title: Spatholobi Caulis extracts promote angiogenesis in HUVECs in vitro and in zebrafish embryos in vivo via up-regulation of VEGFRs.
abstract: BACKGROUND
  Spatholobi Caulis is a traditional blood-activating and stasis-dispelling herb medicine, which has been used to treat diseases related to blood stasis syndrome (BSS) by inhibiting platelet aggregation, stimulate hematopoiesis, etc. It has been demonstrated that pro-angiogenesis could improve BSS. However, the pro-angiogenic activity of Spatholobi Caulis was not well elucidated AIM OF STUDY: To determine the potential pro-angiogenic activity of Spatholobi Caulis and elucidate its underlying mechanism. The active fractions of Spatholobi Caulis were further screened.
  
  
  METHODS
  Gelatin precipitation and reversed-phase liquid chromatography (RPLC) were used to purify the methanol extracts of Spatholobi Caulis, respectively. The RPLC was also used to prepare fractions. Total flavonoids of purified methanol extracts of Spatholobi Caulis (PSC) were determined using ultraviolet spectrophotometry. The morphological observation of subintestinal vessel plexus (SIVs) and tyrosine kinase inhibitor II (VRI)-induced intersegmental blood vessels (ISVs) loss in transgenic zebrafish Tg(fli-1a: EGFP)y1 were selected to evaluate the pro-angiogenic activity of PSC in vivo. Cell proliferation by MTT assay and cell migration assay were used to evaluate the pro-angiogenesis effect of PSC in human umbilical vein endothelial cells (HUVECs) in vitro. Both zebrafish and HUVECs were used in screening active fractions of PSC. The mechanism of PSC promoting angiogenesis were studied by real-time PCR in zebrafish and western blotting in HUVECs.
  
  
  RESULTS
  Co-treatment PSC dramatically rescued VRI-induced ISVs loss in zebrafish embryos in a dose-dependent manner and 80% of the defective vascular recovered at the concentration of 30μg/ml compared with VRI-only group. PSC also concentration-dependently increased average sprouting number and diameter of SIVs in zebrafish embryo. Real-time PCR assay proved that PSC significantly restored the down regulation of VEGFRs including Flt-1, Kdr and Kdrl induced by VRI in zebrafish (P<0.001). Furthermore, PSC not only promoted proliferation and migration of normal HUVECs but also ameliorated VRI-induced HUVECs cytotoxicity. Western blotting assay showed that co-treatment of PSC increased the expression of VEGFRs and phosphorylation of MAPKs which decreased by VRI treatment. In addition, quality evaluation experiments showed that the content of total flavonoids of PSC reached 56.36% and the main pro-angiogenic fractions of PSC were F3, F4 and F5 both in zebrafish and HUVECs.
  
  
  CONCLUSIONS
  Our data demonstrated that PSC presented pro-angiogenic activity both in zebrafish and HUVECs, and principal pro-angiogenic active components were likely flavonoids. Thus, the current study provided evidence for the clinical usage of Spatholobi Caulis in promoting blood circulation and removing stasis in traditional Chinese medicine (TCM).
authors.affiliation: Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Institute of Chinese Medical Sciences, University of Macau, Macao, China. Electronic address: biozzy@126.com.
authors.forename: Zhong-Yan
authors.initials: ZY
authors.lastname: Zhou
authors.affiliation: Engineering Research Center of Pharmaceutical Process Chemistry, Ministry of Education, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Electronic address: sherry132@126.com.
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Huan
authors.affiliation: Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China. Electronic address: witoy17@163.com.
authors.forename: Wai-Rong
authors.initials: WR
authors.lastname: Zhao
authors.affiliation: Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China. Electronic address: tangnuo2002@163.com.
authors.forename: Nuo
authors.initials: N
authors.lastname: Tang
authors.affiliation: Engineering Research Center of Pharmaceutical Process Chemistry, Ministry of Education, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Electronic address: jiny@ecust.edu.cn.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China. Electronic address: dr_tang@163.com.
authors.forename: Jing-Yi
authors.initials: JY
authors.lastname: Tang
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D047108:Embryonic Development / Q000187:drug effects* / Q000502:physiology; D007887:Fabaceae; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D018547:Plant Stems; D040262:Receptors, Vascular Endothelial Growth Factor / Q000819:agonists / Q000096:biosynthesis*; D015027:Zebrafish
chemical_list: D043925:Angiogenesis Inducing Agents; D010936:Plant Extracts; D040262:Receptors, Vascular Endothelial Growth Factor
keywords: Flavonoids; HUVECs; Pro-angiogenesis; Spatholobi Caulis; Zebrafish