DL-3-n-butylphthalide Protects Blood-Brain Barrier In A Rat Model Of Vascular Cognitive Impairment Via Upregulation Of Tight Junction Proteins

Vascular cognitive impairment?VCI?is a cognitive disorder related to cerebrovascular disease,including mild cognitive impairment to dementia.Meanwhile,vascular dementia is the second most common cause of dementia after Alzheimer's disease.The pathogenesis of VCI is complex and effective treatments for VCI are still not available.The integrity of the blood-brain barrier?BBB?plays an important role in maintaining the homeostasis of the central nervous system.Many studies have shown that although increased BBB permeability is an inevitable process of natural aging,patients with VCI or Alzheimer's disease have significantly increased BBB permeability compared with the same age group,and compared with patients with Alzheimer's disease,this phenomenon is more obvious in patients with VCI.The BBB is composed of specialized brain microvascular endothelial cells?BMECs?,astrocyte endfeet,pericytes and the extracellular matrix,and the first defense between the systemic circulation and the central nervous system is BMECs.One characteristic distinguishing BMECs from peripheral cells is the presence of tight junctions?TJs?.The TJs are the intercellular molecular binding systems restricting paracellular flux of hydrophilic molecules across the capillary endothelial cells.The barrier function of the BBB is mainly completed by BMECs and TJs.Therefore,an understanding of the molecular mechanisms which regulate the BBB permeability and the disruption of TJs is essential for establishing future therapeutic strategies to alter dementia disease progression associated with VCI.Although the BMECs and their TJs are the ultimate permeability barrier,astrocytes and pericytes play a major regulatory role.Indeed,the BBB is one part of the‘neurovascular unit',a dynamic structure regulated by these and additional cells including neurons,microglia and even peripheral immune cells.Considering BBB is in a network of neurovascular unit will facilitate a more deep study of the relationship between BBB damage and VCI,and more importantly,expand the strategies for the treatment of VCI.DL-3-n-butylphthalide?NBP?,with the chemical name dl-3-butyl-1?3H?-isobenzofuranone,was approved by China Food and Drug Administration for therapy of ischemic stroke in 2002.Multicenter clinical trials have shown that NBP is effective in improving neurological function in patients with brain ischemia and exhibits good safety.Our team focous on the research of NBP treatment in VCI.Our recent studies proved that NBP improved cognitive impairment in rats with chronic cerebral hypoperfusion?CCH?and in mice with repeated cerebral ischemia-reperfusion.However,the previous studies mainly focused on the direct effects of NBP on neurons or angiogenesis in the advanced stages of brain ischemia,and little is known about the effect of NBP on TJs of endothelial cells.Since disruption of the BBB is one of the most important pathophysiological events contributing to ischemia injury and TJs are the very first structures to be injured,we hypothesized that NBP may exert its protective effect by affecting the expression and distribution of TJs in brain ischemia.To test this hypothesis,we investigated the effect of NBP on the expression and localization of claudin-5,ZO-1 and occludin in a rat model of VCI.An in vitro study was also conducted in primary cultured BMECs to investigate the potential role of NBP on TJs disruption induced by hypoxia or oxygen glucose deprivation?OGD?/reoxygenation?OGD/R?.We further discusse whether the effect of NBP on TJs is related to the activation of protein B?Akt?/glycogen synthase kinase 3??GSK-3??/?-catenin signaling pathway and upregulation the expression of sonic hedgehog?Shh?in astrocytes.Thus,these findings may provide the basic data for the clinical use of NBP in VCI prevention and treatment.Part one NBP protects blood-brain barrier and upregulats tightjunction proteins in a rat model of VCIObjective:To explore the effects of NBP on BBB permeability and the expressions of tight junction proteins in VCI rats.Methods:Adult male Sprague-Dawley?SD?rats were subjected to the permanent occlusion of the bilateral common carotid arteries?BCCAO?to establish the VCI rat model.The rats were randomly divided into 5 groups:the sham group,the model group,NBP-L?20 mg/kg?,NBP-M?40 mg/kg?,and NBP-H?80 mg/kg?group.NBP was administrated by oral gavage,once a day.NBP treatment were administrated for 14 or 28 consecutive days.Behavioral tests by Morris water maze were performed after treatment of NBP for 28 days,and other tests were performed 14 days after NBP treatment.The BBB permeability was assessed by Evans blue assay.The expressions and localization of claudin-5,ZO-1,occludin were evaluated by Western blot or immuohistochemical staining.TJs ultrastructure was observed by transmission electron microscope.Results:1.NBP alleviated learning and memory impairments induced by chronic cerebral hypoperfusionCompared with the sham group,the escape latency of rats in the model group was significantly prolonged?P<0.05?.However,the poor performance was alleviated after NBP treatment.The results of spatial probe test showed that the model group spent significantly less time in the target quadrant than the sham group?P<0.01?,whereas this deficit was significantly improved by NBP treatment?P<0.01?.2.NBP decreased the BBB permeability in VCI ratsRats with BCCAO surgery in the model group showed significantly increased leakage of Evans blue dyes compared with the sham group?P<0.01?.However,with NBP treatment,decreased contents of Evans blue dyes in NBP-M group?P<0.01?and NBP-H groups?P<0.01?were observed compared to that of model group.There was significantly statistical difference between NBP-M and NBP-L group?P=0.03?,which indicated NBP could improve the barrier function of BBB in a dose-dependent manner.3.NBP increased the expressions of claudin-5 and ZO-1Immunohistochemical staining showed that there were more claudin-5or ZO-1 positive cells in NBP-H group as observed than in the model group.The expression of claudin-5 protein was significantly lower in the model group compared with that in the sham group?P<0.01?.In comparison with the model group,the expression of claudin-5 was significantly increased in the NBP-L?P=0.03?,NBP-M?P<0.01?,and NBP-H?P<0.01?groups.There were statistically significant differences among the three groups treated with NBP?P<0.05?,which indicated that the effect of NBP on claudin-5 expression was dose-dependent.The levels of ZO-1 expression in the NBP-M?P<0.05?and NBP-H?P<0.01?groups were also higher than those in the model group.No significant change in occludin expression was observed in different groups?F=2.455,P=0.075?.4.NBP prevented the degradation of TJ ultrastructure in VCI ratsThe alteration in TJ ultrastructure was prevented by NBP treatment,leading to an ultrastructure similar to that of the sham group.Conclusion:NBP improves the barrier function of BBB by upregulating the expression of TJ proteins in VCI rats.Part two NBP ameliorates the permeability of endothelial barrier invitro via upregulation of tight junctions during hypoxiaObjective:To investigate the in vitro effect of NBP on the permeability of BMECs barrier and the expression of TJ proteins under hypoxia condition.Methods:The BBB model was established by culturing primary rat BMECs.Confluent rat BMECs were randomized into four groups:control group,hypoxia group?1%O₂,24 h?,NBP?0.1?mol/L?and NBP?1.0?mol/1?.The flux of horseradish peroxidase?HRP?was used as an indicator of changes in BMECs permeability in vitro.The morphological identification was observed by transmission electron microscopy.The expression and localization of TJ proteins were evaluated by immunofluorescence microscopy and Western blots.Results:1.NBP protected BMECs against hypoxia-induced disruption of paracellular permeability in titroHypoxia increased the leakage of HRP across the BMECs barrier?P<0.01?.Treatment with NBP reduced the HRP leakage?P<0.05?.2.NBP prevented the degradation of TJ ultrastructure after hypoxia injuryTJ ultrastructure showed high electronic density between adjacent cells in the control group,while it was visualized by a lower electron-dense architecture or cellular gap in BMECs in hypoxia group,indicating that ultrastructural injury occurred.The alteration in TJ ultrastructure was prevented by NBP treatment,leading to an ultrastructure similar to that of the control group.3.NBP increased the expressions of TJ proteins and improved the linear distribution in BMECsBMECs in the control group showed continuous staining of claudin-5and ZO-1 on the cell membrane by immunofluorescence.After hypoxia injury,the immunofluorescent intensity of claudin-5 and ZO-1 reduced.With treatment of NBP,the expressions and linear distribution of TJ proteins in the membrane of BMECs were improved partly.Western blot analysis showed that hypoxia decreased the expression of claudin-5?P<0.01?and ZO-1?P<0.01?in comparison with the corresponding values in the control group.NBP?0.1?mol/L?and NBP?1.0?mol/L?treatment increased the expression of claudin-5?P<0.05,P<0.01?and ZO-1?P<0.05,P<0.01?compared with the hypoxia group.There was no significant difference in occludin expression among the four groups?F=0.064,P=0.598?.Conclusion:NBP ameliorates the permeability of endothelial barrier against hypoxia injury via upregulation the tight junction proteins in brain microvascular endothelial cells.Part three NBP protects tight junctions by reducing oxidative stressand activating the Akt/GSK-3?/?-cateninsignaling pathwayObjective:To explore the molecular mechanisms underlying NBP-meadiated protective effects on TJs and whether the protective effects of NBP was related to Akt/glycogen synthase kinase 3??GSK-3??/?-catenin signal pathway.Methods:The in vitro BMECs model and grouping methods were the same as in part two.Reactive oxygen species?ROS?generation was observed under a fluorescence microscope and measured by flow cytometry analysis with DCFH-DA fluorescence probe.The mitochondrial membrane potential was detected by JC-1 fluorescence probe.The activity of antioxidant enzyme SOD and GSH-Px was measured by biochemical methods.The phosphorylation levels of Akt and GSK-3?and protein expression of?-catenin were analyzed with Western blots.Results:1.NBP attenuated ROS generation induced by hypoxia in BMECsNBP decreased the hypoxia-induced increase in DCFH-DA positive cells?green?.According to the results of flow cytometry analysis,treatment with NBP?0.1?mol/L?or NBP?1.0?mol/L?significantly decreased intracellular ROS level in a dose-dependent manner?NBP 0.1?mol/L25.6±3.0%vs.NBP 1.0?mol/L:17.3±2.6%,P<0.01?in comparison with the hypoxia group.2.NBP increased mitochondrial membrane potential of BMECsHypoxia induced a significant increase in intracellular green fluorescence in the hypoxia group in comparison with the control group.NBP treatment increased the ratio of red and green fluorescent intensity of BMECs,which indicated that NBP could increase mitochondrial membrane potential and reduce mitochondrial damage.3.NBP reversed the decreasing activity of SOD and GSH-Px induced by hypoxiaIn compared with the control group,the activity of SOD?P<0.05?and GSH-Px?P<0.01?decreased in hypoxia group,while those increased in0.1?mol/L and 1.0?mol/L NBP group when compared with hypoxia group?P<0.05?.4.NBP inhibited the depression of AKT/GSK-3?/?-catenin pathway induced by hypoxiaHypoxia injury decreased the relative expression of p-Akt/AKT?0.22±0.07 vs.0.64±0.11,P<0.01?,p-GSK-3?/GSK-3??0.36±0.09 vs.0.93±0.10,P<0.01?and?-catenin/?-actin?0.23±0.03 vs.0.54±0.07,P<0.01?in BMECs in comparison with the control group.In compared with hypoxia group,NBP treatment elevated the expression level of p-Akt/Akt,p-GSK-3?/GSK-3?and?-catenin/?-actin?all P<0.05?.Conclusion:The protective effect of NBP on TJs during hypoxia may be related to reducing oxidative stress and activation of the Akt/GSK-3?/?-catenin signaling pathway.Part four Effect of NBP on claudin-5 expressions in a co-culture mod-el of brain microvascular endothelial cells and astrocytes during hypoxic injuryObjective:To explore the effect of NBP on claudin-5 expression in BMECs and sonic hedgehog?Shh?expression in astrocytes in a co-culture model of BBB.Methods:Rat BMECs and astrocytes were isolated and purified respectively and the co-culture model was established though transwell chamber.The co-cultured cells were randomized into three groups:control group,hypoxia group?1%O₂,24h?and NBP group?1.0?mol/1?.Three parallel mono-culture BMECs groups were established as the same time.The claudin-5 expression in BMECs and the Shh expression in astrocytes were analyzed by Western blots.RT-PCR technique was used to detect the mRNA level of Shh gene in astrocytes.The expression of Shh proteins in the brain?paraffin specimens of VCI rats in the first part?was detected by immunofluorescence method.Results:1.Effects of NBP on claudin-5 expression in co-cultured modelThe expression of claudin-5 in co-culture control group was higher than that in mono-culture control group.After 24 h of hypoxic injury,the expressions of claudin-5 were decreased significantly in both co-culture and mono-culture BMECs,and no statistical difference was found between the two groups?P>0.05?.Treatment with NBP?1.0?mol/1?promoted the expression of claudin-5 in both mono-culture and co-culture BMECs,and the claudin-5 expression in co-culture BMECs was higher than that in mono-culture BMECs?P<0.05?.2.Effects of NBP on Shh gene and protein expression in cultured astrocytes in vitroThere was no significant difference in Shh mRNA and protein expression between the hypoxia group and the control group,but Western blots and RT-PCR results showed that mRNA and protein of Shh were higher in NBP group than those in the hypoxia group and the control group?P<0.05?.3.Effects of NBP on Shh protein expressions in brain tissue of VCI ratsIn compared with the sham group and model group,the immunofluorescence intensity of Shh increased in NBP-H group and Shh fluorescence signal surrounding microvessels was enhanced.Conclusion:The protective effects of NBP on TJs may be related to the increased Shh expression of astrocytes.