The Mechanism Of Aβ-receptor For Advanced Glycation End-products Interaction Disrupts Blood-brain Barrier And Intervention Study Of EGb

Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative disease in the occidental world, as well as the most common form of senile dementia in old individuals, characterized by neuronal cell loss, the accumulation of amyloid β-peptide (Aβ) in the brain parenchyma. Changes of cerebral microvasculature have been also reported in the brain of AD individuals and are the major event of AD. Mounting evidence indicates that the deposition of Aβ aggregates in the cerebral blood vessels and dysfunction of blood-brain barrier (BBB) are also important pathologies in the disease progression. The deposition of Aβ in the cerebral blood system lead to BBB disruption, dysfunctional barrier, BBB permeability increasing, cerebral blood flow (CBF) reduction, and accumulation of metabolism products in brain, which together accelerate AD development. Given the important role of BBB in AD progression, our study, based on BBB change in AD, investigated the potential mechanisms of BBB disruption induced Aβ in an in vitro research. What’s more, we observed the effect of a widely used Chinese herb, extract of ginkgo biloba (EGb) in BBB disruption provide an experimental evidence for the prevention of AD.BBB, an important barrier between blood and bran, plays an essential role in maintaining the cerebral parenchyma microenvironment and ensures the physical functions of the brain. Brain microvascular endothelial cell (BMEC) is the basic framework of BBB and has its unique characteristics that are differed from peripheral endothelial cells. In a well differentiated neural vascular system, BMEC plays significant roles in the BBB permeability and the transport of various molecules across BBB. Tight junctions (TJ) are the important prominent feature of brain endothelium and responsible for the BBB integrity, as well as the most important structures that are responsible for the barrier function. Therefore, any damage factors that lead to TJ breakdown could result in BBB disruption, dysfunctional internal environment of central nervous system (CNS) and consequently CNS pathology progression. TJ system is resembled by TJ scaffold proteins such as Claudin (Claudin-1,3,5,12), Occludin, seeming to provide an indirect connection with cytoskeletal protein Actin by cytoplasmic attachment protein named Zonula occluding (ZO-1,2,3). Studies have shown that the alteration of TJ-associated proteins contribute to the changes in TJ structure and the loss of BBB function, leading to an increase in BBB permeability. A recent study showed that TJ disruption and BBB permeability increased were observed in AD individuals, and a down-regulated expression levels of TJ proteins have also been detected. Furthermore, both in vivo and in vitro experimental evidence show that TJ breakdown and BBB integrity induced by A(3 toxicity have been determined.Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor and a transmembrane cell-signaling receptor. RAGE has been recognized as a key molecule in the development of severe chronic pathologies, including diabetic complications, chronic inflammation, atherosclerosis, neurodegeneration and cancer. With the exception of lungs, the basal expression of RAGE is low but increases with the levels of its ligands, such as advanced glycation end-products (AGEs) and at sites of stress and injury. Besides AGEs, RAGE also interacts with several other ligands that are structurally unrelated, including high mobility group box 1 (HMGB-1), S100/calgranulins family of polypeptides, transthyretin, and Aβ.Recently, more and more evidence indicates that the up-regulated expression of RAGE in the brain is intensively associated with AD progression. Aβ-RAGE engagement promotes the generation of reactive oxygen species (ROS) and the activation inflammatory reaction, which lead to neuron damage and result in microglia activation. What’s more important, Aβ-RAGE interaction also results in an increased expression of RAGE via nuclear factor-KB (NF-KB) activation, thereby converting a transient proinflammatory responsible into a chronic pathophysiological state in CNS and leading to A(3-induced positive feedback loop. In addition of these, RAGE is slightly expressed in BMEC but a pivotal cargo transporter mediating A(3 influx into brain, resulting in the deposition and aggregation of Aβ in the brain. Furthermore, LDH receptor related protein (LRP-1), another critical transporter and maintains the physical level of Aβ in brain. LRP-1 mediates Aβ. LRP-1 binds free Aβ and transports it into peripheral circulation, resulting in clearance of Aβ in liver. It has been determined that the expression of LRP-1 in AD brain is down-regulated, while, RAGE is up-regulated. However, the mechanisms that underlie the response to changes in BBB permeability in AD are unknown. There exits little evidence determining which form of Aβ composition, such as monomer, oligomer and fibril, is the major factor inducing BBB disruption.Under the pathology conditions, matrix metalloproteinases (MMPs) degrade extracellular matrix (ECM), which is associated with increasing in BBB permeability induced by many diseases. While inhibition of MMPs shows cerebral protective effects followed by BBB permeability ameliorated. The decreased expression levels of TJ proteins including ZO-1, Claudin-5 and Occludin have been determined in a stroke murine model, while MMP-9 knockout blocked the changes. Another experimental research found that the gene and protein expression of ZO-1 was significantly decreased and BBB permeability increase in rat, while MMP-2 and MMP-9 expression were significantly increased after exposed pulsed electromagnetic field (PMSF). When rats were treated with SB-3CT, a gelatinase inhibitor that inhibiting MMP-2 and MMP-9, before PMSF, BBB breakdown was attenuated and the ZO-1 degradation was reversed. Based on these, we speculate that BBB disruption in AD might be associated with Aβ-induced activation of MMPs that consequently leading to degradation of TJ proteins and BBB permeability.EGb, the extract from the leaves of Ginkgo biloba tree, is one the most popular herbal supplements widely used in clinic. Numerous preclinical studies have shown the neuroprective effects of EGb and support the notion that it may be effective in the treatment and prevention of neurodegenerative disorders such as AD. Recent in vivo and in vitro studies demonstrated that EGb has potent anti-oxidant properties, ameliorates mitochondrial function and inhibits neurotoxicity of Aβ. Recently, an experimental research reported that EGb, the standard EGb produced by Dr. Willar Schwabe Pharmaceuticals, markedly ameliorated the damage from oxygen glucose deprivation (OGD), and significantly reversed OGD-induced up-regulation of RAGE expression. Given to these data, we intend to observe the effects of EGb on BBB disruption induced by AP, and then, we attempt to determine its relationships with RAGE inhibition.We investigated the influence of Aβ on BBB permeability and RAGE expression in a monolayer murine BMEC model, and explored the molecular mechanisms responsible for BBB disruption. Furthermore, we observed the effects of EGb on Aβ-induced BBB damage and RAGE expression. The results were summarized as follows:(一) RAGE-Aβ1-42 interaction leads to BBB disruption through up-regulated MMP-2 and MMP-9 expression in BMEC1. Aβ1-42 induces BMEC injury in vitroAβ1-42-inuced BMEC injury was determined by MTT. The results present that Aβ1-42-treatment decreased the cell viability. All the three main forms of Aβ1-42, Aβ1-42-monomer (Aβ1-42-Mono), Aβ1-oligomer (Aβ1-42-Oligo) and Aβ1-42-monomer (Aβ1-42-Fibril), can induce BMEC damage and damage the cell viability. Our results indicate that the injury effect of Aβ1-42-Oligo is more significant than that of both Aβ1-42-Mono and Aβ1-42-Fibril. Furthermore, it shows that Aβ1-42-Oligo induced cell injury was in a time and concentration dependent manner.2. Aβ1-42 induces an increase of BBB permeability in BMECAβ1-42-inuced an increase of BBB permeability was determined by sodium fluorescein (Na-F) leakage test through detected the absorbance after treatment. The results show that all the three main forms of Aβ1-42 were able to induce BBB leakage. The results detected by the spectrophotometer suggests that, compared with the control, the absorbance in the group of Aβ1-42-Mono was 4.2 times higher, in the group of Aβ1-42-Oligo was 8.2 times and was 1.7 times in the group of Aβ1-42-Fibril. Additionally, our data also indicates that Aβ1-42-Oligo induced BBB leakness was in a time and concentration dependent manner.3. Aβ1-42 treatment decreases the expression levels of tight junctions scaffold proteins in BMEC1) The decreasing in levels of ZO-1, Claudin-5 and Occludin induced by Aβ1-42-treatment was detected by Western Blot. The results present that all the three forms of Aβ1-42 can down-regulate the levels of ZO-1, Claudin-5 and Occludin in BMEC, in which Aβ1-42-Oligo was determined to be the most significant form.2) The result determined by Western Blot indicates that down-regulation of ZO-1, Claudin-5, and Occludin induced by Aβ1-42-Oligo was in a time and concentration dependent manner.4. Aβ1-42 treatment up-regulates the expression level of RAGE in BMEC1) Aβ1-42-treatment induced RAGE protein expression was determined by Western Blot. The results present that all the three forms of Aβ1-42 can up-regulate the levels of RAGE in BMEC, in which Aβ1-42-Oligo was determined to be the most significant form.2) Our result detected by Western Blot indicates that up-regulation of RAGE induced by Aβ1-42-Oligo was in a time and concentration dependent manner.3) RT-PCR shows that Aβ1-42-Oligo up-regulated the mRNA level of RAGE in BMEC.5. Aβ1-42 treatment increases the expression level of MMP-2 and MMP-9 in BMEC1) Western Blot shows that Aβ1-42-Oligo treatment increased the protein levels of MMP-2 and MMP-9 in BMEC.2) RT-PCR shows that Aβ1-42-Oligo up-regulated the mRNA level of MMP-2 and MMP-9 in BMEC.6. Neutralizing anti-RAGE antibody attenuates Aβ1-42-induced alterations in tight junctions scaffold proteins and up-regulation of RAGE, MMP-2 and MMP-9 in BMEC1) Aβ1-42-Oligo-induced alterations in ZO-1, Claudin-5 and Occludin are attenuated by an anti-RAGE antibody in BMEC.2) Inhibition of RAGE by anti-RAGE antibody attenuated Aβ1-42-Oligo-induced up-regulation of RAGE, MMP-2 and MMP-9.(二) EGb ameliorates BBB permeability via inhibition of Aβ1-42-induced cytotoxicity and RAGE expression1. EGb prevents Aβ1-42-Oligo-induced injury in BMECEGb ameliorated Aβ1-42-Oligo-induced cytotoxicity, detected by MTT, in a concentration dependent manner.2. EGb prevented Aβ1-42-Oligo-triggered apoptosis in BMECEGb prevented Aβ1-42-Oligo-triggered apoptosis. EGb protected BMEC against Aβ1-42-Oligo-induced cell apoptosis determined by Hoechst-33258 stains assay.3. EGb ameliorated Aβ1-42-Oligo-triggered ROS production in BMECEGb ameliorated reactive oxygen species (ROS) production. EGb protected BMEC against Aβ1-42-Oligo-induced ROS production determined by H2DCF-DA.4. EGb protects against Aβ1-42-Oligo-induced increase in BBB permeabilityEGb inhibited Aβ1-42-Oligo-induced BBB leakage. Na-F leakage test determined that EGb ameliorated BBB disruption induced by Aβ1-42-Oligo in a concentration dependent manner.5. EGb ameliorates the levels of tight junctions scaffold proteins resulting from Aβ1-42-Oligo treatmentEGb ameliorated alterations in ZO-1, Claudin-5 and Occludin induced by Aβ1-42-Oligo. Western Blot results show that EGb inhibited Aβ1-42-Oligo-induced alternations in ZO-1, Claudin-5 and Occludin in a concentration dependent manner.6. EGb revers the level of RAGE up-regulated by Aβ1-42-Oligo treatmentEGb reversed up-regulation of RAGE resulting in Aβ1-42-Oligo-treatment. Western Blot results indicate that EGb inhibited Aβ1-42-Oligo-induced up-regulation of RAGE in BMEC.Conclusions:1) Aβ-treatment induces TJ scaffold proteins and BBB leakage, in which the oligomer was determined to be the most striking one of the three main forms of AP including monomer, oligomer and fibril.2) RAGE is the essential factor in TJ breakdown induced by Aβ. And its mechanism was associated with up-regulation of RAGE, MMP-2 and MMP-9, resulting in BBB disruption.3) EGb protects against Aβ cytotoxicity to BMEC through inhibition of ROS production and cell apoptosis, and ameliorates the levels of TJ scaffold proteins and BBB permeability via down-regulation of RAGE expression.Taken together, our present determined the effects of different forms of Aβ in BBB permeability. And we, for the first time, reported that Aβ-Oligo might be the main toxicity that leading to BBB disruption. Furthermore, our results indicate that RAGE might be the pivotal link associated with BBB leakage. Additionally, it is the first report that presents the role of EGb in Aβ-induced BBB disruption. Our results suggest that the protective effect of EGb was associated with ameliorating of BMEC cell viability, inhibition of ROS production and cell apoptosis. These data suggest that RAGE might be a potential therapeutic target for AD, and provide an experimental evidence for exploring the protective mechanisms of EGb treatment in AD.