| ObjectiveCerebral ischemia is one of the diseases that severely threaten human health, and mechanisms underlying the injury have been gradually elucidated, including the excitotoxicity, calcium overload, apotosis, inflammation, free radicals, nitro-oxygen (NO) and dysfunction of mitochondria. The breakdown of blood brain barrier (BBB) structure and variation of its function are the important pathological processes and major risk factors of nonage death following focal cerebral ischemia. So it is of great importance to study the variation of BBB permeability after cerebral ischemia and possible protective mechanisms.Green tea polyphenols (GTPs), typically flavonoids or catechins, are polyhydroxy phenolic compounds. Epigallocatechin-3-gallate (EGCG) is the major constituent, accounting for about 80%of the catechin, recognized as the most effective element of GTPs. Recent studies have shown that GTPs have multiple pharmacologic actions such as anti-carcinogenic, antioxidation, anxiolytic, anti-inflammatory, antiradiation damage, lowering blood glucose and blood fat. GTPs have preventive effects on neurodegenerative diseases like Alzheimer's disease and Parkinson's disease due to the neuroprotective mechanisms such as metal-chelating, free radical scavenging, influencing cell survival and apoptosis genes overexpression, regulating signal transduction, mitochondria function and ubiquitin proteasomes system. But the effects of GTPs on BBB permeability after focal cerebral ischemia have not been reported.Brain microvascular endothelial cells are important components of BBB. Caveolae-mediated internalization is one of the classical transcellular pathways. Caveolin-1, a 21-to 24-kDa integral membrane protein within caveolae, plays an important role in modulating BBB permeability, and its expression may be regulated via both extracellular signal-regulated kinase 1/2(ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) signaling in endothelial cells. This study aims to investigate the effects and related mechanisms of GTPs on the permeability of BBB after ischemia, providing experimental data for protection from ischemia damage.Methods1. Animals and groupingAdult male Wistar rats (250-300g) were purchased from the Center for Experimental Animals, China Medical University. NIH Guidelines for the Care and Use of Laboratory Animals were followed in all animal procedures.Animals were randomly assigned to control and GTPs groups. GTPs (400mg/kg/day) were administered by intragastric gavage twice a day for 30 days before operation. Saline was given in the same way in the control group. Then each group included four time points as follows:ischemia 0h, 1h,2h, and 4h. These animals were housed in laboratory cages and maintained on a 12-hr light-dark cycle, with free access to food and water throughout the study period. After 30 days, all groups were performed surgery for middle cerebral artery occlusion (MCAO).2. Cerebral ischemia modelRats were anesthetized with 10%chloral hydrate (350mg/kg, i.p.). An intraluminal filament technique was used to induce MCAO. After a median incision of the neck skin, the left common, external, and internal carotid arteries were exposed. The common and external carotid arteries were permanently ligated with sutures. A filament was inserted into the internal carotid artery via an incision in the common carotid artery and advanced until the rounded tip reached the entrance to the left middle cerebral artery. To confirm proper occlusion of the left middle cerebral artery, a laser-Doppler probe was fixed on the skull (1.5 mm posterior to the bregma and 5 mm from the midline on the left side) before (baseline) and after operation, to measure local cortical blood flow in an area supplied by the middle cerebral artery. The successful occlusion was determined by a 30%decrease to baseline in local cortical blood flow. The experimental animals were deeply anesthetized at ischemia 0h, 1h,2h, and 4h respectively to continue following procedures. In the ischemia 0h group, rats underwent a surgical procedure similar to the others but the left arteries were not occluded.3. Measurement of infarct volume and behavioral testingThe neurological deficit score of each rat was obtained according to Longa's method. Then for detection of the ischemia infarction of the brain, the cross-sectional infarction area on the surfaces of each brain slice was defined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method.4. Evaluation of BBB permeability5. RT-PCR for caveolin-1 mRNA expression6. Immunohistochemical assessment.7. Western blot assessment8. Image analysis9. Statistics analysisData were disposed with SPSS 13.0 statistic software by One-Way ANOVA and they were expressed as mean±standard deviation, P<0.05 was considered statistically significant.Results1. Effects on the cerebral blood flow during MCAOCerebral blood flow (CBF) were determined immediately before and after occlusion. The blood flux was regarded as basal value before operation, and the model was recognized to be successful when the blood flux fell to less than 30%of basal value during ischemia. The rats that didn't fall to less than 30%of basal value or developed cerebral hemorrhage after surgery were excluded.2. Effects on the infarct volume, neurological score and the permeability of BBBGTPs significantly reduced the infarct volume compared to the control ischemia 4h group (P<0.01), and ameliorated the neurological deficit, compared with the control ischemia 2h and 4h (P<0.05, P<0.01). No infarction occurred in control and GTPs ischemia 0h, 1h, and 2h groups. The brain tissue of ischemic hemisphere was stained in blue, whereas, no visible staining was found in control and GTPs ischemia Oh groups. EB content in the ischemic hemisphere increased respectively at cerebral ischemia 1h, 2h and 4h in a time-dependent manner, compared with the control ischemia Oh (P<0.01, P<0.01, P<0.01). There was no significant change between the EB content of control and GTPs ischemia Oh groups (P>0.05). In GTPs groups, at ischemia 1h,2h, and 4h, the exudation of EB decreased significantly, compared to the control group at corresponding time points, i.e. control ischemia 1h,2h, and 4h (P<0.05, P<0.01, P<0.05).3. Effects on the mRNA expression of caveolin-1 in microvessel fragments of ischemia brain tissue by RT-PCRTo determine the alteration of caveolin-1 occurring in the pre-transcriptional or post-transcriptional stage, we measured the expression of caveolin-1 mRNA with RT-PCR. The relative expression level of caveolin-1 mRNA was normalized for actin mRNA expression. The expression level of caveolin-1 mRNA in the control group exposed to 1h,2h, and 4h ischemia significantly increased, compared to the control Oh group (P<0.01, P<0.01, P<0.01), whereas, there was no significant difference between the ischemia lh and 2h groups (P>0.05). On the other hand, in the GTPs group, caveolin-1 mRNA showed no difference, compared to the control ischemia Oh group (P>0.05), but significantly lowered at ischemia 1h,2h, and 4h, compared to the control corresponding time points (P<0.01, P<0.01, P<0.05).4. Effects on the expression of caveolin-1 by immunohistochemisitryThe expression of caveolin-1 protein was investigated in the microvessel fragments of ischemic brain tissue. Caveolin-1 expressed normally in the ischemia Oh brain tissue of control and GTPs groups. Immunohistochemical studies were carried out to examine the localization of caveolin-1. The brown staining represents that the target proteins were observed. The positive immunohistochemical staining of caveolin-1 was distributed in the vascular endothelial cells along the blood vessel of brain tissue. The positive staining of caveolin-1 was increased as the brain tissue was exposed to ischemia lh,2h, and 4h, but declined in the GTPs group, compared to the control respective time points.5. Effects on the expression of caveolin-1 in microvessel fragments of ischemia brain tissue by western blotThe expression of caveolin-1 protein was significantly up-regulated at ischemia 1h,2h, and 4h, compared to Oh in the control group (P<0.01, P<0.01, P<0.01). Caveolin-1 protein expression level showed no difference at baseline between the control and GTPs groups (P>0.05), while in other GTPs groups, caveolin-1 protein showed significantly down-regulation at ischemia 1h,2h, and 4h, compared to the control group at respective ischemia time points (P<0.01, P<0.01, P<0.01). The western blot results are consistent with the findings obtained by immunohistochemical anslysis.6. Effects on the expression of ERK1/2 in microvessel fragments of brain from ischemia rat by western blotResults from western blotting showed that p-ERK1/2 protein expression in the ischemic region was remarkably increased at cerebral ischemia 1h,2h, and 4h, compared to ischemia Oh (P<0.01, P<0.01, P<0.01). There was no significant difference between the control and GTPs groups at basal level (P>0.05). In GTPs groups, at ischemia 1h,2h, and 4h, the expression of p-ERKl/2 protein decreased significantly, compared with the control group at respective time points (P<0.01, P<0.01, P<0.01).DiscussionIn the study, we investigated the effects of GTPs on BBB permeability following focal cerebral ischemia in rats which were pretreated with GTPs by gavage. GTPs significantly reduced infarct volume, ameliorated the neurological deficit and reduced the permeability of BBB after ischemia. The results of EB exudation in ischemic brain tissue suggested that the BBB permeability gradually increased during 4h ischemia. While in GTPs groups, at ischemia 1h,2h, and 4h, the exudation of EB in ischemic brain tissue decreased significantly, compared with the control group at corresponding time points.GTPs also down-regulated caveolin-1 and phosphorylated ERK1/2 expression at ischemia 1h,2h, and 4h, compared to the control respective time point groups. These provided the first protective evidence of GTPs against ischemia-induced increase of BBB permeability. The mechanism may be correlative with the reduced caveolin-1 mRNA and protein expression levels, as well as activated ERK1/2 protein.Cerebral ischemia induced the opening of BBB. GTPs have been confirmed to prevent stroke, and can be served as neuroprotectant after ischemia. GTPs were widely distributed in the body, including the brain tissue through BBB, after absorbed by the digestive tract, and attenuated the postischemic brain edema and neuronal damage induced by ischemia. Present studies about GTPs focus on the central nervous system protection. GTPs groups remarkably reduced the permeability of BBB, and it was the first time to confirm that GTPs could maintain the integrity of BBB.Our study further demonstrated the effects of GTPs on caveolin-1 expression. The result showed that the expression of caveolin-1 mRNA and protein were obviously increased depending on the duration of ischemia, and GTPs significantly lowered the up-regulated caveolin-1 expression, compared with the control corresponding ischemiaIn this study, pretreatment with GTPs significantly decreased the activity of ERK1/2, while the phosphorylation level of ERK1/2 increased after ischemia. GTPs may modulate the expression of caveolin-1 by regulating the activity of NOS, and ERK1/2 pathway may be one of the mechanisms for GTPs.Conclusion1,The current experiment firstly demonstrated that GTPs significantly decreased the permeability of BBB which was increased as a result of ischemia.2,GTPs significantly decreased the mRNA and protein expression level of caveolin-1 in micro vessel fragments of ischemia tissue, this may be one of the mechanisms for GTPs in protecting the integrity of the BBB after cerebral ischemia.3,GTPs significantly decreased the protein expression of p-ERK1/2, which was increased in micro vessel fragments of ischemia tissue.
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