阿苯达唑载药囊泡对细粒棘球蚴原头节活性的影响

doi:10.12140/j.issn.1000-7423.2022.03.007

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (3): 324-329.doi: 10.12140/j.issn.1000-7423.2022.03.007

阿苯达唑载药囊泡对细粒棘球蚴原头节活性的影响

乔世源¹(), 周雪¹, 刘程豪¹, 姜慧娇¹, 卜媛媛¹, 陈雪玲², 吴向未¹^,^*()

1.石河子大学医学院第一附属医院,石河子 832008
2.石河子大学医学院免疫学教研室,石河子 832008

收稿日期:2021-11-22 修回日期:2022-02-09 出版日期:2022-06-30 发布日期:2022-07-06
通讯作者: 吴向未
作者简介:乔世源（1994-）,男,硕士研究生,从事肝胆疾病的基础研究。E-mail： 876179388@qq.com
基金资助:
国家自然科学基金(81760570);国家自然科学基金(81760371);兵团科技发展专项资金(2018CB017);兵团科技发展专项资金(2019AB031)

Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus

QIAO Shi-yuan¹(), ZHOU Xue¹, LIU Cheng-hao¹, JIANG Hui-jiao¹, BU Yuan-yuan¹, CHEN Xue-ling², WU Xiang-wei¹^,^*()

1. Hepatobiliary Surgery, the First Affiliated Hospital of Medical College of Shihezi University, Shehezi 832008, China
2. Department of Immunology, College of Medicine, Shihezi University, Shihezi 832008, China

Received:2021-11-22 Revised:2022-02-09 Online:2022-06-30 Published:2022-07-06
Contact: WU Xiang-wei
Supported by:
National Natural Science Foundation of China(81760570);National Natural Science Foundation of China(81760371);XPCC Special Fund for Science and Technology Development(2018CB017);XPCC Special Fund for Science and Technology Development(2019AB031)

摘要/Abstract

摘要：

目的探讨装载阿苯达唑的细胞外囊泡体外对细粒棘球蚴原头节活性的影响。方法将H22小鼠肝癌细胞分为低、中、高浓度组,分别加入终浓度为200、400、600 μmol/L的阿苯达唑,紫外线（UVB,300 J/m²）照射1 h,孵育18~20 h,然后通过超高速差速离心法制备阿苯达唑载药囊泡;以同样方法制备未加阿苯达唑的空载囊泡。用透射电镜观察载药囊泡的形态、激光粒度仪测量粒径、液相色谱仪检测载药囊泡的最佳载药量。将体外培养的细粒棘球蚴原头节随机分为4组,分别加入培养基（空白对照组）、空载囊泡（空载囊泡组）、载药囊泡（载药囊泡组）、阿苯达唑（阿苯达唑阳性对照组）,其中载药囊泡组和阿苯达唑阳性对照组的阿苯达唑终浓度为13 μmol/L。共培养第1、3、5、7天取原头节进行伊红染色,观察原头节形态与活力,计算各组的存活率;共培养第3、5、7天取原头节,用半胱天冬氨酸蛋白酶-3（caspase-3）检测试剂盒检测原头节caspase-3的表达量,观察原头节凋亡情况。组间比较使用单因素方差分析。结果透射电镜观察结果显示,载药囊泡形态呈大小不一的小囊泡,具有双层膜结构;粒径为200~400 nm。液相色谱检测结果显示,低、中、高浓度组载药囊泡的阿苯达唑有效药物浓度分别为（36.3 ± 2.85）、（79.0 ± 2.30）、（99.5 ± 4.20）μmol/L。有效药物浓度的的提高幅度,中浓度组较低浓度组高于高浓度组较中浓度组,差异有统计学意义（F = 21.43,P < 0.05）,制备载药囊泡的最适加药浓度为400 μmol/L。伊红染色后镜下观察结果显示,共培养第7天,空白对照组及空载囊泡组的原头节活性良好,形态、结构清晰;阿苯达唑阳性对照组原头节活性减弱,结构欠清晰,体积缩小;载药囊泡组原头节结构紊乱、皱缩、死亡;共培养第7天,空白对照组、空载囊泡组、阿苯达唑阳性对照组、载药囊泡组原头节存活率分别为（91.2 ± 1.07）%、（88.9 ± 1.43）%、（64.5 ± 1.19）%、（45.3 ± 0.98）%,载药囊泡组原头节存活率均低于其他各组,差异有统计学意义（F = 1 021.17,P < 0.05）。原头节凋亡检测结果显示,共培养第3天,空白对照组、空载囊泡组、阿苯达唑阳性对照组和载药囊泡组caspase-3的表达量分别为（41.80 ± 3.02)、（40.26 ± 2.78)、（55.20 ± 3.09）和（68.15 ± 3.60）μmol/L;第5天分别为（43.18 ± 2.43）、（43.02 ± 3.13）、（52.17 ± 4.13）和（62.74 ± 3.16）μmol/L,第7天分别为（52.93 ± 1.46)、（53.08 ± 1.60）、（57.32 ± 1.81）和（61.99 ± 1.14）μmol/L;第3、5、7天,各组间差异均有统计学意义（F = 51.97、24.53、23.82,P < 0.05）,载药囊泡组caspase-3的表达量均高于阿苯达唑阳性对照组（F = 22.36、12.43、14.33,P < 0.05）。结论载药囊泡可提高阿苯达唑的溶解度,增强阿苯达唑对细粒棘球蚴原头节的杀伤效果。

关键词: 细粒棘球蚴, 阿苯达唑, 载药囊泡

Abstract:

Objective To investigate the effect of albendazole-loaded extracellular vesicles (drug-loaded vesicles) on the vitality of Echinococcus granulosus protoscolex in vitro. Methods The mouse hepatoma cells H22 culture suspension was assigned into low, medium and high concentration groups, to each of which albendazole was added at final concentrations of 200, 400 and 600 μmol/L, respectively. UV irradiation (UVB, 300 J/m²) was performed for 1 hour, followed by 18-20 hours of incubation. The drug-loaded vesicles were produced by ultra-high speed differential centrifugation. Vesicles without albendazole were prepared using the same method. The shape of the drug-loaded vesicles was observed with a transmission electron microscope, the particle diameter was measured with a laser particle sizer, and the optimal dose determined by liquid chromatography. The E. granulosus protoscolex was divided into 4 groups and was cultured in vitro with pure medium (blank control group), no-loading vesicles (no-loading vesicle group), drug-loaded vesicles (drug-loaded vesicle group) and albendazole (albendazole positive control group), respectively. The final concentration of albendazole in the drug-loaded vesicle group and albendazole positive control group was 13 μmol/L. The protoscolex was stained with Eosin to observe the morphology and activity at 1, 3, 5 and 7 days post-treatment. The survival rate of protoscolex was calculated. The caspase-3 expression level of E. granulosus protoscolex was detected by using caspase-3 detection kit 3, 5, 7 days post-treatment to identify the apoptosis of protoscolex. One-way ANOVA was used for comparisons between groups. Results The transmission electron microscopy showed variations in vesicle of different size with double membrane structure. The particle diameter sizer showed that the particle diameter was 200-400 nm. The liquid chromatography showed that the effective drug concentrations in low, medium and high concentration groups were (36.3 ± 2.85), (79.0 ± 2.30), (99.5 ± 4.20) μmol/L. The increment of albendazole concentration from the low concentration group to the medium concentration group was higher than that from the medium concentration group to the high concentration group. The difference between the groups was statistically significant (F = 21.43, P < 0.05). The optimal concentration of the final drug-loaded vesicles was 400 μmol/L. Eosin staining showed that on day 7 post-treatment, the protoscolex remained active, and the morphology and structure of the protoscolex were clear in the blank control group and the no-loading vesicle group. In the albendazole positive control group, the protoscolex was inactive, and unsharpness and its volume decreased. In the drug-loaded vesicle group, the structure of protoscolex was disordered, shrunken and partially died. On day 7 post-treatment, the survival rate of the protoscolex in the blank control group, no-loading vesicle group, albendazole positive control group and drug-loaded vesicle group were (91.2 ± 1.07)%, (88.9 ± 1.43)%, (64.5 ± 1.19)% and (45.3 ± 0.98)%. The survival rate curves in the drug-loaded vesicle group were lower than that of the the other groups (F = 1 021.17, P < 0.05). The apoptosis of the protoscolex results showed, on day 3 post-treatment, the caspase-3 of E. granulosus protoscolex in blank control group, no-loading vesicle group, albendazole positive control group and the drug-loaded group were (41.80 ± 3.02), (40.26 ± 2.78), (55.20 ± 3.09) and (68.15 ± 3.60) μmol/L, respectively; on day 5 post-treatment, were (43.18 ± 2.43), (43.02 ± 3.13), (52.17 ± 4.13) and (62.74 ± 3.16) μmol/L, respectively; while on day 7 post-treatment, were (52.93 ± 1.46), (53.08 ± 1.60), (57.32 ± 1.81) and (61.99 ± 1.14) μmol/L, respectively. The differences were significant between the groups on 3, 5, 7 days post-treatment (F = 51.97, 24.53, 23.82; P < 0.05) and the caspase-3 in the drug-loaded vesicle group were all higher than that in the albendazole positive control group (F = 22.36, 12.43, 14.33; P < 0.05). Conclusion Albendazole-loaded vesicle could improve the solubility of albendazole, enhancing the killing effect on E. granulosus protoscolex.

Key words: Echinococcus granulosus, Albendazole, Drug-loaded vesicle

中图分类号:

R383.33

乔世源, 周雪, 刘程豪, 姜慧娇, 卜媛媛, 陈雪玲, 吴向未. 阿苯达唑载药囊泡对细粒棘球蚴原头节活性的影响[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 324-329.

QIAO Shi-yuan, ZHOU Xue, LIU Cheng-hao, JIANG Hui-jiao, BU Yuan-yuan, CHEN Xue-ling, WU Xiang-wei. Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(3): 324-329.

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阿苯达唑载药囊泡对细粒棘球蚴原头节活性的影响

Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus

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