Quercetin relaxes rat pulmonary arteries and trachea ex vivo: Role of nitric oxide and K⁺ channels

Quercetin relaxes rat pulmonary arteries and trachea ex vivo: Role of nitric oxide and K⁺ channels | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Quercetin relaxes rat pulmonary arteries and trachea ex vivo: Role of nitric oxide and K⁺ channels Hong-Yan Sun, Mei-Yang Xu, Zi-Yi Chen, Ying Tang, Yu-Fei Ke, Cheng-Wen Fu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7873590/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Quercetin (Que) is a natural compound with potential bioactivity, known for its relaxant effects through modulation of smooth muscle tone. This study aimed to systematically investigate the relaxant effects of Que on rat pulmonary artery and trachea, and to explore the roles of endothelium and potassium ion channels in its mechanism of action. Isolated pulmonary artery rings and tracheal rings from Sprague-Dawley rats were placed in an ex vivo tissue bath system. Relaxation was evaluated by the percentage of vasodilation in response to different concentrations of Que (1-300 µmol/L) against contractions induced by potassium chloride (KCl) or acetylcholine (ACh) in both endothelium-intact and endothelium-denuded rings. The influences of nitric oxide (NO) and prostacyclin was evaluated using L-NAME, methylene blue, indomethacin, and dideoxyadenosine. Endothelium-denuded rings were pretreated with various potassium channel blockers including TEA, 4-AP, linopirdine, RY796, DPO-1, iberiotoxin, TRAM-34, apamin, BaCl₂, glibenclamide, and TASK-1-IN-1. Que induced concentration-dependent relaxation in KCl-precontracted pulmonary artery rings. Removal of the endothelium, as well as pre-incubation with L-NAME and methylene blue, significantly attenuated this relaxant effect. Moreover, the Que-induced relaxation was markedly reduced by potassium channel blockers such as TEA, 4-AP, and iberiotoxin. Similar mechanistic dependencies were observed in the relaxant response of tracheal smooth muscle to Que. In conclusion, Que produces significant relaxation of rat pulmonary artery and trachea, which is dependent on the integrity of the endothelium, activation of the NO signaling pathway, and involvement of multiple potassium channels, including voltage-gated (K V ) and calcium-activated (K Ca ) potassium channels. Biological sciences/Biochemistry Health sciences/Cardiology Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Physiology Quercetin nitric oxide K+ channels Vascular and Tracheal tension Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Vascular and airway contraction and relaxation functions depend on the complex interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) to maintain hemodynamics [ 1 , 2 ] and airway homeostasis [ 3 , 4 ] . ECs regulate smooth muscle tone by releasing key relaxing factors, including nitric oxide (NO) and prostacyclin (PGI₂). NO activates the sGC/cGMP/PKG pathway, while PGI₂ triggers the AC/cAMP/PKA pathway, both resulting in decreased intracellular calcium concentration and smooth muscle relaxation [ 5 , 6 ] . Additionally, the activation of potassium ion channels, including voltage-dependent potassium channels (Kv) and calcium-activated potassium channels (K Ca )-induces membrane hyperpolarization, which in turn inhibits the opening of voltage-dependent calcium channels (VDCC). This process contributes to a reduction in the intracellular Ca²⁺ concentration and constituting a core mechanism that facilitates vascular and airway smooth muscle relaxation [ 5 – 8 ] . Disruption of this regulatory system is closely associated with pathological states such as pulmonary arterial hypertension (PAH) and airway hyperresponsiveness (AHR). PAH is a cardiopulmonary disease characterized by pulmonary vascular remodeling, in which abnormal proliferation of pulmonary artery SMCs (PASMCs) leads to vessel wall thickening and functional alterations, ultimately causing blood flow obstruction and right heart failure [ 9 , 10 ] . Similarly, AHR diseases are characterized by structural and functional dysregulation of airway smooth muscle, with predominant features of airway narrowing and airflow limitation [ 11 , 12 ] . PAH progresses rapidly and has a poor long-term prognosis, whereas severe AHR exhibits heterogeneous and diverse phenotypes that contribute to variable therapeutic outcomes. Quercetin (Que), a widely distributed natural flavonoid compound in plants, exhibits various bioactivities, including antioxidant, anti-inflammatory, antiviral, antitumor, and cardiovascular protective effects [ 13 – 18 ] . Previous studies suggest that Que possesses the potential to modulate smooth muscle tone, positioning it as a candidate for treating PAH and asthma. Research shows that Que not only exerts antioxidant and anti-inflammatory effects [ 14 , 23 , 27 ] but also enhances endothelial function and increases NO bioavailability, thereby inducing membrane hyperpolarization [ 19 , 20 ] and promoting smooth muscle relaxation. Additionally, Que mediates smooth muscle relaxation via potassium ion channels by activating multiple K⁺ channels, such as K V and K Ca channels, inducing membrane hyperpolarization, inhibiting VDCC, and lowering intracellular Ca²⁺ levels [ 14 , 21 , 22 ] . In the airway, Que has also been shown to inhibit calcium channels and inflammation, thereby facilitating tracheal relaxation [ 23 , 24 ] . Notably, studies have reported that Que activates Kv7 channels [ 15 , 25 ] , and voltage-gated potassium channels, especially Kv7, play critical roles in regulating vascular smooth muscle excitability [ 26 ] . Although the vasorelaxant effects of Que have been reported, systematic comparative studies examining its relaxant effects on isolated rat pulmonary artery rings and tracheal rings remain limited. Particularly, the dependency on intact endothelium, the specific modulatory effects on downstream signaling pathways (NO/PGI₂), and the selective activation of multiple K⁺ channels and their subtypes have not been fully elucidated. Therefore, this study aims to systematically investigate the relaxant effects of Que on isolated rat pulmonary artery and tracheal rings and to explore the underlying mechanisms. The study evaluates the concentration-dependent effects of Que on the relaxation of endothelium-intact and endothelium-denuded rings precontracted with KCl or Ach. This will provide crucial experimental evidence to clarify the core mechanisms by which Que induces relaxation in rat pulmonary artery and trachea, highlighting the essential roles of endothelial function, NO/PGI₂ signaling pathways, and multiple K⁺ channels. The findings will provide a theoretical foundation for the potential application of Que in related cardiovascular and respiratory diseases. 2. Materials and methods 2.1 Instruments and Reagents RM6240E multi-channel physiological signal acquisition and processing system SGQ-4, isolated tissue and organ constant-temperature perfusion apparatus, JZJ01 type tension transducer sensor. Que (HY-18085) was acquired from MCE with a molecular formula of C15H10O7, CAS:117-39-5, and purity > 99%, Additional compounds from MCE (China) included acetylcholine chloride (Ach) (HY-B0282), L-NAME hydrochloride (L-NAME) (HY-18729A), methylene blue(HY-B1359), indomethacin (Indo) (HY-14397), 2',5'-dideoxyadenosine (HY-135878), TRAM-34 (HY-13519), apamin (HY-P0256), TASK-1-IN-1 (HY-151891), DPO-1 (HY-100712), RY796 (HY-120033), linopirdine (HY-W020468). Reagents from Sigma (USA) included tetraethylammonium chloride (TEA) (No.86616), barium chloride (BaCl2) (No.202738), glibenclamide (Gli) (No.G0639), iberiotoxin (No.I5904), 4-aminopyridine (4-AP) (No.HY-B0604), 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (No.H3375)。 2.2 Animals Male Sprague-Dawley (SD) rats of specific pathogen-free (SPF) grade, weighing 220–240 g, were provided by the Experimental Animal Center of Hangzhou Medical College, with license number SCXK (Zhejiang) 2024-0002. The rats had free access to food and water. The experimental environment was maintained at room temperature with relative humidity between 45% and 55%. The experiments commenced after an acclimatization period of 7 days. This study was approved by the Ethics Committee of Bengbu Medical College (ethical approval no: 2019-074), and the care and handling of animals strictly followed the Regulations on the Management of Laboratory Animals. 2.3 Preparation of Vascular and Tracheal Rings 2.3.1 Preparation and Treatment of Rat Pulmonary Artery Rings After euthanizing the experimental rats, the thoracic cavity was immediately opened, and the lung lobes were excised and rinsed in PBS. The lung tissue was gently isolated and transferred to a dish containing 4℃ K-H solution, continuously bubbled with a gas mixture of 95% O₂ and 5% CO₂. Subsequently, the connective tissue surrounding the pulmonary artery was carefully removed to obtain vascular rings measuring 3 mm in length. Each vascular ring was mounted between two triangular metal rings, with the upper end connected to a tension transducer and the lower end fixed. Changes in vascular ring tension were recorded using the RM6240E system. The water bath was continuously supplied with 95% O₂ and 5% CO₂ to maintain a volume of 15 mL K-H solution at 37℃. The K-H solution was replaced every 20 minutes, and the baseline tension was adjusted by 0.1 g increments every 5 minutes until a stable baseline tension of 0.75 g was reached. After stabilizing for 2 hours, this tension was recorded as the baseline tension of the vascular ring. Next, the vascular rings were stimulated twice consecutively with 60 mmol/L KCl. If the difference between the contraction amplitudes of the two stimulations was less than 10%, the vascular rings were considered to have adequate vascular activity. To investigate the mechanism of Que on pulmonary artery contraction and relaxation function, endothelial removal was performed by wiping the vascular lumen with cotton wrapped around ophthalmic forceps. Endothelial removal was confirmed by stimulating the rings with Ach at 1×10⁻⁶ mol/L; a relaxation degree of less than 10% indicated successful endothelial denudation, whereas a relaxation degree greater than 80% indicated intact endothelial function. After assessing endothelial function, the vascular rings were returned to baseline state by rinsing with K-H solution, and tension was recorded again. Specific signaling pathway inhibitors and potassium channel blockers were pre-incubated with the rings for 20 minutes. Following this, KCl was used to induce a stable contraction. Que was cumulatively added at concentrations of 1, 3, 10, 30, 100, and 300 µmol/L every 15 minutes. The control group received an equal volume of DMSO. Tension changes were recorded for each vascular ring, and the percentage of vasorelaxation induced by Que was calculated to evaluate the effect of pathway inhibitors and potassium channel blockers on pulmonary artery relaxation. The calculation formula for the percentage of vasorelaxation induced by Que was as follows: Vasorelaxation (%) = (Maximum contraction tension induced by KCl Tension- after Que treatment Maximum contraction tension induced by KCl) / (Maximum contraction tension induced by KCl - Baseline tension) * 100% (n = 6). 2.3.2 Preparation and Treatment of Rat Tracheal Rings The trachea was excised, cleaned, and cut into 3-mm rings, then mounted in the same setup as pulmonary arteries. Baseline tension was adjusted to 1.2 g and equilibrated for 2 h. Contractile activity was confirmed by two consecutive stimulations with 60 mmol/L KCl. Epithelium was mechanically removed and verified by 80% indicated intact epithelium. After functional assessment, rings were washed and pre-incubated with inhibitors or K⁺ channel blockers for 20 min. Contraction was induced with Ach, followed by cumulative addition of Que (1–300 µM) every 15 min; DMSO served as control. The calculation formula for the percentage of Que-induced tracheal relaxation was as follows: Tracheal Relaxation (%) = (Maximum contraction tension induced by Ach Tension- after Que treatment Maximum contraction tension induced by Ach)/ (Maximum contraction tension induced by Ach) -Baseline tension) * 100% (n = 6). 3. Statistical analysis Data were statistically analyzed using GraphPad Prism 9 software, and logarithmic concentration-response relaxation curves were plotted. Quantitative data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for multiple group comparisons, and differences were considered statistically significant at P < 0.05. The maximal vasorelaxant response was expressed as Emax, and "n" represents the number of arteries tested. 4. Results 4.1 Effects of Que on Relaxation of Pulmonary Arteries and Trachea with Intact and Denuded Endothelium. We investigated the relaxant effects of Que at different concentrations (1, 3, 10, 30, 100, and 300 µmol/L) on pulmonary arteries and trachea from normal rats, examining the influence of intact and denuded endothelium on Que-induced relaxation of pulmonary artery and tracheal rings. The results showed that Que induced a concentration-dependent relaxation in both endothelium-intact and endothelium-denuded pulmonary artery and tracheal rings (P < 0.01, n = 6; Fig. 1 A-H). Compared with the DMSO control group, the maximal relaxation (Emax) in pulmonary artery rings was 97.7 ± 1.26% in the endothelium-intact group (Que + End + ) and 84.3 ± 4.2% in the endothelium-denuded group (Que + End − ); in tracheal rings, the Emax was 103.8 ± 3.50% in the epithelium-intact group (Que + End + ) and 83.23 ± 4.43% in the epithelium-denuded group (Que + End − ). These results indicate that Que induces concentration-dependent relaxation of pulmonary artery and tracheal rings regardless of endothelial or epithelial integrity, although denudation attenuates the vasorelaxant effect of Que. This suggests that both endothelium-dependent and endothelium-independent mechanisms contribute to the vasorelaxation induced by Que. 4.2 Que induces relaxation of the pulmonary artery and trachea through NO rather than prostaglandins. To further investigate whether Que-induced vasodilation is related to NO and prostaglandins, experiments were conducted using L-NAME (100 µmol/L), methylene blue (10 µmol/L), Indo (5 µmol/L), and dideoxyadenosine (200 µmol/L). The results demonstrated that, compared with the endothelium-intact group, both L-NAME and methylene blue significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P 0.05, n = 6; Fig. 3 A-H). Specifically, in pulmonary artery rings, the Emax was 80.06 ± 2.82% and 87.93 ± 4.24% for L-NAME and methylene blue, respectively, while Indo and dideoxyadenosine showed Emax values of 96.99 ± 2.78% and 92.21 ± 5.84%. In tracheal rings, the Emax values for L-NAME and methylene blue were 85.26 ± 4.01% and 94.13 ± 4.50%, respectively, whereas Indo and dideoxyadenosine had Emax values of 99.74 ± 4.44% and 101.25 ± 4.47%. These findings suggest that Que induces relaxation of the pulmonary artery and trachea via the NO signaling pathway. 4.3 Que induces relaxation of the pulmonary artery and trachea through potassium ion channels. To investigate whether Que-induced vasodilation is related to potassium channels, experiments were conducted using the non-selective potassium channel inhibitor TEA (5 mmol/L). The results showed that, compared with the endothelium-denuded group, TEA significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P < 0.01, n = 6; Fig. 4 A-F). Specifically, in pulmonary artery rings, the Emax induced by TEA was 57.91 ± 5.75%, while in tracheal rings, the Emax was 59.83 ± 5.31%. These findings indicate that Que induces relaxation of the pulmonary artery and trachea through potassium ion channels. 4.4 Que primarily induces relaxation of the pulmonary artery and trachea through K V channels. To further assess the role of K⁺ channels in Que-induced vasodilation, we first pretreated the pulmonary artery and trachea with the K V channel inhibitor. The experiment showed that pretreatment with the K V channel inhibitor 4-AP (1 mmol/L) significantly inhibited Que-induced relaxation of both the vessels and trachea (G P < 0.01, n = 6; Fig. 5 B). Specifically, in the pulmonary artery, the Emax after 4-AP treatment was 65.78 ± 3.69%, while in the trachea, the Emax was 63.97 ± 4.94%. Given that Que-induced vasodilation is mediated by activation of K V channels (Fig. 5 B, G). we further conducted experiments targeting multiple K V subtypes. Various K V channel subtypes are present in smooth muscle, including those responsible for maintaining vascular and tracheal tone: K V 1.5, K V 2.1, and K V 7. To evaluate these subtypes, specific inhibitors were used: K V 1.5 inhibitor DPO-1 (1×10⁻⁵ mol/L), K V 2.1 inhibitor RY796 (1×10⁻⁵ mol/L), and K V 7 inhibitor linopirdine (10 µmol/L). The results demonstrated that, compared with the endothelium-denuded group, linopirdine significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P 0.05, n = 6; Fig. 5 D-E, I-J). Specifically, in pulmonary artery rings, the Emax values were 71.29 ± 2.85% for linopirdine, 82.09 ± 3.22% for DPO-1, and 86.72 ± 2.39% for RY796; in tracheal rings, the corresponding Emax values were 66.21 ± 2.24% for linopirdine, 76.52 ± 4.13% for DPO-1, and 81.77 ± 1.10% for RY796. These findings indicate that the vasodilatory effect of Que is associated with its activation of voltage-gated potassium channels, particularly the K V 7 subtype, while K V 1.5 and K V 2.1 channels are not involved. 4.5 Que induces relaxation of the pulmonary artery and trachea through large-conductance calcium-activated potassium (BK Ca ) channels. To further evaluate the role of K Ca channels in Que-induced vasodilation, we incubated pulmonary artery and trachea tissues with inhibitors of BK Ca , IK Ca , and SK Ca channels. The experiments revealed that pretreatment with the BK Ca channel inhibitor Iberiotoxin (0.1 µmol/L) significantly attenuated Que-induced relaxation of both the vessels and trachea (P < 0.01, n = 6; Fig. 6 B, F). In contrast, pretreatment with the IK Ca and SK Ca channel inhibitors TRAM-34 (20 nmol/L) and Apamin (100 nmol/L), respectively, had no significant effect on Que-induced vasorelaxation in these tissues (P > 0.05, n = 6; Fig. 6 C-D, G-H). Specifically, in the pulmonary artery, the Emax was 70.91 ± 2.06% with Iberiotoxin, compared to 82.17 ± 2.88% and 82.39 ± 2.84% with TRAM and Apamin, respectively. In the trachea, the Emax values were 68.41 ± 3.59% for Iberiotoxin, and 80.41 ± 2.50% and 82.24 ± 2.84% for TRAM-34 and Apamin, respectively. These results indicate that Que-induced vasodilation is associated with the activation of K Ca channels, particularly the BK Ca subtype, while IK Ca and SK Ca subtypes do not appear to be involved. 4.6 The effects of inwardly rectifying potassium channels (K ir ), ATP-sensitive potassium channels (K ATP ), and two-pore domain potassium channels (K 2P ) on que-induced relaxation of the pulmonary artery and trachea. We further investigated the effects of inhibitors of three other types of potassium channels (K ir , K ATP , and K 2P ) on que-induced relaxation of the pulmonary artery and trachea. The experiments showed that pretreatment with the K ir , K ATP and K 2P channel inhibitors BaCl₂ (0.1 mmol/L), Gli (1×10⁻⁵ mol/L), and TASK-1-IN-1 (1×10⁻⁶ mol/L) respectively had no significant effect on the vasorelaxant and tracheal relaxant actions induced by Que (P > 0.05, n = 6; Fig. 7 , Fig. 8 , Fig. 9 ). Specifically, in the pulmonary artery, the maximum relaxation rates induced by BaCl₂, Gli, and TASK-1-IN-1 were 81.35 ± 2.97%, 80.20 ± 3.46% and 82.89 ± 3.46%, respectively. In the trachea, the maximum relaxation rates were 83.28 ± 3.26%, 81.22 ± 3.29%, and 80.59 ± 2.53%. Discussion In recent years, the regulation of contraction and relaxation of vascular and airway smooth muscle has become a focal area of research, especially for the treatment of diseases such as PAH and AHR. Modulation of smooth muscle tone holds significant clinical importance in these conditions. Previous studies have confirmed that flavonoids, a class of natural polyphenolic compounds derived from plants, exhibit diverse biological activities, particularly showing potential in vascular relaxation and airway dilation [ 27 , 28 ] . Among them, Que has attracted considerable attention due to its multitarget and multipathway mechanisms. Its vasodilatory effects are mediated by promoting NO release from ECs [ 29 ] , inhibiting the production of endothelin-1 and angiotensin II [ 30 – 32 ] , and modulating multiple ion channels, enzymes, and signaling pathways. According to the literature, Que can relax rat aorta [ 19 , 33 ] , coronary arteries [ 21 ] , and mesenteric arteries [ 34 ] . Additionally, it activates potassium ion channels and facilitates the release of anticholinergic neurotransmitters, thereby reducing gastrointestinal smooth muscle contraction [ 14 ] . In AHR-related diseases, Que inhibits the release of pro-contractile mediators, such as histamine and leukotrienes, while regulating cyclic adenosine monophosphate (cAMP) levels and activating phosphodiesterase enzymes to promote airway smooth muscle relaxation [ 17 , 23 ] . These effects not only alleviate airway obstruction but also reduce airway hyperreactivity, providing a potential pharmacological basis for the treatment of asthma and related disorders. This study systematically elucidates, for the first time, that Que induces concentration-dependent relaxation of rat pulmonary arteries and trachea through a dual mechanism involving NO signaling and activation of potassium channels (Fig. 1 ). In a series of isolated tissue experiments, removal of the endothelium or epithelium attenuated, but did not completely abolish, the relaxant effect of Que, indicating that it exerts relaxation via both endothelium-dependent and epithelium-independent pathways. Notably, even after endothelial or epithelial denudation, a residual relaxation response persisted (Figs. 1 G-H), suggesting that Que directly targets smooth muscle ion channels, consistent with mechanisms reported for other flavonoids [ 15 , 22 , 35 ] . Further investigation employing inhibitors of endothelium-dependent relaxant factors-including the NO synthase inhibitor L-NAME, guanylate cyclase inhibitor methylene blue, cyclooxygenase inhibitor indomethacin, and adenylate cyclase inhibitor 2',5'-dideoxyadenosine-demonstrated that L-NAME markedly reduced Que-induced vasorelaxation, and methylene blue similarly diminished its relaxant effect, whereas indomethacin and 2',5'-dideoxyadenosine had no significant impact (Fig. 2 ). These findings underscore the primary role of the NO signaling pathway in Que-mediated relaxation of pulmonary arteries and trachea. Previous studies also indicate that Que enhances the expression and activity of endothelial NO synthase, increases NO production, and thereby activates the adenylate cyclase pathway, culminating in smooth muscle relaxation [ 29 ] . It is noteworthy that the NO/cGMP/PKG signaling cascade, through modulation of potassium channels, plays a crucial role in vascular relaxation [ 36 , 37 ] , cardioprotection [ 38 , 39 ] and neural regulation [ 40 ] . SMCs express various potassium channels, including K V , K Ca , K ATP , inwardly rectifying potassium channels (K ir and K 2P ) [ 7 , 41 ] . In this study, non-specific K⁺ channel blocker TEA, the K V channel blocker 4-AP, and BK Ca channel blocker iberiotoxin all attenuated Que-induced relaxation, further indicating that Que mediates its effects primarily through activation of multiple potassium channel types. Beyond promoting K⁺ efflux to cause membrane hyperpolarization and inhibit Ca²⁺ channel opening, potassium channels enhance endothelial K⁺ flux, which facilitates NO release and vasodilation by modulating membrane potential and smooth muscle tone [ 42 ] . Conversely, the K ATP channel blocker glibenclamide, the acid-sensitive potassium channel inhibitor TASK-1-IN-1, and the SK Ca channel blocker apamin did not affect Que's relaxant action. This implies that K ATP and TASK-1 channels are not primary targets of Que, which may also relate to tissue- and cell-specific distribution and function of different potassium channels, reflecting certain organ-specific molecular targeting by Que. Vasorelaxation of blood vessels and trachea can occur via both endothelium-dependent and endothelium-independent mechanisms. In endothelium-independent vasorelaxation, potassium channels play a pivotal role. Collectively, this study confirms that Que mediates pulmonary artery and tracheal smooth muscle relaxation through synergistic activation of the NO signaling pathway and multiple potassium channels, delineating its underlying mechanism. These findings provide a theoretical foundation for the potential therapeutic utility of Que in PAH and AHR disorders. Notably, this study has several limitations. All experiments were performed using isolated tissue preparations from healthy rats, which may not fully replicate the complex in vivo environment, including neurohumoral regulation, blood flow, and disease-specific alterations in pulmonary arteries or airways. Therefore, the effects of Que in pathological conditions such as PAH or AHR remain to be confirmed. Moreover, although we identified the involvement of NO signaling and specific potassium channels, the detailed downstream molecular pathways and potential crosstalk between different ion channels warrant further investigation. Conclusion This study demonstrates that Que induces potent, concentration-dependent relaxation of rat pulmonary artery and tracheal smooth muscle. The relaxant effects of Que are significantly reliant on the presence of an intact endothelium and the activation of the NO signaling pathway, as evidenced by the attenuation of vasodilation following endothelial removal and inhibition of NO synthase and guanylate cyclase. Additionally, our findings reveal that multiple potassium channels, particularly K V and BK Ca channels, play crucial roles in mediating Que-induced relaxation, as blockers targeting these channels markedly reduced its effects. The interplay between NO signaling and potassium channel activation likely underpins the mechanism by which Que modulates smooth muscle tone in both vascular and airway tissues. Overall, this research provides important mechanistic insights into Que’ s bioactivity and supports its potential therapeutic application for conditions characterized by pulmonary arterial and airway hypercontractility, such as PAH and AHR. Declarations CRediT authorship contribution statement Hong-Yan Sun: Writing original draft, Formal analysis, Data curation. Mei-Yang Xu: Data curation. Zi-Yi Chen: Data curation. Ying Tang: Methodology, Data curation. Yu-Fei Ke: Data curation. Cheng-Wen Fu: Data curation. Rui-Yang Chen: Data curation. Guo-Qing Lu: Methodology, Data curation. Le-Qiang Liu: Data curation. Qin Gao: Writing review & editing. Bi Tang: Writing review & editing, Funding acquisition, Conceptualizaion. Pin-Fang Kang: Writing review & editing, Funding acquisition. Ethics statement All studies were approved by the Medical Ethics Committee of Bengbu Medical University has passed the ethics examination and approval. The reference number is as follows: [2019] number 074. Declaration of competing interest The authors declare no conflict of interest. Funding This work was supported by the National Natural Science Foundation of China (82500426); the Natural Science Foundation of Anhui Province (2208085MH192); the Anhui Province Outstanding Youth Project (2022AH030141); and the Excellent Scientific Research and Innovation Team of Anhui Province (2022AH010083). Acknowledgements National Outstanding Youth Science Fund Project of NationalNatural Science Foundation of china (82500426), Anhui Province Natural Foundation of China (Grant nos. 2208085MH192), Anhui Province Outstanding Youth Project of China (2022AH030141), Anhui Province excellent scientific research and innovation team of China (2022AH010083). References FRISMANTIENE A, PHILIPPOVA M, ERNE P, et al.Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity[J].Cell Signal,2018,5248-64. CHEN Y L, DANEVA Z, KUPPUSAMY M, et al.Novel Smooth Muscle Ca(2+)-Signaling Nanodomains in Blood Pressure Regulation[J].Circulation,2022,146(7) 548-64. 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07:06:21","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86566,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/a0d2ebb5a1c13156c7eba565.png"},{"id":96239380,"identity":"900abf58-b571-4623-9cc9-aab64458288d","added_by":"auto","created_at":"2025-11-19 07:06:28","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171702,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/b8e99afea5516576d4658bc7.png"},{"id":96239745,"identity":"1b0efff3-2da4-4b3a-b0c0-895b502fb3cc","added_by":"auto","created_at":"2025-11-19 07:07:32","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144908,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/3f556d92f2c0db6acfff35ff.png"},{"id":95827221,"identity":"91ac275c-a256-4128-8a63-173732e2309e","added_by":"auto","created_at":"2025-11-13 11:25:33","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82429,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/9cd1a263ec22e943adc7e955.png"},{"id":95827236,"identity":"fb2ebcb7-a6c3-406a-be9a-55d11ae5be09","added_by":"auto","created_at":"2025-11-13 11:25:33","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83448,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/eec86e6d92dfc27cb32dbdb7.png"},{"id":95827223,"identity":"893d66bd-2c5d-44db-b3cf-2f75bba37694","added_by":"auto","created_at":"2025-11-13 11:25:33","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86489,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig9.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/653a0b586d9b79b066cb9763.png"},{"id":96239640,"identity":"f8c0b451-3c7e-420a-908b-be0d6a1dbb49","added_by":"auto","created_at":"2025-11-19 07:07:15","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99098,"visible":true,"origin":"","legend":"","description":"","filename":"639624e23b0744fbbb8a000b4519c6b41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/ac8e7a1d77da2a799451c6ba.xml"},{"id":95827231,"identity":"191559af-55ec-4fd3-871b-476a65c366cb","added_by":"auto","created_at":"2025-11-13 11:25:33","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111039,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/c6296417d9f37734b41a6924.html"},{"id":95827195,"identity":"95da41c8-4397-46c3-b3c2-b5b79bff5ca2","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":826669,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of preconstricted endothelium-intact (End\u003csup\u003e+\u003c/sup\u003e) and endothelium- denuded (End\u003csup\u003e-\u003c/sup\u003e)isolated pulmonary artery and tracheal. A, D: DMSO group. The representative tracings show the effect of Que on End\u003csup\u003e+\u003c/sup\u003e (B, E) and End\u003csup\u003e-\u003c/sup\u003e (C, F) artery and tracheal rings preconstricted with KCl (60 mmol/L) or Ach(10\u003csup\u003e-5\u003c/sup\u003emmol/L). G, H: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs DMSO group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/14d99d7867b8e03ca016423e.png"},{"id":96240317,"identity":"61875368-3516-4c48-816d-ed8547ce9941","added_by":"auto","created_at":"2025-11-19 07:08:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":847544,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e+\u003c/sup\u003e pulmonary artery and tracheal rings incubated with L-NAME (eNOS inhibitor) and methylene blue (guanylate cyclase inhibitor). A, D: Que+ End\u003csup\u003e+ \u003c/sup\u003egroup. BCEF: Inhibitor Intervention group. G, H: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que+ End\u003csup\u003e+\u003c/sup\u003e group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/85d8b137e008cd53ccea9ffc.png"},{"id":95827196,"identity":"cf6147e8-983f-4668-8e70-9953d35c7cea","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":849386,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e+\u003c/sup\u003e pulmonary artery and tracheal rings incubated with indomethacin (Indo) and dideoxyadenosine. A, D: Que+ End\u003csup\u003e+ \u003c/sup\u003egroup. BCEF: Inhibitor Intervention group. G, H: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que+ End\u003csup\u003e+\u003c/sup\u003e group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/05839ece38d8016d1de5b99e.png"},{"id":95827205,"identity":"84180818-c400-40e3-bd71-9e908e25f6ee","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":598086,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e-\u003c/sup\u003e pulmonary artery and tracheal rings incubated with TEA. A, C: Que+ End\u003csup\u003e-\u003c/sup\u003e group. BD: Inhibitor Intervention group. E, F: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que+ End\u003csup\u003e-\u003c/sup\u003e group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/3ba88796d29e15d7a66edb0e.png"},{"id":96240553,"identity":"a1f5a136-04f6-4953-9e95-b5d545b14009","added_by":"auto","created_at":"2025-11-19 07:09:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1248306,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e- \u003c/sup\u003epulmonary artery and tracheal rings incubated with 4-AP, DPO-1, RY796 and Linopirdine. AF: Que +End\u003csup\u003e-\u003c/sup\u003egroup. B-E, G-J: Inhibitor Intervention group. K, M: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que +End\u003csup\u003e- \u003c/sup\u003egroup (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/9a979862bc4cec13aff68cd0.png"},{"id":95827203,"identity":"d2df3f59-b111-4461-ab77-257ced988817","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1043593,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e-\u003c/sup\u003e pulmonary artery and tracheal rings incubated with Iberiotoxin, TRAM and apamin. A, C: Que +End\u003csup\u003e-\u003c/sup\u003e group. B-D, F-H: Inhibitor Intervention group. I, J: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que +End\u003csup\u003e-\u003c/sup\u003e group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/20e1039cc239e16792a3e1ca.png"},{"id":96239749,"identity":"61465572-910a-4ef5-90a1-16a845864f21","added_by":"auto","created_at":"2025-11-19 07:07:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":571922,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e-\u003c/sup\u003e pulmonary artery and tracheal rings incubated with Bacl\u003csub\u003e2\u003c/sub\u003e. A, C: Que+ End\u003csup\u003e- \u003c/sup\u003egroup. BD: Inhibitor Intervention group. E, F: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que+ End\u003csup\u003e-\u003c/sup\u003e group (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/057cf493f616fbc82e82af62.png"},{"id":95827200,"identity":"2854d1fa-8191-4a20-9501-c938998b4112","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":576179,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End+ pulmonary artery and tracheal rings incubated with Gli. A, C: Que+ End\u003csup\u003e-\u003c/sup\u003e group. BD: Inhibitor Intervention group. E, F: concentration-response curve. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 vs Que+ End\u003csup\u003e- \u003c/sup\u003egroup (x±s, n=6).\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/84e7c47ea6cbefa005ca05a7.png"},{"id":96239502,"identity":"4c38a14a-1d15-4f40-bf20-ed6ccb0eaa24","added_by":"auto","created_at":"2025-11-19 07:06:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":605089,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Que on relaxation of isolated End\u003csup\u003e-\u003c/sup\u003e pulmonary artery and tracheal rings incubated with TASK-1-IN. A, C: Que+ End\u003csup\u003e+\u003c/sup\u003e group. BD: Inhibitor Intervention group. E, F: concentration-response curve.\u003csup\u003e *\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001 vs Que+ End\u003csup\u003e- \u003c/sup\u003egroup (x±s, n=6)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/60d59cbe92a73c7bd53a877b.png"},{"id":96452959,"identity":"1c412966-7823-45b9-87cc-163ffcd41da6","added_by":"auto","created_at":"2025-11-21 09:55:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8993692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/255b6153-d35a-465b-96b4-b87ae562c15a.pdf"},{"id":96239571,"identity":"48592503-2e76-40a7-8461-489c2f43286d","added_by":"auto","created_at":"2025-11-19 07:07:00","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4157879,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigurespulmonaryartery.zip","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/13deeeae75811aa18faa6c81.zip"},{"id":96239030,"identity":"4f173dcc-208b-4b47-9e2b-9c09d80955c2","added_by":"auto","created_at":"2025-11-19 07:01:31","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4136890,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigurestracheal.zip","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/4a7296cf95ec397fcb63e881.zip"},{"id":95827198,"identity":"1ef693fe-3376-4f79-a920-37ed7918ea99","added_by":"auto","created_at":"2025-11-13 11:25:32","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32551,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarystatisticaldatas.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7873590/v1/46dc63b039f4a23903f38331.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quercetin relaxes rat pulmonary arteries and trachea ex vivo: Role of nitric oxide and K⁺ channels","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVascular and airway contraction and relaxation functions depend on the complex interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) to maintain hemodynamics\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and airway homeostasis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. ECs regulate smooth muscle tone by releasing key relaxing factors, including nitric oxide (NO) and prostacyclin (PGI₂). NO activates the sGC/cGMP/PKG pathway, while PGI₂ triggers the AC/cAMP/PKA pathway, both resulting in decreased intracellular calcium concentration and smooth muscle relaxation\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Additionally, the activation of potassium ion channels, including voltage-dependent potassium channels (Kv) and calcium-activated potassium channels (K\u003csub\u003eCa\u003c/sub\u003e)-induces membrane hyperpolarization, which in turn inhibits the opening of voltage-dependent calcium channels (VDCC). This process contributes to a reduction in the intracellular Ca\u0026sup2;⁺ concentration and constituting a core mechanism that facilitates vascular and airway smooth muscle relaxation\u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Disruption of this regulatory system is closely associated with pathological states such as pulmonary arterial hypertension (PAH) and airway hyperresponsiveness (AHR). PAH is a cardiopulmonary disease characterized by pulmonary vascular remodeling, in which abnormal proliferation of pulmonary artery SMCs (PASMCs) leads to vessel wall thickening and functional alterations, ultimately causing blood flow obstruction and right heart failure\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Similarly, AHR diseases are characterized by structural and functional dysregulation of airway smooth muscle, with predominant features of airway narrowing and airflow limitation\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. PAH progresses rapidly and has a poor long-term prognosis, whereas severe AHR exhibits heterogeneous and diverse phenotypes that contribute to variable therapeutic outcomes.\u003c/p\u003e\u003cp\u003eQuercetin (Que), a widely distributed natural flavonoid compound in plants, exhibits various bioactivities, including antioxidant, anti-inflammatory, antiviral, antitumor, and cardiovascular protective effects\u003csup\u003e[\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Previous studies suggest that Que possesses the potential to modulate smooth muscle tone, positioning it as a candidate for treating PAH and asthma. Research shows that Que not only exerts antioxidant and anti-inflammatory effects\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e but also enhances endothelial function and increases NO bioavailability, thereby inducing membrane hyperpolarization\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e and promoting smooth muscle relaxation. Additionally, Que mediates smooth muscle relaxation via potassium ion channels by activating multiple K⁺ channels, such as K\u003csub\u003eV\u003c/sub\u003e and K\u003csub\u003eCa\u003c/sub\u003e channels, inducing membrane hyperpolarization, inhibiting VDCC, and lowering intracellular Ca\u0026sup2;⁺ levels\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In the airway, Que has also been shown to inhibit calcium channels and inflammation, thereby facilitating tracheal relaxation\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Notably, studies have reported that Que activates Kv7 channels\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and voltage-gated potassium channels, especially Kv7, play critical roles in regulating vascular smooth muscle excitability\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the vasorelaxant effects of Que have been reported, systematic comparative studies examining its relaxant effects on isolated rat pulmonary artery rings and tracheal rings remain limited. Particularly, the dependency on intact endothelium, the specific modulatory effects on downstream signaling pathways (NO/PGI₂), and the selective activation of multiple K⁺ channels and their subtypes have not been fully elucidated. Therefore, this study aims to systematically investigate the relaxant effects of Que on isolated rat pulmonary artery and tracheal rings and to explore the underlying mechanisms. The study evaluates the concentration-dependent effects of Que on the relaxation of endothelium-intact and endothelium-denuded rings precontracted with KCl or Ach. This will provide crucial experimental evidence to clarify the core mechanisms by which Que induces relaxation in rat pulmonary artery and trachea, highlighting the essential roles of endothelial function, NO/PGI₂ signaling pathways, and multiple K⁺ channels. The findings will provide a theoretical foundation for the potential application of Que in related cardiovascular and respiratory diseases.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Instruments and Reagents\u003c/h2\u003e\u003cp\u003eRM6240E multi-channel physiological signal acquisition and processing system SGQ-4, isolated tissue and organ constant-temperature perfusion apparatus, JZJ01 type tension transducer sensor. Que (HY-18085) was acquired from MCE with a molecular formula of C15H10O7, CAS:117-39-5, and purity\u0026thinsp;\u0026gt;\u0026thinsp;99%, Additional compounds from MCE (China) included acetylcholine chloride (Ach) (HY-B0282), L-NAME hydrochloride (L-NAME) (HY-18729A), methylene blue(HY-B1359), indomethacin (Indo) (HY-14397), 2',5'-dideoxyadenosine (HY-135878), TRAM-34 (HY-13519), apamin (HY-P0256), TASK-1-IN-1 (HY-151891), DPO-1 (HY-100712), RY796 (HY-120033), linopirdine (HY-W020468). Reagents from Sigma (USA) included tetraethylammonium chloride (TEA) (No.86616), barium chloride (BaCl2) (No.202738), glibenclamide (Gli) (No.G0639), iberiotoxin (No.I5904), 4-aminopyridine (4-AP) (No.HY-B0604), 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) (No.H3375)。\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animals\u003c/h2\u003e\u003cp\u003eMale Sprague-Dawley (SD) rats of specific pathogen-free (SPF) grade, weighing 220\u0026ndash;240 g, were provided by the Experimental Animal Center of Hangzhou Medical College, with license number SCXK (Zhejiang) 2024-0002. The rats had free access to food and water. The experimental environment was maintained at room temperature with relative humidity between 45% and 55%. The experiments commenced after an acclimatization period of 7 days. This study was approved by the Ethics Committee of Bengbu Medical College (ethical approval no: 2019-074), and the care and handling of animals strictly followed the Regulations on the Management of Laboratory Animals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation of Vascular and Tracheal Rings\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Preparation and Treatment of Rat Pulmonary Artery Rings\u003c/h2\u003e\u003cp\u003eAfter euthanizing the experimental rats, the thoracic cavity was immediately opened, and the lung lobes were excised and rinsed in PBS. The lung tissue was gently isolated and transferred to a dish containing 4℃ K-H solution, continuously bubbled with a gas mixture of 95% O₂ and 5% CO₂. Subsequently, the connective tissue surrounding the pulmonary artery was carefully removed to obtain vascular rings measuring 3 mm in length. Each vascular ring was mounted between two triangular metal rings, with the upper end connected to a tension transducer and the lower end fixed. Changes in vascular ring tension were recorded using the RM6240E system. The water bath was continuously supplied with 95% O₂ and 5% CO₂ to maintain a volume of 15 mL K-H solution at 37℃. The K-H solution was replaced every 20 minutes, and the baseline tension was adjusted by 0.1 g increments every 5 minutes until a stable baseline tension of 0.75 g was reached. After stabilizing for 2 hours, this tension was recorded as the baseline tension of the vascular ring.\u003c/p\u003e\u003cp\u003eNext, the vascular rings were stimulated twice consecutively with 60 mmol/L KCl. If the difference between the contraction amplitudes of the two stimulations was less than 10%, the vascular rings were considered to have adequate vascular activity. To investigate the mechanism of Que on pulmonary artery contraction and relaxation function, endothelial removal was performed by wiping the vascular lumen with cotton wrapped around ophthalmic forceps. Endothelial removal was confirmed by stimulating the rings with Ach at 1\u0026times;10⁻⁶ mol/L; a relaxation degree of less than 10% indicated successful endothelial denudation, whereas a relaxation degree greater than 80% indicated intact endothelial function. After assessing endothelial function, the vascular rings were returned to baseline state by rinsing with K-H solution, and tension was recorded again. Specific signaling pathway inhibitors and potassium channel blockers were pre-incubated with the rings for 20 minutes. Following this, KCl was used to induce a stable contraction. Que was cumulatively added at concentrations of 1, 3, 10, 30, 100, and 300 \u0026micro;mol/L every 15 minutes. The control group received an equal volume of DMSO. Tension changes were recorded for each vascular ring, and the percentage of vasorelaxation induced by Que was calculated to evaluate the effect of pathway inhibitors and potassium channel blockers on pulmonary artery relaxation. The calculation formula for the percentage of vasorelaxation induced by Que was as follows: Vasorelaxation (%) = (Maximum contraction tension induced by KCl Tension- after Que treatment Maximum contraction tension induced by KCl) / (Maximum contraction tension induced by KCl - Baseline tension) * 100% (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Preparation and Treatment of Rat Tracheal Rings\u003c/h2\u003e\u003cp\u003eThe trachea was excised, cleaned, and cut into 3-mm rings, then mounted in the same setup as pulmonary arteries. Baseline tension was adjusted to 1.2 g and equilibrated for 2 h. Contractile activity was confirmed by two consecutive stimulations with 60 mmol/L KCl. Epithelium was mechanically removed and verified by \u0026lt;\u0026thinsp;10% relaxation to adrenaline; relaxation\u0026thinsp;\u0026gt;\u0026thinsp;80% indicated intact epithelium. After functional assessment, rings were washed and pre-incubated with inhibitors or K⁺ channel blockers for 20 min. Contraction was induced with Ach, followed by cumulative addition of Que (1\u0026ndash;300 \u0026micro;M) every 15 min; DMSO served as control. The calculation formula for the percentage of Que-induced tracheal relaxation was as follows: Tracheal Relaxation (%) = (Maximum contraction tension induced by Ach Tension- after Que treatment Maximum contraction tension induced by Ach)/ (Maximum contraction tension induced by Ach) -Baseline tension) * 100% (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Statistical analysis","content":"\u003cp\u003eData were statistically analyzed using GraphPad Prism 9 software, and logarithmic concentration-response relaxation curves were plotted. Quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance (ANOVA) was used for multiple group comparisons, and differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The maximal vasorelaxant response was expressed as Emax, and \"n\" represents the number of arteries tested.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Effects of Que on Relaxation of Pulmonary Arteries and Trachea with Intact and Denuded Endothelium.\u003c/h2\u003e\u003cp\u003eWe investigated the relaxant effects of Que at different concentrations (1, 3, 10, 30, 100, and 300 \u0026micro;mol/L) on pulmonary arteries and trachea from normal rats, examining the influence of intact and denuded endothelium on Que-induced relaxation of pulmonary artery and tracheal rings. The results showed that Que induced a concentration-dependent relaxation in both endothelium-intact and endothelium-denuded pulmonary artery and tracheal rings (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-H). Compared with the DMSO control group, the maximal relaxation (Emax) in pulmonary artery rings was 97.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26% in the endothelium-intact group (Que\u0026thinsp;+\u0026thinsp;End\u003csup\u003e+\u003c/sup\u003e) and 84.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2% in the endothelium-denuded group (Que\u0026thinsp;+\u0026thinsp;End\u003csup\u003e\u0026minus;\u003c/sup\u003e); in tracheal rings, the Emax was 103.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50% in the epithelium-intact group (Que\u0026thinsp;+\u0026thinsp;End\u003csup\u003e+\u003c/sup\u003e) and 83.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43% in the epithelium-denuded group (Que\u0026thinsp;+\u0026thinsp;End\u003csup\u003e\u0026minus;\u003c/sup\u003e). These results indicate that Que induces concentration-dependent relaxation of pulmonary artery and tracheal rings regardless of endothelial or epithelial integrity, although denudation attenuates the vasorelaxant effect of Que. This suggests that both endothelium-dependent and endothelium-independent mechanisms contribute to the vasorelaxation induced by Que.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Que induces relaxation of the pulmonary artery and trachea through NO rather than prostaglandins.\u003c/h2\u003e\u003cp\u003eTo further investigate whether Que-induced vasodilation is related to NO and prostaglandins, experiments were conducted using L-NAME (100 \u0026micro;mol/L), methylene blue (10 \u0026micro;mol/L), Indo (5 \u0026micro;mol/L), and dideoxyadenosine (200 \u0026micro;mol/L). The results demonstrated that, compared with the endothelium-intact group, both L-NAME and methylene blue significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-H), whereas Indo and dideoxyadenosine had no significant effect on the relaxation induced by Que in pulmonary artery and tracheal rings (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). Specifically, in pulmonary artery rings, the Emax was 80.06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82% and 87.93\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24% for L-NAME and methylene blue, respectively, while Indo and dideoxyadenosine showed Emax values of 96.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78% and 92.21\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84%. In tracheal rings, the Emax values for L-NAME and methylene blue were 85.26\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01% and 94.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50%, respectively, whereas Indo and dideoxyadenosine had Emax values of 99.74\u0026thinsp;\u0026plusmn;\u0026thinsp;4.44% and 101.25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47%. These findings suggest that Que induces relaxation of the pulmonary artery and trachea via the NO signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Que induces relaxation of the pulmonary artery and trachea through potassium ion channels.\u003c/h2\u003e\u003cp\u003eTo investigate whether Que-induced vasodilation is related to potassium channels, experiments were conducted using the non-selective potassium channel inhibitor TEA (5 mmol/L). The results showed that, compared with the endothelium-denuded group, TEA significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). Specifically, in pulmonary artery rings, the Emax induced by TEA was 57.91\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75%, while in tracheal rings, the Emax was 59.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31%. These findings indicate that Que induces relaxation of the pulmonary artery and trachea through potassium ion channels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Que primarily induces relaxation of the pulmonary artery and trachea through K\u003csub\u003eV\u003c/sub\u003e channels.\u003c/h2\u003e\u003cp\u003eTo further assess the role of K⁺ channels in Que-induced vasodilation, we first pretreated the pulmonary artery and trachea with the K\u003csub\u003eV\u003c/sub\u003e channel inhibitor. The experiment showed that pretreatment with the K\u003csub\u003eV\u003c/sub\u003e channel inhibitor 4-AP (1 mmol/L) significantly inhibited Que-induced relaxation of both the vessels and trachea (G P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Specifically, in the pulmonary artery, the Emax after 4-AP treatment was 65.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69%, while in the trachea, the Emax was 63.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94%. Given that Que-induced vasodilation is mediated by activation of K\u003csub\u003eV\u003c/sub\u003e channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, G). we further conducted experiments targeting multiple K\u003csub\u003eV\u003c/sub\u003e subtypes. Various K\u003csub\u003eV\u003c/sub\u003e channel subtypes are present in smooth muscle, including those responsible for maintaining vascular and tracheal tone: K\u003csub\u003eV\u003c/sub\u003e1.5, K\u003csub\u003eV\u003c/sub\u003e2.1, and K\u003csub\u003eV\u003c/sub\u003e7. To evaluate these subtypes, specific inhibitors were used: K\u003csub\u003eV\u003c/sub\u003e1.5 inhibitor DPO-1 (1\u0026times;10⁻⁵ mol/L), K\u003csub\u003eV\u003c/sub\u003e2.1 inhibitor RY796 (1\u0026times;10⁻⁵ mol/L), and K\u003csub\u003eV\u003c/sub\u003e7 inhibitor linopirdine (10 \u0026micro;mol/L). The results demonstrated that, compared with the endothelium-denuded group, linopirdine significantly inhibited Que-induced relaxation of pulmonary artery and tracheal rings (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, H), whereas other subtype inhibitors did not significantly affect Que-induced relaxation in these tissues (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E, I-J). Specifically, in pulmonary artery rings, the Emax values were 71.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85% for linopirdine, 82.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22% for DPO-1, and 86.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39% for RY796; in tracheal rings, the corresponding Emax values were 66.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24% for linopirdine, 76.52\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13% for DPO-1, and 81.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10% for RY796. These findings indicate that the vasodilatory effect of Que is associated with its activation of voltage-gated potassium channels, particularly the K\u003csub\u003eV\u003c/sub\u003e7 subtype, while K\u003csub\u003eV\u003c/sub\u003e1.5 and K\u003csub\u003eV\u003c/sub\u003e2.1 channels are not involved.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Que induces relaxation of the pulmonary artery and trachea through large-conductance calcium-activated potassium (BK\u003csub\u003eCa\u003c/sub\u003e) channels.\u003c/h2\u003e\u003cp\u003eTo further evaluate the role of K\u003csub\u003eCa\u003c/sub\u003e channels in Que-induced vasodilation, we incubated pulmonary artery and trachea tissues with inhibitors of BK\u003csub\u003eCa\u003c/sub\u003e, IK\u003csub\u003eCa\u003c/sub\u003e, and SK\u003csub\u003eCa\u003c/sub\u003e channels. The experiments revealed that pretreatment with the BK\u003csub\u003eCa\u003c/sub\u003e channel inhibitor Iberiotoxin (0.1 \u0026micro;mol/L) significantly attenuated Que-induced relaxation of both the vessels and trachea (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, F). In contrast, pretreatment with the IK\u003csub\u003eCa\u003c/sub\u003e and SK\u003csub\u003eCa\u003c/sub\u003e channel inhibitors TRAM-34 (20 nmol/L) and Apamin (100 nmol/L), respectively, had no significant effect on Que-induced vasorelaxation in these tissues (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D, G-H). Specifically, in the pulmonary artery, the Emax was 70.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06% with Iberiotoxin, compared to 82.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88% and 82.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84% with TRAM and Apamin, respectively. In the trachea, the Emax values were 68.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59% for Iberiotoxin, and 80.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50% and 82.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84% for TRAM-34 and Apamin, respectively. These results indicate that Que-induced vasodilation is associated with the activation of K\u003csub\u003eCa\u003c/sub\u003e channels, particularly the BK\u003csub\u003eCa\u003c/sub\u003e subtype, while IK\u003csub\u003eCa\u003c/sub\u003e and SK\u003csub\u003eCa\u003c/sub\u003e subtypes do not appear to be involved.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e4.6 The effects of inwardly rectifying potassium channels (K\u003c/b\u003e\u003csub\u003e\u003cb\u003eir\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e), ATP-sensitive potassium channels (K\u003c/b\u003e\u003csub\u003e\u003cb\u003eATP\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e), and two-pore domain potassium channels (K\u003c/b\u003e\u003csub\u003e\u003cb\u003e2P\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) on que-induced relaxation of the pulmonary artery and trachea.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe further investigated the effects of inhibitors of three other types of potassium channels (K\u003csub\u003eir\u003c/sub\u003e, K\u003csub\u003eATP\u003c/sub\u003e, and K\u003csub\u003e2P\u003c/sub\u003e) on que-induced relaxation of the pulmonary artery and trachea. The experiments showed that pretreatment with the K\u003csub\u003eir\u003c/sub\u003e, K\u003csub\u003eATP\u003c/sub\u003e and K\u003csub\u003e2P\u003c/sub\u003e channel inhibitors BaCl₂ (0.1 mmol/L), Gli (1\u0026times;10⁻⁵ mol/L), and TASK-1-IN-1 (1\u0026times;10⁻⁶ mol/L) respectively had no significant effect on the vasorelaxant and tracheal relaxant actions induced by Que (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Specifically, in the pulmonary artery, the maximum relaxation rates induced by BaCl₂, Gli, and TASK-1-IN-1 were 81.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97%, 80.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46% and 82.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46%, respectively. In the trachea, the maximum relaxation rates were 83.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26%, 81.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29%, and 80.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, the regulation of contraction and relaxation of vascular and airway smooth muscle has become a focal area of research, especially for the treatment of diseases such as PAH and AHR. Modulation of smooth muscle tone holds significant clinical importance in these conditions. Previous studies have confirmed that flavonoids, a class of natural polyphenolic compounds derived from plants, exhibit diverse biological activities, particularly showing potential in vascular relaxation and airway dilation\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Among them, Que has attracted considerable attention due to its multitarget and multipathway mechanisms. Its vasodilatory effects are mediated by promoting NO release from ECs\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, inhibiting the production of endothelin-1 and angiotensin II\u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, and modulating multiple ion channels, enzymes, and signaling pathways. According to the literature, Que can relax rat aorta\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, coronary arteries\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, and mesenteric arteries\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Additionally, it activates potassium ion channels and facilitates the release of anticholinergic neurotransmitters, thereby reducing gastrointestinal smooth muscle contraction\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In AHR-related diseases, Que inhibits the release of pro-contractile mediators, such as histamine and leukotrienes, while regulating cyclic adenosine monophosphate (cAMP) levels and activating phosphodiesterase enzymes to promote airway smooth muscle relaxation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. These effects not only alleviate airway obstruction but also reduce airway hyperreactivity, providing a potential pharmacological basis for the treatment of asthma and related disorders.\u003c/p\u003e\u003cp\u003eThis study systematically elucidates, for the first time, that Que induces concentration-dependent relaxation of rat pulmonary arteries and trachea through a dual mechanism involving NO signaling and activation of potassium channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In a series of isolated tissue experiments, removal of the endothelium or epithelium attenuated, but did not completely abolish, the relaxant effect of Que, indicating that it exerts relaxation via both endothelium-dependent and epithelium-independent pathways. Notably, even after endothelial or epithelial denudation, a residual relaxation response persisted (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H), suggesting that Que directly targets smooth muscle ion channels, consistent with mechanisms reported for other flavonoids\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Further investigation employing inhibitors of endothelium-dependent relaxant factors-including the NO synthase inhibitor L-NAME, guanylate cyclase inhibitor methylene blue, cyclooxygenase inhibitor indomethacin, and adenylate cyclase inhibitor 2',5'-dideoxyadenosine-demonstrated that L-NAME markedly reduced Que-induced vasorelaxation, and methylene blue similarly diminished its relaxant effect, whereas indomethacin and 2',5'-dideoxyadenosine had no significant impact (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings underscore the primary role of the NO signaling pathway in Que-mediated relaxation of pulmonary arteries and trachea. Previous studies also indicate that Que enhances the expression and activity of endothelial NO synthase, increases NO production, and thereby activates the adenylate cyclase pathway, culminating in smooth muscle relaxation\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt is noteworthy that the NO/cGMP/PKG signaling cascade, through modulation of potassium channels, plays a crucial role in vascular relaxation\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, cardioprotection\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e and neural regulation\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. SMCs express various potassium channels, including K\u003csub\u003eV\u003c/sub\u003e, K\u003csub\u003eCa\u003c/sub\u003e, K\u003csub\u003eATP\u003c/sub\u003e, inwardly rectifying potassium channels (K\u003csub\u003eir\u003c/sub\u003e and K\u003csub\u003e2P\u003c/sub\u003e)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. In this study, non-specific K⁺ channel blocker TEA, the K\u003csub\u003eV\u003c/sub\u003e channel blocker 4-AP, and BK\u003csub\u003eCa\u003c/sub\u003e channel blocker iberiotoxin all attenuated Que-induced relaxation, further indicating that Que mediates its effects primarily through activation of multiple potassium channel types. Beyond promoting K⁺ efflux to cause membrane hyperpolarization and inhibit Ca\u0026sup2;⁺ channel opening, potassium channels enhance endothelial K⁺ flux, which facilitates NO release and vasodilation by modulating membrane potential and smooth muscle tone\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Conversely, the K\u003csub\u003eATP\u003c/sub\u003e channel blocker glibenclamide, the acid-sensitive potassium channel inhibitor TASK-1-IN-1, and the SK\u003csub\u003eCa\u003c/sub\u003e channel blocker apamin did not affect Que's relaxant action. This implies that K\u003csub\u003eATP\u003c/sub\u003e and TASK-1 channels are not primary targets of Que, which may also relate to tissue- and cell-specific distribution and function of different potassium channels, reflecting certain organ-specific molecular targeting by Que.\u003c/p\u003e\u003cp\u003eVasorelaxation of blood vessels and trachea can occur via both endothelium-dependent and endothelium-independent mechanisms. In endothelium-independent vasorelaxation, potassium channels play a pivotal role. Collectively, this study confirms that Que mediates pulmonary artery and tracheal smooth muscle relaxation through synergistic activation of the NO signaling pathway and multiple potassium channels, delineating its underlying mechanism. These findings provide a theoretical foundation for the potential therapeutic utility of Que in PAH and AHR disorders.\u003c/p\u003e\u003cp\u003eNotably, this study has several limitations. All experiments were performed using isolated tissue preparations from healthy rats, which may not fully replicate the complex in vivo environment, including neurohumoral regulation, blood flow, and disease-specific alterations in pulmonary arteries or airways. Therefore, the effects of Que in pathological conditions such as PAH or AHR remain to be confirmed. Moreover, although we identified the involvement of NO signaling and specific potassium channels, the detailed downstream molecular pathways and potential crosstalk between different ion channels warrant further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that Que induces potent, concentration-dependent relaxation of rat pulmonary artery and tracheal smooth muscle. The relaxant effects of Que are significantly reliant on the presence of an intact endothelium and the activation of the NO signaling pathway, as evidenced by the attenuation of vasodilation following endothelial removal and inhibition of NO synthase and guanylate cyclase. Additionally, our findings reveal that multiple potassium channels, particularly K\u003csub\u003eV\u003c/sub\u003e and BK\u003csub\u003eCa\u003c/sub\u003e channels, play crucial roles in mediating Que-induced relaxation, as blockers targeting these channels markedly reduced its effects. The interplay between NO signaling and potassium channel activation likely underpins the mechanism by which Que modulates smooth muscle tone in both vascular and airway tissues. Overall, this research provides important mechanistic insights into Que\u0026rsquo; s bioactivity and supports its potential therapeutic application for conditions characterized by pulmonary arterial and airway hypercontractility, such as PAH and AHR.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHong-Yan Sun: Writing original draft, Formal analysis, Data curation. Mei-Yang Xu: Data curation. Zi-Yi Chen: Data curation. Ying Tang: Methodology, Data curation. Yu-Fei Ke: Data curation. Cheng-Wen Fu: Data curation. Rui-Yang Chen: Data curation. Guo-Qing Lu: Methodology, Data curation. Le-Qiang Liu: Data curation. Qin Gao: Writing review \u0026amp; editing. Bi Tang: Writing review \u0026amp; editing, Funding acquisition, Conceptualizaion. Pin-Fang Kang: Writing review \u0026amp; editing, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll studies were approved by the Medical Ethics Committee of Bengbu Medical University has passed the ethics examination and approval. The reference number is as follows: [2019] number 074.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82500426); the Natural Science Foundation of Anhui Province (2208085MH192); the Anhui Province Outstanding Youth Project (2022AH030141); and the Excellent Scientific Research and Innovation Team of Anhui Province (2022AH010083).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;National Outstanding Youth Science Fund Project of NationalNatural Science Foundation of china (82500426), \u0026nbsp;Anhui Province Natural Foundation of China (Grant nos. 2208085MH192), \u0026nbsp;Anhui Province Outstanding Youth Project of China (2022AH030141), Anhui Province excellent scientific research and innovation team of China (2022AH010083).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFRISMANTIENE A, PHILIPPOVA M, ERNE P, et al.Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity[J].Cell Signal,2018,5248-64.\u003c/li\u003e\n\u003cli\u003eCHEN Y L, DANEVA Z, KUPPUSAMY M, et al.Novel Smooth Muscle Ca(2+)-Signaling Nanodomains in Blood Pressure Regulation[J].Circulation,2022,146(7) 548-64.\u003c/li\u003e\n\u003cli\u003eARTEAGA-SOLIS E, ZEE T, EMALA C W, et al.Inhibition of leptin regulation of parasympathetic signaling as a cause of extreme body weight-associated asthma[J].Cell Metab,2013,17(1) 35-48.\u003c/li\u003e\n\u003cli\u003eAMRANI Y, PANETTIERI R A.Airway smooth muscle: contraction and beyond[J].Int J Biochem Cell Biol,2003,35(3) 272-6.\u003c/li\u003e\n\u003cli\u003eKERR P M, WEI R, TAM R, et al.Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback[J].Vascul Pharmacol,2015,74130-8.\u003c/li\u003e\n\u003cli\u003eGUNTUR D, OLSCHEWSKI H, ENYEDI P, et al.Revisiting the Large-Conductance Calcium-Activated Potassium (BKCa) Channels in the Pulmonary Circulation[J].Biomolecules,2021,11(11) \u003c/li\u003e\n\u003cli\u003eTYKOCKI N R, BOERMAN E M, JACKSON W F.Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles[J].Compr Physiol,2017,7(2) 485-581.\u003c/li\u003e\n\u003cli\u003eSCHUBERT R, NELSON M T.Protein kinases: tuners of the BKCa channel in smooth muscle[J].Trends Pharmacol Sci,2001,22(10) 505-12.\u003c/li\u003e\n\u003cli\u003eRUOPP N F, COCKRILL B A.Diagnosis and Treatment of Pulmonary Arterial Hypertension: A Review[J].Jama,2022,327(14) 1379-91.\u003c/li\u003e\n\u003cli\u003eSHEN H, GAO Y, GE D, et al.BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension[J].Circulation,2024,150(2) 132-50.\u003c/li\u003e\n\u003cli\u003eMARTIN J, DUGUET A, EIDELMAN D.The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease[J].European Respiratory Journal,16(2) 349-54.\u003c/li\u003e\n\u003cli\u003eCAMORETTI-MERCADO B, LOCKEY R F.Airway smooth muscle pathophysiology in asthma[J].J Allergy Clin Immunol,2021,147(6) 1983-95.\u003c/li\u003e\n\u003cli\u003eREN K, LIU H, GUO B, et al.Quercetin relieves D-amphetamine-induced manic-like behaviour through activating TREK-1 potassium channels in mice[J].Br J Pharmacol,2021,178(18) 3682-95.\u003c/li\u003e\n\u003cli\u003eMODZELEWSKA B, DRYGALSKI K, KLESZCZEWSKI T, et al.Quercetin relaxes human gastric smooth muscles directly through ATP-sensitive potassium channels and not depending on the nitric oxide pathway[J].Neurogastroenterol Motil,2021,33(7) e14093.\u003c/li\u003e\n\u003cli\u003eDIAS P, SALAM R, POUROV\u0026aacute; 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This study aimed to systematically investigate the relaxant effects of Que on rat pulmonary artery and trachea, and to explore the roles of endothelium and potassium ion channels in its mechanism of action. Isolated pulmonary artery rings and tracheal rings from Sprague-Dawley rats were placed in an ex vivo tissue bath system. Relaxation was evaluated by the percentage of vasodilation in response to different concentrations of Que (1-300 \u0026micro;mol/L) against contractions induced by potassium chloride (KCl) or acetylcholine (ACh) in both endothelium-intact and endothelium-denuded rings. The influences of nitric oxide (NO) and prostacyclin was evaluated using L-NAME, methylene blue, indomethacin, and dideoxyadenosine. Endothelium-denuded rings were pretreated with various potassium channel blockers including TEA, 4-AP, linopirdine, RY796, DPO-1, iberiotoxin, TRAM-34, apamin, BaCl₂, glibenclamide, and TASK-1-IN-1. Que induced concentration-dependent relaxation in KCl-precontracted pulmonary artery rings. Removal of the endothelium, as well as pre-incubation with L-NAME and methylene blue, significantly attenuated this relaxant effect. Moreover, the Que-induced relaxation was markedly reduced by potassium channel blockers such as TEA, 4-AP, and iberiotoxin. Similar mechanistic dependencies were observed in the relaxant response of tracheal smooth muscle to Que. In conclusion, Que produces significant relaxation of rat pulmonary artery and trachea, which is dependent on the integrity of the endothelium, activation of the NO signaling pathway, and involvement of multiple potassium channels, including voltage-gated (K\u003csub\u003eV\u003c/sub\u003e) and calcium-activated (K\u003csub\u003eCa\u003c/sub\u003e) potassium channels.\u003c/p\u003e","manuscriptTitle":"Quercetin relaxes rat pulmonary arteries and trachea ex vivo: Role of nitric oxide and K⁺ channels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 11:25:27","doi":"10.21203/rs.3.rs-7873590/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00c3ca87-1944-4e42-bfe2-56f7af14f3e2","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57818982,"name":"Biological sciences/Biochemistry"},{"id":57818983,"name":"Health sciences/Cardiology"},{"id":57818984,"name":"Biological sciences/Drug discovery"},{"id":57818985,"name":"Health sciences/Medical research"},{"id":57818986,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-11-14T13:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 11:25:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7873590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7873590","identity":"rs-7873590","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}