| Objective:People are seriously damaged by ischemic cerebrovascular disease. The leakage of blood brain barrier in acute ischemic stroke results in cerebral edema. Brain edema is aggravated by the continuously leakage of blood brain barrier during the course of disease and increases the risk of hemorrhage transformation. Thus, Early treatment of stroke is focused on reducing effusion of blood brain barrier. However, there is little effective therapy currently available to treat stroke. Despite thrombolysis treatment is recognized as a useful measures in the acute phase, its strictly time-window has limited the workable treatment among patients in the acute phase so that stroke still causes permanent disability of varying degrees. During the recovery, promoting axon growth is the crucial treatment. The concept of Neurovascular unit (NVU) makes the researchers put focus not only on vascular endothelial cells, neurons and other hot research subjects but on the importance of extracellular matrix and peripheral structures. ECM, functionally helping cells to bind together and regulating a number of cellular functions, supports all the cells in NVU. As the main composition of ECM, Fibulin-5 promotes adhesion of endothelial cells by interaction with integrin receptor. Meanwhile, this kind of vertical adhesion maintains the integrity of the BBB by combination of enhancing the expression of tight junction (TJ) protein between the horizontal position of the vascular endothelial cell. In addition, Fibulin-5 competitive binding with fibronectin and integrins receptor a5β1 of endothelial cells, suppresses the signaling way of integrins, reducing the generation of reactive oxygen species (ROS). ROS generated after cerebral ischemia can induce apoptosis of endothelia cells, activate Matrix metalloproteinase (MMPs) and promote TJ degradation, deteriorating the damage of BBB. Therefore, overexpressed Fibulin-5 has the important role in protecting the BBB. ROS is also a critical second messenger molecule on the signal transduction pathways. Fibulin-5 overexpression mediates ROS which can initiate GSK-3β/CRMP-2 pathway and alter activity by phosphorylated modification. GSK-3β/CRMP-2 pathway has close relation with axonal regeneration, as well as facilitating assembly of neuronal microtubules and extending axons. As a multifunctional molecule, Fibulin-5 can’t only maintain the structure of the BBB, but also participate in signaling pathways of axonal regeneration through decreasing ROS after ischemic stroke. ROS induced by integrin signal pathway contributes to the GTPase Rac-1, so the mechanism of regulating ROS by Fibulin-5 participation needs further exploring. Meanwhile, Fibulin-5 can be combined with extracellular superoxide dismutase (eSOD) to mediate degradation of extracellular ROS. To sum up, Fibulin-5 has the critical effect on antioxidants by regulating the generation of ROS from two aspects of source and remove. Therefore, we assume that overexpression of Fibulin-5 reduces the damage after cerebral ischemia reperfusion. We use overexpressed Fibulin-5 recombinant adenovirus vector transfected experimental rats of cerebral ischemia reperfusion, and farther discuss the mechanism.Methods:Part I:1. Overexpression of Fibulin-5(Ad-FBLN) animal models were established. Three titers of adenovirus vector, Ad-HK and normal saline were injected by intracranial stereotaxic instrument. The distribution and intensity of green fluorescent protein (GFP) was obtained with laser confocal microscope. Using PCR and Western-blot to measure the level of Fibulin-5 mRNA and protein. Combining these results and inflammation around the injection, so as to choose the best adenovirus vector titer.2. SD rats were randomly grouped:sham group, I/R group, Ad-FBLN+I/R group, Ad-HK+I/R group. Operation of middle cerebral artery occlusion (MCAO)/reperfusion was performed at 7 days after transfection adenovirus vector. The measurement of neurological scores was performed at 1 day and 3 days.3. Brain and endothelial cells apoptosis were tested by using Tunel staining, immunofluorescence and Western blot at 1 day and 3 days after MCAO/reperfusion.4. MRI was used to detect cerebral edema at 1 day and 3 days after ischemia-reperfusion. Brain barrier permeability was measured via the rate of Evans Blue leakage. The ultrastructure of BBB was tested under the electron microscopy5. Detecting morphological changes of TJ by Immunofluorescence at 1 day and 3 days after MCAO/reperfusion and expression of TJ and MMP-9 were measured by Western blot analyses.6. Detecting the contents of ROS by ELISA at 1 day and 3 days after cerebral reperfusion, T-SOD activity was detected by using SOD kits.7. Detecting the expression of NADPH and NOX2 mRNA level by immunohistochemistry analyses and q-PCR. Rac-1 activity was detected by using Rac-1 kits.Part Ⅱ:1. The model and groups were same to part I. GAP-43, Fibulin-5, p-GSK-3p, GSK-3β and p-CRMP-2, CRMP-2 protein were measured by Western blot at 3 days and 7 days after MCAO/reperfusion.2. The mRNA of GAP-43 and Fibulin-5 were detected by q-PCR at 3 days and 7 days after cerebral ischemia reperfusion.3. Immunofluorescence tested GAP-43 expression in ischemia tissue at 3 and 7 days after MCAO/reperfusion.4. BDA was used to mark axons at 6 weeks after cerebral ischemia reperfusion. Number of positive labeled nerve fibers were calculated in each model group to determine the role of overexpressed Fibulin-5 in axonal regeneration after MCAO/reperfusion.5. Detecting the content of ROS by ELISA at 3 days and 7 days after cerebral reperfusion and T-SOD activity was detected by using SOD kit.6. Neurological function scores, balance beam walking and stair experiment were used to estimate motor function at 3 days and 7 days after cerebral ischemia reperfusion in each model group.Results:PartⅠ:1. Different titers of recombinant adenovirus were transfected in rat. Combined with these results of GFP, IL-1β and Fibulin-5 expression,9.5 × 1010 pfu/ml was considered to the optimum transfection titer.2. Compared with I/R and Ad-HK+I/R group, neurological scores were significantly improved at 1 day and 3 days in Ad-FBLN+I/R group after cerebral ischemia reperfusion(P<0.01, P<0.05).3. Overexpression of Fibulin-5 reduced the brain cells apoptosis at 1 day and 3 days after MCAO/reperfusion, especially the vascular endothelial cells. Western blot results also showed apoptosis-related proteins Caspase-3 and p-AKT had significantly differences in Ad-FBLN+I/R group compared to Ad-HK+I/R group and I/R group(P< 0.05).4. Compared with Ad-HK+I/R group and I/R group, the leakage of BBB and cerebral edema were significantly decreased in Ad-FBLN+I/R group at 1 day and 3 days after cerebral ischemia reperfusion(P<0.05, P< 0.01; P<0.05). The level of MMP-9 and occludin proteins were decreased and increased respectively in Ad-FBLN+I/R group at 1 day and 3 days after cerebral ischemia reperfusion(P<0.055 P<0.05).5. Overexpression of Fibulin-5 reduced the production of ROS in brain tissue at 1 day and 3 days after cerebral ischemia reperfusion(P< 0.05, P< 0.01), and enhanced the combination of SOD3 on vascular endothelial cell. However, there was no significant differences in the activity of T-SOD in all groups(P>0.05).6. The level of NADPH and NOX2 mRNA were significantly increased in other groups compared to sham group(P<0.05). There was no significant differences among I/R group, Ad-FBLN+I/R group and Ad-HK+I/R group (P>0.05). Interestingly, expression of GTPase Rac-1 was increased in Ad-FBLN+I/R group compared with Ad-HK+I/R and I/R group (P<0.05).Part Ⅱ1. The level of GAP-43 and Fibulin-5 were significantly increased in other groups at 3 days and 7 days after cerebral ischemia reperfusion compared to sham group(P<0.01). Compared with Ad-HK+I/R and I/R group, the expression of GAP-43 was significantly increased in Ad-FBLN+I/R group(P< 0.05). The level of p-GSK-3β protein was increased after ischemia reperfusion, especially in Ad-FBLN+I/R group(P < 0.01). The protein level of p-CRMP-2 was increased after ischemia reperfusion(P< 0.01), but overexpression of Fibulin-5 reduced the p-CRMP-2 protein expression(P< 0.01). An opposite tendency was observed in the expression of CRMP-2 in all groups compared to p-CRMP-2.2. The mRNA level of Fibulin-5 and GAP-43 were significantly increased in other groups at 3 days and 7 days after cerebral ischemia reperfusion compared to sham group(P< 0.05). Compared with Ad-HK+I/R and I/R group, the level of GAP-43 was significantly increased in Ad-FBLN+I/R group(P< 0.01). The results were similar to immunofluorescence images.3. We observed nerve fibers from ischemic contralateral across the midline on red nucleus plane in all groups at 6 weeks after cerebral ischemia reperfusion. The number of positive fibers was larger in Ad-FBLN+I/R group than in Ad-HK+I/R and I/R group (P<0.05). However, no statistical difference was observed between Ad-HK+I/R and I/R group(P>0.05).4. The level of ROS was significantly increased in other groups at 3 days and 7 days after cerebral ischemia reperfusion compared to sham group(P<0.01). The level of ROS was significantly increased in Ad-FBLN+I/R group compared to Ad-HK+I/R group and I/R group(P<0.01). Interestingly, the expression of GTPase Rac-1 was increased in Ad-FBLN+I/R group compared with Ad-HK+I/R and I/R group (P<0.05).5. Neurological function scores, balance beam walking and stair experiment were used to estimate motor function at 3 days,7 days and 28 days after MCAO/reperfusion in all groups. Compared with Ad-HK+I/R and I/R group, the motor function of rats in Ad-FBLN+I/R group was significantly ameliorated (P<0.05).Conclusions:1. Overexpression of Fibulin-5 alleviates the BBB injure and brain edema after MCAO/reperfusion and improves neurological scores by reducing the apoptosis of endothelial cells, the degradation of occludin and the activation of MMP-9.2. The protection mechanism of overexpressed Fibulin-5 on BBB dues to the decreased ROS, which attributes to reduce the activity of Rac-1 protein and increase the combination of SOD3 and blood vessels.3. Overexpression of Fibulin-5 enhances the expression of GAP-43 around ischemic cortex and lateral branch sprout of contralateral cortex efferent fibers.4. Overexpression of Fibulin-5 initiates GSK-3β/CRMP-2 pathway by mediating the production of ROS. The production of reduced ROS after MCAO/reperfusion promotes the expression of p-GSK-3β and decreases the expression of p-CRMP-2 in order to promote axonal regeneration and neurological function recovery. |
PDF Full Text Request