氯硝柳胺对光滑双脐螺氧化磷酸化的影响  

Affect of niclosamide on the oxidative phosphorylation of Biomphalaria glabrata

作　　者：张苏阳 邢云天[1] 袁轩 曲国立[1] 姚甲凯 戴建荣[1] ZHANG Su-yang;XING Yun-tian;YUAN Xuan;QU Guo-li;YAO Jia-kai;DAI Jian-rong(Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology,Wuxi 214064,China)

机构地区：[1]江苏省血吸虫病防治研究所,国家卫生健康委员会寄生虫病预防与控制技术重点实验室,江苏省寄生虫与媒介控制技术重点实验室,无锡214064

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第1期61-67,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家重点研发计划(2020YFC1200100)。

摘　　要：目的研究氯硝柳胺对光滑双脐螺氧化磷酸化的影响,探讨氯硝柳胺的杀螺机制。方法用线粒体提取试剂盒提取光滑双脐螺成螺线粒体,考马斯蓝染色法检测提取线粒体蛋白含量,用线粒体复合体Ⅳ活性检测试剂盒检测提取过程中组织破碎液、丢弃上清和线粒体悬液中复合体Ⅳ活性以评价提取线粒体的纯度。用线粒体复合物活性检测试剂盒提取光滑双脐螺的线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ,每种复合物各分成0.2μg/ml、1.0μg/ml组和二甲基亚砜(DMSO)组,分别加入终浓度为0.2和1.0μg/ml的氯硝柳胺、0.5%DMSO,分别测定不同波长处的吸光度(A值),计算复合体的活力值。在96孔板中每孔加入蛋白浓度为33μg/ml的线粒体悬液,分别加入终浓度为0.2和1.0μg/ml的氯硝柳胺(0.2μg/ml组和1.0μg/ml组)或0.5%DMSO (DMSO组),测定25℃条件下10、20、30 min的A;值,计算线粒体膜通透性转运孔(m PTP)开放度。在96孔板中每孔加入含终浓度为5μg/ml的JC-1染料、蛋白浓度为0.1 mg/ml的线粒体悬液,0.2、0.4、0.6、0.8、1.0μg/ml组分别加入终浓度为0.2、0.4、0.6、0.8和1.0μg/ml的氯硝柳胺,氰化羰基-3-氯苯腙(CCCP)组加入20μg/ml的CCCP作阳性对照组,DMSO组加入0.5%的DMSO作阴性对照组,在激发光485 nm、发射光590 nm、25℃条件下连续检测荧光强度30 min。组间比较采用单因素方差分析。结果提取获得的线粒体总蛋白浓度为(4.24±0.11) mg/ml,组织破碎液、丢弃上清和线粒体悬液中电子传递链复合物Ⅳ活性分别为(14.88±1.80)、(5.60±0.96)、(24.19±3.53) U/mg,3组间差异有统计学意义(F=46.922,P<0.01)。0.2μg/ml组、1.0μg/ml组与DMSO组的复合物Ⅰ活性分别为(523.98±120.37)、(559.74±238.48)、(796.64±218.79) U/mg;复合物Ⅱ各组活性分别为(3.70±0.36)、(3.54±1.90)、(5.47±2.18) U/mg;复合物Ⅲ各组活性分别为(6.03±0.79)、(5.01±0.80)、(5.82±0.69) U/mg;复合物Ⅳ各组活性分别为(31Objective To study the affect of niclosamide on the oxidative phosphorylation of Biomphalaria glabrata,and to explore its molluscicide mechanisms.Methods The mitochondria of B.glabrata were extracted using the mitochondrial extraction kit,and the protein content of the extracted mitochondria was detected by Bradford method.The mitochondrial complex Ⅳactivity assay kit was used to measure the complex Ⅳactivity in the tissue fragment fluid,the discarded supernatant and the mitochondrial suspension during the extraction process to evaluate the purity of the extracted mitochondria.Mitochondrial complexesⅠ,Ⅱ,Ⅲ,Ⅳand Ⅴof B.glabrata were extracted with the mitochondrial complex activity assay kit,and each complex was divided into 0.2μg/ml,1.0μg/ml group and dimethyl sulfoxide (DMSO) group,which were added with a final concentration of 0.2 and 1.0μg/ml of niclosamide or 0.5%DMSO respectively,then the absorbance (A) of the groups were determined at different wavelengths to calculate the viability of the complexes.Mitochondrial suspensions with a protein concentration of 33μg/ml were transferred to each well of a 96-well plate,and niclosamide at final concentrations of 0.2 and 1.0μg/ml (0.2μg/ml and 1.0μg/ml groups) or 0.5%DMSO (DMSO group) were added respectively,the A;value at 10,20,30 min at 25℃was measured to calculate the mitochondrial permeability transition pore (mPTP) openness degree.To each well of the 96-well microplate containing JC-1 dye and mitochondrial suspension with a final concentrations of 5μg/ml and 0.1 mg/ml protein,respectively,niclosamide was added at a final concentration of 0.2,0.4,0.6,0.8 and 1.0μg/ml as the niclosamide groups,20μg/ml carbonylcyanide-m-chlorophenylhydrazone (CCCP) was added as the positive control group,and 0.5%DMSO was added as the negative control group,and then the fluorescence intensity was continuously detected for 30 min under the conditions of excitation wavelength 485 nm,emission wavelength of 590 nm,and 25℃.One-way ANOVA was used for comparison

关 键 词：光滑双脐螺 氯硝柳胺 线粒体膜电位 氧化磷酸化 

分 类 号：R383.241[医药卫生—医学寄生虫学]