Primary Study On Molecular Base Underlying Hydroxysafflor Yellow A On Angiogenesis Of Brain Tissue In Rats With Focal Cerebral Ischemia

AIM:Angiogenesis therapy after cerebral ischemia has come into fashion in recentyears. Hydroxysaffior yellow A is one of the main active ingredients extracted fromCarthamus tinctorius. L. Our preliminary work suggested that it exerted a goodprotective effect on cerebral ischemia in rats by enhancement on angiogenesisaround cerebral ischemic infarct area. This study is designed to investigate theprimary molecular base underlying Hydroxysaffior Yellow A on angiogenesis ofbrain tissue in rats with focal cerebral ischemia.METHODS:The establishment of focal cerebral ischemia in rat was followed by thedescription of Zea Longa, et al. Healthy adult male Sprague-Dawley rats, weightingfrom 260g to 280g, were randomly divided into four groups as follows: thesham-operated group (n=8), the model group (n=8), the positive control group(MK801 0.5mg/kg, n=8) and HSYA group (n=8). 12mg/kg of HSYA was injectedvia sublingular vein immediately after the onset of MCA occlusion and followed bythe presence of HSYA at the dose of 12 mg/kg at the interval of every 30 minutes,respectively, adding up to 60 mg/kg dosage. At 6, 12, 24-hour time point post MCAocclusion, the behavior deficits were observed, and then infarction was stained byTTC.For the analysis of the differential expression of the multiple angiogenesis-associated genes, the Oligo GEArray(?) Rat angiogenesis microarray which profilesthe expression of 113 key genes involved in modulating the biological processes ofangiogenesis were used. The microarray includes the growth factors and receptors,the adhesion molecules and matrix proteins, as well as proteases and their inhibitors.Besides, cytokines and chemokines related to angiogenesis are contained along withtranscription factors and other related genes. Forty-five rats were sacrificed at theindicated time points (1.5, 3, 6, 12, 24 hours) after the onset of MCA occlusion for microarray analysis. Rat cortical penumbra from ipsilateral cortices was isolated andtotal RNA was extracted, total RNA prepared from 3 rats each group was pooled forhybridization to minimize variation. Thus 15 arrays were used in total. Correctedspot intensities were normalized to averages of the housekeeping genes and the finalvalues were presented as a ratio.To validate the microarray data, the mRNA level of several angiogenesisintimately related genes (VEGF, Flt, bFGF, aFGF and HIF-1α) was analyzed byreverse transcription polymerase chain reaction (RT-PCR). Rat GAPDH gene wasused as control for determining the quantity of RNA. Additionally, Real timeRT-PCR was also used to detect mRNA levels of VEGF and HIF-1αgenes forconfirmation of the microarray data.Besides, total protein from brain cortex penumbra tissue was extracted at theindicated time points (1.5, 3, 6, 12, 24 hours) after the onset of MCA occlusion. Theexpressions of VEGF, Flt, bFGF, aFGF and HIF-1αprotein were analyzed byWestern Blotting, rat GAPDH was used as housekeeper.RESULTS:The rats in the model group showed significant neurobehavioral obstacles andischemic brain infarction at 6, 12, 24-hour time point after the onset of MCAocclusion compared with those in the sham-operated groups. HSYA as well as thepositive drug (MK801) by intravenous infusion significantly improvedischemia-induced behavioral impairment and reduced the infarct volume (P＜0.05).DNA microarray data indicated that within 24 hours, there were 75 geneschanged notably, 19 of which increased significantly, while 56 significantly reducedin the indicated rats brain tissue in the model group compared with those of thesham-operated group; treatment with the intervention of HSYA, the changes of geneexpression apparently trended to exert the beneficial effects on improvement ofcerebral ischemia in the rats. In the HSYA-treated group, the notable changes in 85genes expression has occurred, 58 of which increased, while 27 reduced markedly.Comparing with the two sets of chips, the most significant genes might be angiogenesis factors (such as HIF-1, VEGF, aFGF, Ang2) and inflammatorycytokine factors (such as IL-6, IL-1, IL-10). HSYA could increase mRNAexpression of the most of angiogenesis genes, such as angiopoietin, acid fibroblastgrowth factor (aFGF), epidermal growth factor (EGF), insulin-like growth factor-1(IGF1), which were down-regulated induced by ischemic insult. Meanwhile, theexpression of the inhibitory angiogenesis gene thrombospondin, had been inhibitedby the presence of HSYA. In addition, it was also found that HSYA reversed the twodownward phases of anti-inflammatory cytokine IL-10.The validation of microarray implicated that down regulated expression of aFGF,HIF-1α, Flt, bFGF gene in the cortex penumbra of rats with MCAO wassignificantly reversed by administration of HSYA at immediate early stage (3h) postcerebral ischemia. Similarly, HSYA promoted mRNA expression levels of bFGFand VEGF at 24h point (P＜0.05).On the other hand, ischemic cortex penumbra protein expression of HIF-1, Flt,aFGF, bFGF were significantly increased at the indicated time points after treatmentof HSYA (P＜0.05) within 24h after MCAO. However, there was no significantdifference showing that HSYA afforded an effect on up-regulation the expression ofVEGF protein in comparsion with that in the model group.CONCLUSION:In general, DNA microarray observation suggested that endogenous mechanismsof angiogenesis in ischemic brain tissue might experience a first decline before anupward dynamic process within 24 hours, implicating that the mechanismsunderlying HSYA on revascularization in the earlier stage might be associated with amulti-access network which involved in, at least in part, the regulation in the positiveor negative angiogenesis-related genes (HIF-1α, VEGF, Ang2, Ephrin-A1)expression.These data obtained from the combination with Functional Gene Chip and Realtime PCR technique, etc, gave us a good understanding of the molecular basis ofangiogenic effect afforded by the presence of HSYA in vivo study, might be relatedwith the beneficial regulation of the transcriptional and protein levels of HIF-1α, aFGF, bFGF, Flt and VEGF, positive growth factors on angiogenesis in rats corticalpenumbra. Therefore, the present findings not only enlightened us a primarymolecular explanation for the enhancement on therapeutic angiogenesis stimulatedby the presence of HSYA, but also contributed to the further investigation on theexact mechanisms underlying angiogenesis exerted by treatment of HSYA.