基于斑马鱼模型的蟾酥甲醇提取物抑制血管生成和抗炎活性研究

Antiangiogenic and anti-inflammatory activities of methanol extract of Bufonis Venenum based on zebrafish model

目的对蟾酥甲醇提取物中化学成分进行初步分析,并利用斑马鱼模型评价其抑制血管生成及抗炎活性。方法采用液质联用(LC-MS)技术,用反相C_(18)色谱柱,以0.1%甲酸溶液-乙腈为流动相进行梯度洗脱,采用电喷雾-三重四级杆飞行时间质谱正/负离子双重扫描方式,对提取物中的样品进行分析;采用受精后24 h(24 hpf)绿色荧光标记血管的转基因斑马鱼作为实验动物模型,荧光显微镜下观察背部节间血管生成情况,计算节间血管长度,研究PTK787(阳性药,0.2μg/mL)和蟾酥甲醇提取物2、4、6μg/mL浓度对斑马鱼血管生成的影响;采用受精后3 d(3 dpf)健康绿色荧光标记炎症细胞的转基因斑马鱼作为实验动物模型,设置对照组、模型(CuSO_(4),20μmol/L)组、布洛芬(阳性药,20μmol/L)组和蟾酥甲醇提取物(0.4、0.8μg/mL)组,计数神经丘周围炎症细胞个数。结果蟾酥甲醇提取物中共检测出33个色谱峰,并鉴定出其中7个为结合型蟾蜍二烯酸内酯。与对照组比较,蟾酥甲醇提取物4、6μg/mL浓度组斑马鱼血管生成受到抑制,血管长度变短,具有显著性差异(P<0.05、0.01);与对照组比较,CuSO_(4)模型组斑马鱼迁移到神经丘侧线部位的炎症细胞数量显著增多(P<0.01);与模型组比较,蟾酥甲醇提取物0.8μg/mL组炎症细胞迁移减少,具有显著差异(P<0.01)。结论通过LC-MS法从蟾酥甲醇提取物中鉴定出7个结合型蟾蜍二烯酸内酯,蟾酥甲醇提取物具有抑制血管生成和抗炎活性。

Objective To preliminarily analyze the chemical constituents in methanol extract of Bufonis Venenum(MEBV),and to evaluate its anti-angiogenesis and anti-inflammatory activities in zebrafish.Methods A reversed-phase C_(18) column with 0.1%formic acid solution-acetonitrile as mobile phase was used for gradient elution by liquid-mass spectrometry(LC-MS).The samples were analyzed by electrospray and triple quadrupole time-of flight mass spectrometry(TFMS)with positive/negative ion dual scanning.The transgenic zebrafish with green fluorescent protein(GFP)labeled blood vessels at 24 hpf(hours post fertilization)were used as the experimental animal model.The angiogenesis of back internode was observed under fluorescent microscope,the development of dorsal longitudinal anastomotic vessel(DLAV)and the length of inter-segmental blood vessels(ISV)were measured to study the effects of MEBV(2,4,and 6μg/mL)and PTK787(positive medicine,0.2μg/mL)on zebrafish angiogenesis at different concentrations.The transgenic zebrafish with GFP labeled inflammatory cells on 3 dpf(days post fertilization)were used to study the effect of of MEBV on inflammatory response by counting the number of inflammatory cells around the neuromast.The control group,model group(CuSO_(4),20μmol/L),ibuprofen group(positive drug,20μmol/L)and MEBV(0.4 and 0.8μg/mL)groups were set.Results A total of 33 chromatographic peaks were detected in methanol extract of toad venom,and seven of them were conjugated bufadienolides.In the experiment of antiangiogenesis,the formation of new blood vessels were inhibited at the concentration of 4 and 6μg/mL,and the vascular development was not complete and the length of ISV was significantly decreased(P<0.05 and 0.01).The results of anti-inflammatory experiment showed that compared with model group,the migration of inflammatory cells in zebrafish at the concentration of 0.8μg/mL was significantly decreased(P<0.01).Conclusion Seven Bufonadienolides were identified by LC-MS from the MEBV.Bufonis Venenum has anti-angiogenesis and anti-inflammatory activities.