没食子酸与氯硝柳胺联合灭螺作用研究  

Molluscicidal efficacy of gallic acid in combination with niclosamide

作　　者：郑涛 刘佳豪 李彬 李佳珊 聂娟 熊涛 ZHENG Tao;LIU Jiahao;LI Bin;LI Jiashan;NIE Juan;XIONG Tao(Department of Microbiology,School of Medicine,Hunan University of Chinese Medicine)

机构地区：[1]湖南中医药大学医学院病原生物学教研室,长沙410006

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第2期251-258,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：中国博士后科学基金委面上项目(2021M791078);湖南省自然科学基金青年项目(2022JJ40311);湖南省教育厅科学研究项目青年项目(21B0388);湖南省卫健委科研项目一般项目(202201054363);湖南省中医药管理局科研计划项目青年项目(2021163);湖南中医药大学创新训练项目。

摘　　要：目的本研究旨在探讨具有交替氧化酶(AOX)抑制活性的中药单体分子没食子酸和氯硝柳胺的联合灭螺效果及其灭螺机制。方法阴性钉螺采自湖北省公安县,随机分为7组,空白对照组(H_(2)O),实验组分别为氯硝柳胺(N1组:0.06 mg/L,N2组:0.1 mg/L)和没食子酸(G1组:0.8 g/L,G2组:1.6 g/L)单独使用,及联合使用(M1组:0.06 mg/L氯硝柳胺+0.8 g/L没食子酸,M2组:0.06 mg/L氯硝柳胺+1.6 g/L没食子酸)。检测各组钉螺经药物浸杀12、24和48 h后的存活率,液氮包埋后切片并染色,光学显微镜下观察并定量分析,检测钉螺软体切片中细胞色素C氧化酶(CCO)和乳酸脱氢酶(LDH)的相对酶活力值,以平均光密度值表示。钉螺匀浆后离心、取上清,DCFH-DA探针共孵育,用BCA蛋白定量试剂盒测定总蛋白含量,多功能酶标仪检测荧光强度,ROS值以样品测得的荧光强度(FI)除以蛋白质浓度(μg)的值表示,用总超氧化物歧化酶(SOD)活性检测试剂盒测定SOD水平。钉螺死亡率的差异分析采用卡方检验;CCO活性和氧化应激水平的数据用Levene’s Test确定方差齐性后,用Tukey HSD的多重比较方法进行不同组间的两两比较。结果G1和G2组浸杀后未表现出显著的灭螺效应。M1组和M2组在浸杀48 h后的钉螺死亡率分别达到70.0%(56/80)和84.2%(101/120),均高于N1组(44.4%,32/72)(χ2=9.13、32.52,均P<0.05);与N2组相比,钉螺死亡率差异均无统计学意义(χ2=0.11、2.58,均P>0.05)。N1和N2组钉螺的LDH活性呈下降趋势;M1组和M2组钉螺体内CCO和LDH活性均降低(均P<0.05),48 h后M1组的LDH活性在肌肉组织和肝脏分别为0.152±0.002和0.172±0.016,CCO活性分别为0.180±0.022和0.335±0.014;M2组的LDH活性为0.166±0.008和0.173±0.022,CCO活性为0.199±0.009和0.294±0.015,与空白对照组LDH活性(0.229±0.006和0.227±0.010)、CCO活性(0.259±0.008和0.428±0.024)的差异均有统计学意义(均P<0.05)。M1组在处理后24 h的SOD活性为(5.56±0.91)UI/g,高于空�Objective To investigate the combined snail killing effect and mechanism of traditional Chinese medicine monomers gallic acid and niclosamide,which have alternating oxidase(AOX)inhibitory activity.Methods Negative snails were collected from Gong’an County,Hubei Province and randomly divided into 7 groups:a blank control group(H_(2)O),experimental groups were treated with niclosamide(N1 group:0.06 mg/L,N2 group:0.1 mg/L)and gallic acid(G1 group:0.8 g/L,G2 group:1.6 g/L)alone or in combination(M1 group:0.06 mg/L niclosamide+0.8 g/L gallic acid,M2 group:0.06 mg/L niclosamide+1.6 g/L gallic acid).Detect the survival rates of each group of snails after drug immersion for 12,24,and 48 hours.Slice and stain them after liquid nitrogen embedding,observe and quantitatively analyze them under an optical microscope.Measure the relative enzyme activity values of cytochrome C oxidase(CCO)and lactate dehydrogenase(LDH)in the soft sections of snails,expressed as average optical density values.After homogenizing the snail,centrifuge and take the supernatant.Incubate with DCFH-DA probe,determine the total protein content using a BCA protein quantification kit,detect fluorescence intensity(FI)using a multifunctional enzymelinked immunosorbent assay(ELISA),and divide the ROS value by the FI measured in the sample by the protein concentration(μg).The value represents the determination of superoxide dismutase(SOD)levels using a total SOD activity detection kit.The difference analysis of snail mortality rate was conducted usingχ2 test.After determining the homogeneity of variance using Levene’s Test,the data on CCO activity and oxidative stress levels were compared pairwise between different groups using Tukey HSD’s multiple comparison method.Results The G1 and G2 groups did not show significant snail killing effects after immersion.The mortality rates of snails in the M1 and M2 groups after 48 h of immersion reached 70.0%(56/80)and 84.2%(101/120),respectively,higher than those in the N1 group(44.4%,32/72)(χ2=9.13,32.52;P<0.05

关 键 词：湖北钉螺 没食子酸 氯硝柳胺 灭螺效果 葡萄糖代谢酶活性 氧化应激水平 

分 类 号：R978.82[医药卫生—药品]