Research Center for High Altitude Medicine, Laboratory for High Altitude Medicine of Qinghai Province, Key Laboratory for High Altitude Medicine (Ministry of Education), Key Laboratory of Application and Foundation for High Altitude Medicine Research Qinghai Province (Qinghai-Utah Joint Research Key Lab for High Altitude Medicine), Qinghai University, Xining, 810001, China; Qinghai Health Institute of Sciences, Xining, 810016, China.
Central Laboratory, Clinical Medical College & Affiliated Hospital of Chengdu University, Sichuan, 610086, China.
Phytomedicine. 2024 Sep;132:155840. doi: 10.1016/j.phymed.2024.155840. Epub 2024 Jun 22.
Hypoxic pulmonary vascular remodeling (HPVR) is a key pathological feature of hypoxic pulmonary hypertension (HPH). Oxygen-sensitive potassium (K) channels in pulmonary artery smooth muscle cells (PASMCs) play a crucial role in HPVR. Luteolin (Lut) is a plant-derived flavonoid compound with variety of pharmacological actions. Our previous study found Lut alleviated HPVR in HPH rat.
To elucidate the mechanism by which Lut mitigated HPVR, focusing on oxygen-sensitive voltage-dependent potassium channel 1.5 (Kv1.5).
HPH rat model was established using hypobaric chamber to simulate 5000 m altitude. Isolated perfused/ventilated rat lung, isolated pulmonary arteriole ring was utilized to investigate the impact of Lut on K channels activity. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was assessed. CyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-3 levels in lung tissue of HPH rat were tested. The effect of Lut on Kv1.5, cytoplasmic free calcium concentration ([Ca]), CyclinD1, CDK4, PCNA, Bax/Bcl-2 was examined in PASMCs under hypoxia, with DPO-1 as a Kv1.5 specific inhibitor. The binding affinity between Lut and Kv1.5 in PASMCs was detected by drug affinity responsive target stability (DARTS). The overexpression of KCNA5 gene (encoding Kv1.5) in HEK293T cells was utilized to confirm the interaction between Lut and Kv1.5. Furthermore, the impact of Lut on mitochondrial structure, SOD, GSH, GSH-Px, MDA and HIF-1α levels were evaluated in lung tissue of HPH rat and PASMCs under hypoxia.
Lut dilated pulmonary artery by directly activating Kv and Ca-activated K channels (K) in smooth muscle. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was upregulated by Lut. Lut downregulated CyclinD1, CDK4, PCNA while upregulating Bax/Bcl-2/caspase-3 axis in lung tissue of HPH rat. Lut decreased [Ca], reduced CDK4, CyclinD1, PCNA, increased Bax/Bcl-2 ratio, in PASMCs under hypoxia, by upregulating Kv1.5. The binding affinity and the interaction between Lut and Kv1.5 was verified in PASMCs and in HEK293T cells. Lut also decreased [Ca] and inhibited proliferation via targeting Kv1.5 of HEK293T cells under hypoxia. Furthermore, Lut protected mitochondrial structure, increased SOD, GSH, GSH-Px, decreased MDA, in lung tissue of HPH rat. Lut downregulated HIF-1α level in both lung tissue of HPH rat and PASMCs under hypoxia.
Lut alleviated HPVR by promoting vasodilation of pulmonary artery, reducing cellular proliferation, and inducing apoptosis through upregulating of Kv1.5 in PASMCs.
低氧性肺血管重塑（HPVR）是低氧性肺动脉高压（HPH）的关键病理特征。肺动脉平滑肌细胞（PASMC）中的缺氧敏感钾（K）通道在 HPVR 中起着至关重要的作用。木犀草素（Lut）是一种具有多种药理作用的植物衍生类黄酮化合物。我们之前的研究发现 Lut 可减轻 HPH 大鼠的 HPVR。
阐明 Lut 减轻 HPVR 的机制，重点研究氧敏感电压依赖性钾通道 1.5（Kv1.5）。
使用低压舱模拟 5000 米海拔高度来建立 HPH 大鼠模型。使用离体灌注/通气的大鼠肺和分离的肺动脉环来研究 Lut 对 K 通道活性的影响。评估 HPH 大鼠肺组织和肺动脉中的 Kv1.5 水平。检测 HPH 大鼠肺组织中细胞周期蛋白 D1（CyclinD1）、细胞周期蛋白依赖性激酶 4（CDK4）、增殖细胞核抗原（PCNA）、Bax、Bcl-2、caspase-3 的水平。在缺氧条件下，使用 DPO-1 作为 Kv1.5 特异性抑制剂，研究 Lut 对 PASMC 中 Kv1.5、细胞质游离钙浓度（[Ca]）、CyclinD1、CDK4、PCNA、Bax/Bcl-2 的影响。通过药物亲和反应靶标稳定性（DARTS）检测 Lut 与 PASMC 中 Kv1.5 的结合亲和力。利用 HEK293T 细胞中转染 KCNA5 基因（编码 Kv1.5）来确认 Lut 与 Kv1.5 之间的相互作用。此外，还评估了 Lut 对 HPH 大鼠肺组织和缺氧 PASMC 中线粒体结构、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）和低氧诱导因子 1α（HIF-1α）水平的影响。
Lut 通过直接激活平滑肌中的 Kv 和钙激活钾通道（K）来扩张肺动脉。Lut 上调了 HPH 大鼠肺组织和肺动脉中的 Kv1.5 水平。Lut 下调了 HPH 大鼠肺组织中的 CyclinD1、CDK4、PCNA，同时上调了 Bax/Bcl-2/caspase-3 轴。Lut 在缺氧条件下通过上调 Kv1.5，降低[Ca]，减少 CDK4、CyclinD1、PCNA，增加 Bax/Bcl-2 比值，从而抑制 PASMC 的增殖。在 PASMC 和 HEK293T 细胞中验证了 Lut 与 Kv1.5 的结合亲和力和相互作用。Lut 还通过靶向 Kv1.5 降低缺氧条件下 HEK293T 细胞中的[Ca]并抑制增殖。此外，Lut 还保护了 HPH 大鼠肺组织中的线粒体结构，增加了 SOD、GSH、GSH-Px，降低了 MDA。Lut 下调了 HPH 大鼠肺组织和缺氧 PASMC 中的 HIF-1α 水平。
Lut 通过在 PASMC 中上调 Kv1.5，促进肺动脉扩张、减少细胞增殖和诱导细胞凋亡，从而减轻 HPVR。
