补阳还五汤减轻氧化应激保护脑缺血再灌注损伤大鼠血脑屏障的作用研究
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河北中医学院

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中央引导地方科技发展资金项目(No.206Z7706G);河北省科技研发平台与新型研发机构建设专项(No.20567626H),河北中医学院科技能力提升重点项目(No.KTZ2019018)


Study on the effect of Buyang Huanwu Decoction in reducing oxidative stress and protecting cerebral ischemia-reperfusion injury in rats' blood-brain barrier
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Hebei University of Chinese Medicine

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    摘要:

    目的:探究补阳还五汤(Buyang Huanwu Decoction, BYHWD)降低氧化应激水平保护脑缺血再灌注损伤(cerebral ischemia/reperfusion injury,CIRI)大鼠血脑屏障(blood-brain barrier,BBB)的机制。方法:采用线栓法建立大鼠大脑中动脉栓塞再灌注(middle cerebral artery occlusion/ reperfusion,MCAO/R)模型,应用PeriCam PSI激光散斑血流成像系统检测脑血流量判定模型建立是否成功。通过Zea Longa评分评价大鼠神经功能缺损情况;HE染色观察大鼠脑组织病理学改变;干湿重法检测脑水肿程度;伊文思蓝(Evans blue, EB)染色检测BBB通透性;透射电镜观察BBB超微结构改变;试剂盒检测氧化应激相关指标活性氧(ROS)、丙二醛(MDA)的含量及总超氧化物歧化酶(SOD)活性;免疫组织化学染色及Western blot检测基质金属蛋白酶-9(MMP-9)的表达水平;免疫荧光双染及Western blot检测紧密连接蛋白(tight junction protein,TJP)中Occludin、ZO-1、Claudin-5蛋白的表达水平。结果:BYHWD能够降低MCAO/R大鼠神经功能缺损评分,减轻脑组织病理学损伤,减轻血脑屏障结构破坏,延长紧密连接结构致密区,减轻缺血侧大脑脑水肿情况,降低BBB通透性。BYHWD可以降低ROS与MDA水平,提高SOD活性,降低MMP-9表达水平,提高Occludin、Claudin-5、ZO-1蛋白表达水平。结论:BYHWD可以升高BBB紧密连接蛋白的表达水平,降低血脑屏障通透性,保护血脑屏障超微结构,减轻脑水肿,其机制可能与BYHWD抗氧化应激,抑制MMP-9的激活相关。

    Abstract:

    Objective To explore the mechanism of Buyang Huanwu Decoction (BYHWD) in reducing oxidative stress levels to protect the blood-brain barrier (BBB) in cerebral ischemia/reperfusion injury (CIRI) rats. Methods: Middle cerebral artery occlusion / reperfusion (MCAO/R)model was established by wire embolization method in rats, and PeriCam PSI laser speckle flow imaging system was applied to detect whether the model establishment was successful. The neurological deficits of rats were evaluated by Zea longa score; The histopathological changes in the rat brain were observed by HE staining; The degree of brain edema was detected by dry and wet weight method; The BBB permeability was detected by Evansblue (EB) staining; Ultrastructural changes of BBB were observed by transmission electron microscopy; The levels of ROS, MDA, and SOD activities, which are related to oxidative stress, were detected by kits; The expression levels of MMP-9 were detected by immunohistochemical staining and Western blot; The protein expression levels of Occludin, ZO-1, and Claudin-5 in TJP were determined by double staining of immunofluorescence and Western blot. Results:BYHWD can reduce neurological deficit scores, alleviate the brain histopathological damage, alleviate the disruption of blood-brain barrier structure, prolonged the dense region of tight junction structure, attenuated the edema of the brain in the ischemic side, and reduce the BBB permeability in MCAO / R rats. BYHWD can decrease the levels of ROS and MDA, increase the activity of SOD, decrease the expression levels of matrix metalloproteinase-9 (MMP-9), and increase the protein expression levels of Occludin, Claudin-5, and ZO-1. Conclusions: BYHWD can increase BBB tight junction protein expression levels, reduce the permeability of the BBB, protect the ultrastructure of the BBB, and reduce brain edema, and its mechanism may be related to the antioxidant stress of BYHWD and inhibition of MMP-9 activation.

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  • 收稿日期:2023-04-20
  • 最后修改日期:2023-07-13
  • 录用日期:2024-02-26
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