基于秀丽隐杆线虫模型的去氢骆驼蓬碱衍生物1-(4-甲氧基)苯基-9-丁基-β-咔啉神经毒性效应

Neurotoxic effect of harmine derivatives 1-(4-methoxy)phenyl-9-butyl-carboline based on Caenorhabditis elegans model

目的探讨1-(4-甲氧基)苯基-9-丁基-β-咔啉(DH-004)对秀丽隐杆线虫(简称线虫)的神经毒性。方法采用野生型线虫为模式生物,分别以DH-0040.0(溶剂对照组),12.5,25.0,50.0,100.0和200.0μmol·L^(-1)连续培养,分别于不同时间显微镜下观察线虫的存活率、头部摆动频次、有效产卵总数、趋向和趋避行为及摄食行为。吖啶橙染色荧光显微镜下观察细胞凋亡;试剂盒检测虫体组织乙酰胆碱酯酶(AChE)活性。结果与溶剂对照组相比,不同浓度DH-004染毒处理5 d后,线虫存活率随药物浓度的增加而显著降低(R^(2)=0.949,P<0.01)。DH-00425.0μmol·L^(-1)处理3 d(P<0.01)、50.0μmol·L^(-1)处理1~3 d(P<0.05,P<0.01)及100.0和200.0μmol·L^(-1)处理1~3 d(P<0.01)组线虫头部摆动频次均显著降低。与溶剂对照组相比,DH-00425.0,50.0,100.0和200.0μmol·L^(-1)组有效虫卵总数明显减少(P<0.01),趋向系数显著下降(P<0.05,P<0.01),趋避系数显著上升(P<0.01);DH-00412.5μmol·L^(-1)组处理2~3 d(P<0.01)及25.0,50.0,100.0和200.0μmol·L^(-1)组处理1~3 d(P<0.01)线虫咽部收缩次数均显著减少。吖啶橙染色结果显示,与溶剂对照组相比,DH-00450.0,100.0和200.0μmol·L^(-1)组细胞凋亡水平显著升高(P<0.01)。与溶剂对照组相比,DH-004浓度依赖性增高线虫体内AChE活性抑制率(R^(2)=0.851,P<0.01),IC50为(94.7±3.7)μmol·L^(-1)。结论DH-004对线虫具有一定的神经毒性效应,其作用机制可能与促进细胞凋亡和抑制AChE活性有关。

OBJECTIVE To investigate the neurotoxic effect of 1-(4-methoxy)phenyl-9-butyl-β-carboline(DH-004)on Caenorhabditis elegans(C.elegans).METHODS Wild type C.elegans were used as model organisms,and consecutively treated with DH-0040.0(solvent control group),12.5,25.0,50.0,100.0 and 200.0μmol·L^(-1).The survival rate,head swing frequency,effective total number of eggs,tendency and avoidance behavior and feeding behavior of C.elegans were observed under a microscope at different times of treatment.After acridine orange staining,cell apoptosis was observed under a fluorescence microscope.The activity acetylcholinesterase(AChE)was detected by spectrophotometry.RESULTS Compared with the solvent control group,the survival rate of C.elegans decreased significantly with the increase of DH-004 concentration(R^(2)=0.949,P<0.01).Treatment with DH-00425.0μmol·L^(-1)3 d(P<0.01),50.0μmol·L^(-1) for 1-3 d(P<0.05,P<0.01)and 100.0 and 200.0μmol·L^(-1) treated for 1-3 d(P<0.01)significantly reduced the frequency of head swing.Compared with the solvent control group,the total number of effective eggs in DH-00425.0,50.0,100.0 and 200.0μmol·L^(-1) groups decreased significantly(P<0.01),so did the tendency coefficient groups(P<0.05,P<0.01),while the avoidance coefficient increased significantly(P<0.01).DH-00412.5μmol·L^(-1) treatment for 2-3 d(P<0.01)and 25.0,50.0,100.0 and 200.0μmol·L^(-1) treatment for 1-3 d(P<0.01)significantly reduced the number of times of throat contraction.The results of acridine orange staining showed that DH-00450.0,100.0 and200.0μmol·L^(-1) increased the level of apoptosis significantly(P<0.01).Compared with the solvent control group,DH-004 increased the inhibitory rate of ACh E activity in a dose-dependent manner(R^(2)=0.851,P<0.01),and the IC50 was(94.7±3.7)μmol·L^(-1).CONCLUSION DH-004 has some neurotoxic effect on C.elegans,and the mechanism may be related to promoting cell apoptosis and inhibiting AChE activity.