none mesylate for 30 min, cells were stimulated without or with PA for 30 min and then washed thrice with PBS. For another experiment, cells were pretreated with tectorigenin for 30 min. After loading with 5 mM DAF-FM DA at 37uC for 30 min and kept in the dark, the HUVECs were rinsed three times with PBS. After that, cells were incubated with insulin at 37uC for 5 min, and fixed in 2% paraformaldehyde at 4uC for another 5 min. Fixed cells were examined using an order Piceatannol Olympus IX81 inverted microscope with attached charge-coupled device camera Tectorigenin Attenuates Endothelial Dysfunction using appropriate filters with a peak excitation wavelength of 495 nm and a peak emission wavelength of 515 nm. Determination of the decreased mitochondrial membrane potential Mitochondrial membrane potential production was assessed using the fluorescent dye 5,59,6,69-tetrachloro-1,19,3,39-tetraethylimidacarbocyanineiodide. When HUVECs grown to conuence in 48-well plates, cells were washed three times with PBS, then starved in serum-free medium for 12 h. Pretreated with tectorigenin, glutathione reduced or Mitoquinone mesylate for 30 min, cells were stimulated with PA for additional 30 min and then washed with PBS. After loading with 25 mg/ml JC-1 at 37uC for 30 min and kept in the dark, the HUVECs were rinsed three times with PBS and fixed in 2% paraformaldehyde at 4uC for 5 min. In addition, a positive control which will induce potent collapse of mitochondrial membrane potential was designed. In this group, HUVECs were treated with 10 mM carbonylcyanide-m-chlorophenylhydrazone for 20 min without PA stimulation before loading with JC-1. Fixed cells were examined using an Olympus IX81 inverted microscope with attached charge-coupled device camera using appropriate filters with a peak excitation wavelength of 585 nm and a peak emission wavelength of 590 nm. ence in six-well plates and then incubated with tectorigenin, salicylate, wortmannin or PD98059 for 0.5 10608278 h, respectively, and then stimulated with PA for 2 h. After incubation, cells were treated with insulin for 20 min in the continued presence of the inhibitors or tectorigenin. Total RNA was extracted from the cells using TRNzol Plus and its concentration was calculated from spectrophotometric measurements at 260 nm. Total RNA from each RNA sample was reverse-transcribed into cDNA with the RT kit. Comparison to b-actin values was carried out for normalization. Relative expression levels in each sample were determined by comparison with the standard. The PCR products were electrophoresed in 1.5% agarose gel with 0.5 mg/mL ethidium bromide and visualized under UV light, then the signal intensity was detected by Bio-Rad image system and analyzed with computer program Quantity One. Preparation of aortic rings and assessment of endothelium-dependent relaxation Rats were killed by cervical dislocation. The thoracic aorta was immediately removed and placed in 4uC Krebs-Henseleit solution 0.026, glucose 5.0, mM, pH 7.4) and gassed with 95% O2 5% CO2. The dissected aortae were cleaned of connective tissue and cut into rings. Take care to avoid 
abrading the intimal surface to maintain the integrity of the endothelial layer. For measurement of vascular responses, aortic rings were suspended in an organ bath containing 30 mL of 18004284 K-H solution maintained at 37uC, pH 7.4, and continuously aerated with 95% O2 and 5% CO2. A resting tension of 2.0 g was applied to the aortic ring and changes in tension were measured with a