| Background and objectiveStroke is one of the most disabling and fatal diseases over the world,divided into ischemic and hemorrhagic stroke,and ischemic sroke takes an account of more than 80%.Ischemic stroke is the consequese of reversible or permanent deficiency of local infusion in cereberal arterial areas,there exsits complicated physiopathologic events after stroke,furthermore,with the development of stroke,the changes like neuronal death、glia cells’proliferation and axonal degeneration would diffuse to the surrounding regions from the ischemic center.This complexity determines that targeted therapy both aiming at neurons and glias is required.Along with immunity、inflammation and regeneration becoming the concerning center of stroke research,the role that glia cells play in the pathogenesis of stroke gains much attention increasingly.Ginkgolide B(GB)is the specific component of gingko,existent evidences show that GB can improve the neurological functional defects which accompany stroke,but there also exsits some contradictory datas in the reporting literature.PAD2 takes part in the pathogenesis of several nervous system diseases,however,whether GB can exert the regulation over PAD2 is not clear.This experiment aims to investigate GB’s protective effects in ischemic stroke and the possible mechanism how GB mediates the degradation of glia scar through PAD2.Experimental methods:Use cellular immunity fluorescence and MTT to identify and detect the living activity of newborn rats’ primary cerebral cortex astrocytes cultured in vitro;use confocal microscope to detect the effects of GB on calcium in astrocytes;the prepration of rat MCAO model;use transmission electron microscope to observe the ultramicro-structure of astrocytes and the rat cerebrum;use immunohistochemical frozen slices to detect the expression of GFAP and PAD2 in the rat cerebrum of different groups.Results1.In vitro testAdministration group:(1)GB in the concentration between 25 and 200μmol/L does not have obvious effects on the living activity of normal cortex astrocytes.In this concentration range,GB can increase the living activity of excitotoxicity injured ASTs(p<0.05),and 200μmol/L GB exerts the most obvious effects,accordingly,choosing 200μmol/L GB as the treating concentration for the following experiment in vitro.(2)The expression of GFAP in ASTs of GB administration group decreased than that in the excitotoxicity group,GFAP in normal group distributed evenly in reticulate shape.In excitotoxicity model group,the form of ASTs retracted,GFAP presented a scattered distribution;in contrast,the changes of GFAP in GB administration group were lighter than model group,tending to recover.(3)PAD2 exsited basic expression activity in normal ASTs,but PAD2 expressed in excitotoxicity ASTs was lower than that in normal group ASTs;In the excitotoxicity injured ASTs treated by GB,the expression of PAD2 increased obviously,higher than the basic expression in normal ASTs.(4)The changes of calcium concentration in ASTs of group treated by GB were close to that in normal group.(5)Use electron microscope to observe the ultramicro-structure of astrocytes in different groups,finding that the form of mitochondria in the astrocytes treated by GB was almostly normal,but the mitochondria’s form in the astrocytes of excited toxical injured group was irregular,part of the mitochondria presented in sphericity because of edema,the cristae broke into pieces and overlapped,even were blurred.Pretreatment group:(1)The expression of GFAP in ASTs of GB pretreatment group decreased than that in the excitotoxicity group,GFAP in normal group distributed evenly in reticulate shape.In excitotoxicity model group,the form of ASTs retracted,GFAP presented a scattered distribution;the astrocytes in GB pretreatment group appeared slight injury,however,the changes of GFAP is lighter than that of model group,tending to recover.(2)PAD2 expressed in GB pretreatment group increased than that in excitotoxicity group ASTs;the basic expression of PAD2 in normal ASTs was higher than PAD2 expressed in model group,and lower than that in the group treated by GB.(3)The intracellular calcium concentration increased quickly,and the increasing degree is bigger than that of excitotoxicity group.(4)Use electron microscope to observe the ultramicro-structure of astrocytes in different groups,finding that the result of GB pretreatment group is similar to the result of GB treatment group(5)mentioned above.2.In vivo test(1)The cerebral infarction areas of MCAO rats after 24h was detected by TTC staining.Control group did not form the white infarction lesions,but in MCAO model group,there was a large area of infarction in the cortex and striatum of rats.The relative infarction areas of 5mg/kg GB treated group and 30mg/kg GB treated group decreased compared with MCAO model group.(2)HE staining showed that the brain damage of MCAO rats treated by GB after 24h was significantly lighter than that of MCAO model group.(3)Observe the ultramicro-structure of rats’ brain cortex in each treatment group and find that mitochondrial morphology of GB administration group and pretreatment group was normal,but in the astrocytes of excitotoxicity model group,there was irregular shape of mitochondria,the cristae broke into pieces and overlapped,even were blurred.Observing brain capillary vessels found that the endothelial cells were flat,had smooth surface and integritive membrane,close and clear connection,and foot processes of astrocytes were normal in control group.Cross section of micro-blood vessels in model group showed that endothelial cells were swelling obviously,basement membrane blurred,the surroundings of capillary exsited edema,podocytes around the capillaries were swelling,and there exsited visible pericytes’ nuclear fragmentation,microglias around the blood vessels and visible platelets in the lumina can also be found.GB administered 24h group:edema slightly alleviated,capillary lumen was normal,basement membrane was continued,podocytes around the capillaries presented alleviative swelling.GB pretreatment group seemed to be worse,because of the notable vascular collapse.(4)The expression of GFAP in the cerebral cortex and cingulate cortex of GB administration group and pretreatment group was significantly lower than that in the MCAO group,which was slightly higher than that in the control group.(5)The expression of PAD2 was significantly higher in the cerebral cortex and cingulate cortex of the GB administration group and the control group than that in the MCAO group,which was slightly higher than that in the control group.Conclusion1.The administration of Ginkgolide B can reduce the damage of astrocytes induced by excitotoxicity in a certain degree,improve the survival rate of astrocytes,and there exsits an effective dose range.2.Ginkgolide B may activate PAD2 through increasing astrocytes intracellular calcium concentration,then inhibit GFAP expression to exert protective effects. |
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