| Therapeutic angiogenesis, also known as " a drug promoting self heart bypass grafting",can promote the formation and open of coronary collateral circulation, reduce myocardial ischemia, prevent cell necrosis, prevent and reverse ventricular remodeling, and improve clinical treatment effect and patient outcomes.It is believed that therapeutic angiogenesis provides new ideas and broad application prospects for the treatment of various ischemic diseases. As the molecular "switches" of gene regulation, microRNA plays an important role in the regulation of angiogenesis process;it may regulate the expression levels of some important angiogenic factors. miR-126 is one of the most expressed miRNAs in cardiovascular diseases, and it is specifically expressed in the vascular endothelial.miR-126 plays a key role in ischemic heart disease therapeutic angiogenesis. Thus, the research on miR-126 as the target of therapeutic angiogenesis is of great significance.Objective:To observe the impact of Blood activation and Qi Supplement prescription(BAQS) on the vascular endothelial-specific miR-126 and angiogenetic negative regulator Spredl, and clarify the relationship between miR-126,Spredl and angiogenesis, and the mechanism and compatibility of the compound decoction of BAQS mediating ischemic heart disease therapeutic angiogenesis through miR-126 and Spredl.Methods:Model rats of acute myocardial infarction (AMI) were established by legation of left anterior descending coronary artery, and randomly divided into 4 groups. Therapeutic groups were treated with BAQS, Blood Activation prescription (BA), or Qi Supplement prescription (QS) and model group was treated with saline. Rats of sham group treated with saline were operated without ligation. Animals were sacrificed and the myocardial infarction border areas were taken as indicators after 4 weeks treatment. Microvascular density (MVD) was assessed by detecting the mature vascular marker a-SMA and the endothelial specific marker CD31 using Immunohistochemistry, and the mean microvessel diameter (MMVD) was measured to observe the effects of angiogenesis in infarcted zone. The expression of miR-126, Spredl mRNA and VEGF mRNA were detected by quantitative Real-time PCR (qRT-PCR), and Western Blot was used to observe the expression of Spredl and VEGF.Resμlts:1. By detecting a-SMA, the MVD in model group was higher than in sham group (P< 0.01). Compared with model group, BAQS, BA and QS group can improve MVD, but only BAQS (P＜0.01), BA (P＜0.05) group had significant differences. The MVD in BAQS group was higher than in the other two therapeutic groups, but there was no significant difference (P< 0.01, P< 0.05).2. The Immunohistochemistry results of CD31 showed that the MVD in model group was higher than in sham group (P< 0.01). Compared with model group, BAQS, BA and QS group can significantly improve MVD (P< 0.01). The MVD in BAQS group was higher than in the other two therapeutic groups, but there was no significant difference (P> 0.05).3. And the MMVD in model group was higher than in sham group (P< 0.01). Compared with model group, BAQS, BA and QS group can improve MMVD,but only BAQS group had a significant difference (P< 0.01). The MMVD in BAQS group was higher than in the other two therapeutic groups(P< 0.01).4. The mRNA level of VEGF in model group was higher than in sham group, but there was no significant difference (P> 0.05). Compared with model group, BAQ, BA and QS group can improve the mRNA level of VEGF,but only BAQS, BA group had significant differences (P＜0.01). The mRNA level of VEGF in BAQS group was higher than in QS or BA group. However it is only significantly higher than in QS group (P<0.05)5. The expression of VEGF in model group was higher than in sham group, but there was no significant difference (P> 0.05). Compared with model group, BAQ and QS group can increase the expression of VEGF, but there was no significant difference (P> 0.05). The expression of VEGF in BAQS group was higher than in QS or BA group, but there was no significant difference (P> 0.05).6. The mRNA level of Spredl in model group was higher than in sham group (P<0.05). Compared with model group, BAQ, BA and QS group can significantly improve the mRNA level of Spredl (P<0.05). The mRNA level of Spredl in BAQS group was lower than in QS group or BA group. However it is only significantly lower than in BA group (P< 0.01).7. The expression of Spredl in model group was higher than in sham group, but there was no significant difference (P> 0.05). Compared with model group, BAQS, BA and QS group can significantly reduce the expression of Spredl (P< 0.05). The expression of Spredl in BAQS group was higher than in QS group and lower than in BA group, but there was no significant difference (P> 0.05).8. The expression of miR-126 in model group decreased significantly compared with sham group (P＜0.01). BAQS, BA and QS group can significantly reduced the expression of miR-126 (P<0.05). The level of miR-126 in BAQS group was lower than in QS or BA group. However it is only significantly lower than in QS group (P< 0.01).Conclusion:To varying degrees, BAQS and its decomposed recipes can promote angiogenesis in myocardial infarction border areas in rats after AMI. The effect of BAQS is the most significant. Its two decomposed recipes work synergistically. The mechanisms may be related with their effects on down-regulating the expression of miR-126, up-regulating the mRNA level of VEGF directly or indirectly, and on down-regulating Spredl and up-regulating the mRNA level of Spredl. In addition, we found that the mRNA level of Spred1 increased while the expression of Spred1 decreased. It is suggesting that Spred1 mRNA may be regulated at the post-transcriptional level by other factors, therefore further researches regarding the specific mechanism may be needed. |
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