关键词: 光滑双脐螺, 氯硝柳胺, 线粒体膜电位, 氧化磷酸化

Abstract:

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.

Key words: Biomphalaria glabrata, Niclosamide, Mitochondrial membrane potential, Oxidative phosphorylation