| Blood brain barrier (BBB) is a natural barrier between blood and brain tissue, with its strictly selective screening effect, BBB can effectively guarantee the material exchange between blood and brain tissue fluid, and the inflammatory response triggered by ischemia reperfusion has been considered as one of the key links in BBB damage.After cerebral ischemia, as the resident immune response cells, microglial cells were first activated, produce neurotrophic factors to protect neurons, while release the inflammatory factor, produce reactive oxygen species (ROS) against intracellular proteins, nucleic acids and other ingredients, cause oxidative stress damage. This topic focuses on ischemia-reperfusion injury of inflammation, early microglia response to BBB structure and function change were set to be the research target, the experiment is divided into two parts, in vitro and in vivo studies. in vitro part, by using the matured rat middle cerebral artery block model, observation group after building neural function in rats, observe the cerebral ischemia in rats after treatment with QingKaiLing injection. infarcts surrounding pathological changes in morphology, use immunehistochemical method to observe the microglia and ZO-1, COX-2, gp91phox expression changes, preliminary understand the protection function of QingKaiLing injection for excessive activated microglial cells after cerebral ischemia. In vitro experimental section, innovative uses two kinds of cell culture in vitro models, research about relations btween QingKaiLing injection and TLR4 signaling pathways, its downstream protein and the BBB damage, and also study the influence of probe microglia activation mechanism and the toxic effect of BBB may produce.Objective:1 The relatively matured rat model of middle cerebral artery obstruction will be used, observation group after building neural function in rats, observe the cerebral ischemia in rats after treatment with QingKaiLing injection infarcts surrounding pathological changes in morphology, further using immunohistochemical method to observe the microglia and ZO-1 and COX-2, gp91phox expression changes, preliminary understand QingKaiLing injection excessive the activated microglial cells after cerebral ischemia inhibition and protection of BBB.2 The method of cell culture, establish and study the microglia and were cultured in vitro BBB model method. In-depth study after hypoxia and reoxygenation activated microglial cells may through the activation of TLR4 pathway of BBB damage mechanism, open for QingKaiLing injection based on detoxification method treatment of ischemic encephalopathy provide new theoretical basis and train of thought.3 Discuss QingKaiLing injection to block the microglia activation link, QingKaiLing injection open way to reduce toxic effect and regulation mechanism, the role of detoxification method,provide experimental basis for clinical treatment of stroke, further reflected in the clinical treatment of "toxin impairing brain collaterals" academic point of view.Methods:1100 SPF Sprague-Dawley rats were randomly divided into control group,the rest of the rats using modified Longa method preparation of focal cerebral ischemia (MCAO) model, building a successful rats randomly divided into model group, the minocycline group, QingKaiLing injection group, all the animals in the postoperative observation record general status and the function score, to build model by intraperitoneal injection of success after 2 h, respectively, after 24 h,48 h the brain were taken, to observe staining pathology change in HE, immunohistochemical staining after microglia, ZO-1, COX-2 and gp91phox changes.2 Two mice BV2 microglial cells in vitro culture can be divided into 6 groups:(1) the control group, using serum-free DMEM-H culture medium; (2) the model group, using serum-free medium DMEM-H, hypoxia and reoxygenation. (3) QingKaiLing low, medium and high concentration groups, respectively in the anoxic when join in serum-free DMEM-H culture medium preparation of different concentrations of QingKaiLing injection; (4) the minocycline group, when a lack of oxygen to join prepared with serum-free medium DMEM-H minocycline;3 In vitro culture BV2 cell hypoxia and reoxygenation model:to hypoxia (1.0% O2, serum free medium DMEM-H), then after oxygen (5% CO2, do not change the culture method of simulation model of brain ischemia-reperfusion injury; With CCK-8 colorimetric method, NO method of evaluation model.4:In vitro BBB model trained model 6 groups of BV2 cells in cell interpolator, spirit to open different doses, microglia activation inhibitors minocycline intervention,24 h under anoxic environment cause activation, anoxic end, insert the microglia cells into the device in the endothelial cells of 6 orifice, disregards incubator (95% of air+5% CO2) in the trained after 24 h of microglia activation toxic effects on endothelial cells and the influence of QingKaiLing injection of ischemia-reperfusion injury co-culture model of oxidative stress and inflammation in the intervention effect of research:(1) the CCK-8 testing the change of cell vitality. (2) NO method to detect cell co-culture on the change of NO concentrations in the supernatant. (3) Western blot method to detect, TLR-4, ZO-1, gp91phox protein expression changes.Results:1 Pathology change:control tissue morphological structure is normal, normal nerve cell morphological structure; Model group to the larger necrosis of the visible area, necrotic area is visible to the size of cavity, scattered remnants of the nerve cells, the visible part of the nerve cells in the nucleus pycnosis, hyperchromatic, did not see nucleoli, visible part of the nerve cells in the nucleus dissolved, cytoplasm loose, cell boundaries blurred, edema, neutrophils in necrosis in peripheral area; The necrosis area of clear open spirit group compared with model group, the nerve cell edema have eased in the model group, cytoplasm and nucleolus is clear. Necrotic area of minocycline group was obviously reduced, small vacuoles are still visible in the infarction area.2. Immunohistochemical staining results:24 h,48 h control group and model group rats the ischemic side of brain tissue BSI-B4 positive cells was detected. Visible BSI-B4 model 24 h BSI-B4 positive cells expression than the normal group significantly increased (P< 0.01), the main distribution around the infarction area and cortical areas, most of the circular or branch of the cell. Visible BSI-B4 model 48h group-a huge increase in BSI-B4 positive cells (P< 0.01), accompanied by the cell body expands, mostly round or amoebic sample cells. Minocycline 24 h group of 24 h, qingkailing injection can reduce the BSI-B4 after MCAO rats had group BSI-B4 the expression of positive cells, compared with the model group had significant difference (P< 0.01). QingKaiLing Injection 48 h can reduce rats after MCAO group within the organization BSI-B4 the expression of positive cells, compared with the model group had significant difference (P< 0.01).ZO-1 immune positive material show puce, microscopically in granular distribution or dot distribution. Control in 24 h,48 h group rats had detected ZO-1 immune positive dot or granular product rendering a large number of expression, the dyeing depth, clearly visible.24 h,48 h after MCAO model group organization in central infarction rats, the hippocampus and cortex in the scope of ZO-1 immune positive result compared with control group rats significantly decreased (P< 0.01), and the dyeing is shallow. Minocycline 24 h group, qing ling injection 24 h after MCAO group in central infarction rats, the hippocampus and cortex ZO-1 immune positive product within the scope of model group significantly increased (P< 0.01). QingKaiLing Injection in infarction 48 h after MCAO rats had the potential within the scope of the central area, the hippocampus and cortex ZO-1 immune positive product increased significantly in the model group (P< 0.01).Cox-2 protein immune positive material show puce, microscopically in granular distribution or dot distribution. Control in 24 h,48 h group rats had the potential to detect COX-2 immune positive dot or granular product for a small amount of expression, dyeing shallow.24h,48h after MCAO model group organization in central infarction rats, the hippocampus and cortex ZO-1 immune positive product within the scope of a large number of expression, the dyeing depth, clearly visible. Compared with control group rats increased significantly (P< 0.01). Minocycline 24h group, qingkailing injection 24h after MCAO group in central infarction rats, the hippocampus and cortex in the scope of COX-2 immune positive product were less than model group (P< 0.05). QingKaiLing Injection in infarction 48h after MCAO rats had center, the hippocampus and cortex COX-2 immune positive product within the scope of model group significantly decreased (P< 0.01).Control of 24 h,48 h group rats had detected gp91phox immune positive dot or granular product for a small amount of expression, dyeing shallow.24h,48h after MCAO model group organization in central infarction rats, the hippocampus and cortex gp91phox immune positive within the scope of products have a large number of expression, the dyeing depth, clearly visible. Compared with control group rats increased significantly (P< 0.01). Minocycline 24h group, QingKaiLing Injection 24h after MCAO group, in central infarction rats, the hippocampus and cortex gp91phox immune positive product within the scope of reduction in the model group (P<0.05). QingKaiLing Injection in infarction 48 h after MCAO rats had center, the hippocampus and cortex gp91phox immune positive product within the scope of model group significantly decreased (P< 0.01).3 Hypoxia (1.0% O2, serum free medium DMEM-H) 24 H after oxygen (5% CO2, not replace the broth) of 12 H method to establish in vitro model of cerebral ischemia reperfusion injury in CCK-8 method results: the model group A value than the normal group decreased significantly (P< 0.01); NO method to detect cell culture supernatant NO concentration results:the model group the NO concentration higher than the normal group significantly (P< 0.01).4 CCK 8 method to detect the change of each cell survival activity,QingKaiLing Injection were 0.0625%, 0.125%, 0.25% concentration group, concentration of minocycline 200 nmol/L set A value compared with the model group significantly increased (P< 0.01), suggesting the drugs in this concentration have a certain activity of cell survivodel5. Group of cell culture supernatant on NO concentration significantly increased than the normal group (P< 0.05),0.0625%, 0.125% and 0.25% of the QingKaiLing Injection, 200 nmol/L minocycline can significantly reduce the hypoxia reoxygenation cause NO BV2 cells, compared with the model group had obvious difference (P< 0.05).6 Western results:normal group has a large number of ZO-1 protein expression, the expression of model group compared with normal group had significantly lower (P< 0.01), 0.0625%, 0.125% and 0.25% of the QingKaiLing Injection, 200 nmol/L minocycline can reduce hypoxia reoxygenation damage, there are differences between the compare model group (P< 0.05), with 0.25% of the QingKaiLing Injection group, 200 nmol/L minocycline compared with model group had significant difference (P< 0.01). Normal group with a small amount of TLR4 protein expression, the expression of model group significantly increased than the normal group (P< 0.05),0.25% of the QingKaiLing Injection can reduce hypoxia reoxygenation in Balb/c cells the expression of TLR4 proteins, there are differences between the compared with model group (P< 0.05), QingKaiLing Injection of 0.0625% ,0.125%, and 200 nmol/L m ring is to reduce the trend, but no statistical difference (P> 0.05).gp91phox protein expression little in the normal group, the expression of model group significantly increased than the normal group (P< 0.05),0.25%, 0.125% of the QingKaiLing injection injection can significantly reduce hypoxia reoxygenation BV2 cells gp91phox protein expression, compared with the model group had significant difference (P< 0.01),200 nmol/L minocycline also significantly reduced gp91phox protein expression (P< 0.05),0.0625% of the QingKaiLing Injection has a tendency to reduce, but no statistical difference (P> 0.05). Conclusion:1.From the point of pathological change, shape and structure of the control group is normal, normal nerve cell morphological structure; Model group microscopically conform to the pathological characteristics of ischemic infarction, all the changes suggest building success.2 Immunohistochemical staining results suggest:QingKaiLing Injection can effectively reduce postoperative microglia MCAO expression, and increase the expression of ZO-1, resume the function of the nervous system, protect the BBB integrity; By reducing the expression of COX-2 and gp91phox after cerebral ischemia, decrease the infarction area, reduce inflammation, protect brain tissue, has a positive guidance for clinical treatment.3 USES gas incubator to simulate the hypoxia environment, lack of oxygen (1.0% O2, serum free medium DMEM-H) 24 H after oxygen (5% CO2, not replace the broth) 24h method to establish simulation model of brain ischemia-reperfusion injury in vitro, successful simulated cerebral ischemia reperfusion damage to the microglia.4 Cells co-culture model test that the supernatant fluid NO expression, prompt BV2 cells after ischemia reperfusion injury will produce a lot of NO, stimulate the endothelial cells, and QingKaiLing injection inhibit NO express way to inhibit microglia activation, thus reducing ischemia-reperfusion injury.5 Western blot shows that BV2 cells ischemia-reperfusion injury after activation induced by the high expression of TLR4, gp91phox protein, and at the same time can protect BBB main constitute ZO-1 protein, thus protecting the brain environment.
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