4种中草药单体对嗜热四膜虫生长和凋亡的影响

Effects of four kinds of Chinese herbal monomers on growth and apoptosis of Tetrahymena thermophila

利用Hoechst 33258荧光染色、活细胞计数、CCK-8细胞活力测定、抗氧化酶活力测定等技术,研究了青蒿素、大黄素、芦荟大黄素、小檗碱(又名黄连素)等4种中草药单体对嗜热四膜虫(Tetrahymena thermophila)生长和凋亡的影响,为应用嗜热四膜虫筛选抗鱼寄生虫中草药提供参考。结果表明,采用Hoechst荧光染色,4种单体在高浓度(20~200 mg·L^(−1))作用下均可使嗜热四膜虫凋亡,显著着色发亮。4种单体对嗜热四膜虫的半抑制浓度(IC50)和95%置信区间(95%confidence interval,95%CI),用细胞计数法测定依次为21.23(16.71~28.23)mg·L^(−1)、3.77(1.91~5.54)mg·L^(−1)、1.70(0.18~3.82)mg·L^(−1)和44.56(29.82~98.66)mg·L^(−1),CCK-8细胞活力测定法依次为20.03(13.02~28.45)mg·L^(−1)、6.07(4.39~7.65)mg·L^(−1)、3.67(2.61~4.72)mg·L^(−1)和107.39(66.78~267.17)mg·L^(−1),CCK-8法与细胞计数法所呈现的趋势较为一致。在单体质量浓度为1.25~50.0 mg·L^(−1),随着药物浓度的增加,过氧化氢酶(CAT)活力有升高趋势,而丙二醛(MDA)呈下降趋势,超氧化物歧化酶(SOD)活力先升高后降低。上述结果表明,4种中草药单体对嗜热四膜虫的生长和凋亡有剂量效应关系,凋亡的产生可能与细胞氧化应激状态改变有关。

To provide references for screening Chinese herbal medicine against fish parasites by Tetrahymena thermophila,we studied the effects of Chinese herbal monomers(CHM)such as artemisinin,emodin,aloe-emodin and berberine on the growth and apoptosis of T.thermophila by Hoechst 33258 fluorescence staining,living cell counting,CCK-8(Cell Counting Kit-8)cell viability test and antioxidase activity assay.The results show that with Hoechst 33258 fluorescence staining,T.thermophila cells in the CHM groups appeared significantly stronger fluorescence than those of the control groups at high concentration(20−200 mg·L^(−1)),which indicates that the four kinds of CHMs can cause apoptosis of T.thermophila cells.The 50%inhibitory concentration(IC50)and 95%confidence intenal(95%CI)of the four monomers to T.thermophila cells determined by living cell counting method was 21.23(16.71−28.23)mg·L^(−1),3.77(1.91−5.54)mg·L^(−1),1.70(0.18−3.82)mg·L^(−1) and 44.56(29.82−98.66)mg·L^(−1),respectively;and 20.03(13.02−28.45)mg·L^(−1),6.07(4.39−7.65)mg·L^(−1),3.67(2.61−4.72)mg·L^(−1) and 107.39(66.78−267.17)mg·L^(−1) determined by CCK-8 cell viability test,respectively,consistent with the results by living cell counting method.When the monomer concentration was 1.25−50.0 mg·L^(−1),with the increase of drug concentration,the activities of catalase(CAT)increased,malondialdehyde(MDA)decreased,and superoxide dismutase(SOD)increased first and then decreased.In conclusion,the four kinds of CHMs affect the proliferation and the growth and apoptosis of T.thermophila were dose-dependent.The occurrence of apoptosis may be related to the change of cellular oxidative stress.