芳香新塔花总黄酮保护大鼠缺血/再灌注心肌损伤中miRNAs的作用机制研究

The confirms that flavonoids has protection of ischemic myocardial injury, but can control the injury of ischemic myocardium in the expression of microRNA is a new research field. In our research Z.clinopodioides flavonoids (ZCF) was found to could significantly resist the symptom of ischemia myocardial and increase the potential of antioxidant. And after the treatment of ZCF on the pattern of cardiac muscle schemia-reperfusion injury, 13 miRNAs with difference in expression were detected by high-throughput sequencing. And it predicted that miRNAs might protect cardiac muscle through regulating the expression of its combined target genes. Here, based on the pattern of cardiac muscle schemia-reperfusion injury, the miRNAs changes after treatment by ZCF in cardiac muscle tissues were focused. The transcripts of miRNAs would be systematically detected by high-throughput sequencing, Realtime-PCR and Chip-seq methods, and then to found the complete ZCF binding elements in the miRNA precursors. This research would be helpful for explaining the molecular mechanism of miRNAs in ischemia myocardial and finding the regulation pathways of ZCF on miRNAs. It will provide novel insights for prevention of ischemic myocardial disease.

已证实黄酮类化合物具有保护缺血心肌损伤的作用，但能否调控缺血心肌损伤中microRNA(miRNA)表达是一个全新的研究领域。项目组从芳香新塔花中分离得到了具有显著抗心肌缺血作用的总黄酮有效部位(Z.clinopodioides flavonoids，ZCF)，ZCF处理大鼠心肌缺血再灌注损伤模型后，高通量测序发现13个差异表达miRNAs，推测miRNA可能通过与心肌靶基因结合，调控其靶基因的表达，进而起心肌保护作用。本项目采用大鼠心肌缺血再灌注损伤模型，以ZCF处理心肌损伤miRNAs为切入点，借助miRNA高通量测序、Realtime-PCR等技术方法检测ZCF对靶miRNAs表达的影响；采用Chip-seq检测miRNAs的前体序列中是否有完整的ZCF结合元件。本项目有助于阐释ZCF对大鼠心肌损伤保护作用的miRNAs调控方式及目标蛋白，为缺血性心肌疾病的防治提供新的思路和实验依据

芳香新塔花的干燥地上部分为维吾尔医和哈萨克医习用药材，课题组前期已证明芳香新塔花总黄酮（ZCF）能有效的抗心肌缺氧复氧损伤对大鼠心肌缺血再灌注损伤起保护作用。项目进一步开展如下研究：1.芳香新塔花总黄酮的含量测定及抗氧化活性分析，采用紫外可见分光光度比色法测定ZCF的含量为933.33 mg∙g-1；其抗氧化实验表明，ZCF在一定程度上可以清除DPPH∙、∙OH和O2-∙，同时表现出还原性。2.芳香新塔花总黄酮ZCF对大鼠心肌缺血再灌注损伤保护作用，大鼠预处理给药ZCF建立心肌缺血再灌注损伤模型后，发现给药组ZBFL、ZBFM、ZBFH中大鼠心肌缺血和梗死面积明显减少，心肌损伤病理学形态有所改善，心肌细胞凋亡数减少；血清中标志物SOD、GSH-Px的活性增加，MDA、LDH、CK-MB的活性降低；心肌组织中NO、NOS的含量增高；预给药ZCF能有大鼠心肌中抑制炎症因子TNF-α、IL-6的释放。3.ZCF保护大鼠心肌缺血再灌注损伤中miRNA表达谱分析，通过高通量测序成功构建各组心肌组织miRNA表达谱，差异性表达的共有671种miRNAs，新发现miRNAs有261种，差异性miRNA有41种，采用DEGseq，DESeq2分析差异小RNA筛选（P value<0.05），然后选择显著差异的miRNA共有19种miRNA（log2 fold Change＞2），其中有10种表达量上调，有9种表达量下调。4.芳香新塔花总黄酮中蒙花苷的含量测定及保护心肌缺氧复氧损伤的机制研究，项目采用超高效液相色谱（UPLC）对ZCF中主要的成分蒙花苷进行了含量测定，UPLC检测出ZCF中主要活性成分蒙花苷的含量为772.69 mg∙g-1；蒙花苷预给药H9c2心肌细胞，在缺氧复氧4h后，与模型组相比，LDH和MDA显著增加，SOD则显著降低( *p<0.05)。Western blot检测结果显示，与模型组相比p-PI3K 表达量及 AKT 磷酸化程度及凋亡蛋白bcl-2表达水平升高，Bax表达水平降低（*p<0.05）。加抑制剂LY294002后，蒙花苷对缺氧复氧损伤H9c2细胞的保护作用被抑制；结果显示蒙花苷可以通过PI3K /Akt/m-TOR信号通路抑制H9c2细胞凋亡。