Phenylephrine pe

These rats were anesthetized with intraperitoneal injection of 25% urethane (Sigma-Aldrich Chemical Company). Once they were completely anesthetized, the thoracic aortae of rats were rapidly taken out and stored at precooled Krebs–Henseleit solution for later arterial circle preparation (2–3 nm). These prepared circles were mixed with 5% CO₂/95% O₂, followed by a water bath at 37°C. Data were recorded and processed by BL-420F bio-functional experiment system (Chengdu Thai Union Biological Technology Co, Ltd, Sichuang, China). These arterial circles were given a tension of 1.0 g, and balanced for 60 minutes. After maximum amplitude of vasoconstriction was induced by phenylephrine (PE, 10 μmol/L, Sigma), endothelial vasodilation function was detected at different concentrations of sodium nitroprusside (10⁻⁷–10⁻³ mol/L, Beijing Double-Crane Pharmaceutical Co, Ltd, Beijing, China) or at different concentrations of acetylcholine (10⁻⁶–10⁻³ mol/L, Sigma-Aldrich Chemical Company).

Next, the thoracic aorta was carefully removed and stripped of adherent tissue. Starting at the diaphragm, the aorta was cut in 6 segments of 2 mm width using a calibrated stereomicroscope. Starting from the aortic arch, thoracic aorta (TA) segments TA2 to TA5 were immersed in KR solution, aerated with a 95% O₂/5% CO₂ gas mixture. Isometric contractions and relaxations were measured on segments TA4-TA5 by means of a Statham UC2 force transducer (Gould, United States). The aortic segments were stretched and stabilized to a preload of 25 mN. VSMCs were stimulated using the α₁-adrenergic agonist phenylephrine (PE, 2 × 10^-6 M) (Sigma-Aldrich, Belgium) and NΩ-nitro-l-arginine methyl ester (L-NAME, 3 × 10^-4 M) (Sigma-Aldrich, Belgium) was used to inhibit the endothelial nitric oxide synthase (eNOS).

PQQ was obtained in the form of a disodium salt from Nascent Health Sciences LCC (NY, USA). Phenylephrine (PE) was from Sigma-Aldrich (MO, USA). Primary antibodies specific for YAP, GPX4, FSP1, and CoQ10 were from ABclonal (A1002, A1933, A12128, and A15193), while antibodies specific for p-YAP, BNP, β-actin, GAPDH, and Histone H3, as well as secondary goat anti-rabbit and goat anti-mouse IgG were from Wuhan Servicebio Technology Co., Ltd. (GB114060, GB11667, GB12001, GB15002, GB11026, GB23303, and GB23301). The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (S0033) and the mitochondrial membrane potential assay kit JC-1 (C2006) were purchased from the Beyotime Institute of Biotechnology. A test kit for measuring ferrous ion concentration (E-BC-K773-M) was purchased from Elabscience (Wuhan, China), and the kits for measuring malondialdehyde (MDA) and glutathione (GSH) levels were obtained from Nanjing Jiancheng Institute of Biological Engineering (A003-4-1, A006-2–1). Ferrostatin-1(Fer-1) was purchased from Selleck Chemicals (USA). Gibco (CA, United States) was the source of all cell culture reagents.

The thoracic segments of the rat aorta were dissected and the surrounding connective tissues were cleaned off. Each aorta was cut into ring segments of 2~3 mm in length. The aortic rings were mounted in organ chambers filled with Krebs-Henseleit (KH) solution at 37°C with constant bubbling of 95% oxygen/5% carbon dioxide. KH solution contained (mM) 133 NaCl, 4.75 KCl, 1.5 CaCl₂, 1.25 MgCl₂, 25 NaHCO₃, and 11 D-glucose. Isometric tension was recorded with a force transducer (RM6240C, Chengdu Instrument Factory). A basal tension of 20 mN was applied to each vascular ring. After being placed in organ baths for 90 min, 0.5 μM phenylephrine (PE, Sigma) was first administered to the rings to test their contractility and then 1 μM acetylcholine (ACh, Sigma) was administered to assess the integrity of the endothelial layer. Rings with less than 80% relaxation response to ACh were discarded. The aortic rings were pretreated with or without 10 μM SAL for 30 min before H₂O₂ 100 μM was added to the bath. After precontracting with PE (0.1 μM), ACh (1 × 10⁻⁸~1 × 10⁻⁴M) was added cumulatively to the bath to evoke the endothelium-dependent relaxation. The relaxation rate is the ratio between the tension relaxed by ACh and the tension contracted by PE.

ET-1 was from Merck Millipore, prepared as a 200 μM stock in 5% acetic acid and applied at 100 nM. Phenylephrine (PE) was from Sigma-Aldrich, prepared as a 10 mM stock in water and applied at 10 μM. Inhibitor or vehicle was applied 30 min prior to cell stimulation with agonist. BQ123 was from Merck Millipore, prepared in water and was used at 1 μM. Prazosin was from Tocris, prepared in DMSO and was applied at 100 nM. Dynole (Dynole 34-2) was from Tocris, was prepared in Ethanol and was applied at 10 μM. PD184352 (PD) was from Sigma-Aldrich, prepared in DMSO and used at 10 μM. Actinomycin D (Act D) was from Tocris, prepared in DMSO and applied at 2 μg/ml. Controls were exposed to vehicle.