去氢骆驼蓬碱对细粒棘球蚴的线粒体Drp1、Mfn2蛋白表达以及线粒体途径凋亡的影响

The effects of Harmine on mitochondrial Drp1 and Mfn2 protein expressions and of Echinococcus granulosus

目的 探讨去氢骆驼蓬碱对细粒棘球蚴的线粒体Drp1、Mfn2蛋白表达以及线粒体途径凋亡的影响。方法 无菌采集细粒棘球蚴,随机分为空白对照组4个不同药物干预组。共干预5 d,采用亚甲蓝拒染试验观察细粒棘球蚴存活率,采用活性氧试剂盒检测ROS生成水平,结合Mito-Tracker Red CMXRos、Mito-Tracker Green荧光探针染色技术及JC-1染色技术综合检测线粒体膜电位和线粒体损伤情况,Western blot法检测细粒棘球蚴融合分裂蛋白Drp1、Mfn2、凋亡蛋白Bcl-2、Cyt-C表达水平变化。结果 与空白对照组相比,25μg/mLABZSO组、25μg/mLHM组、12.5μg/mLMdivi-1组、HM:Mdivi-1组(25μg/mL:12.5μg/mL)细粒棘球蚴平均存活率显著降低(P<0.05或P<0.01)。与阳性药组相比,HM:Mdivi-1组(25μg/mL:12.5μg/mL)细粒棘球蚴平均存活率显著降低(P<0.05)。ROS试验显示,各干预组平均荧光强度与空白对照组相比均增强(均P<0.01)。Mito-Tracker Red CMXRos、Mito-Tracker Green试验显示,随着药物干预每组平均荧光强度均较空白对照组显著降低(均P<0.01)。JC-1染色显示,阳性对照组和药物组绿色荧光增强。Western blot显示,各给药组Drp1蛋白水平与空白对照组相比均呈上调趋势(均P<0.01),Mdivi-1组呈下降趋势(P<0.01);Mfn2蛋白均呈下调趋势(P<0.05或P<0.01);Cyt-C蛋白均呈上调趋势(P<0.05或P<0.01),Mdivi-1组呈下降趋势(P<0.01);Bcl-2蛋白均呈下调趋势(均P<0.01),Mdivi-1组呈上降趋势(P<0.01)。结论 去氢骆驼蓬碱通过线粒体途径使细粒棘球蚴凋亡,线粒体分裂蛋白Drp1和融合蛋白Mfn2的表达证实存在线粒体融合分裂机制。

Objective To explore the effects of Harmine on the expression of mitochondrial Drp1 and Mfn2 proteins and the apoptosis of mitochondrial pathway in Echinococcus granulosus. Methods The E. granulosus was aseptically collected and randomly divided into the blank control group, the 25 μg/mL ABZSO group, the 25 μg/mL HM group, the 12.5 μg/mL Mdivi-1 group, and the HM:Mdivi-1 group(25 μg/mL:12.5 μg/mL). After intervention for 5d, the survival rate of E. granulosus was observed by methylene blue staining exclusion test. Reactive oxygen species assay kit was used to qualitatively observe and photograph ROS in the insect body under fluorescence microscope. Mito-tracker Red CMXRos and Mito-Tracker Green were used to observe the mitochondrial damage of E. granulosus under the fluorescence microscope and take photos. The active mitochondria in E. granulosus were observed under fluorescence microscope using Mitochondrial Membrane Potential Detection Kit(JC-1). After drug treatment, the total protein of E. granulosus was extracted, and the protein concentration was measured. Western blot was used to detect the protein expression levels of Drp1,Mfn2,Bcl-2 and Cyt-C in E. granulosus of each group. Results Compared with the blank control group, the average survival rate of E. granulosus in each administration group was significantly reduced(P<0.05 or P<0.01). Compared with the positive drug group, the average survival rate of E. granulosus in the HM:Mdivi-1 group was significantly reduced(P<0.05). ROS test showed that the average fluorescence intensity of each intervention group was higher than that of the blank control group(all P<0.01). The Mito-Tracker Red CMXRos and Mito-Tracker Green experiments showed that the average fluorescence intensity of each group was significantly lower than that of the blank control group with the drug intervention(all P<0.01). JC-1 staining showed that the green fluorescence of the positive control group and the medication group was enhanced. Western blot data showed that the protein levels of Drp1 in all administration groups were up-regulated compared with the blank control group(all P<0.01),and Mdivi-1 group was down-regulated(P<0.01). Mfn2 proteins tended to be down-regulated(P<0.05 or P<0.01). Cyt-C protein was up-regulated(P<0.05 or P<0.01),while Mdivi-1 group showed a downward trend(P<0.01). Bcl-2 proteins were down-regulated(all P<0.01),and Mdivi-1 group showed an up-down trend(P<0.01). Conclusion Harmine can induce apoptosis of E. granulosus through mitochondrial pathway. that expression of mitochondrial divide protein Drp1 and fusion protein Mfn2 confirm the existence of mitochondrial fusion and division mechanism.