Cheng Lin-Hui, Chen Lan-Ying, Shou Bin-Yao, Luo Ying-Ying, Cui Ya-Ru, Liu Rong-Hua
School of Pharmacy, Jiangxi University of Chinese Medicine Nanchang 330004, China.
School of Pharmacy, Jiangxi University of Chinese Medicine Nanchang 330004, China National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine Nanchang 330006, China.
Zhongguo Zhong Yao Za Zhi. 2022 Dec;47(24):6696-6708. doi: 10.19540/j.cnki.cjcmm.20220902.702.
Dalbergia cochinchinensis(DC) is chemically similar to the valuable and scarce Chinese herb Dalbergiae Odoriferae Lignum, and both of them belong to the Dalbergia Leguminosae. DC is used for treating cardiovascular diseases and cancer. However, its potent active ingredient groups and molecular mechanisms in anti-myocardial ischemia are not fully clarified. In this study, the active ingredient groups, targets, and signaling pathways of DC heartwood for the treatment of myocardial ischemia were screened out based on network pharmacology and molecular docking technology, and the effects were verified by the rat model of acute myocardial ischemia induced by isoprenaline(ISO). The molecular mechanism of DC heartwood was elucidated based on the target of multi-ingredient and multi-target pathways. The crossing targets of DC heartwood for the treatment of myocardial ischemia were identified through the screening of active ingredients in DC heartwood and the prediction of targets. The Kyoto Encyclopedia of Genomes(KEGG) pathway enrichment and Gene Ontology(GO) functional annotation were performed. AutoDock was used to bind the active ingredient groups to the pathway targets. Finally, the molecular mechanism of myocardial ischemia treatment by DC heartwood extracts in the treatment of myocardial ischemia was revealed through the rat model of ISO-induced acute myocardial ischemia by performing electrocardiogram(ECG), hemodynamic, cardiac enzymes, hematoxylin-eosin(HE) staining, high-energy phosphate compounds, reverse transcription polymerase chain reaction(RT-PCR), and Western blot pharmacodynamic experiments, based on the multi-ingredient and multi-target action of active ingredient groups and pathway targets. The network pharmacology showed that the 18 ingredients of DC heartwood corresponded to 510 targets, 629 myocardial ischemia-related targets, and 101 cross-targets. GO and KEGG enrichment analyses showed that DC heartwood was involved in the hypoxic response, vasoconstriction, and nitric oxide biosynthesis, and had effects on the molecular functions of hemoglobin binding, protein binding, and adenosine triphosphate(ATP) binding. It regulated the signaling pathways such as hypoxia-inducible factor 1(HIF-1), vascular endothelial growth factor(VEGF), and phosphatidylinositol-3-kinase/protein kinase B(PI3 K/AKT) to act on myocardial ischemia. Experimental studies showed that DC heartwood slowed down the heart rate and ST segment change(ΔST), and increased systolic blood pressure(SBP), diastolic blood pressure(DBP), and mean arterial pressure(MBP) in rats with ISO-induced acute myocardial ischemia. It also reduced plasma lactate dehydrogenase(LDH), creatine kinase isoenzyme MB(CK-MB), and glutamate transaminase(AST) levels, relieved myocardial fiber disorders and inflammatory cell infiltration, and increased ATP and cellular energy(EC) levels. DC heartwood increased the mRNA expressions of calmodulin-dependent protein kinase kinase(CAMKK) in the myocardial tissue, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3), mammalian target of rapamycin(mTOR), PI3 K, VEGF, endothelial nitric oxide synthase(eNOS), HIF-1α in the myocardial tissue. It decreased the mRNA expression of pyruvate dehydrogenase(PDH), and increased the protein expressions of PFKFB3, VEGFA, and eNOS. Molecular docking showed that liquiritigenin, stigmasterol, isodalbergin, latifolin, 4-methoxydalbergione, dibutyl terephthalate, 2,4-dihydroxy-5-methoxybenzophenone in DC heartwood produced bio-binding activities with epidermal growth factor receptor(EGFR), HIF-1α, CAMKK, PI3 K, mTOR, and PDH, respectively. Therefore, the active ingredient groups of DC heartwood act on the HIF-1 signaling pathway, regulate cardiomyocyte energy metabolism, and increase ATP energy charge in a multi-ingredient and multi-target manner, improving cardiac function and histopathological changes to protect rats with acute myocardial ischemia induced by ISO.
越南黄花梨（Dalbergia cochinchinensis，DC）在化学成分上与珍贵稀缺的中药材降香（Dalbergiae Odoriferae Lignum）相似，二者均属于豆科黄檀属。越南黄花梨用于治疗心血管疾病和癌症。然而，其抗心肌缺血的有效活性成分组及分子机制尚未完全阐明。本研究基于网络药理学和分子对接技术，筛选出越南黄花梨心材治疗心肌缺血的活性成分组、靶点及信号通路，并通过异丙肾上腺素（ISO）诱导的大鼠急性心肌缺血模型进行验证。基于多成分多靶点途径的靶点，阐明越南黄花梨心材的分子机制。通过筛选越南黄花梨心材中的活性成分及预测靶点，确定越南黄花梨心材治疗心肌缺血的交叉靶点。进行京都基因与基因组百科全书（KEGG）通路富集和基因本体论（GO）功能注释。利用AutoDock将活性成分组与通路靶点进行结合。最后，基于活性成分组和通路靶点的多成分多靶点作用，通过对ISO诱导的急性心肌缺血大鼠模型进行心电图（ECG）、血流动力学、心肌酶、苏木精-伊红（HE）染色、高能磷酸化合物、逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法等药效学实验，揭示越南黄花梨心材提取物治疗心肌缺血的分子机制。网络药理学研究表明，越南黄花梨心材的18种成分对应510个靶点，其中629个与心肌缺血相关，有101个交叉靶点。GO和KEGG富集分析表明，越南黄花梨心材参与缺氧反应、血管收缩和一氧化氮生物合成，对血红蛋白结合、蛋白质结合和三磷酸腺苷（ATP）结合等分子功能有影响。它调节缺氧诱导因子1（HIF-1）、血管内皮生长因子（VEGF）和磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/AKT）等信号通路作用于心肌缺血。实验研究表明，越南黄花梨心材可使ISO诱导的急性心肌缺血大鼠心率减慢、ST段变化（ΔST）减小，收缩压（SBP）、舒张压（DBP）和平均动脉压（MBP）升高。还可降低血浆乳酸脱氢酶（LDH）、肌酸激酶同工酶MB（CK-MB）和谷氨酸转氨酶（AST）水平，减轻心肌纤维紊乱和炎性细胞浸润，并提高ATP和细胞能量（EC）水平。越南黄花梨心材可增加心肌组织中钙调蛋白依赖性蛋白激酶激酶（CAMKK）、6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3（PFKFB3）、雷帕霉素靶蛋白（mTOR）、PI3K、VEGF、内皮型一氧化氮合酶（eNOS）、HIF-1α的mRNA表达。降低丙酮酸脱氢酶（PDH）的mRNA表达，并增加PFKFB3、VEGFA和eNOS的蛋白表达。分子对接显示，越南黄花梨心材中的甘草素、豆甾醇、异黄檀素、阔果黄檀素、4-甲氧基黄檀醌、对苯二甲酸二丁酯、2,4-二羟基-5-甲氧基二苯甲酮分别与表皮生长因子受体（EGFR）、HIF-1α、CAMKK、PI3K、mTOR和PDH产生生物结合活性。因此，越南黄花梨心材的活性成分组以多成分多靶点方式作用于HIF-1信号通路，调节心肌细胞能量代谢，增加ATP能量电荷，改善心脏功能和组织病理学变化，保护ISO诱导的急性心肌缺血大鼠。
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