土荆皮乙酸的抗多房棘球蚴作用及其机制研究  

Study on the efficacy and mechanism of pseudolaric acid B against Echinococcus multilocularis

作　　者：陈青 达哇卓玛 刘川川 樊海宁[2,3] CHEN Qing;DAWA Zhuoma;LIU Chuanchuan;FAN Haining(School of Clinical Medicine,Qinghai University,Xining 810001,P.R.China;Department of Hepatopancreatobiliary Surgery,The Affiliated Hospital of Qinghai University,Xining 810001,P.R.China;Qinghai Province Research Key Laboratory for Echinococcosis,Xining 810001,P.R.China)

机构地区：[1]青海大学临床医学院,西宁810001 [2]青海大学附属医院肝胆胰外科,西宁810001 [3]青海省包虫病研究重点实验室,西宁810001

出　　处：《中国普外基础与临床杂志》2024年第6期695-705,共11页Chinese Journal of Bases and Clinics In General Surgery

基　　金：青海省科技厅项目(项目编号:2023-ZJ-992Q)。

摘　　要：目的探讨土荆皮乙酸(pseudolaric acid B,PAB)体外对原头节细胞凋亡的影响,以及体内对病灶-宿主微环境组织中血管生成和细胞凋亡的调节作用及其可能机制,为临床开发多房棘球蚴病新型替代药物提供依据。方法体外实验:原头节、囊泡、生发层细胞、人包皮成纤维细胞(human foreskin fibroblasts,HFFs)和正常人肝细胞经不同浓度(0、2.5、5、10、20、40、80、160和320μmol/L)的PAB分别处理7、5、5、5、5 d后,评价原头节存活率、囊泡磷酸葡萄糖异构酶(phosphoglucose isomerase,PGI)释放水平、生发层细胞活力及HFFs和正常人肝细胞活力;原头节和囊泡经2.5%戊二醛固定后用于扫描电镜和透射电镜观察。动物实验:从保种沙鼠腹腔病灶中分离原头节,然后腹腔注射感染18只C57BL/6J小鼠建立模型,随机分为3组,每组6只:模型组(Model组)每日给予0.3 mL PBS缓冲液灌胃;阿苯达唑(albendazole,ABZ)组每日给予0.3 mL ABZ(100 mg/kg)灌胃;PAB组每日予以0.3 mL PAB(40 mg/kg)灌胃。连续灌胃6周后,取病灶-宿主微环境组织,采用ELISA法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)和半胱氨酸蛋白酶3(cysteinyl aspartate specific proteinase3,caspase3)的表达水平,采用生化检测试剂盒检测一氧化氮(nitric oxide,NO)的表达水平,采用Western blot法检测B淋巴细胞瘤-2相关X蛋白(BCL2-associated X protein、Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl2)、caspase3、活化caspase3(cleaved cysteinyl aspartate specific proteinase3,cleaved-caspase3)、VEGF、血管内皮细胞生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)、磷脂肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)、磷酸化PI3K(phosphorylated phosphatidylinositol 3 kinase,p-PI3K)、蛋白激酶B(protein kinase B,AKT)及磷酸化AKT(phosphorylated protein kinase B,p-AKT)蛋白的表达水平。结果体外实验:体外�Objective To investigate the in vitro effect of pseudolaric acid B(PAB)on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo,as well as its possible mechanisms,in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis.Methods In vitro experiments:the protoscoleces,vesicles,germinal cells,human foreskin fibroblasts(HFFs)and normal human liver cells were treated with different concentrations of PAB(0,2.5,5,10,20,40,80,160 and 320μmol/L)for 7,5,5,5 and 5 days,then evaluated the survival rate of the protoscoleces,the release level of phosphoglucose isomerase(PGI)from the vesicles,the viability of the germinal cells,as well as the viability of HFFs and normal human liver cells.The protoscoleces and vesicles were fixed with 2.5%glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation.Animal experiments:the protoscoleces were isolated from the abdominal lesions of the protected gerbils,and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models,dividing into 3 groups with 6 mice in each group.The model group was given 0.3 mL of PBS by gavage daily,the albendazole(ABZ)group was given 0.3 mL ABZ(100 mg/kg)daily by gavage,the PAB group was given 0.3 mL of PAB(40 mg/kg)by gavage daily.After continuous gavage for 6 weeks,the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor(VEGF),endothelial nitric oxide synthase(eNOS)and cysteinyl aspartate specific proteinase3(caspase3),the expression levels of nitric oxide(NO)was detected using a biochemical detection kit,Western blot was used to detect the expression levels of BCL2-associated X protein(Bax),B-cell lymphoma-2(Bcl2),caspase3,cleaved-caspase3,VEGF,vascular endothelial growth factor receptor 2(VEGFR2),phosphatidylinositol 3 kinase(PI3K),phosphorylated PI3K(p-PI3K),pro

关 键 词：多房棘球绦虫 土荆皮乙酸 原头节 囊泡 生发层细胞 细胞凋亡 血管生成 磷脂肌醇3激酶/蛋白激酶B信号通路 

分 类 号：R285[医药卫生—中药学]