利用斑马鱼模型和代谢组学技术研究丹红注射液促进血管生成作用及潜在代谢机制

Effect and potential metabolic mechanism of Danhong Injection on angiogenesis based on zebrafish model and metabonomics technology

目的基于斑马鱼模型研究丹红注射液促进血管生成作用,并利用代谢组学和qRT-PCR技术探索其代谢机制。方法利用血管特异标记绿色荧光蛋白的转基因品系Tg(fli1:EGFP)斑马鱼作为模式动物,观察丹红注射液对斑马鱼节间血管的促进作用。利用基于超高效液相色谱-四极杆飞行时间质谱联用技术(ultra performance liquid chromatography quadrupole time of flightmass spectrometry,UPLC-Q-TOF-MS)的非靶向代谢组学检测斑马鱼在血管生成表型变化过程中的内源性差异代谢物,进一步利用qRT-PCR技术探索与丹红注射液促进血管生成作用相关的潜在代谢机制。结果丹红注射液能够呈剂量相关性地逆转血管内皮细胞生长因子受体抑制剂PTK787对斑马鱼节间血管的抑制作用(P<0.01)。与模型组比较,丹红注射液组有7个内源性代谢物水平发生了显著变化,主要与戊糖磷酸途径、精氨酸和鸟氨酸代谢途径相关。qRT-PCR结果显示,丹红注射液显著逆转了斑马鱼体内与戊糖磷酸通路关键酶6-磷酸葡萄糖脱氢酶(glucose-6-phosphate dehydrogenase,g6pd)、转醛缩酶(transaldolase 1,taldo1)、转酮醇酶(transketolase a,tkta)、核糖-5-磷酸异构酶A(ribose 5-phosphate isomerase A,rpia)和5-磷酸核酮糖3-差向异构酶(ribulose-5-phosphate-3-epimerase,rpe)基因的表达水平(P<0.05、0.01)。结论利用斑马鱼模型验证了丹红注射液的显著血管生成作用,代谢组学和qRT-PCR研究结果表明其血管生成主要与磷酸戊糖途径有关。

Objective To study the effect of Danhong Injection(丹红注射液,DHI)on angiogenesis based on zebrafish model and explore its metabolic mechanism by metabonomics and qRT-PCR.Methods The transgenic strain Tg(flk1:EGFP)was used as model animal to observe the promoting effect of DHI on the blood vessels of zebrafish internodes.The endogenous differential metabolites of zebrafish in the process of angiogenic phenotype change were detected by non-targeted metabonomics based on ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS),and the potential metabolic mechanism related to the angiogenic effect of DHI was further explored by qRT-PCR.Results DHI could reverse the inhibitory effect of vascular endothelial growth factor receptor inhibitor PTK787 on interganglia vessels of zebrafish in a dose-dependent manner(P<0.01).Compared with model group,seven endogenous metabolites levels were found to have significant changes in DHI group,mainly related to the pentose phosphate pathway,arginine and ornithine metabolic pathway.qRT-PCR results showed that DHI significantly reversed the expression levels of glucose-6-phosphate dehydrogenase(g6pd),transaldolase 1(taldo1),transketolase a(tkta),ribose 5-phosphate isomerase A(rpia)and ribulose-5-phosphate-3-epimerase(rpe)genes in the pentose phosphate pathway in zebrafish(P<0.05,0.01).Conclusion Zebrafish model was used to verify the remarkable angiogenesis effect of DHI.Metabonomics and qRT-PCR studies showed that the angiogenesis of DHI was mainly related to pentose phosphate pathway.