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1 Chinese Journal of Applied Entomology 2015, 52(6): DOI: /j.issn 摘 * 绿僵菌素对四种昆虫细胞的毒性何广位 1, 2** 胡维娜 1, 2** 胡琼波 1,2*** 要目的 Destruxins Bombyx mori Bm12 Ostrinia furnacalis Ofh S2 Sf9 方法 MTT A B DA DB DABM 4 IC 50 结果 24 h < 25 μg /ml Ofh Bm12 S2 Sf9 DA DB DABM DA DB DABM Ofh IC μg/ml Bm12 S2 Sf9 IC μg/ml DA DB Ofh Sf9 Bm12 S μg/ml Bm12 Ofh S2 Sf9 结论 Ofh S2 4 关键词 Toxicity of destruxins to four insect cell lines HE Guang-Wei 1, 2** HU Wei-Na 1, 2** 1, 2*** HU Qiong-Bo (1. The Key Laboratory of Natural Pesticide and Chemical Biology, South China Agricultural University, Guagzhou , China; 2. Key Laboratory of Integrated Pests Management of Southern China, Ministry of Agriculture, Guagzhou , China) Abstract [Objectives] Destruxins are insecticidal secondary compounds isolated from Metarhizium anisopliae. In this study, the toxicity of different destruxins was tested on four insect cell lines, i.e., Bombyx mori Bm12, Ostrinia furnacalis hemocytes Ofh, Drosophila melanogaster S2 and Spodoptera frugiperda Sf9. [Methods] MTT and microscopy analysis were employed to evaluated the effects of destruxins A and B (DA and DB), and a 1/1 (m/m) mixture of these compounds (DABM), on the four insect cell lines. Relative toxicity was assessed by comparing IC 50 s values and morphological changes in the cell lines. [Results] The results indicate that Ofh cells were more susceptible to lower dosages (< 25 μg/ml) of DA and DB, with IC 50 values of , and μg/ml for DA, DB and DABM, respectively, compared to IC 50 values > 250 μg/ml in the other cell types. Furthermore, DA and DB had a synergistic effect on Ofh cells, but an antagonistic effect on Sf9 cells, and an additive effect on Bm12 and S2 cells. Microscopy revealed morphological changes in the different cell lines, even at dosages as low as 6.25 μg/ ml. Moreover, these morphological changes became more apparent at higher dosages. Ruptured cells, strumae, aggregation, expanded cells, vacuoles and reduced cell numbers were found in Bm12 cells. In Ofh cells, cells * Supported projects AA10A204-2 ** First author [email protected]; @qq.com *** Corresponding author [email protected] Received Accepted

2 6 : 1467 became expanded but appeared to return to normal with aggregation. Vacuoles and a few expanded, ruptured and aggregated cells were found in the S2 cell line, and membrane shrinkage, broken cells and a decrease in cell numbers were recorded in the Sf9 cell line. [Conclusion] The Ofh cell line was the most sensitive to destruxins, and the non-host insect Drosophila melanogaster S2 cell line the least sensitive. The results suggest that although relatively large doses of destruxins are required to kill insect cells, smaller dosages are sufficient to cause changes in cell morphology. Key words destruxins, insect cells, toxicity Destruxins Metarhizium anisopliae Pedras et al A B DA DB Liu and Tzeng 2012 Galleria mellonella Vilcinskas et al. 1997a 1997b Aspergillus niger μg/ml Vey et al Drosophila melanogaster Escherichia coli IMD Pal et al Fan et al Spodoptera litura SL-1 Chen et al A B 4 1 材料与方法 1.1 细胞与培养 Bombyx mori (Linnae) Bm12 TNM-FH Hyclone Ostrinia furnacalis Guenee Ofh EX-CELL 420 Sigma Drosophila melanogaster (Meigen) S2 Schneider Sigma Spodoptera frugiperda (J.E.Smmith) Sf9 SFX Hyclone 4 10% Gibco 27 Bm12 S2 Sf9 2~4 d Ofh 3~4 d 1.2 绿僵菌素与处理 A B DA DB Hu et al DA DB m/m DABM 1 mg 100 μl DMSO Sigma μg/ml 1-80

1468 Chinese Journal of Applied Entomology 52 4 000 2 000 1 000 500 250 125 62.5 μg/ml 1 1.

3 1468 Chinese Journal of Applied Entomology μg/ml MTT 法检测绿僵菌素对 4 种细胞的毒性 MTT MTT Formazan 臢 OD OD % =1 OD OD / OD OD Corning Incorporated 90 μl 24 h /ml 10 μl μg/ml 1% DMSO h 50 μl 1 MTT 27 4 h r/min 150 ml DMSO imark 490 nm (OD) SPSS IBM IC 形态学观察 24 h 2 结果与讨论 2.1 绿僵菌素对 4 种细胞的毒性 MTT 24 h Bm12 Ofh S2 Sf9 1 Bm12 Ofh Sf9 S2 DA DB 6.25~25.00 μg/ml 3 35%~48% S2 20% 1 4 DA DB DABM Ofh DABM 12.5 μg/ml Ofh 45% Bm12 Sf9 S2 13% 35% 15% 400 μg/ml Bm12 Sf9 S2 55% 46% 56% IC 50 IC 50 IC 50 1 DA DB Bm12 Sf9 Fig. 1 图 1 绿僵菌素处理 24 h 后 4 种昆虫细胞的死亡率 Mortalities of four cell lines after 24 h treated with destruxins DA: A DB: B DABM: A B 1/1 DA: Destruxin A; DB: Destruxin B; DABM: The 1/1 mixture of destruxin A and destruxin B.

4 6 : 1469 Cell line Treatment Table 1 表 1 绿僵菌素对 4 种细胞的毒性回归方程和 IC 50 值 IC-p equations and IC 50 values of destruxins against four cell lines IC-p equation Chi-Square test (P) IC 50 95% mg/l IC 50 (95% confident interval) Bm12 DA y= x ( ) - CTC DB y= x ( ) - DABM y= x ( ) Ofh DA y= x DB y= x DABM y= x S2 DA y= x ( ) - DB y= x ( ) - DABM y= x ( ) Sf9 DA y= x ( ) - DB y= x ( ) - DABM y= x DA: A DB: B DABM: A B 1/1 DA: Destruxin A; DB: Destruxin B; DABM: The 1/1 mixture of destruxin A and destruxin B. IC 50 Ofh S2 DB DA IC 50 4 Ofh DA DB DABM Ofh IC μg/ml μg/ml 3 IC μg/ml 1 DA DB 4 DABM Ofh CTC DA DB S2 CTC DA DB CTC 80~ 绿僵菌素对 4 种细胞形态的影响 Bm12 Ofh S2 Sf9 6.25~400 μg/ml DA DB DABM Bm12 24 h 2 DA DB DABM Ofh Plastohemocytes Oenocytoide 3 S2 4 Sf9 5 3 讨论 4 Ofh IC 50 DA DB Ofh IC μg/ml DABM IC μg/ml 6.25 μg/ml

1470 应用昆虫学报 Chinese Journal of Applied Entomology Fig. 2 52 卷图 2 绿僵菌素处理 Bm12 细胞 24 h 后的显微图片 Profiles of Bm12 cells after 24 h treated with destruxins CK.

5 25 50 100 200 和 400 μg/ml 处理细胞 ag 细胞聚集 db 碎片 ex 膨胀 str 瘤状突起 va 空泡比例尺=100 μm 下图同 CK: 1% DMSO control; A/B/AB1 7: The treatments of DA, DB and DABM at the concentrations of

5 1470 应用昆虫学报 Chinese Journal of Applied Entomology Fig 卷图 2 绿僵菌素处理 Bm12 细胞 24 h 后的显微图片 Profiles of Bm12 cells after 24 h treated with destruxins CK. 1% DMSO 对照 A/B/AB1~7. DA DB 和 DABM 分别以和 400 μg/ml 处理细胞 ag 细胞聚集 db 碎片 ex 膨胀 str 瘤状突起 va 空泡比例尺=100 μm 下图同 CK: 1% DMSO control; A/B/AB1 7: The treatments of DA, DB and DABM at the concentrations of 6.25, 12.5, 25, 50, 100, 200, 400 μg/ml. ag: Aggregation; db: Debris; ex: Expand; str: Strumae; va: Vacuole. Scale bar =100 μm. The same below. Fig. 3 图 3 绿僵菌素处理 Ofh 细胞 24 h 后的显微图片 Profiles of Ofh cells after 24 h treated with destruxins pl 浆血细胞 oe 类绛色细胞比例尺=100 μm pl: Plastohemocytes; oe: Oenocytoide. Scale bar =100 μm.

destruxins br 细胞破碎比例尺=50 μm br: Broken. Scale bar= 50 μm. Fig.

6 6期何广位等: 绿僵菌素对四种昆虫细胞的毒性 Fig. 4 图 4 绿僵菌素处理 S2 细胞 24 h 后的显微图片 Profiles of S2 cells after 24 h treated with destruxins br 细胞破碎比例尺=50 μm br: Broken. Scale bar= 50 μm. Fig. 5 图 5 绿僵菌素处理 Sf9 细胞 24 h 后的显微图片 Profiles of Ofh cells after 24 h treated with destruxins sh 细胞收缩比例尺=100 μm. sh: Shrinkage. Scale bar =100 μm. 1471

7 S2 IC μg/ml S2 Bm12 Sf9 Bm12 Sf9 A B S μg/ml Vey et al Fan et al S2 A B S2 4 参考文献 (References) Chen XR, Hu QB, Yu XQ, Ren SX, Effects of destruxins on free calcium and hydrogen ions in insect hemocytes. Insect Science, Doi / Fan JQ, Chen XR, Hu QB, Effect of destruxins A on hemocytes morphology of Bombyx mori. Journal of Integrative Agriculture, 12(6): Hu QB, Ren SX, Review of destruxins of Metarhizium anisopliae Sorokin. Chinese Journal of Biological Control, 20(4): [,, , 20(4): ] Hu QB, Ren SX, Wu JH, Chang JM, Musa DP, Investigation of destruxin A and B from 80 Metarhizium starins in China, and the optimization of cultural conditions for the strain MaQ10. Toxicon, 48(5): Liu BL, Tzeng YM, Development and applications of destruxins: a review. Biotechnology Advances, 30: Pal S, St Leger RJ, Wu LP, Fungal peptide destruxin a plays a specific role in suppressing the innate immune response in Drosophila melanogaster. Journal of Biological Chemistry, 282: Pedras MSC, Zaharia LI, Ward DE, The destruxins: synthesis, biosynthesis, biotransformation, and biological activity.

8 6 : 1473 Phytochemystry, 59(6): Vey A, Matha V, Dumas C, Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction. Journal of Invertebrate Pathology, 80(3): Vilcinskas A, Matha V, Gotz P, 1997a. Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth Galleria mellonella. J. Insect Physiol., 43(12): Vilcinskas A, Matha V, Gotz P, 1997b. Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites. J. Insect Physiol., 43(5):

- 14 - Drug Evaluation Research 第 36 卷第 1 期 2013 年 2 月料 ), 山西皇城相府药业有限公司, 批号 20111003 ( 人口服推荐剂量为每日 3 次, 每次 4 粒, 成人体质量按 60

- 14 - Drug Evaluation Research 第 36 卷第 1 期 2013 年 2 月料 ), 山西皇城相府药业有限公司, 批号 20111003 ( 人口服推荐剂量为每日 3 次, 每次 4 粒, 成人体质量按 60 Drug Evaluation Research 第 36 卷第 1 期 2013 年 2 月 - 13 - 柴青消癖胶囊对乳腺增生模型大鼠的治疗作用研究 * 刘聪, 岳永花, 郝旭亮山西省中医药研究院, 山西太原 030012 摘要 : 目的探讨柴青消癖胶囊对乳腺增生模型大鼠的治