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0.2 μg/ml、1.0 μg/ml组和二甲基亚砜（DMSO）组,分别加入终浓度为0.2和1.0 μg/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33 μg/ml的线粒体悬液,分别加入终浓度为0.2和1.0 μg/ml的氯硝柳胺（0.2 μg/ml组和1.0 μg/ml组）或0.5% DMSO（DMSO组）,测定25 ℃条件下10、20、30 min的A₅₂₀值,计算线粒体膜通透性转运孔（mPTP）开放度。在96孔板中每孔加入含终浓度为5 μg/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、1.0 μg/ml组分别加入终浓度为0.2、0.4、0.6、0.8和1.0 μg/ml的氯硝柳胺,氰化羰基-3-氯苯腙（CCCP）组加入20 μg/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）。0.2 μg/ml组、1.0 μg/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（31.20 ± 3.99）、（32.08 ± 3.20）、（30.82 ± 4.21）U/mg;复合体Ⅴ各组活性分别为（22.38 ± 3.83）、（23.08 ± 6.50）、（25.84 ± 6.86）U/mg。0.2 μg/ml组、1.0 μg/ml组线粒体复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ活性与DMSO组差异均无统计学意义（F = 1.658、1.215、1.181、0.138、0.298,P > 0.05）。加入氯硝柳胺10 min时,0.2 μg/ml组、1.0 μg/ml组和DMSO组mPTP开放度分别为（0.040 ± 0.005）、（0.041 ± 0.002）、（0.039 ± 0.036）;加入20 min时分别为（0.069 ± 0.008）、（0.067 ± 0.002）、（0.065 ± 0.015）;加入30 min时分别为（0.090 ± 0.009）、（0.088 ± 0.002）、（0.087 ± 0.012）。加入氯硝柳胺10、20、30 min,0.2 μg/ml组、1.0 μg/ml组与DMSO组mPTP开放度差异均无统计学意义（F = 0.025、0.094、0.060,P > 0.05）。相对于DMSO组（1.000）,在加入氯硝柳胺15 min时0.6、0.8和1.0 μg/ml组的线粒体膜电位分别为（0.874 ± 0.008）、（0.843 ± 0.018）、（0.773 ± 0.027）,均低于DMSO组（F = 44.285,P < 0.05）;加入30 min时,0.2、0.4、0.6、0.8和1.0 μg/ml组的线粒体膜电位分别为（0.951 ± 0.051）、（0.886 ± 0.022）、（0.766 ± 0.019）、（0.746 ± 0.016）、（0.675 ± 0.021）,除0.2 μg/ml组,其余各组均低于DMSO组（F = 125.738,P < 0.01）。 结论 浓度低于1.0 μg/ml的氯硝柳胺对光滑双脐螺线粒体电子传递链复合物活性无影响,也未造成线粒体膜通透性转运孔的开放,但可引起光滑双脐螺线粒体膜电位的剧烈下降,影响光滑双脐螺的氧化磷酸化过程。

Objective 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 Ⅳ activity assay kit was used to measure the complex Ⅳ activity 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 complexes Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ of B. glabrata were extracted with the mitochondrial complex activity assay kit, and each complex was divided into 0.2 μg/ml, 1.0 μg/ml group and dimethyl sulfoxide (DMSO) group, which were added with a final concentration of 0.2 and 1.0 μg/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 33 μg/ml were transferred to each well of a 96-well plate, and niclosamide at final concentrations of 0.2 and 1.0 μg/ml (0.2 μg/ml and 1.0 μg/ml groups) or 0.5% DMSO (DMSO group) were added respectively, the A₅₂₀ 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 5 μg/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 1.0 μg/ml as the niclosamide groups, 20 μg/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 between groups. Results The total protein concentration of the extracted mitochondria was (4.24 ± 0.11) mg/ml, and the electron transport chain complex Ⅳ activity in the tissue fragmentation solution, discarded supernatant and mitochondrial suspension was (14.88 ± 1.80), (5.60 ± 0.96) and (24.19 ± 3.53) U/mg, respectively, with statistically significant differences between the three groups (F = 46.922, P < 0.01). The complexⅠactivity was (523.98 ± 120.37), (559.74 ± 238.48) and (796.64 ± 218.79) U/mg in the 0.2, 1.0 μg/ml and DMSO groups, respectively; the complex Ⅱ activity in the three groups was (3.70 ± 0.36), (3.54 ± 1.90) and (5.47 ± 2.18) U/mg; the complex Ⅲ activity in the groups was (6.03 ± 0.79), (5.01 ± 0.80) and (5.82 ± 0.69) U/mg; the complex Ⅳ activity in each group was (31.20 ± 3.99), (32.08 ± 3.20), (30.82 ± 4.21) U/mg; while the complex Ⅴ activity was (22.38 ± 3.83), (23.08 ± 6.50), (25.84 ± 6.86) U/mg, respectively. The differences between the mitochondrial complexes Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ activities in the 0.2 and 1.0 μg/ml groups and the DMSO group were not statistically significant (F = 1.658, 1.215, 1.181, 0.138, 0.298; P > 0.05). At 10 min of niclosamide addition, the mPTP openness in the 0.2 and 1.0 μg/ml and DMSO groups were (0.040 ± 0.005), (0.041 ± 0.002) and (0.039 ± 0.036), respectively; at 20 min of addition, those openness were (0.069 ± 0.008), (0.067 ± 0.002) and (0.065 ± 0.015); at 30 min of addition, those openness were (0.090 ± 0.009) and (0.088 ± 0.002) and (0.087 ± 0.012), respectively. The differences in mPTP openness between the 0.2 and 1.0 μg/ml groups and the DMSO group at niclosamide addition for 10, 20 and 30 min were not statistically significant(F = 0.025, 0.094, 0.060; P > 0.05). Compared to the DMSO group (1.000), the mitochondrial membrane potentials of the 0.6, 0.8 and 1 μg/ml groups at 15 min of addition were (0.874 ± 0.008), (0.843 ± 0.018) and (0.773 ± 0.027), respectively, which were lower than those of the DMSO group (F = 44.285, P < 0.05). When added for 30 min, the mitochondrial membrane potentials of 0.2, 0.4, 0.6, 0.8, and 1.0 μg/ml groups were (0.951 ± 0.051), (0.886 ± 0.022), (0.766 ± 0.019), (0.746 ± 0.016), (0.675 ± 0.021), respectively, except for the 0.2 μg/ml group, all other groups were lower than those of DMSO group (F = 125.738, P < 0.01). Conclusion Niclosamide below 1.0 μg/ml was not found to affect the mitochondrial electron transport chain complex activity of B. glabrata, nor did the openness of mPTP, but caused a dramatic decrease in the mitochondrial membrane potential of B. glabrata, and affected its oxidative phosphorylation process as well.