黄腐酚对体外泡状棘球蚴原头节活性的影响

Effect of xanthohumol on the activity of protoscoleces of Echinococcus multilocularis in vitro

目的探讨不同浓度黄腐酚在体外对泡状棘球蚴原头节活性及形态学影响。方法将体外培养的泡状棘球蚴原头节随机分为6组:空白对照组,溶剂对照组和黄腐酚10、20、40、80μmol/L组,培养7 d,每24 h用0.1%伊红染色后在倒置显微镜下观察原头节的活性并计算成活率;黄腐酚作用泡状棘球蚴原头节3 d后,应用半胱天冬氨酸蛋白酶-3(caspase-3)检测试剂盒检测caspase-3活性;Western blot检测体外培养3d后的原头节Bcl-2、Bax蛋白表达量;使用活性氧检测试剂盒检测体外作用24 h后6组原头节内活性氧(ROS)变化水平。结果随着药物浓度升高,泡状棘球蚴原头节活力降低,空白对照组、溶剂对照组泡状棘球蚴原头节在第7 d的活力分别为(97.26±0.208)%、(97.33±0.305)%,第7 d 10μmol/L、20μmol/L、40μmol/L黄腐酚组泡状棘球蚴原头节的活力分别为(42.06±0.404)%、(24.10±0.200)%、(11.83±0.416)%,均低于对照组(P均<0.05)。80μmol/L黄腐酚组杀伤作用较强,原头蚴的活力为(0.96±0.208)%。不同浓度黄腐酚组作用原头蚴3 d,caspase-3活性与对照组相比显著增高(P<0.05)。黄腐酚不同浓度作用原头蚴3 d后,凋亡抑制蛋白Bcl-2表达下降,而凋亡蛋白Bax表达随着药物浓度的增加而显著上调(P<0.05)。不同浓度黄腐酚作用24 h后原头节体内活性氧(ROS)水平与对照组相比显著升高(P<0.05)。结论黄腐酚在体外具有一定杀灭泡球蚴原头节作用,并表现出时间依赖性和浓度依赖性。其机制可能与caspase-3活性增强、Bax表达水平升高、Bcl-2表达水平降低、ROS水平增高诱导头节细胞的凋亡有关。

Objectives To investigate the effect of different concentrations of xanthohumol on the activity and morphology of Echinococcus multilocularis protoscoleces in vitro.Methods E.multilocularis protoscoleces cultured in vitro were randomly divided into 6 groups:a blank control group,a solvent control group,and groups treated with 10,20,40,or 80μmol/L xanthohumol.Protoscoleces were cultured for 7 d,stained with 0.1%eosin,and then their activity was observed under an inverted microscope and their survival rate was calculated.After protoscoleces were treated with xanthohumol for 3 d,a caspase-3 detection kit was used to detect caspase-3 activity.Three d after culturing,Western blotting was used to detect the expression of Bcl-2 and Bax protein in the protoscoleces.One d later,a reactive oxygen species(ROS)detection kit was used to detect changes in levels of ROS in the 6 groups of protoscoleces.Results The vitality of protoscoleces decreased as the drug concentration increased.On day 7,the viability of protoscoleces in the blank control group was(97.26±0.208)% and the viability of protoscoleces in the solvent control group was(97.33±0.305)%.On day 7,the viability of protoscoleces in the 10μmol/L xanthohumol group was(42.06±0.404)%,that in the 20μmol/L group was(24.10±0.200)%,and that in the 40μmol/L group was(11.83±0.416)%.Viability in all of these groups was lower than that in the control group(P<0.05 for all).A strong killing effect was noted in the 80μmol/L xanthohumol group,and the vitality of protocercariae was(0.96±0.208)%.After 3 d of xanthohumol treatment,caspase-3 activity in the blank control group was 12.413±0.519μmol/L,which was not significantly different from that in the solvent control group(12.463±0.758μmol/L)(P>0.05).Caspase-3 activity in the protoscoleces in the 10μmol/L xanthohumol group was 16.584±0.754μmol/L,that in the 20μmol/L xanthohumol group was 23.696±0.635μmol/L,that in the 40μmol/L xanthohumol group was 32.562±0.309μmol/L,and that in the 80μmol/L xanthohumol group was 42.279±0.958μmol/L.The level of caspase-3 activity differed significantly compared to that in the control group,and enzyme activity increased significantly as the drug concentration increased(F=924.20,P<0.05 for all).After protocercariae were treated with different concentrations of xanthohumol for 3 d,expression of the apoptosis inhibitor protein Bcl-2 decreased while expression of the apoptosis protein Bax increased significantly as the drug concentration increased(P<0.05).The level of ROS in the protoscoleces increased significantly compared to that in the control group(P<0.05)after exposure to different concentrations of xanthohumol for 24 h.Conclusion Xanthohumol kills E.multilocularis protoscoleces in vitro in a time-and concentration-dependent manner.The mechanism may be related to the apoptosis of scolex cells induced by increased caspase-3 activity,an increased level of Bax expression,a decreased level of bcl-2 expression,and an increased level of ROS.