藏茵陈水提物对斑马鱼新生血管的抑制效果及调控机制

Inhibitory effect of aqueous extract of Capillary wormwood herb on neovascularization of zebrafish and its regulatory mechanism

目的探讨藏茵陈水提物对斑马鱼新生血管的抑制效果及调控机制。方法选取TG品系的fli1a-EGFP转基因斑马鱼为实验动物,收集90个22hpf发育正常的斑马鱼胚胎,分为空白对照组、阳性对照组、藏茵陈水提物组,每组各30例。空白对照组予以0.1%二甲基亚砜(DMSO)、阳性对照组予以瓦他拉尼(工作浓度为5μg/ml)、藏茵陈水提物组予以藏茵陈水提取物(工作浓度为200μg/ml)。在体式荧光显微镜下观察藏茵陈提取物处理的鱼胚血管表型并拍照,分析各样品对斑马鱼体节间血管(ISVs)生成的影响。利用实时荧光定量PCR技术检测处理后鱼胚的ptp-rb、nr2f1a、VE-cadherin、S1P1、CD146、pik3r2、netrin1a、fgfr1a、fgfr2、fgfr3、fgfr4基因的表达情况。结果与空白对照组及阳性对照组相比较,藏茵陈水提取物组鱼胚ISVs变薄,背主动脉出芽减少,显示出抑制斑马鱼血管生成的作用(P<0.05)。藏茵陈水提物组的nr2f1a基因出现下调,但与空白对照组及阳性对照组相比较差异无统计学意义(P>0.05),ptp-rb基因出现下调,差异有统计学意义(P<0.0001),VE-cadherin、S1P1、CD146、pik3r2、netrin1a、fgfr1a、fgfr2、fgfr3、fgfr4基因出现上调,但与空白对照组及阳性对照组比较差异无统计学意义(P>0.05)。结论藏茵陈水提取物有抑制斑马鱼新生血管的作用,其机制与下调ptp-rb的基因表达有关。

Objective To explore the inhibitory effect and regulatory mechanism of water extract of Capillary wormwood herb on zebrafish neovascularization. Methods 90 embryos of 22 HPF normal development zebrafish were collected from fli1 a EGFP transgenic zebrafish of TG strain. They were divided into blank control group, positive control group and Capillary wormwood herb water extract group. The blank control group was given 0.1% DMSO, the positive control group was given 5 μg/ml vatarani, the water extract group was given 200 μg/ml. The vascular phenotypes of zebrafish embryos treated with the extract of Artemisia Capillaria were observed and photographed under the body fluorescence microscope, and the effects of the samples on the formation of the internode vessels(ISVs) of zebrafish were analyzed. The expression of PTP Rb, nr2 f1 a, VE cadherin, S1 P1, CD146, pik3 r2, netrin1 a, fgfr1 a, FGFR2, FGFR3, FGFR4 genes in the treated fish embryos was detected by real-time fluorescence quantitative PCR. Results Compared with the blank control group and the positive control group, the ISVs of the fish embryo in the water extract group of Artemisia Capillaria became thinner and the sprouting of the dorsal aorta decreased, showing the inhibition effect on the angiogenesis of zebrafish(P<0.05). The nr2 f1 a gene of the water extract group was down regulated, but there was no significant difference compared with the blank control group and the positive control group(P>0.05), PTP Rb gene was down regulated, the difference was statistically significant(P<0.0001). The expression of VE-cadherin, S1 P1, CD146, pik3 r2, netrin1 a, fgfr1 a, fgfr2, fgfr3 and fr4 increased, But there was no significant difference between the two groups(P>0.05). Conclusion The water extract of Capillary wormwood herb can inhibit the neovascularization of zebrafish, and its mechanism is related to the down-regulation of PTP Rb gene expression.