Polygonum viviparum L. (PV) was obtained from Tibet. Its authenticity was confirmed by Dr Shin-Ming Ku (Herbarium, Biodiversity Research Center, Academia Sinica, Taipei, Taiwan). The herb (PV 100 g) was extracted with 3 L of 2-propanol for 7 days, then the extract was filtered and centrifuged at 13 000 × g for 10 min. The extract supernatant was passed through a 0.22 μm sterile filter (Millipore, Billerica, MA, USA) and first concentrated using a vacuum rotary evaporator (Yamato, Tokyo, Japan) at 40°C. Normally, 8.76 g of dried powder could be obtained from 100 g of PV. The dried extract yield from the crude material was thus approximately 8.76% .
Drugs and chemicals
Phenylephrine (PE), acetylcholine (Ach), N
G-nitro-l-arginine methyl ester (l-NAME), N
G-monomethyl-l-arginine acetate (l-NMMA), methylene blue (MB), hemoglobin (Hb), and all chemicals of the Krebs solution were from Sigma Chemical (St. Louis, MO, USA). Culture materials (M199 medium, fetal bovine serum (FBS), and trypsin-EDTA) were obtained from Life Technologies (Gibco, Grand Island, NY, USA). Endothelial cell growth supplement (ECGS) was purchased from Millipore (Billerica, MA, USA). A cyclic guanosine 3′,5′-monophosphate (cGMP) enzyme immunoassay kit was purchased from R & D System (Minneapolis, MN, USA). All other agents of cell culture were obtained from Sigma Chemical.
Preparation of rat aorta and tension recording ex vivo
Male Sprague-Dawley rats weighing about 250 ~ 350 g were purchased from BioLASCO (Taipei, Taiwan). All animal procedures were approved by the institutional animal care and using committee of Taipei Medical University. Animals were housed in polycarbonate cages in a room at 22 ± 2°C on a 12-h light-dark cycle. The procedure described by Hu et al.  was used to isolate rat aortic rings. When the experiment began, the rats were sacrificed by exsanguination from the carotid artery under lose consicousness by knocking medulla; the thoracic aorta of rats was carefully removed, and the fat and tissue were dissected away in normal Krebs’ buffer at an adjusted pH of 7.4. The composition of this buffer was as follows: 118.5 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM NaHCO3, 11.1 mM glucose, and 2.5 mM CaCl2. The aorta was cut into rings about 5 mm long in Krebs buffer which was constantly gassed with 95% O2 + 5% CO2 at 37 ± 0.5°C. Two “L”-type stainless steel hooks were inserted into the aortic lumen; one side was fixed in the bottom bath and the other side was connected to a force transducer using a cotton thread. The aortic rings were equilibrated in Krebs buffer and maintained under a 1-g tension for 60 ~ 90 min, with three changes of buffer, before the experimental procedures began. Contractions were recorded isometrically via an iWorx FT-302 force transducer connected to an iWorx 304 T recorder (iWorx System, Dover NH, USA). In denuded aorta, the endothelium was removed by rubbing with a cotton ball, and the absence of ACh-induced relaxation was taken as an indicator of successful denudation. After PE-induced contraction and ACh-induced relaxation twice, PV (1, 3, 10, 30, 100 and 300 μg/ml) was treated after PE-induced contraction. The effects of PV as percentage of relaxation considering the maximum contraction elicited by phenylephrine in preparations with at least 80% of relaxation to ACh.
Cyclic guanosine 3′,5′-monophosphate (cGMP) measurement
Rat aorta cGMP was analyzed using the method of Kauffman et al. . Briefly, the aorta was immediately isolated from a rat, and cut into segments of about 20 mg/tissue. First, the rat aortic rings were pre-incubated in Krebs’ solution with 3-isobutyl-1-methylxathine (IBMX, 10 μM) for 5 min. Then, the aortic segments were placed in Ca2+-free Krebs’ (EGTA 2.5 mM) buffer or pretreatment inhibitors of l-NAME (50 μM) and MB (10 μM) for 10 min; then PV (100 μg/ml), sodium nitroprusside (SNP, 10 μM), and ACh (10 μM), as positive reagents, were added for a further 2 min. After incubation with PV and ACh, the aortic segments were rapidly frozen in liquid nitrogen and stored at -80°C until homogenized in 0.5 ml of 10% trichloroacetic acid using a motor-driven glass homogenizer. The homogenate was centrifuged at 10,000 × g for 5 min, and the supernatant was removed and extracted three times with 1.5 ml of water-saturated diethyl ether. The cGMP content was then assayed using enzyme immunoassay kits (R & D System, Minneapolis, MN, USA). Protein was measured by dissolving the trichloroacetic acid precipitate in 1 ~ 2 ml of 5 N NaOH followed by analysis using the method of Lowry et al. .
Human umbilical vein endothelial cells (HUVECs, confluent second passage, P = 2) were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were grown at 37 ± 0.5°C in a humidified 5% CO2 atmosphere in M199 medium (pH 7.4) supplemented with 10% FBS, 25 U/ml heparin, 30 μg/ml ECGS, 2 mM glutamine, 1.5 g/l NaHCO3, 10,000 units/l of penicillin, and 100 mg/l of streptomycin. Culture plates were coated with 1% gelatin before use. Confluent cells were detached by trypsin-EDTA (0.05%: 0.02%, v/v), and cells from passages 3 to 7 were used in the experiment.
Determination of nitric oxide (NO) production
HUVECs (5 × 105 cells/well) in 6-well plates were incubated with or without various concentrations of PV (10, 30, and 100 μg/ml) and ACh (30 μM), as a positive control, for 1 h. The supernatants of conditioned cells were deproteinized by zinc-sulfate (30%, v/v) and passed through a copper cadmium reduction column to reduce NO3
- to NO2
-. As an indicator of NO production, the nitrite concentration in the culture medium was determined using the Griess reagent, as previously described . The culture supernatant (100 μl) was mixed with 100 μl of the Griess reagent (1% sulfanilamide and 0.1% N-1-naphthyl ethylenediamine) for 10 min, and then the chromophoric azo-derivative molecule’s absorbance was measured in a microplate reader at 540 nm. Fresh culture medium was used as the blank in all experiments. A range of dilutions of sodium nitrite (NaNO2) was used to create a standard curve with the amount of nitrite in each sample. The final NO production was expressed as μmol/l (μM).
Preparation of total cell lysates
HUVECs (5 × 105 cells/well) in 6-well plates were incubated with or without concentrations of PV (3, 10, and 30 μg/ml) or ACh (30 μM) for 0.5 or 24 h. Total cell lysates were obtained using a lysis buffer (250 mM Tris-HCl (pH 6.8), 1% Triton-100, 0.1% sodium dodecylsulfate (SDS), 1 mM Na3VO4, 1 mM EDTA, 5 mM sodium fluoride, 1 mM PMSF, and 1 mg/ml leupeptin), and cell debris was removed using a centrifuge at 10,000 × g for 10 min at 4°C and stored at -80°C until required. The protein content of the cell lysates was determined using the Bradford assay .
Western blot analysis
Equal amounts of cell lysates (30 μg) were electroblotted onto a nitrocellulose membrane (Millipore), following separation using 8% ~ 12% SDS-polyacrylamide gel electrophoresis (PAGE). The blot was probed using a primary antibody against p-eNOS, total-eNOS (Millipore), Nrf2, HO-1, and β-actin (Santa Cruz Biochemicals, Santa Cruz, CA, USA). The intensity of each band was quantified using density analysis software (Biospectrum 500 Imaging System; Vision Works LS 6.5.2v, UK), and the density ratio represented the relative intensity of each band against controls in each experiment.
Data and statistical analyses
Results of all experiments are expressed as the mean ± S.E. of multiple experiments (n ≥ 3). Data were compared using a one-way analysis of variance (ANOVA) with a post-hoc Bonferroni analysis when applicable, and p values of < 0.05 were considered statistically significant. Values of 50% effective concentration (EC50) were calculated and obtained from 5 regression lines; each regression line was constructed from 3 ~ 5 points. Values of inhibition of these points ranged 20% ~ 80%.