作者：马瑞雪,张綦慧

单位：

1.100029 北京市，北京中医药大学 2.100078 北京市，北京中医药大学东方医院脑病二科 通信作者：张綦慧，E-mail：qihuizhang@vip.sina.com

Units：

1.Beijing University of Chinese Medicine, Beijing 100029, China 2.The Second Department of Encephalopathy, Dongfang Hospital, Beijing University of Chinese Medicine, Beijing 100078,China Corresponding author: ZHANG Qihui, E-mail: qihuizhang@vip.sina.com

关键词：

卒中; 急性缺血性脑卒中; 再灌注损伤; 血瘀毒损证; 苦碟子注射液; 神经功能; 海马神经元;

Keywords：

Stroke; Acute ischemic stroke; Reperfusion injury; Syndrome of blood stasis and toxin damage; Kudieziinjection; Nerve function; Hippocampal neurons

CLC：

DOI：

10.12114/j.issn.1008-5971.2021.00.285

Funds：

国家自然科学基金资助项目（81704051）；国家重点研发计划项目（2018YFC1312300）

摘要：

背景急性缺血性脑卒中(AIS)后脑缺血再灌注损伤血瘀毒损证患者的神经功能损伤具有不可逆的特点,因此探讨苦碟子注射液预处理对其神经功能及海马神经元的影响具有一定价值。目的 分析苦碟子注射液预处理对AIS后脑缺血再灌注损伤血瘀毒损证模型大鼠神经功能及海马神经元的影响。方法 本实验时间为2020年10月至2021年2月。采用随机数字表法将64只SPF级成年雄性Wistar大鼠分为假手术组、模型组和治疗组,每组18只,余10只备用。治疗组构建AIS后脑缺血再灌注损伤血瘀毒损证模型,并进行苦碟子注射液预处理。模型组构建AIS后脑缺血再灌注损伤血瘀毒损证模型,不进行苦碟子注射液预处理。假手术组不构建AIS后脑缺血再灌注损伤血瘀毒损证模型。观察各组大鼠整个实验过程中的症状、体征;分别于造模后1、3、6、24、72 h及7 d选取各组大鼠4只,评估其Zea Longa评分以观察其神经功能缺损情况;分别于造模后1、3、6、24、72 h及7 d选取各组大鼠3只,透射电镜下观察其海马神经元形态及超微结构。结果 症状、体征:假手术组大鼠活动灵活,四肢对称;模型组大鼠随着造模时间的延长,逐渐出现爪甲肿胀,鼠尾黑紫甚至出现断尾,易激惹,活动受限;治疗组大鼠爪甲肿胀及黑尾、断尾程度较模型组减轻,活动轻微受限,四肢稍显不对称。假手术组大鼠Zea Longa评分均为0分。治疗组大鼠造模后24h、72 h、7 d Zea Longa评分低于模型组(P <0.05)。模型组大鼠造模后24 h、72 h、7 d Zea Longa评分高于本组造模后1 h,造模后72 h、7 d Zea Longa评分高于本组造模后3、6 h(P <0.05);治疗组大鼠造模后7 d Zea Longa评分高于本组造模后1 h(P <0.05)。海马神经元形态及超微结构:假手术组大鼠海马神经元及细胞器结构完整。模型组大鼠造模后6 h海马神经元体积缩小;造模后24、72 h可见细胞核破碎,细胞器大量减少,线粒体肿胀,高尔基体和粗面内质网囊性扩张、肿胀;造模后7 d可见神经元大量减少,形态不规则,细胞器结构破坏严重,线粒体呈空泡状态。治疗组大鼠造模后6 h海马神经元与模型组相比细胞器增多,细胞器结构轻度改变;造模后72 h可见线粒体肿胀程度减轻,高尔基体、粗面内质网形态较好,空泡减少甚至消失,各细胞器结构状态明显好于同期模型组。结论 苦碟子注射液预处理可以减轻AIS后脑缺血再灌注损伤血瘀毒损证模型大鼠的神经功能缺损情况,抑制大鼠患侧海马神经元形态及超微结构的改变,对脑组织具有一定的保护作用,且在造模后6～72 h的作用最明显。

Abstract：

【Abstract】 Background Patients with blood stasis and toxin syndrome of cerebral ischemia-reperfusion injuryafter acute ischemic stroke (AIS) have irreversible neural function injury. Therefore, it is of certain value to explore the effect ofKudiezi injection pretreatment on their neural function and hippocampal neurons. Objective To investigate the effect of Kudieziinjection pretreatment on neural function and hippocampal neurons in rats with blood stasis and toxin syndrome of cerebralischemia-reperfusion injury after AIS. Methods This study was conducted from October 2020 to February 2021. A total of 64SPF adult male Wistar rats were divided into sham group, model group and treatment group according to random number tablemethod, with 18 rats in each group and the other 10 rats as spares. In the treatment group, the blood stasis and toxin syndromemodel of cerebral ischemia-reperfusion injury after AIS was constructed, and Kudiezi injection was pretreated. In the modelgroup, the blood stasis and toxin syndrome model of cerebral ischemia-reperfusion injury after AIS was constructed withoutKudiezi injection pretreatment. The sham group did not construct the blood stasis and toxin syndrome model of cerebral ischemiareperfusion injury after AIS. The symptoms and signs of rats in each group during the whole experiment were observed. Four ratsin each group were selected at 1, 3, 6, 24, 72 h and 7 d after modeling, and their Zea Longa score was evaluated to observe theirneurological deficit. Three rats in each group were selected at 1, 3, 6, 24, 72 h and 7 d after modeling, and the morphology andultrastructure of hippocampal neurons were observed under transmission electron microscope. Results Symptoms and signs:rats in the sham group moved flexibly and had symmetrical limbs. In the model group, with the prolongation of modeling time,the rats gradually appeared paw nail swelling, black and purple tail and even broken tail, irritable, limited movement. Comparedwith model group, the degree of paw nail swelling, black and broken tail of the rats in the treatment group were reduced, theiractivities were slightly restricted, and the limbs were slightly asymmetrical. Zea Longa score of rats in sham group was 0. TheZea Longa scores of rats in the treatment group were lower than those in the model group at 24 h, 72 h, and 7 d after modeling(P < 0.05) . The Zea Longa score of the model group at 24 h, 72 h and 7 d after modeling was higher than that at 1 h aftermodeling, and the Zea Longa score at 72 h and 7 d after modeling was higher than that at 3 and 6 h after modeling (P < 0.05) .The Zea Longa score of rats in the treatment group at 7 d after modeling was higher than that at 1 h after modeling (P < 0.05) .Morphology and ultrastructure of hippocampal neurons: the structures of hippocampal neurons and organelles in sham group wereintact. In model group, volume of hippocampal neuron decreased at 6 h after modeling; at 24 and 72 h after modeling, the nucleuswas broken, the organelles were greatly reduced, the mitochondria were swollen, and the Golgi apparatus and rough endoplasmicreticulum were expanded and swollen; at 7 d after modeling, a large number of neurons were reduced, the shape was irregular,the organelle structure was seriously damaged, and the mitochondria were in a vacuole state. Compared with the model group,the organelles of hippocampal neurons in the treatment group increased and the structure of organelles changed slightly at 6 hafter modeling; at 72 h after modeling, the swelling of mitochondria was reduced, the morphology of Golgi and rough endoplasmicreticulum was better, vacuoles were reduced or even disappeared, and the structure of each organelle was significantly better thanthat of the model group in the same period. Conclusion Kudiezi injection pretreatment can reduce the neurological deficit ofrats with blood stasis and toxin syndrome of cerebral ischemia-reperfusion injury after AIS, inhibit the changes of morphology andultrastructure of hippocampal neurons on the affected side, and have a certain protective effect on brain tissue, and the effect ismost obvious at 6-72 h after modeling.

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