丹参酮IIA对多房棘球蚴原头节体外活性及生长的影响

Effects of tanshinone IIA on the Activity and growth of Echinococcus multilocularis protoscoleces in vitro

目的探究丹参酮IIA对多房棘球蚴原头节体外活性及生长的影响。方法从沙鼠体内获取多房棘球蚴原头节并在体外培养3d后分别与250、500、1000μmol/L丹参酮IIA共培养7d,以DMEM为空白对照组,DMSO为溶剂对照组,显微镜下观察各组原头节形态及活性(活性良好的原头节不被伊红染色),计算存活率并绘制活力曲线;共培养24h后用活性氧(ROS)测定试剂盒测定各组原头节的ROS水平;共培养2d后用Westernblot检测各组原头节凋亡相关蛋白Bcl-2、Bax和Caspase-3相对表达量;扫描电镜观察共培养2d时各组原头节表面显微结构。多房棘球蚴原头节与大鼠肝癌细胞共培养8~9周形成多房棘球蚴囊泡,囊泡分别与两对照组、250、500、1000μmol/L丹参酮IIA共培养2 d,扫描电镜观察囊泡生发层显微结构。结果原头节与250、500、1000μmol/L丹参酮IIA共培养2d,随着药物浓度的升高原头节形态结构发生改变,虫体模糊不清且红染率升高,原头节活性受抑制;共培养2d时空白对照组、溶剂对照组活力分别为100%、(98.53±0.503)%,250、500和1000μmol/L组多房棘球蚴原头节活力分别为(81.00±3.606)%、(63.97±3.275)%、(37.07±3.296)%(F=298.1,P<0.05);第7d时空白对照组和溶剂对照组多房棘球蚴原头节活力分别为(98.33±0.577)%、(93.90±0.529)%,250和500μmol/L组多房棘球蚴原头节活力分别为(51.67±7.638)%、(9.37±6.075)%,均低于对照组(P<0.05)。1000μmol/L丹参酮IIA组杀伤作用最强,原头节均死亡。共培养24h,各浓度丹参酮IIA组原头节ROS水平显著高于对照组(均P<0.05);共培养2d,随药物浓度增加,各浓度丹参酮IIA组原头节Bcl-2表达减少,Bax和Caspase-3表达升高(均P<0.05)。扫描电镜观察各浓度丹参酮IIA组原头节显微结构出现不同程度破坏甚至破裂,空白对照组和溶剂对照组原头节结构正常;不同浓度丹参酮IIA与囊泡共培养2d后扫描电镜观察囊泡生发层显微结构,可见生发层细胞不同程度地出现脱落、破裂。空白对照组和溶剂对照组生发层细胞包膜完整、形态饱满,并可见大量小的生发囊。结论丹参酮IIA对多房棘球蚴原头节体外活性及生长具有抑制作用,该抑制作用可能与丹参酮IIA打破原头节抗氧化防御系统的平衡、诱导细胞凋亡及抑制生发层细胞生长有关。

Objective To investigate the effects of tanshinoneIIA on the activity and growth of Echinococcus multilocularis protoscoleces in vitro.Methods The protoscoleces were extracting from girbil and cultured in vitro for 3 days,and then co-cultured with 250,500,1000 mol/L tanshinone IIA for 7days,by using DMEM as a blank control group and DMSO as solvent control group.The morphology,activity of protoscoleces were observed through a microscope(The active protoscoleces did not stain by eosin),and the survival rate was calculated.The ROS were tested by a Reactive Oxygen Species(ROS)test kit after 24 hours.Test the expression of apoptosisrelated proteins Bcl-2,Bax and Caspase-3 protein in each group by western blotting after 2 days.Scanning electron microscope was used to observe the surface microstructure of protoscoleces after 2 days.The protoscoleces were co-cultured with rat hepatoma cells for 8-9 weeks to form E.multilocularis vesicles,The vesicles were co-cultured with blank control group,solvent control group,and 250,500,1000 mol/L tanshinone IIA groups for 2 days,and the micros-tructure of The germinal layer of vesicles was observed by scanning electron microscope.Results Tanshinone IIA at concentrations of 250,500,and 1000 mol/L changed the morphology and structure of protoscoleces,blurred the larvae and increased the red staining rate,and inhibited the activity of protoscoleces.After co-cultured for 2 days,the viability ofprotoscoleces inthe blank control group was 100%and the viability ofprotoscoleces in the solvent control group was(98.53±0.503)%,the viability ofprotoscoleces in 250,500 and 1000μmol/L groups were(81.00±3.606)%,(63.97±3.275)%and(37.07±3.296)%(F=298.1,P<0.05).On day 7,the viability ofprotoscoleces in blank control group and solvent control groupwere(98.33±0.577)%and(93.90±0.529)%,and than in the 250 and 500μmol/L groups were(51.67±7.638)%,(9.37±6.075)%,respectively,which were significantly lower than those in the control group(P<0.05).The 1000μmol/L tanshinone IIA group had the strongest killing effect,and all protoscoleces died.After co-cultured for 24 hours,the ROS was significantly higher in protoscoleces than control group.After co-cultured for 2 days,Bcl-2 expression decreased as the concentration of the drug increase,while Bax and Caspase-3 increased(P<0.05 for all).Scanning electron microscope showed that the microstructure of prot-oscoleces were damaged to varying degrees or even ruptured,while the structure of protoscoleces were normal in the blank control group and the solvent control group.After 2 days of co-culture with different concentrations of tanshinone IIA,the microstructure of the germinal layer cells of vesicles were observed by scanning electron microscope show that the germinal layer cells were exfoliated and ruptured to varying degrees.The germinal layer cells in the blank and the solvent control group had complete envelope,full shape,and a large number of small germinal vesicles.Conclusion Tanshinone IIA inhibited the activity and growth of the E.multilocularis protoscoleces in vitro,this inhibitory effect may be related to tanshinone IIA breaking the balance of the antioxidant defense system of protoscoleces,inducing cell apoptosis and inhibiting the growth of germinal layer cells.