背景:低氧性肺血管重构(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(PASMCs)中的氧敏感钾(K)通道在HPVR中起着至关重要的作用。木犀草素(Lut)是一种植物来源的黄酮类化合物,具有多种药理作用。我们先前的研究发现Lut减轻了HPH大鼠的HPVR。
目的:为了阐明Lut减轻HPVR的机制,关注氧敏感电压依赖性钾通道1.5(Kv1.5)。
方法:采用低压舱模拟海拔5000m建立HPH大鼠模型。隔离灌注/通气大鼠肺,利用分离的肺小动脉环来研究Lut对K通道活性的影响。评估了HPH大鼠肺组织和肺小动脉中的Kv1.5水平。CyclinD1,CDK4,PCNA,Bax,检测HPH大鼠肺组织中Bcl-2、caspase-3的水平。Lut对Kv1.5,细胞质游离钙浓度([Ca2]cyt)的影响,CyclinD1,CDK4,PCNA,Bax/Bcl-2在缺氧条件下检测PASMCs,DPO-1作为Kv1.5特异性抑制剂。通过药物亲和力响应性靶标稳定性(DARTS)检测PASMC中Lut与Kv1.5之间的结合亲和力。利用KCNA5基因(编码Kv1.5)在HEK293T细胞中的过表达来证实Lut和Kv1.5之间的相互作用。此外,Lut对线粒体结构的影响,SOD,GSH,GSH-Px,测定缺氧条件下HPH大鼠和PASMCs肺组织中MDA和HIF-1α的含量。
结果:Lut通过直接激活平滑肌中的Kv和Ca2激活的K通道(KCa)来扩张肺动脉。Lut上调了HPH大鼠肺组织和肺小动脉中的Kv1.5水平。Lut下调HPH大鼠肺组织中的CyclinD1,CDK4,PCNA,同时上调Bax/Bcl-2/caspase-3轴。Lut降低了[Ca2+]cyt,减少CDK4,CyclinD1,PCNA,Bax/Bcl-2比值增加,在缺氧条件下的PASMC中,通过上调Kv1.5。在PASMC和HEK293T细胞中验证了Lut和Kv1.5之间的结合亲和力和相互作用。Lut还通过在缺氧下靶向HEK293T细胞的Kv1.5降低[Ca2]cyt并抑制增殖。此外,Lut保护的线粒体结构,增加SOD,GSH,GSH-Px,MDA降低,在HPH大鼠的肺组织中。Lut下调缺氧条件下HPH大鼠和PASMC肺组织HIF-1α水平。
结论:Lut通过促进肺动脉舒张减轻HPVR,减少细胞增殖,并通过上调PASMC中Kv1.5诱导细胞凋亡。
BACKGROUND: 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.
OBJECTIVE: To elucidate the mechanism by which Lut mitigated HPVR, focusing on oxygen-sensitive voltage-dependent potassium channel 1.5 (Kv1.5).
METHODS: 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 ([Ca2+]cyt), 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.
RESULTS: Lut dilated pulmonary artery by directly activating Kv and Ca2+-activated K+ channels (KCa) 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 [Ca2+]cyt, 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 [Ca2+]cyt 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.
CONCLUSIONS: Lut alleviated HPVR by promoting vasodilation of pulmonary artery, reducing cellular proliferation, and inducing apoptosis through upregulating of Kv1.5 in PASMCs.