砂生槐总生物碱水剂和片剂体外体内抗多房棘球蚴的效果

Anti-Echinococcus multilocularis effect of total alkaloids of Sophora moorcroftiana in water solution and tablet forms in vitro and in vivo

目的评价砂生槐总生物碱(TA-SM)水剂和片剂体外体内抗多房棘球蚴的效果。方法从保种的蒙古长爪沙鼠中分离多房棘球蚴原头节,置培养瓶中(10000个原头节/瓶)培养。将原头节培养瓶随机分组为培养基对照组、溶剂对照组、阿苯达唑(ABZ)阳性对照组、阿苯达唑亚砜(ABZ-SO)阳性对照组和低、中、高浓度TA-SM水剂组和片剂组,各组分别加RPMI 1640完全培养基、终浓度为0.1%的二甲基亚砜(DMSO)、10μg/ml的ABZ、10.60μg/ml的ABZ-SO或终浓度为5(低浓度)、10(中浓度)、20μg/ml(高浓度)的水剂或片剂TA-SM,37℃、5%CO_(2)培养7 d,每天取样台盼蓝染色,倒置显微镜下观察原头节存活情况,计算存活率;共培养至第5天检测培养上清中的碱性磷酸酶(ALP)和原头节中的半胱天冬氨酸蛋白酶3(caspase 3)的相对表达水平。取54只雌性昆明小鼠,每鼠经腹腔注射0.2 ml多房棘球蚴原头节(密度为104个/ml),感染4个月后随机分为生理盐水组、片剂组和水剂组,每组18只,水剂组和片剂组每鼠灌服28 mg/(kg·d)的水剂或片剂TASM,生理盐水组灌服等量的生理盐水,每天给药1次。给药治疗15、30、60 d后,每组随机抽取6只小鼠剖取包囊,称重,计算抑囊率;取肝组织切片,苏木素-伊红(HE)染色后观察肝组织的病理变化,扫描电子显微镜(SEM)观察肝组织的超微结构。组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果体外共培养至第7天,培养基和溶剂对照组的原头节形态呈椭圆形,各部分完整清晰,未受损伤,表现为典型的内陷型;ABZ和ABZ-SO阳性对照组的原头节虫体变长,可见吸盘,表现为典型的外翻型;低、中浓度TA-SM水剂组和片剂组原头节以内陷型为主,偶有外翻型;高浓度TA-SM水剂组和片剂组原头节形态多为外翻型。体外共培养至第7天,低、中、高浓度TA-SM水剂组和片剂组原头节存活率分别为(34.7±2.75)%、(31.5±10.12)%、(27.6±2.51)%和(30.8±4.15)%、(29.8±8.11)%、(25.2±4.30)%,二者存活率差异无统计学意义(t=1.36、0.22、0.83,均P>0.05);TA-SM水剂组和片剂组的存活率均低于培养基对照组[(78.4±9.68)%](F=32.30、38.53,均P<0.01)和溶剂对照组[(82.7±5.45)%](F=55.15、67.50;均P<0.01),与ABZ阳性对照组[(23.85±19.63)%](F=0.53、0.29,均P>0.05)和ABZ-SO阳性对照组[(18.93±3.08)%](F=4.44、3.17;均P>0.05)差异无统计学意义。体外共培养至第5天,低、中、高浓度TA-SM水剂组和片剂组,ABZ和ABZ-SO阳性对照组,培养基和溶剂对照组培养上清中ALP的相对表达水平分别为0.15±0.01、0.17±0.03、0.18±0.03和0.21±0.04、0.29±0.04、0.32±0.04,0.17±0.01和0.15±0.01,0.15±0.01和0.13±0.01,其中TA-SM片剂组均高于培养基和溶剂对照组、ABZ和ABZ-SO阳性对照组(F=18.49、22.34、14.86、18.06,P<0.01)以及TA-SM水剂组(t=6.86、3.78、5.19,均P<0.05)。体外共培养至第5天,低、中、高浓度TA-SM水剂组和片剂组、ABZ和ABZSO阳性对照组,培养基和溶剂对照组原头节中caspase 3的相对表达水平分别为0.14±0.01、0.13±0.01、0.14±0.01和0.21±0.02、0.36±0.04、0.42±0.04,0.10±0.01和0.17±0.01,0.13±0.01和0.10±0.01,其中TA-SM片剂组均高于培养基和溶剂对照组、ABZ和ABZ-SO阳性对照组(F=58.97、69.18、71.81、46.77,均P<0.01)以及TA-SM水剂组(t=4.99、10.24、10.82,P<0.01)。小鼠体内药效实验结果显示,感染多房棘球蚴小鼠治疗60 d后,TA-SM片剂组和水剂组小鼠的抑囊率分别为(24.4±4.15)%、(17.2±3.71)%,二者差异有统计学意义(t=3.15,P<0.05);肝切片HE染色光学显微镜观察结果显示,生理盐水组小鼠肝细胞肿大,周围有大量炎性细胞浸润,细胞质有大量空泡样变性;TA-SM水剂组和片剂组肝细胞形态明显趋于正常,炎性细胞明显减少,细胞质空泡样变性减少;SEM观察结果显示,感染小鼠治疗15 d后,生理盐水组小鼠肝组织病灶中出现典型的囊泡状结构,TA-SM片剂组和水剂组小鼠肝组织病灶中典型的囊泡状结构显著减少。结论TA-SM水剂和片剂在体内、体外均有一定的抗多房棘球蚴的效果,体外的疗效与ABZ基本一致,TA-SM片剂的疗效优于水剂。

Objective To evaluate the anti-Echinococcus multilocularis effect of total alkaloids of Sophora moorcroftiana(TA-SM)in water solution and tablet forms in vitro and in vivo.MethodsThe protoscolices of E.multilocularis were isolated from the Mongolian gerbils and cultured in a culture flask(10000 protoscolices/flask).The culture flasks were randomly assigned into the culture medium control group,solvent control group,the albendazole(ABZ)positive control group,the albendazole sulfoxide(ABZ-SO)positive control group,and the low,medium and high concentration groups of TA-SM as water solution groups and as tablet form groups,in which the protoscolices were co-cultured with RPMI 1640 complete medium,or the final concentration of 0.1%dimethyl sulfoxide(DMSO),10μg/ml ABZ,10.60μg/ml ABZ-SO,or the final concentration of 5μg/ml(low concentration),10μg/ml(medium concentration)and 20μg/ml(high concentration)of TA-SM in water solution and tablet forms at 37℃and 5%CO_(2)for 7days.The samples collected every day were stained with trypan blue and observed under inverted microscope to check the survival and calculate the survival rate.The relative expression level of alkaline phosphatase(ALP)in the culture supernatant and caspase 3 in protoscolices was detected on the fifth day of co-culture.Fifty-four female Kunming mice were injected with protoscolices of E.multilocularis(0.2 ml/mouse,104 protoscolices/ml)intraperitoneally and were randomly assigned into the normal saline group,tablet group and water solution group 4 months post-infection.In the water solution group and tablet group,mice were treated with 28 mg/(kg·d)of TA-SM in water solution or tablet forms,respectively,by oral gavage,once a day.In the normal saline group mice were given the same volume of normal saline only.On the 15th,30th and 60th days of treatment,six mice in each group were randomly selected to dissect,the mass of cyst was weighed,and the rate of cyst inhibition was calculated.The liver tissue sections were prepared,and the pathological changes of the liver tissue were observed using HE staining,the ultrastructure of liver tissue was observed by scanning electron microscope(SEM).One-way ANOVA was used for inter-group comparison,and LSD-t test was used for pairwise comparison.ResultsOn the 7th day of co-culture in vitro,the protoscolices in the medium and in the solvent control groups were oval in shape,and the protoscolices were intact and clear without damage,showing typical invagination.In the ABZ and ABZ-SO and positive control group,the protoscolices became longer,and the sucker was visible,showing the typical eversion type.In the low and medium concentration groups of TA-SM in water solution and tablet forms,the protoscolices were mainly invaginated,and occasionally everted.The morphology of the protoscolices in the high concentration groups of TA-SM in water solution and tablet forms was mostly eversion type.On the 7th day of co-culture in vitro,the survival rates of the protoscolices in the low,medium and high concentration groups of TA-SM in water solution and tablet forms were(34.7±2.75)%,(31.5±10.12)%,(27.6±2.51)%and(30.8±4.15)%,(29.8±8.11)%,(25.2±4.30)%,respectively,with no statistically significant difference between the TA-SM in water solution group and tablet form group(t=1.36,0.22,0.83,all P>0.05).The survival rates in the TA-SM in water solution group and tablet form group were all lower than that of the medium control group[(78.4±9.68)%](F=32.30,38.53;all P<0.01)and the solvent control group[(82.7±5.45)%](F=55.15,67.50;all P<0.01),but there were no significant differences with ABZ positive control group[(23.85±19.63)%](F=0.53,0.29;all P>0.05)or the ABZ-SO positive control group[(18.93±3.08)%](F=4.44,3.17;all P>0.05).On the fifth day of co-culture in vitro,the relative expression levels of ALP in the culture supernatant in the low,medium and high concentration groups of the TA-SM in water solution groups and the tablet form groups,the ABZ and ABZ-SO positive control groups,the medium and solvent control groups were 0.15±0.01,0.17±0.03,0.18±0.03 and 0.21±0.04,0.29±0.04,0.32±0.04;0.17±0.01 and 0.15±0.01;0.15±0.01 and 0.13±0.01,respectively,in which the TA-SM in tablet form groups were higher than medium and solvent control groups,ABZ and ABZ-SO positive control groups(F=18.49,22.34,14.86,18.06;P<0.01)and the TA-SM in water solution groups(t=6.86,3.78,5.19;P<0.05).On the fifth day of co-culture in vitro,the relative expression levels of caspase 3 in protoscolices in the low,medium and high concentration groups of TA-SM in water solution groups and tablet form groups,ABZ and ABZ-SO positive control groups,medium and solvent control groups were 0.14±0.01,0.13±0.01,0.14±0.01 and 0.21±0.02,0.36±0.04,0.42±0.04;0.10±0.01 and 0.17±0.01;0.13±0.01 and 0.10±0.01,respectively,in which the TA-SM in tablet form groups were higher than the medium and solvent control groups,ABZ and ABZ-SO positive control groups(F=58.97,69.18,71.81,46.77;all P<0.01)and the TA-SM in water solution group(t=4.99,10.24,10.82;P<0.01).The results of the in vivo experiment showed that after 60 days of treatment,the cyst inhibition rates of the tablet group and the water solution group were(24.4±4.15)%and(17.2±3.71)%,respectively,with a statistically significant difference between them(t=3.15,P<0.05).The HE staining results showed that after 60 days of treatment,the hepatocytes in the saline group were swollen,there were a large number of inflammatory cells infiltrating around,and there were a large number of vacuolar degeneration in the cytoplasm.The morphology of hepatocytes in the water solution group and the tablet group tended to be normal,the inflammatory cells were significantly reduced,and the vacuolar degeneration in the cytoplasm was reduced.SEM results showed that after 15 days of treatment,the typical vesicular structure appeared in the liver tissue lesions of mice in the saline group,while the typical vesicular structures in the liver tissue lesions of mice in the tablet group and the water solution group were significantly reduced.ConclusionTA-SM in water solution and tablet forms have certain suppressive effects on protoscolices of E.multilocularis in vitro and in vivo.The efficacy in vitro test is essentially the same as that of ABZ,and the efficacy of TA-SM in tablet form is better than that in water solution.