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、100 μg/ml）与RI-1（0、5、10、20、40、80 μg/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、100 μg/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、80 μg/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40 μg/ml∶10 μg/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（12.98 ± 0.67）、去氢骆驼蓬碱组（39.79 ± 1.11）相比均增强（F = 339.795、773.96、76.99，均P < 0.01）。混合药物组Olive尾矩值（17.73 ± 1.11）高于空白对照组（0.03 ± 0.02）、阳性对照组（6.46 ± 0.56）和去氢骆驼蓬碱组（8.80 ± 0.61）（F = 230.32、165.59、151.44，均P < 0.01）。混合药物组总超氧化物歧化酶（SOD）含量为（0.71 ± 0.07）U/mg，与空白对照组[（2.32 ± 0.24）U/mg]、去氢骆驼蓬碱组[（1.26 ± 0.12）U/mg]相比均降低（F = 39.57、 15.74，均P < 0.01）；混合药物组丙二醛（MDA）含量为（0.62 ± 0.03）mmol/mg，与空白对照组[（0.82 ± 0.05）mmol/mg]、去氢骆驼蓬碱组[（0.70 ± 0.03）mmol/mg]相比均降低（F = 22.78、28.37，P < 0.01）；混合药物组Caspase-3水平为（1.82 ± 0.05）U/mg，与空白对照组[（0.70 ± 0.02）U/mg]、去氢骆驼蓬碱组为[（1.36 ± 0.19）U/mg]相比均升高（F = 26.24，P < 0.01；F = 7.52，P < 0.05）。混合药物组细粒棘球蚴线粒体ATP水平为（112.21 ± 2.72）µmol/ml，与空白对照组[（157.50 ± 5.23）µmol/ml]、去氢骆驼蓬碱组[（123.10 ± 4.73）µmol/ml]相比均降低（F = 60.73，P < 0.01；F = 67.42，P < 0.05）。混合药物组细粒棘球蚴LDH释放量为（1.83 ± 0.09）mU，与空白对照组[（0.70 ± 0.05）mU]相比升高（F = 146.79，P < 0.01），与去氢骆驼蓬碱组[（1.51 ± 0.05）mU]相比差异无统计学意义（F = 62.78，P > 0.05）。混合药物组细粒棘球蚴h2ax、rad51、brca1、mre11基因相对表达水平分别为2.34 ± 0.15、1.19 ± 0.14、1.35 ± 0.08、1.43 ± 0.10，与空白对照组（均为1.00 ± 0.00）相比均上调（F = 23.79，P < 0.01；F = 8.96，P < 0.05；F = 30.75，P < 0.05；F = 39.81，P < 0.01）。 结论 去氢骆驼蓬碱联合rad51抑制剂RI-1可增强去氢骆驼蓬碱体外抗细粒棘球蚴的作用，同时增强细粒棘球蚴DNA损伤。

Objective 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, 100 μg/ml) and RI-1 (0, 5, 10, 20, 40, 80 μg/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 100 μg/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 80 μg/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 ratio of 40 μg/ml∶10 μg/ml as the optimal synergistic ratio for E. granulosus, the survival rate of the mixed drug group was (48.67 ± 2.62)%, which was lower than that of the blank control group [(96.33 ± 1.25)%], positive control group [(91.67 ± 1.25)%], and harmine group [(72.67 ± 4.50)%] (F = 78.85, P < 0.01; F = 437.92, P < 0.05; F = 42.49, P < 0.01). The structure of the mixed drug group’s worm body collapsed, with severe morphological damage, significant shrinkage of the worm body, and insect like changes in the epidermal layer. The average fluorescence intensity of reactive oxygen species in the mixed drug group (45.71 ± 1.52) was enhanced compared with the blank control group (3.40 ± 0.55), the positive control group (12.98 ± 0.67), and the dehydroberberine group (39.79 ± 1.11) (F = 339.795, 773.96, 76.99, all P < 0.01). The Olive tail moment value of the mixed drug group (17.73 ± 1.11) was higher than that of the blank control group (0.03 ± 0.02), positive control group (6.46 ± 0.56), and dehydrocamel base group (8.80 ± 0.61) (F = 230.32, 165.59, 151.44, all P < 0.01). The SOD content in the mixed drug group was (0.71 ± 0.07) U/mg, which was lower than that in the blank control group [(2.32 ± 0.24) U/mg] and the dehydroberberine group [(1.26 ± 0.12) U/mg] (F = 39.57, 15.74, both P < 0.01). The MDA content in the mixed drug group was (0.62 ± 0.03) mmol/mg, which was lower than that in the blank control group [(0.82 ± 0.05) mmol/mg] and the dehydroberberine group [(0.70 ± 0.03) mmol/mg] (F = 22.78, 28.37, P < 0.01). The level of Caspase-3 in the mixed drug group was (1.82 ± 0.05) U/mg, which was higher than that in the blank control group [(0.70 ± 0.02) U/mg] and the dehydroberberine group [(1.36 ± 0.19) U/mg] (F = 26.24, P < 0.01; F = 7.52, P < 0.05). The mitochondrial ATP level of E. granulosus in the mixed drug group was (112.21 ± 2.72) µmol/ml, which was lower than that of the blank control group [(157.50 ± 5.23) µmol/ml] and the dehydrocoumarin group [(123.10 ± 4.73) µmol/ml] (F = 60.73, P < 0.01; F = 67.42, P < 0.05). The release of LDH from E. granulosus in the mixed drug group was (1.83 ± 0.09) mU, which was higher than that in the blank control group [(0.70 ± 0.05) mU] (F = 146.79, P < 0.01), but there was no statistically significant difference compared with the dehydrocoumarin group [(1.51 ± 0.05) mU] (F = 62.78, P > 0.05). The relative expression levels of h2ax, rad51, brca1 and mrel1 genes in the mixed drug group of E. granulosus were 2.34 ± 0.15, 1.19 ± 0.14, 1.35 ± 0.08, and 1.43 ± 0.10, respectively, which were upregulated compared with the blank control group (all 1.00 ± 0.00) (F = 23.79, P < 0.01; F = 8.96, P < 0.05; F = 30.75, P < 0.05; F = 39.81, P < 0.01). Conclusion The combination of harmine and rad51 inhibitor RI-1 can enhance the in vitro efficacy of harmine against E. granulosus, while also increasing DNA damage in E. granulosus.