加味通窍活血汤下调PI3K/Akt通路抗基底动脉延长扩张症的机制  

Modified Tongqiao Huoxuetang Down-regulates PI3K/Akt Pathway to Treat Basilar Artery Dolichoectasia

作　　者：刘飞祥 张道培 吴兆鑫 张怀亮[1,2,3] 张运克 苗晋鑫[5] 张振强[5] 孙瑞芹[5] 王理想 LIU Feixiang;ZHANG Daopei;WU Zhaoxin;ZHANG Huailiang;ZHANG Yunke;MIAO Jinxin;ZHANG Zhenqiang;SUN Ruiqin;WANG Lixiang(The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China;Henan Vertigo Disease Diagnosis and Treatment Center,Zhengzhou 450000,China;Institute of Vertigo Disease,Henan University of Chinese Medicine,Zhengzhou 450000,China;Henan University of Chinese Medicine,Zhengzhou 450046,China;College of Traditional Chinese Medicine,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学第一附属医院,郑州450000 [2]河南省眩晕病诊疗中心,郑州450000 [3]河南中医药大学眩晕病研究所,郑州450000 [4]河南中医药大学,郑州450046 [5]河南中医药大学中医药研究院,郑州450046

出　　处：《中国实验方剂学杂志》2024年第1期87-94,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82104730);河南省科学技术研究与发展计划联合基金(优势学科培育类)项目(222031420080);中国博士后科学基金第72批面上资助二等项目(2022M721065);河南省中医药科学研究专项(2019ZY1013,2019JDZX2032,2021ZPZX021)。

摘　　要：目的:建立基底动脉延长扩张症(BAD)小鼠模型,探讨加味通窍活血汤(JTQHX)对BAD模型小鼠磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路的影响。方法:将60只C57/BL6雌性小鼠随机分为假手术组(注射10 U·mL^(-1)灭活弹性蛋白酶)、模型组、阿托伐他汀钙片组、JTQHX低、高剂量组(注射10 U·mL^(-1)弹性蛋白酶)。造模14 d,假手术组和模型组给予等体积的纯水灌胃,阿托伐他汀钙片组给予2.6 mg·kg^(-1)·d^(-1)阿托伐他汀钙片灌胃,JTQHX低、高剂量组分别给予含生药3.4、17 g·kg^(-1)·d^(-1)的JTQHX颗粒剂,连续药物干预2个月。酶联免疫吸附测定法(ELISA)检测血清中小鼠白细胞介素-6(IL-6)和小鼠钙蛋白酶(Lp A)的变化;伟郝夫范吉森(EVG)染色观察血管的病理变化;原位末端标记法(TUNEL)检测血管平滑肌细胞(VSMCs)凋亡率;Image-Pro Plus软件观察并计算小鼠基底动脉血管的弯曲指数、延长长度、血管直径增加百分比及弯曲度情况;蛋白免疫印迹法(Western blot)检测小鼠血管组织中PI3K、Akt蛋白的表达水平。结果:与假手术组比较,模型组小鼠血清中IL-6水平显著降低(P<0.01);Lp A水平差异无统计学意义;VSMCs细胞凋亡显著升高(P<0.01);基底动脉血管的弯曲指数、延长长度、血管直径增加百分比及弯曲角度升高(P<0.01);血管中PI3K、Akt蛋白表达显著升高(P<0.01);基底动脉血管管壁中膜内弹力层破坏、肌层萎缩和结缔组织透明样改变严重程度升高。与模型组比较,JTQHX干预2个月后,血清中IL-6水平显著升高(P<0.01);基底动脉血管的延长度、弯曲角度、血管直径增加百分比和迂曲指数明显降低(P<0.05,P<0.01);VSMCs的凋亡显著降低(P<0.01);PI3K、Akt蛋白的表达显著降低(P<0.01);基底动脉血管管壁中膜的内弹力层的破坏、肌层的萎缩及结缔组织透明样变严重程度降低。结论:JTQHX抑制基底动脉血管延长、扩张、迂曲,改善病理变化的机制,可能Objective:To establish a mouse model of basilar artery dolichoectasia(BAD)and explore the mechanism of modified Tongqiao Huoxuetang(JTQHX)in regulating BAD via phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Method:Sixty C57/BL6 female mice were randomized into sham operation(injected with 10 U·mL^(-1) inactivate elastase),model,atorvastatin calcium tablets(2.6 mg·kg·d^(-1)),and low-and high-dose(crude drug 3.4,17 g·kg^(-1)·d^(-1),respectively)JTQHX groups.The mouse model of BAD was established by injection with 10 U·mL^(-1) elastase.After 14 days of modeling,the sham operation group and model group were administrated with equal volumes of pure water by gavage,and other groups with corresponding drugs for 2 months.The levels of interleukin-6(IL-6)and calpain(LpA)in the serum were measured by enzyme-linked immunosorbent assay(ELISA).Verhoeff's Van Gieson(EVG)staining was employed to observe the pathological changes of blood vessels.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)was employed to examine the apoptosis rate of vascular smooth muscle cells(VSMCs).Image Pro Plus was used to observe and calculate the curvature index,elongation length,percentage increase in vessel diameter,and curvature angle of the basilar artery vessels in mice.Western blot was employed to determine the expression levels of PI3K and Akt in the vascular tissue.Result:Compared with the sham operation group,the model group showed lowered IL-6 level(P<0.01),no significant change in LpA level,increased apoptosis of VSMCs(P<0.01),and increased curvature index,elongation length,percentage increase in vessel diameter,and curvature angle(P<0.01).Furthermore,the modeling up-regulated the protein levels of PI3K and Akt in blood vessels(P<0.01)and aggravated the destruction of the inner elastic layer,atrophy of the muscular layer,and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall.Compared with the model group,2 months of treatment with JTQHX elevated the IL-6

关 键 词：基底动脉延长扩张症 加味通窍活血汤 磷脂酰肌醇3-激酶 蛋白激酶B 

分 类 号：R2-0[医药卫生—中医学] R22R242R2-031R285.5