补气活血合剂对脑缺血再灌注模型大鼠血清外泌体及Nestin、GFAP蛋白表达的影响.pdf
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1、中医康复2024年2月 第1卷 第2期Traditional Chinese Medicine Rehabilitation,Feb.2024,Vol.1,No.02补气活血合剂对脑缺血再灌注模型大鼠血清外泌体补气活血合剂对脑缺血再灌注模型大鼠血清外泌体及及NestinNestin、GFAPGFAP蛋白表达的影响蛋白表达的影响*刘通1,2,廖翔宇1,陈玉宁1,蒋颖1,陈琼君1,熊亮1,邝伟川1,2,文希1,2,刘悦1,2(1.广州中医药大学第五临床医学院,广东 广州 510095;2.广东省第二中医院(广东省中医药工程技术研究院),广东 广州 510095)摘摘要要 目的目的:观察补气活血合剂
2、对脑缺血再灌注模型大鼠血清外泌体及巢蛋白(Nestin)、胶质纤维酸性蛋白(glial fibrillaryacidic protein,GFAP)表达的影响。方法方法:将40只雄性SD大鼠随机分为假手术组、模型组、补气活血合剂组及补气活血合剂+外泌体抑制剂GW4869组(后简称合剂+GW4869组)各10只,模型组、补气活血合剂组及合剂+GW4869组均采用大脑中动脉线栓法复制大鼠大脑中动脉梗塞模型,假手术组仅给予颈内动脉、颈外动脉及颈总动脉的解剖分离而不进行线栓插入,缺血2h后拔除线栓进行再灌注;补气活血合剂及合剂+GW4869组分别在再灌注2h后开始灌胃补气活血合剂(10mL/kg,3次
3、/日),合剂+GW4869组在每次灌胃前给予外泌体抑制剂GW4869腹腔注射(2.5mg/kgd),假手术组及模型组给予灌胃等量生理盐水,四组均连续干预7天。7天后,分别记录各组大鼠干预前后的神经功能缺损评分,TTC染色法计算干预后各组大鼠脑梗死体积,HE染色法评估各组大鼠神经组织形态学变化,Western Blot法检测大鼠梗死区组织Nestin、GFAP蛋白及血清外泌体CD63蛋白的表达水平。结果结果:干预7天后,假手术组未表现出神经功能缺损症状,模型组神经功能缺损评分显著升高(P0.01),补气活血合剂组神经功能缺损评分较模型组显著降低(P0.05),应用GW4869后,神经功能缺损评分
4、较补气活血合剂组显著升高(P0.01)。TTC染色结果显示:假手术组大鼠脑组织成正常红色,未见肉眼苍白梗死灶,模型组、补气活血合剂组、合剂+GW4869组均可见右侧大脑苍白梗死灶;与假手术组相比,模型组、补气活血合剂组、合剂+GW4869组大鼠的脑梗死体积比均显著增加(P0.01);与模型组相比,补气活血合剂组的大鼠脑梗死体积比显著降低(P0.05);与补气活血合剂组相比,合剂+GW4869组大鼠的脑梗死体积比均显著增加(P0.01)。HE结果显示:假手术组染色均匀,组织完整,神经细胞形状为圆锥状,排列整齐,核大居中,有明显的核仁;模型组脑皮层神经细胞排列不规则,神经元明显肿胀,核仁不明显,出
5、现核固缩、细胞间隙存在不同大小的空泡样改变,出现间质水肿;与模型组相比,补气活血合剂组、合剂+GW4869组神经元坏死减少、组织水肿的形态表现较轻;与合剂+GW4869组相比,补气活血合剂组坏死神经元和脑组织水肿等病理损伤较轻。Western Blot结果显示:与假手术组相比,模型组GFAP、CD63表达显著降低(P0.05);与模型组相比,补气活血合剂组GFAP、CD63表达显著升高(P0.05);加用GW4869后,补气活血合剂组GFAP、CD63表达显著下降(P0.05)。结论结论:补气活血合剂可缩小脑梗死体积,提高脑梗死组织中GFAP蛋白的表达,改善大鼠缺血再灌注后神经功能缺损程度,该
6、作用可能与促进外泌体分泌有关。关键词关键词 补气活血合剂;缺血再灌注;神经功能缺损;外泌体;Nestin;GFAP;CD63 中图分类号中图分类号 R R285285.5 5 文献标识码文献标识码 B B 文章编号文章编号 20972097-31283128(20242024)0202-00080008-0606DOIDOI:1010.1978719787/j/j.issnissn.20972097-31283128.20242024.0202.002002Effects of Buqihuoxue Compounds on Serum Exosomes and the Expression
7、ofEffects of Buqihuoxue Compounds on Serum Exosomes and the Expression ofNestin and GFAP Proteins in Rats with Cerebral Ischemia/Reperfusion ModelNestin and GFAP Proteins in Rats with Cerebral Ischemia/Reperfusion ModelLIU Tong1,2,LIAO Xiang-yu1,CHEN Yu-ning1,JIANG Ying1,CHEN Qiong-jun1,XIONG Liang1
8、,KUANG Wei-chuan1,2,WEN Xi1,2,LIU Yue1,2(1.The Fifth Clinical Medical School of Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510095;2.Guangdong Second Traditional Chinese Medicine Hospital(Guangdong Research Institute of Traditional Chinese Medicine Manuracturing Technology),Guangzho
9、u,Guangdong 510095)AbstractObjectiveAbstractObjective:To observe the effect of Buqihuoxue compounds on serum exosomes and the expression of Nestin and GFAP Proteins in ratswith cerebral ischemia/reperfusion model.MethodsMethods:40 male SD rats were randomly divided into the sham operation group,mode
10、l group,Buqihuoxue compounds group and Buqihuoxue compounds+exosome inhibitor group,with 10 rats in each group.In the model group,the Buqihuoxue compounds group and the Buqihuoxue compounds+exosome inhibitor group,the rat model of middle cerebral artery occlusion was madeby suturing of the middle ce
11、rebral artery,while rats in the sham operation group was just given the dissection and isolation of the internal carotid artery,external carotid artery and common carotid artery.After 2 hours of cerebral ischemia,the middle artery suture was removed for reperfusion.Ratsin Buqihuoxue compounds group
12、and Buqihuoxue compounds+exosome inhibitor group were given Buqihuoxue compounds after 2 hours ofreperfusion(10mL/kg,3 times/day),the exosome inhibitor GW4869 was given intraperitoneally before each gavage in the Buqihuoxue compounds+exosome inhibitor group(2.5mg/kgd).The sham operation group and th
13、e model group were given an equal amount of saline by gavage,andall four groups were intervened for 7 consecutive days.The neurological deficit scores of the rats in each group were recorded before and after the in*基金项目基金项目:广东省中医药局专项研究项目,编号:20203001;国家自然科学基金,编号:82174482;广东省自然科学基金,编号:2022A1515011676作
14、者简介作者简介:刘通(1987-),男,博士,副主任中医师、博士研究生导师,研究方向:脊柱术后康复、脑卒中的临床及机制研究。通讯作者通讯作者:刘悦(1968-),男,硕士,主任中医师、博士研究生导师,研究方向:神经康复。8第2期刘通 等 补气活血合剂对脑缺血再灌注模型大鼠血清外泌体及Nestin、GFAP蛋白表达的影响tervention,respectively;HE staining method was used to assess the morphology changes of the neurological tissues;the volume of cerebral infa
15、rctionwas calculated by TTC staining method;Western Blot were used to detect the expression of Nestin,GFAP in the infarcted brain tissue and CD63 inthe serum.ResultsResults:After 7d of intervention,the sham operation group did not show symptoms of neurological deficits,the neurological deficits scor
16、eof the model group was significantly higher(P0.01),the neurological deficits score of the Buqihuoxue compounds group was significantly lowerthan that of the model group(P0.05),and the score was significantly higher after the application of GW4869(P0.01);TTC staining showed thatthe brain tissue in t
17、he sham operation group was normal red,and no pale infarct foci were seen,while pale infarct foci were seen on the right side ofthe brain in the model group,Buqihuoxue compounds group and Buqihuoxue compounds+exosome inhibitor group;the infarct volume of themodel group,the Buqihuoxue compounds group
18、 and Buqihuoxue compounds+exosome inhibitor group was significantly increased comparedwith that of the sham operation group(P0.01);and the volume in Buqihuoxue compounds group was significantly lower than that in the modelgroup(P0.05).Compared with the Buqihuoxue compounds group,the volume in the Bu
19、qihuoxue compounds+exosome inhibitor group was significantly higher(P0.01);HE showed that the sham operation group was uniformly stained,tissue intacted,and the neuronal cells were neatly arranged with conical shape,the nucleus was large in the middle with a pronounced nucleoli.In the model group,th
20、e brain nerve cells irregularly arranged,neurons obviously swollen,the nuclei consolidation.Vacuole-like changes of different size existed in the cell space,combined with interstitial edema.Compared with the model group,neuronal necrosis reduction and tissue edema were milder in the Buqihuoxue compo
21、unds and Buqihuoxue compounds+exosome inhibitor group.Compared with the Buqihuoxue compounds+exosome inhibitor group,the neuronal necrosis reduction and tissue edema was milder in the Buqihuoxue compounds group;Western Blot showed that the expression of GFAP and CD63 was significantly lower in the m
22、odel group compared with the sham operation group(P0.05),while the expression in Buqihuoxue compounds group were siginificantly improved(P0.05).ConclusionConclusion:Buqihuoxue compounds can reduce the volume of cerebral infarction,increase the expression of GFAP proteins in cerebral infarcted tissue
23、s,andimprove the degree of neurological deficits after ischemia/reperfusion in rats,which may be related to the promotion of exosome secretion.KeywordsKeywordsBuqihuoxue compounds;ischemia-reperfusion;neurologic deficits;exosomes;Nestin;GFAP;CD63脑卒中严重危害人类健康和生命安全,其发病率高、病死率高、致残率高、复发率高。2019年,发表于柳叶刀子刊 L
24、ancet Neurology 的一项研究对1990年至2019年的204个国家和地区进行了调查,调查结果显示:全球有1220万中风发病病例,1.01亿中风患病病例,1.43亿伤残调整寿命年中风病例,以及655万中风死亡病例。该结果表明:中风仍是世界第二大致死原因、第三大致残原因(按伤残调整生命年衡量)1。在我国,缺血性脑卒中在所有脑卒中患者中所占比例可高达85%,缺血性脑卒中幸存者中约有3/4的患者遗留有不同程度的劳动能力丧失,给社会、家庭带来极大的负担2-3。因此,进一步探讨如何促进脑卒中后肢体功能障碍的康复仍是全球亟待解决的问题。补气活血合剂(原名:中风复元合剂)是我院院内制剂,对治疗气
25、虚痰瘀型缺血性脑卒中有显著的临床疗效4-5,然而其具体起效机制仍不明确。近期,有多项研究表明小胶质细胞、少突胶质细胞、星形胶质细胞和神经干细胞等多种中枢神经系统细胞均能分泌外泌体,并调节神经系统疾病的发生和发展,这些中枢神经系统细胞衍生的外泌体在促进血管生成、调节炎症和中风后大脑重塑方面发挥着关键作用6-7。巢蛋白(Nestin)是神经干细胞的特征性标志物,胶质纤维酸性蛋白(glial fibrillary acidicprotein,GFAP)是星形胶质细胞活化的标志物,卒中后神经细胞的再生多伴随有Nestin和GFAP的表达升高8-9。因此,本研究拟观察补气活血合剂对脑缺血再灌注模型大鼠血
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