基于NOX4调控网络的心衰方干预心衰氧化应激的效应成分及机制研究

Nox4 is a novel target of the inhibition of ROS generation for the treatment of heart failure, but none of the drugs under research has be proved by clinical trial. Our former researches indicate that Xinshuai Recipe can improve the heart functions mainly related on the inhibition of NOX4 expression in heart failure pigs. The analyses of network pharmacology and bioinformatics show that Glycyrrhizic acid, Glycyrrhetinic acid, Kaempferol and Formononetin may be the effective components contained in Xinshuai Recipe which can inhibit the NOX4 expression, and STAT3、HIF1a、GCR and PPARr may be the candidate targets. By using the primary cultured rats cardiomyocytes induced by hypoxia , first to verify the effect of four components on the expression of NOX4. Secondly, in order to obtain the key target of those effective components for the NOX4 expressing regulation in heart failure, we will observe the effect of effective components on activities of those candidate targets , as well as combine it with restraining (RNA interference) or other methods to elucidate the relation between candidate targets and NOX4. Thirdly, the Uniform Design will be introduced, By using heart failure mice after myocardial infarction, to screen the optimum proportion of those four components for the NOX4 expressing regulation. And then we will elucidate the mechanisms of this optimally proportioned component based on the network of NOX4 expressing regulation. At last, we can provide a new way to treat heart failure and develop a unique research approach, which focuses on the key pathophysiological changes of a disease and use specific cells as a screening tool, to clarify the network within ‘Component- Targets- Effective’ of Chinese herbal compounds .

氧化应激是心力衰竭发病的重要环节，NOX4型NADPH氧化酶为其关键酶，是防治心衰的新靶点，尚无有效药物上市。经验心衰方针对心衰核心病机立法，以解毒为特色，可显著改善心衰动物心功能，抑制NOX4表达。网络药理学与生物信息学分析显示，甘草酸、甘草次酸、山奈酚和芒柄花黄素可能是心衰方抑制NOX4表达的效应成分，STAT3、HIF1a、GCR、PPARr为候选靶点。本课题以缺氧诱导的原代乳鼠心肌细胞为工具，验证效应成分调控NOX4的作用，联合应用候选靶点的RNA干扰等技术，筛选其调控NOX4表达的关键环节；采用小鼠心梗后心衰为动物模型，以均匀设计为分组方案，筛选调控NOX4表达的最佳配比组合；结合细胞实验和动物实验，阐明该组合基于NOX4表达调控网络的抗心力衰竭作用机制。为心衰的治疗提供新手段，为以关键病理环节为核心、细胞为工具的中药复方“成分-靶点-效应”网络作用机制的逐级解析探索新路径。

氧化应激是心力衰竭发病的重要环节，NOX4型NADPH氧化酶为其关键酶，是防治心衰的新靶点，网络药理学与生物信息学分析显示，甘草酸、甘草次酸、山奈酚和芒柄花黄素可能是心衰方调控NOX4的效应成分。本课题①基于缺氧诱导的H9C2心肌细胞氧化应激模型，验证网络药理学分析预测的效应成分对NOX4表达水平的影响，发现山奈酚和芒柄花黄素可降低缺氧诱导的ROS水平。山萘酚可以下调Nox4、p67phox、p47phox 的mRNA 和蛋白水平，芒柄花黄素可以下调p67phox、p47phox 的mRNA转录水平，明显下调Nox4、p67phox 蛋白的表达水平。对山萘酚和芒柄花黄素配伍进行了初步考察，对组合效应有了初步认识。②利用angⅡ诱导的H9C2氧化应激模型，发现甘草酸、甘草次酸可抑制angⅡ诱导的ROS升高，本研究中angⅡ对Nox4及其亚基的上调作用不明显，甘草酸、甘草次酸对Nox4及其亚基调控作用不明显,对STAT3、HIF1a、GCR、PPARr的蛋白水平也无明显影响。③NADPH氧化酶酶活试验表明，心衰方中多个单体对酶活有显著抑制作用。④通过超氧阴离子清除能力检测除外单体的直接抗氧化作用，发现丹参酮I是较好的NADPH氧化酶抑制剂候选成分。研究初步揭示了心衰方基于NOX4调控心衰氧化应激的效应成分和作用机制。本研究发现的多个对NOX4有直接或间接作用的单体有望开发为NOX4抑制剂，为心衰的治疗提供新手段，为以关键病理环节为核心、细胞为工具的中药复方“成分-靶点-效应”网络作用机制的逐级解析探索新路径。发表相关论文4篇，其中SCI收录1篇，中文核心期刊2篇。培养博士后1名，已顺利出站，培养硕士生2名，均已经取得硕士学位。项目投入经费23万元，支出20.12万元，各项支出基本与预算相符。剩余经费2.88万元，剩余经费计划用于本项目研究后续支出。