Rhizoma Chuanxiong regulates vascular endothelial growth factor production in hypoxic human umbilical vein endothelial cells in vitro and in peri-infarct rat brain tissue

摘要

BACKGROUND:Vascular endothelial growth factor(VEGF) acts as'molecular bridge'following ischemic stroke to improve and restore blood supply and reduce infarction volume.Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong(Chuanxiong) in the treatment of ischemic cerebrovascular diseases.However,whether it promotes endogenous VEGF expression in ischemic stroke remains unknown. OBJECTIVE:To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis. DESIGN,TIME AND SETTING:In vitro basic comparison of traditional Chinese drug-containing serum pharmacology;in vivo randomized,controlled,animal experiment.This study was performed at the Medical Laboratory of West China Hospital,Sichuan University between December 2002 and April 2004. MATERIALS:Two Chinese rabbits were selected.One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum.The remaining rabbit was intragastrically perfused with the same volume of normal saline twice per day for three consecutive days.Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute,predominantly composed of ligustrazine,ligustilide,and ferulic acid.ChemiKine^(TM) human VEGF Kit was purchased from Chemicon,USA;mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology.Inc.,USA. METHODS:(1) In vitro experiment:in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10%Rhizoma Chuanxiong extract,normal medium without rabbit serum,and rabbit serum without Rhizoma Chuanxiong extract(blank control).In addition,cells from the three groups were incubated under normoxia(5%CO_2,95%air) and hypoxia (1%O_2,5%CO_2,94%N_2),respectively,for 24 hours.(2) In vivo experiment:a total of 4/44 Sprague Dawley rats were selected for the sham-operated group(no occlusion),and the remaining rats were used to establish a cerebral ischemia/reperfusion model by suture occlusion.32 animals with ischemia/reperfusion injury were randomly divided into treatment and model groups,with 16 rats in each group.Both groups were intraperitoneally infused with 0.58 g/kg Rhizoma Chuanxiong extract and normal saline two hours following reperfusion.The sham-operated group was administrated normal saline.Animals were treated with saline or Chuanxiong extracts(0.58 g/kg) twice per day for three consecutive days. MAIN OUTCOME MEASURES:In vitro experiment:VEGF concentration was detected in each group by enzyme-linked immunosorbent assay.In vivo experiment:behavioral alterations of rats were evaluated by neurological function scale;infarct volume was assessed by hematoxylin-eosin staining;VEGF protein expression in the infarct regions was determined by immunohistochemistry. RESULTS:(1) VEGF levels were similar between the three groups under normoxic condition(P>0.05);while hypoxia induced VEGF production(P<0.01).VEGF levels in the drug-containing serum group were particularly higher compared with the other groups(P<0.01).(2) Compared with normal saline treatment,Rhizoma Chuanxiong extract significantly improved the neurological scale and reduced cerebral infarct volumes(P<0.05).The percent of VEGF-positive cells was significantly greater than the model group(P<0.05).The sham-operated group exhibited normal neurological function,with no infarct focus. CONCLUSION:Rhizoma Chuanxiong extract-containing rabbit serum effectively promoted in vitro cultured VEGF production under hypoxia.Rhizoma Chuanxiong extract upregulated VEGF expression in the infarct region,improved neurological function,and reduced infarct size.