| Objective1. In the previous study, we had found that microRNA-223-3p(mir-223-3p) inhibited angiogenesis of ischemic myocardium microvascular endothelial cells(CMECs) in rats by affecting RPS6KB1/HIF-1α signaling pathway. Based on the results, we will explore the microRNA mechanism of Qi-replenishing and blood-activating Traditional Chinese Medicine(TCM) intervening in angiogenesis of rats ischemic CMECs in further.2. To observe the effect of plasma mir-223-3p of Qi-replenishing and blood-activating TCM on acute myocardial infarction(AMI) with heart failure(HF) patients. Methods1. Experimental Study(1) On the basis of preliminary study, according to Qi-replenishing and blood-activating TCM, Qi-Shen-Yi-Qi dripping pills(QSYQ), biological characteristics of ischemic CMECs in different concentrations of culture, we set the optimal dose and the best window period of ischemic CMECs as intervention group. Trizol was extracted of total RNA from intervention group, ischemia model group(MX) and normal control group(ZC). Use gene chip to detect the dynamic microRNA(miRNA) expression, choose significantly differentially expressed miRNA, verify by real-time PCR, and confirm the core miRNA of QSYQ intervention in angiogenesis of ischemic CMECs.(2) Real-time PCR was used to test mRNA expression of target genes and other genes, including HIF-1α, VEGF, MAPK, PI3 K and AKT.(3) Western blot was used to test protein expression of target genes, HIF-1α, VEGF, MAPK, PI3 K and AKT.2. Clinical Research Select 35 cases of AMI patients, including 23 cases of patients with HF. The patients of AMI with HF were randomly divided into treatment group 12 cases and control group 11 cases. The control group was treated with conventional western medicine, and the treatment group was treated by routine western medicine combined with Qi-replenishing and blood-activating TCM. The expressions of plasma mir-223-3p and BNP were detected respectively before and after treating 7 days and 14 days. Results1. Experimental Study(1) Compared with ZC group, there were 8 up-regulated and 15 down-regulated miRNAs in MX group, 16 up-regulated and 18 down-regulated in intervention group; compared with MX group, miRNAs of intervention group had 26 up-regulated and 21 down-regulated. The mir-223-3p expression of intervention group decreased 0.117 times than MX group, which had a statistically significant difference, and mir-223-3p was determined as the core miRNA of QSYQ intervention in angiogenesis of ischemic CMECs. Real-time PCR were consistent with the gene chip in results.(2) Compared with ZC group, the mRNA expressions of target gene RPS6KB1 were significantly up-regulated in MX group and intervention group(P=0.000, P=0.051); compared with MX group, the intervention group was significantly reduced(P=0.000). Compared with ZC group, the protein expression of RPS6KB1 was down-regulated in MX group(P=0.001), and up-regulated in intervention group(P=0.491); compared with MX group, the intervention group was significantly increased(P=0.001).(3) The mRNA and protein expression of related genes located RPS6KB1/HIF-1α signaling pathway, including HIF-1α, VEGF, MAPK, PI3 K and AKT, were significantly up-regulated in intervention group compared with MX group.2. Clinical Research(1) The comparison of plasma mir-223-3p in AMI combined HF patients before and after treating 7 days and 14 days Before treatment, mir-223-3p expression of control group was 1.022 ± 0.218, and treatment group was 1.012± 0.154, with no difference between them(P>0.05). After treating 7 days, mir-223-3p expression of control group was 0.507 ± 0.095, and treatment group was 0.390 ± 0.058, with a significant difference between them(P<0.01). After treating 14 days, mir-223-3p expression was 0.296 ± 0.046 in control group, and treatment group was 0.197 ± 0.040, with a significant difference between them(P<0.01). Compared with before treatment, there were all significantly difference(P<0.01).(2) The correlation analysis between mir-223-3p and BNP mir-223-3p expression was positively correlated with BNP. The correlation coefficient for the control group was r=0.941(P<0.01), and treatment group was r=0.964(P<0.01).(3) The comparison of plasma mir-223-3p in AMI patients and AMI combined HF patients before and after treating 14 days Before treatment, mir-223-3p expression was AMI group 1.011 ± 0.148, and AMI combined HF group 1.012 ± 0.154, with no difference between them(P>0.05); after treating 14 days, mir-223-3p expression was AMI group 0.137 ± 0.025, and AMI combined HF group 0.197 ± 0.040, with a significant difference between them(P<0.05); comparing with before treatment, the expression level of mir-223-3p was significantly lower(P<0.01). Conclusion1. It was confirmed that mir-223-3p was the core miRNA of QSYQ intervention in angiogenesis of ischemic CMECs.2. Segregation of gene and protein expression of target gene RPS6KB1 was consistent with miRNA negative regulation mechanism, while it was significantly improved after Qi-Replenishing and Blood-activating Traditional Chinese Medicine intervention.3. QSYQ activated RPS6KB1/ HIF-1α signaling pathway by down-regulating the mir-223-3p expression, increasing the levels of VEGF, MAPK, PI3 K and AKT, and promoted angiogenesis of ischemic myocardium, which will provide a theoretical basis for intervention mechanism of traditional Chinese medicine.4. Qi-replenishing and blood-activating TCM is better than pure western medicine in treating AMI combined HF patients, which may be related with the down-regulation of plasma mir-223-3p expression. The expression of plasma mir-223-3p and BNP show a significant positive correlation, suggesting that mir-223-3p may become a new plasma marker in diagnosis of heart failure. |
