基于蛋白质组学的生脉注射液抗脑缺血作用机制  

Mechanism of Shengmai Injection Against Cerebral Ischemia Based on Proteomics

作　　者：刘憬曈 胡少伟 常梦丽 许静 蔡晴晴 李兴洪 唐力英[1] 王欢欢 吴宏伟[1] LIU Jingtong;HU Shaowei;CHANG Mengli;XU Jing;CAI Qingqing;LI Xinghong;TANG Liying;WANG Huanhuan;WU Hongwei(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Pharmacy,Zunyi Medical University,Zunyi 563099,China;Hospital of Integrated Chinese and Western Medicine,Tianjin 300100,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]遵义医科大学药学院,贵州遵义563099 [3]天津市中西医结合医院,天津300100

出　　处：《中国实验方剂学杂志》2025年第9期57-67,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：北京市自然科学基金项目(7242249);天津市卫生健康委员会中医中西医结合科研课题(2023165)。

摘　　要：目的:评价生脉注射液抗脑缺血的药理效应,并对其神经保护机制进行研究。方法:SPF级雄性SD大鼠按照随机数字表法分为假手术组,模型组,生脉注射液低、中、高剂量组(3、6、12 mL·kg^(-1))和阳性药金纳多注射液组(4 mL·kg^(-1)),每组12只。采用线栓法制备大鼠脑缺血再灌注(MCAO/R)模型,再灌注后给药,连续给药3 d。通过神经功能评分、脑梗死面积、苏木素-伊红(HE)染色、尼氏染色、原位末端标记法(TUNEL)染色和蛋白免疫印迹法(Western blot)评价生脉注射液对MCAO/R大鼠脑损伤的保护作用;利用蛋白质组学技术探索生脉注射液改善大鼠脑损伤的优势环节和关键蛋白;基于肾上腺素嗜铬细胞瘤(PC12)细胞氧糖剥夺/复氧复糖(OGD/R)模型及MCAO/R大鼠模型,采用酶联免疫吸附测定法(ELISA)、Western blot和氯离子荧光探针法,进一步对相关机制进行验证。结果:与假手术组比较,模型组大鼠神经功能评分、脑梗死面积、神经元凋亡率及凋亡相关蛋白的表达水平明显升高(P<0.05,P<0.01);尼氏小体和神经元密度显著下降(P<0.01);与模型组比较,生脉注射液给药后明显降低大鼠的神经功能评分、脑梗死面积、神经元凋亡率及凋亡相关蛋白的表达量(P<0.05,P<0.01),增加尼氏小体和神经元密度(P<0.05)。蛋白质组学分析结果表明,氧化应激和炎症反应为生脉注射液的主要作用环节,蛋白氯化物细胞内通道蛋白1(CLIC1)为其作用网络中的关键蛋白之一。并且基于MCAO/R大鼠模型,生脉注射液给药后明显改善大鼠脑组织中氧化应激和炎症反应相关指标(P<0.05,P<0.01),回调CLIC1蛋白和下游NOD样受体蛋白3(NLRP3)蛋白的表达水平(P<0.01);基于体外PC12细胞OGD/R模型,生脉注射液显著提高细胞生存率(P<0.01),并降低细胞内氯离子浓度(P<0.05)。结论:生脉注射液具有神经保护作用,氧化应激和炎症反应为其干预脑缺血再灌注损伤的主要作�Objective:To evaluate pharmacological effects of Shengmai injection(SMI)on cerebral ischemia and study its neuroprotective mechanism.Methods:Male specific pathogen-free(SPF)Sprague-Dawley(SD)rats were randomly divided into a sham group,a model group,a low-dose SMI group(3 mL·kg^(-1)),a middle-dose SMI group(6 mL·kg^(-1)),a high-dose SMI group(12 mL·kg^(-1)),and a Ginaton group(4 mL·kg^(-1))according to the random number table method,with 12 rats in each group.The rat model of cerebral ischemia-reperfusion(MCAO/R)was prepared via the suture method.The administration groups were intraperitoneally injected with corresponding concentrations of SMI or Ginaton injection after reperfusion,which was conducted for 3 consecutive days.The sham group and model group were administered the equivalent volume of physiological saline.The pharmacological effects of SMI on brain injury in MCAO/R rats were evaluated by neurological function scores,cerebral infarction area,hematoxylin-eosin(HE)staining,Nissl staining,terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining,and Western blot.The dominant link and key protein of SMI treating cerebral injury were explored using proteomic analysis.The related mechanisms of SMI were further validated using enzyme-linked immunosorbent assay(ELISA),Western blot,and chloride ion fluorescence probe with oxygen-glucose deprivation/reoxygenation(OGD/R)-treated PC12 cells and MCAO/R rats.Results:Compared with the sham group,the model group showed significantly increased neurological function scores,cerebral infarction area,neuronal apoptosis rate,and expression levels of apoptosis related proteins(P<0.05,P<0.01)and significantly decreased density of Nissl bodies and neurons(P<0.01).Compared with the model group,the SMI groups exhibited significantly decreased neurological function scores,cerebral infarction area,neuronal apoptosis rate,and expression levels of apoptosis related proteins(P<0.05,P<0.01)and significantly increased density of Nissl bodies and neurons(P<0.05).Th

关 键 词：生脉注射液 脑缺血再灌注损伤 氧化应激 炎症反应 氯化物细胞内通道蛋白1(CLIC1)蛋白 

分 类 号：R285[医药卫生—中药学] R269[医药卫生—中医学] R289