栝楼桂枝汤治疗脑卒中后肌肉痉挛的药效物质基础研究

发布时间：2018-04-09 21:35

本文选题：栝楼桂枝汤　切入点：脑卒中后肌肉痉挛　出处：《福建中医药大学》2016年博士论文

【摘要】：背景：脑卒中,具有患病率高、致残率高、死亡率高等特点。脑卒中后肌肉痉挛是其常见的后遗症,严重影响患者的康复和日常生活能力。目前临床上针对脑卒中后肌肉痉挛的药物,相对匮乏。中医对中风病的认识源远流长,在治疗慢性、多因素的复杂疾病中显示出良好的疗效优势。栝楼桂枝汤,处方来源于《金匮要略》卷上,由栝楼、白芍、桂枝、生姜、甘草及大枣六味药物组成,具有解肌祛邪,舒缓筋脉。主治太阳柔痉体强证。课题组前期临床研究发现,栝楼桂枝汤对治疗中风后肢体痉挛有较好的疗效,然而其药效物质基础尚不明确。本课题获得了福建省科技厅重点项目(项目编号：2012Y0041)的支持。目的：本课题旨在阐释栝楼桂枝汤治疗脑卒中后肌肉痉挛的药效物质基础,探讨其脑靶向优势,为复方药效物质基础研究和脑源性疾病的多靶点治疗提供有力的科学数据支持。方法：(1)采用液质联用方法,对栝楼桂枝汤中的两种氨基酸(瓜氨酸和Y-氨基丁酸)及其他24种活性成分进行分析、测定。(2)采用大脑中动脉栓线阻断法制备局灶性脑缺再灌注损伤模型(MCAO),缺血2h后进行再灌注,分析测定MCAO模型大鼠的神经行为学、脑梗塞面积、病理组织形态、脑脊液内神经递质含量变化,探讨栝楼桂枝汤治疗脑卒中后肌肉痉挛的药效作用。(3)采用超高效液相色谱-质谱联用(UPLC-MS)方法,分析检测栝楼桂枝汤给药后脑缺血再灌注模型大鼠的入脑成分。采用气相色谱-质谱联用(GC-MS)方法,分析检测栝楼桂枝汤给药后脑缺血再灌注模型大鼠的入脑的挥发性成分。(4)采用伊文思蓝染色方法,观察给药后BBB通透性的变化；采用免疫组织化学技术,检测脑组织中紧密连接蛋白ZO-1、Claudin-5、水通道蛋白AQP-4蛋白的变化；采用ELISA方法,检测脑组织中基质金属蛋白酶MMP-2、MMP-9的变化。结果：(1)分别建立了HPLC-MS、UPLC-MS/MS法测定栝楼桂枝汤中两种氨基酸(瓜氨酸和Y-氨基丁酸)及其他24种活性成分(没食子酸、原儿茶酸、氧化芍药苷、儿茶素、对羟基苯甲酸、没食子酸甲酯、香草酸、芍药内酯苷、芍药苷、4-羟基肉桂酸、甘草苷、紫云英苷、3-羟基肉桂酸、异甘草苷、2-羟基肉桂酸、甘草素、酸枣仁皂苷A、肉桂酸、甘草酸、6-姜酚、甘草查尔酮A、8-姜酚、6-姜烯酚、甘草次酸)的含量的测定方法。(2)药效学结果表明,在神经功能症状评分方面,栝楼桂枝汤低、中、高剂量组第7天评分低于MCAO模型组(P＜0.05)；在运动功能障碍发面,与模型组比较,栝楼桂枝汤低、中、高剂量组,功能障碍评价分数明显升高,有显著性差异(P＜0.05)；在脑梗死体积方面,MCAO模型组与栝楼桂枝汤中、高剂量组比较有显著性差异(P0.05)；在脑组织病理方面,给药组与MCAO模型组相比,病理改变明显减轻。虽出现神经元变性、坏死,组织水肿,细胞排列散乱,核溶解及胞核固缩等现象,但程度和数量均较MCAO模型组减少。在氨基酸类神经递质方面,与模型组比较,栝楼桂枝汤高剂量组能降低脑组织内氨基酸类神经递质(天冬氨酸、谷氨酸、甘氨酸)含量。(3)通过UPLC-MS方法,在灌胃给药后模型大鼠的脑脊液和脑组织中鉴定出11个成分(瓜氨酸、没食子酸、芍药内酯苷、芍药苷、芹糖甘草苷、甘草苷、芹糖异甘草苷、异甘草苷、甘草素、异甘草素、甘草酸)；通过GC-MS方法,在栝楼桂枝汤复方中共鉴别93个挥发性成分,并初步鉴定出54个挥发性入脑成分。(4)在脑组织EB含量方面,与MCAO模型组相比,栝楼桂枝汤组的脑组织EB含量降低(P＜0.01)。免疫组化结果表明；Zo-1、Claudin-5、AQP-4阳性主要定位在胞浆中,模型组中Zo-1、Claudin-5的表达显著低于假手术组(P0.01),模型组中AQP-4蛋白的表达显著高于假手术(P0.01),而栝楼桂枝汤组Zo-1、Claudin-5表达均显著高于模型组(PD.D1),AQP-4蛋白的表达显著低于模型组(P0.01)；在MMP-2、MMP-9含量方面,与假手术组比较,模型组MMP-2、MMP-9含量显著增加(P＜0.01,P＜0.001)；与模型组比较,栝楼桂枝汤MMP-2、MMP-9含量显著降低(P＜0.01,P＜0.001)。结论：(1)本文建立的HPLC-MS、UPLC-MS/MS法分别测定栝楼桂枝汤中两种氨基酸(瓜氨酸和Y-氨基丁酸)及其他24种活性成分的方法,分析时间短、检测灵敏度高,可为复方的药效物质基础研究奠定扎实的基础。(2)栝楼桂枝汤能降低MCAO模型大鼠的神经功能缺损、改善运动功能障碍,缩小脑梗死体积、降低脑组织内氨基酸类神经递质(天冬氨酸、谷氨酸、甘氨酸)含量,减轻脑缺血再灌注的病理组织损伤。(3)栝楼桂枝汤中的瓜氨酸、没食子酸、芍药内酯苷、芍药苷、芹糖甘草苷、甘草苷、芹糖异甘草苷、异甘草苷、甘草素、异甘草素、甘草酸及鉴定出的54种挥发性成分,可以进入脑组织中,提示这些成分可能是复方治疗脑卒中后肌肉痉挛的药效物质基础。(4)栝楼桂枝汤能够降低BBB的通透性,其作用机制可能是通过上调ZO-1、Claudin-5的表达,恢复紧密连接的功能；下调水通道蛋白AQP4的表达,抑制基质金属蛋白MMP-2、MMP-9,减轻缺血再灌注后脑水肿和神经细胞损伤程度,发挥多靶点的脑保护作用,进而改善脑卒中后肌肉痉挛。
[Abstract]:Objective : To study the effect of Guizhi Tang in the treatment of post - stroke muscle spasm .
The expression of AQP - 4 protein in brain tissue was detected by immunohistochemistry , and the expression of AQP - 4 protein in brain tissue was detected .
The results showed that : ( 1 ) HPLC - MS , UPLC - MS / MS method was used to determine the contents of two kinds of amino acids ( including gallic acid , chlorogenic acid , peonionic acid , catechin , p - hydroxybenzoic acid , methyl - 4 - hydroxy cinnamic acid , vanillic acid , penonionic acid , liquiritin , 4 - hydroxy cinnamic acid , vanillic acid , penonionic acid , 6 - gingerol , 3 - hydroxy cinnamic acid , isoliquiritin , 2 - hydroxycinnamic acid , 6 - gingerol , liquiritin A , 8 - gingerol , 6 - gingerol , etc . ) .
Compared with model group , the score of functional dysfunction was significantly higher in the lower , middle and high dosage groups than in model group ( P < 0.05 ) .
There was a significant difference in the volume of cerebral infarction ( P0.05 ) .
( 3 ) In the aspect of amino acid neurotransmitters , 11 components were identified in cerebrospinal fluid and brain tissue of the model rats after the administration of stomach . ( 3 ) Compared with the model group , the high - dose group of Guizhi Tang could reduce the content of amino acid neurotransmitters ( aspartic acid , glutamic acid , glycine ) in brain tissue .
A total of 93 volatile components were identified by GC - MS , and 54 volatile components were identified . ( 4 ) The EB content in brain tissue of Guizhi decoction group decreased ( P < 0.01 ) .
Zo - 1 , Claudin - 5 , AQP - 4 were mainly located in the cytoplasm . The expression of Zo - 1 and Claudin - 5 in the model group was significantly lower than that in sham operation group ( P0.01 ) . The expression of AQP - 4 protein in the model group was significantly higher than that in sham operation group ( P0.01 ) .
Compared with sham operation group , MMP - 2 and MMP - 9 in model group increased significantly ( P & lt ; 0.01 , P & lt ; 0.001 ) .
Compared with model group , the content of MMP - 2 and MMP - 9 in Guizhi decoction decreased significantly ( P & lt ; 0.01 , P & lt ; 0.001 ) . Conclusion : ( 1 ) The HPLC - MS , UPLC - MS / MS method established in this paper can be used to determine the contents of two kinds of amino acids ( melon and Y - aminobutyric acid ) and other 24 active ingredients in Guizhi decoction of the thes kirilowii Maxim .
down - regulate the expression of AQP4 , inhibit MMP - 2 and MMP - 9 of matrix metal protein , reduce the degree of cerebral edema and nerve cell injury after ischemia and reperfusion , exert the brain protection function of multiple target points , and further improve muscle spasm after stroke .

【学位授予单位】：福建中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R277.7

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