眼针联合运动疗法改善脑缺血再灌注损伤大鼠脑水肿干预机制的实验研究

Experimental Study on Mechanism of Eye Acupuncture Combined with Exercise Therapy to Improve Brain Edema in CIRI Rats

目的 观察眼针联合被动运动疗法对脑缺血再灌注损伤(Cerebral ischemia-reperfusion injury, CIRI)大鼠神经功能缺损评分的改善作用,探讨眼针联合运动疗法对CIRI大鼠血脑屏障保护作用的机制。方法 SPF级SD大鼠80只,采用随机数字法将大鼠随机分为两组:假手术组(15只)和造模组(65只)。然后采用线栓法对造模组大鼠进行CIRI模型复制,模型评价后,剔除造模失败大鼠,将符合成模标准的50只大鼠再次随机分为模型对照组(13只)、眼针组(12只)、运动组(12只)、眼针联合运动组(13只)。造模后,待大鼠完全清醒后,对各组大鼠施加相应的干预因素,每8 h干预1次,连续3 d。末次干预后,进行神经功能缺损评分,各组随机抽取3只大鼠进行血脑屏障通透性检测;随机抽取3只大鼠进行脑含水量检测;随机抽取3只大鼠,采用过量的10%水合氯醛腹腔注射麻醉处死,迅速将大鼠置于冰面上,开颅取全脑,置于4%多聚甲醛溶液中固定,最后各组剩余的大鼠处死后,采集大鼠脑皮质缺血灶周围半暗带脑组织,冻存于-80℃冰箱中,采用免疫组化法和免疫印迹法(Western blot)检测脑组织中缺氧诱导因子(hypoxia inducible factor-1,HIF-1)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、紧密连接蛋白(zonula occludens 1,ZO-1)、紧密连接蛋白5(Claudin-5)的表达水平。结果 与假手术组比较,其余各组大鼠神经功能缺损评分、脑组织含水量、依文思蓝渗出量、HIF-1α、VEGF、MMP-2、MMP-9表达水平均显著升高(P<0.01),ZO-1、Claudin-5表达水平显著降低(P<0.01),差异有统计学意义;与模型对照组比较,眼针组、运动组和眼针联合运动组大鼠神经功能缺损评分、依文思蓝渗出量、HIF-1α、VEGF、MMP-2、MMP-9表达水平均显著降低(P<0.05),ZO-1、Claudin-5表达水平显著升高(P<0.01);眼针联合运动组大鼠依文思蓝渗出量显著降低(P<0.05),差异有统计学意义;与眼针组和运动组比较,眼针联合运动组大鼠神经功能缺损评分、脑组织含水量、依文思蓝渗出量、HIF-1α、VEGF、MMP-2、MMP-9均显著降低(P<0.05),ZO-1、Claudin-5表达水平显著升高(P<0.01),差异有统计学意义。结论 眼针联合运动疗法对CIRI大鼠的血脑屏障保护作用机制与其调控HIF-1α关键靶点及其下游蛋白表达水平密切相关。

Objective To observe the improvement effect of eye acupuncture combined with passive exercise therapy on neurological deficit score in rats with cerebral ischemia-reperfusion injury(CIRI)and explore the mechanism of protective effect of eye acupuncture combined with exercise therapy on blood-brain barrier in CIRI rats.Methods Eighty SD rats of SPF grade were randomly divided into two groups:sham operation group(15 rats)and model group(65 rats).After the model was evaluated,the CIRI rats were eliminated and randomly divided into model control group,eye acupuncture group,exercise group and eye acupuncture combined exercise group.After modeling,after the rats were completely awake,the corresponding intervention factors were applied to rats of each group,once every 8 h for 3 days.After the last intervention,3 rats were randomly selected for blood-brain barrier permeability test.Three rats were randomly selected for brain water content detection,three rats were randomly selected and killed with excessive 10%chloral hydrate intraperitoneal injection anesthesia.The rats were quickly placed on the ice surface,the whole brain was opened and fixed in 4%paraformaldehyde solution.After the remaining rats in each group were put to death,the semi-dark brain tissue around the ischemic foci of the cerebral cortex was collected and frozen in the-80℃refrigerator.The expression levels of hypoxia inducible factor-1(HIF-1α),vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),zonula occludens 1(ZO-1)and Claudin-5 in brain tissue were detected by immunohistochemistry and Western blot.Results Compared with those of the sham operation group,the scores of neurological deficit,water content of brain tissue,vessel leakage and expression levels of HIF-1α,VEGF,MMP-2 and MMP-9 were significantly increased(P<0.01)and the expression levels of ZO-1 and Claudin-5 were significantly decreased(P<0.01).The difference was statistically significant.The scores of nerve function defect,the amount of evans blue exudate and the expression levels of HIF-1α,VEGF,MMP-2 and MMP-9 in the eye acupuncture group,exercise group and eye acupuncture combined exercise group were significantly decreased(P<0.05)and the expression levels of ZO-1 and Claudin-5 were significantly increased(P<0.01).Compared with those of the model control group,the scores of neurological deficit,brain water content,evans blue exudate and the expression levels of HIF-1α,VEGF,MMP-2 and MMP-9 in the eye acupuncture combined exercise group were significantly decreased(P<0.05)and the expression levels of ZO-1 and Claudin-5 were significantly increased(P<0.01)in the eye acupuncture combined exercise group.Conclusion The protective mechanism of eye acupuncture combined with exercise therapy on blood-brain barrier in CIRI rats is closely related to its key target HIF-1αregulation and its downstream protein expression level.