnd.3cells by phenolic compounds3#,6#treatment.2. Phenolic compounds from ethyl acetate extraction of Gastrodia elata Blumeprotect the astrocytes of blood-brain barrier2.1Compared with the control, astrocytes exposed to different concentration ofH2O2(0.125mM,0.25mM,0.5mM,0.75mM) for4h and exposed to0.5mM H2O2for2h,4h,6h resulted in a significantly increasing of the LDH leakage (P<0.01). Thereasonable condition of H2O2induced damage in astrocyte is set up as exposure thecells to0.5mM H2O2for4h.2.2The LDH leakage of damaged astrocyte induced by H2O2were reducedsignificantly (P<0.01) by pre-treatment with different concentrations (C1:12.5μg/ml;C2:25μg/ml; C3:50μg/ml) of phenolic compounds5#.2.3Compared with the control, the expression of BDNF in astrocytes treatedwith phenolic compound5#50ug/ml was increased significantly (P<0.05).2.4compared to control, pre-treatment with phenolic compounds5#can increasethe expression of Nrf2, HO-1and NQO1(P<0.05) in astrocyte after exposed to0.25mM H2O2for1h. compared with the model group, the expression of HO-1inastrocytes was increased significantly (P<0.05).Conclusions:1. The phenolic compounds5#from ethyl acetate extraction of Gastrodia elataBlume showed a significant protective effect on the blood brain barrier may be byincreasing the tight junction protein including occludin, claudin-1and claudin-5, promoting the proliferation of the endothelial cells as well as activating the Nrf2pathwayand increasing its downstream proteins BDNF, HO-1and NQO1.2.Phenolic compounds3#,6#,8#from ethyl acetate extraction of Gastrodiaelata Blume have the protective effect on blood brain barrier, the mechanism may viaother pathway but Nrf2/ARE, further study will be needed to clarify it.3. Phenolic compounds1#,2#,4#,7#from ethyl acetate extraction of Gastrodiaelata Blume, didn’t show any protective effect on blood brain barrier by preliminaryscreening. |
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