| Objective:Damage of the blood brain barrier(BBB) during the process of cerebral ischemic injury is not only an important pathophysiological change which results in the brain edema and cerebral infarction but also a key factor which significantly influences the therapeutic efficacy to the cerebral ischemic injury. Our previous studies have shown that the ethyl acetate extracts(Et OAc) from Gastrodia elata Blume significantly protected the cerebrum from the ischemic injury in a MCAO/R animal model. In following studies, we also found that a group of ethyl acetate extracted phenolic compounds, especially compound C, contributed to the cerebral protective effect of Gastrodia, through attenuating the oxidative stress injury or protecting the BBB. Therefore, the present study was designed to verify the mechanisms underlying the protective effects of the Et OAc extraction and the phenolic compound C from Gastrodia elata Blume on the BBB by developing a model of cerebral ischemia-reperfusion in rats.Methods:1. The model of focal ischemic reperfusion in rats:Male Sprague Dawley(SD) rats(SPF level, weighing 250-300g) were anaesthetized with intraperitoneal injections of 10% chloral hydrate(0.3m L/100g).Then, a rat model of MCAO/R was developed by using a suture method. Animals in each group under :went surgery ischemia for 2h and followed by reperfusion for 24 h.Subsequently, the neurological scales, the contents of EB and the changes of the ultrastructures of BBB were detected for the evaluation of the model.2. The protective effect of the Et OAc extract and the phenolic compound C from Gastrodia elata Blume on the BBB:In certain groups, rats were administrated with the Et OAc extracts or the phenolic compound C from Gastrodia elata Blume. Then the neurological scales, the contents of EB and the changes of the ultrastructures of BBB were detected for evaluating the effects of the Et OAc extract and the phenolic compound C from Gastrodia elata Blume. on this focal ischemic reperfusion model.3. Mechanisms involved in the effects of Et OAc extracts and the phenolic compound C from Gastrodia elata Blume on the BBB:3.1 Mesurements of the release of nitric oxide(NO) and the activity of nitric oxide synthase(NOS): After the adminstrations of the rats with the Et OAc extractive or the phenolic compound C from Gastrodia elata Blume., the release of NO in brain tissues and activities of NOS, including the endothelial NOS(e NOS), neuronal NOS(n NOS)and the inducible NOS(i NOS), were determine by using ELISA kits.3.2 Detections of the expressions of BBB-related proteins: Western blot analyses were performed to assess the protein expressions of AQP-4, Occludin, Claudin-5, MMP-2,MMP-3 and MMP-9 in brain tissue after rats were administrated with the Et OAc extractive or the phenolic compound C from Gastrodia elata Blume.Results:1. Development and evaluation of the model of focal ischemic reperfusion in rats:Compared with the sham-operated group, rats of the MCAO/R model group had obvious neurological deficit. Meanwhile, the neurologic scores and the EB contents in the brain tissues were increased significantly(P<0.001). Moreover, the transmission electron microscope(TEM) results showed that the oultrastructures of BBB damaged and changed in varying degree. Therefore, the model was reliable and could be used in the following studies.2. The protective effect of the Et OAc extract and the phenolic compound C from Gastrodia elata Blume on the BBB:2.1 Protective effect of the Et OAc extracts from Gastrodia elata Blume. on the BBB:Compared with the sham-operated group, After rats were pre-treated with different concentrations(102.57mg/kg and 11.397mg/kg) of the Et OAc extracts from Gastrodia elata Blume., the EB contents in the brain tissues(P<0.01), the neurologic scores(P<0.01) and the moisture of the brains(P<0.05) were decreased significantly.2.2 Protective effect of the phenolic compound C from Gastrodia elata Blume. on theBBB: Compared with the sham-operated group, after rats were pre-treated with different concentrations(20mg/kg and 10mg/kg) of the phenolic compound C from Gastrodia elata Blume, the neurologic scores and the EB contents in brain tissue were decreased significantly(P<0.01).3. Mechanisms involved in the effects of Et OAc extracts and the phenolic compound C from Gastrodia elata Blume. on the BBB:3.1 Mechanisms involved in the effects of Et OAc extracts from Gastrodia elata Blume. on the BBB:3.1.1 Compared with the sham-operated group, the release of NO were decreased significantly(P<0.001 and P<0.01, respectively) after rats were pre-treated with different concentrations(102.57mg/kg and 11.397mg/kg) of the Et OAc extracts from Gastrodia elata Blume. In addition, the activities of n NOS and i NOS were decreased significantly(P<0.01 and P<0.05, respectively) but the activity of e NOS had no significant change.3.1.2 Compared with the sham-operated group, the protein expression of AQP-4 was significantly decrease(P<0.05), whereas the protein expression of Claudin-5 was markedly increased(P<0.01) after rats were pre-treated with the Et OAc extracts(102.57mg/kg) from Gastrodia elata Blum. After rats were pre-treated with the Et OAc extracts(102.57mg/kg and 11.397mg/kg) from Gastrodia elata Blum., the protein expression of Occludin was statical notably increased(P<0.05), but the expressions of MMPs(MMP-2, MMP-3 and MMP-9) were not significantly influenced.3.2 Mechanisms involved in the effects of the phenolic compound C from Gastrodia elata Blume on the BBB:3.2.1 Compared with the sham-operated group, the release of NO(P<0.05 and P<0.001, respectively) and the activities of n NOS and i NOS(P<0.01 and P<0.001,respectively) were decreased significantly after rats were pre-treated with different concentrations(20mg/kg and 10mg/kg, respectively) of the phenolic compound C from Gastrodia elata Blume. However, the activity of e NOS was not markedly affected.3.2.2 Compared with the sham-operated group, the expressions of AQP-4 were significantly decreased(P<0.01 and P<0.001, respectively) but the expression of Occludin was notably increased(P<0.05) after rats were pre-treated with the phenolic compound C(20mg/kg and 10mg/kg, respectively) from Gastrodia elata Blume.; andthe The expression of Claudin-5 was signicantly(P<0.01) enhanced after the pre-treatment of the phenolic compound C(20mg/kg). could increse the The expressions of MMPs(MMP-2, MMP-3 and MMP-9) were not significantly influenced.Conclusion:1. The Et OAc extracts and the phenolic compound C from Gastrodia elata Blume.significantly decrease the permeability of BBB in a focal cerebral ischemia rat model.Therefore, they show protective effects on the BBB.2. The inhibitions of the NOS, the enhancement of the expressions of the tight junction proteins, including Occludin, Claudin-5, and the attenuation of the protein expression of AQP-4 contribute the protective effects of the Et OAc extracts and the phenolic compound C from Gastrodia elata Blume. on the BBB. |
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