去氢骆驼蓬碱联合rad51抑制剂对细粒棘球蚴体外作用及DNA损伤水平的影响  

Effect of harmine combined with rad51 inhibitor on the Echinococcus granulosus larvae and its DNA damage in vitro

作　　者：巩月红[1,2,3] 潘美驰 孙佳佳 马瑞佳 吾斯曼江·艾买提 赵一聪 赵军 王建华[1,2,3] GONG Yuehong;PAN Meichi;SUN Jiajia;MA Ruijia;WUSMANJIANG Aimaiti;ZHAO Yicong;ZHAO Jun;WANG Jianhua(Department of Pharmacy,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang,China;State Key Laboratory of Causes and Prevention of High Morbidity in Central Asia,Xinjiang Medical University,Urumqi 830011,Xinjiang,China;Xinjiang Key Laboratory of Clinical Drug Research,Urumqi 830011,Xinjiang,China;Department of Pharmacognosy,School of Pharmacy,Xinjiang Medical University,Urumqi 830054,Xinjiang,China;Department of Medicine,School of Pharmacy,Xinjiang Medical University,Urumqi 830054,Xinjiang,China;Department of Pharmacy,The Seventh Affiliated Hospital of Xinjiang Medical University,Urumqi 830092,Xinjiang,China;Department of Pharmacy,The Second People’s Hospital of Kashgar,Xinjiang,Kashi 844000,Xinjiang,China)

机构地区：[1]新疆医科大学第一附属医院药学部,新疆乌鲁木齐830011 [2]新疆医科大学省部共建中亚高发病成因与防治国家重点实验室,新疆乌鲁木齐830011 [3]新疆药物临床研究重点实验室,新疆乌鲁木齐830011 [4]新疆医科大学药学院,新疆乌鲁木齐830054 [5]新疆医科大学临床医学部,新疆乌鲁木齐830054 [6]新疆医科大学第七附属医院药学部,新疆乌鲁木齐830092 [7]新疆喀什地区第二人民医院药学部,新疆喀什844000

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第6期726-736,共11页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：新疆维吾尔自治区科学技术厅自然科学基金重点项目(2021D01D15);新疆维吾尔自治区天山英才青年骨干项目(TSYC202301B095);省部共建国家重点实验室开放课题(SKL-HIDCA-2022?9)。

摘　　要：目的探讨去氢骆驼蓬碱联合rad51抑制剂(RI-1)对细粒棘球蚴体外作用及DNA损伤水平的影响。方法从绵羊肝脏的棘球蚴包囊中收集细粒棘球蚴,随机分组,各组分别用去氢骆驼蓬碱(0、20、30、40、50、100μg/ml)与RI-1(0、5、10、20、40、80μg/ml)培养5 d,0.1%亚甲基蓝染色计算存活率,选择接近去氢骆驼蓬碱抗细粒棘球蚴半抑制浓度组别的浓度为固定浓度,分别以去氢骆驼蓬碱与RI-1比例分别为40∶5、40∶10、40∶20的混合药物干预细粒棘球蚴,筛选最佳协同比例。设置去氢骆驼蓬碱和RI-1混合药物组,同浓度单独作用的去氢骆驼蓬碱组和RI-1组,并设置阳性对照组(添加阿苯达唑亚砜)与空白对照组,观察干预3 d后细粒棘球蚴与囊泡形态。DCFH-DA法检测各组干预后细粒棘球蚴活性氧含量;各组进行彗星实验(阳性对照为阿霉素),分析并测定Olive尾矩;试剂盒检测药物干预后细粒棘球蚴氧化应激水平及Caspase-3含量,以及三磷酸腺苷(ATP)和乳酸脱氢酶(LDH)含量;qRT-PCR反应检测DNA损伤修复相关基因的mRNA相对表达水平。结果0、20、30、40、50、100μg/ml的去氢骆驼蓬碱干预5 d后细粒棘球蚴存活率分别为(94.67±1.25)%、(83.00±3.27)%、(64.67±3.86)%、(55.33±3.40)%、(29.67±2.49)%、(9.33±1.70)%,0、5、10、20、40、80μg/ml的RI-1干预5 d后细粒棘球蚴存活率分别为(94.33±0.94)%、(70.00±2.94)%、(55.67±3.86)%、(30.00±5.09)%、(2.67±1.25)%、0。以40μg/ml∶10μg/ml作为混合药物最佳协同比例干预细粒棘球蚴3 d后,混合药物组存活率为(48.67±2.62)%,与空白对照组[(96.33±1.25)%]、阳性对照组[(91.67±1.25)%]、去氢骆驼蓬碱组[(72.67±4.50)%]相比均下降(F=78.85,P<0.01;F=437.92,P<0.05;F=42.49,P<0.01)。混合药物组虫体结构塌陷,形态破坏严重,虫体显著缩小且表皮层呈现虫蚀样改变。混合药物组活性氧平均荧光强度(45.71±1.52)与空白对照组(3.40±0.55)、阳性对照组(12.9Objective To investigate the effect of harmine combined with rad51 inhibitor(RI-1)on Echinococcus granulosus in vitro and the level of DNA damage.Methods E.granulosus from the cysts of sheep liver were collected and randomly divided into groups.Each group contains a mixture of harmine(0,20,30,40,50,100μg/ml)and RI-1(0,5,10,20,40,80μg/ml),cultured for 5 days,and stained with 0.1%methylene blue to calculate the survival rate.The group which is close to the half inhibitory concentration of harmine against E.granulosus was selected as the fixed concentration.The groups were treated against E.granulosus with a mixture of harmine and RI-1 in ratios of 40∶5,40∶10 and 40∶20,respectively.The optimal synergistic ratio was screened.Set up a mixed drug group of harmine and RI-1,a harmine group and RI-1 group acting alone at the same concentration,and set up a positive control group(adding albendazole sulfoxide)and a blank control group.Observe the morphology of E.granulosus and vesicles after 3 days of intervention.DCFH-DA method was used to detect the reactive oxygen species content of E.granulosus after intervention in each group.Each group underwent comet assay(the positive control was doxorubicin),and the Olive tail moment was analyzed and measured.The reagent kit is used to detect the oxidative stress level and Caspase-3 content of E.granulosus after drug intervention,as well as the levels of adenosine triphosphate(ATP)and lactate dehydrogenase(LDH).qRT-PCR reaction is used to detect the mRNA expression levels of DNA damage repair related genes.Results The survival rates of E.granulosus after 5 days of intervention with 0,20,30,40,50 and 100μg/ml harmine were(94.67±1.25)%,(83.00±3.27)%,(64.67±3.86)%,(55.33±3.40)%,(29.67±2.49)%,and(9.33±1.70)%,respectively.The survival rates of E.granulosus after 5 days of intervention with 0,5,10,20,40 and 80μg/ml RI were(94.33±0.94)%,(70.00±2.94)%,(55.67±3.86)%,and(30.00±1.70)%,5.09%,(2.67±1.25)%and 0,respectively.After 3 days of intervention with a mixed drug rati

关 键 词：细粒棘球蚴 去氢骆驼蓬碱 rad51抑制剂 存活率 DNA损伤 

分 类 号：R383.3[医药卫生—医学寄生虫学]