Dried roots of Polygonum multiflorum were purchased from Hwa Lim Natural Drug Co., Ltd. (Busan, Korea) in September 2010 and authenticated by one of the authors (Jin Woo Hong). A voucher specimen (accession number, PDRLCW-1) was deposited in the Plant Drug Research Laboratory of Pusan National University (Miryang, Korea). The dried roots of Polygonum multiflorum (1.0 kg) were ground to a fine powder, after which they were subjected to successive extraction at room temperature with n-hexane, ethyl acetate and methanol. Briefly, filtration and evaporation of HEPM was performed under reduced pressure at 45°C, followed by lyophilization, which yielded a white powder of hexane extracts (2.59 g). Sequential extraction of the remaining powder was performed using ethyl acetate and methanol to yield ethyl acetate extracts (EAEPM; 9.30 g) and methanol extracts (MEPM; 150.40 g) of Polygonum multiflorum, respectively. Finally, the solid form of extract was dissolved with dimethyl sulfoxide (DMSO) for use in subsequent experiments.

Male mice (C57BL/6J and eNOS deficient; weighing, 20–25 g) were housed under diurnal lighting conditions and allowed food and tap water ad libitum. All animal procedures were conducted in accordance with the institutional guidelines for animal research, and were approved by the Pusan National University - Institutional Animal Care and Use Committee (PNU-IACUC; Busan, Republic of Korea) on ethical procedures and scientific care (PNU-2011-000367). Anesthesia was achieved by face mask-delivered isoflurane (2% induction and 1.5% maintenance, in 70% N₂O and 30% O₂). Sufficient depth of anesthesia was confirmed by the absence of cardiovascular changes in response to a tail pinch. Rectal temperature was kept at 36.5–37.5°C using a Panlab thermostatically controlled heating mat (Harvard Apparatus, Holliston, MA, USA). To determine whether Polygonum multiflorum was able to protect against ischemic stroke, EAEPM, MEPM and HEPM (100 mg/kg, intraperitoneally) were separately administered to the mice 30 min prior to ischemic insult. Focal cerebral ischemia was then induced using the photothrombotic cortical ischemia model (13). Briefly, 0.1 ml of a 10 mg/ml solution of Rose bengal (Sigma-Aldrich, St. Louis, MO, USA) in sterile saline was injected intraperitoneally 5 min prior to illumination. The mice were then placed in a stereotaxic frame and the midline scalp was incised, pericranial tissues were dissected, and the bregma and lambda points were identified. A fiber optic bundle with a KL1500 LCD cold light source (Carl Zeiss, Jena, Germany) and a 4 mm aperture was then centered 2 mm laterally from the bregma using a micromanipulator. Following this, the brain was illuminated through the intact skull for 15 min, the surgical wound was then sutured and the mice were allowed to recover from anesthesia. Brains were removed 24 h following ischemic insult. Cerebral infarct size was determined based on analysis of 2,3,5-triphenyltetrazolium chloride (TTC)-stained, 2-mm-thick brain sections and infarction areas were quantified using the iSolution full image analysis software (Image & Microscope Technology, Vancouver, Canada). To account for and eliminate the effects of swelling/edema, infarction volume was calculated using an indirect measurement by summing the volumes of each section according to the following formula: contralateral hemisphere (mm³) − undamaged ipsilateral hemisphere (mm³).

Neurological deficit was scored in each mouse 24 h after the ischemic insults in a blinded fashion according to a 0–4 scoring system in which 0 = no deficit; 1 = forelimb weakness and torso turning to the ipsilateral side when held by the tail; 2 = circling to the affected side; 3 = unable to bear weight on the affected side and 4 = no spontaneous locomotor activity or barrel rolling (14).

Vestibulo-motor function was assessed using a wire-grip test 24 h after cerebral ischemia (15). Briefly, mice were placed on a metal wire (45 cm long) suspended 45 cm above protective padding and allowed to traverse the wire for 60 sec. The latency for which the mice remained on the wire within a 60 sec interval was measured, and the wire grip score was quantified using the following 5-point scale in which 0 = unable to remain on the wire for 30 sec; 1 = failure to hold on to the wire with fore paws and hind paws together; 2 = holding on to the wire with the fore and hind paws but not the tail; 3 = holding on to the wire using the tail along with the fore and hind paws; 4 = moving along the wire on four paws plus tail and finally 5 = also ambulating down one of the posts used to support the wire. Tests were administered in triplicate and the average value was calculated for each mouse on each test day.

A membrane-permeable fluorescent indicator for NO, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; Molecular Probes, Eugene, OR, USA) was utilized to detect HEPM-induced changes in NO production. DAF-FM DA is converted via a NO-specific mechanism to an intensely fluorescent triazole derivative (16). HBMECs were obtained from the Applied Cell Biology Research Institute (Kirkland, WA, USA) and cultured in endothelial growth medium-2 (EGM-2) using a MV Bullet kit system (Cambrex, Walkersville, MD, USA). Experiments were performed after 4–6 cell passages. Following reaching sub-confluency, the cells were incubated in endothelial cell basal medium (EBM; Cambrex) with HEPM and L-arginine for 10 min, after which 5 μmol/l DAF-FM DA was loaded into the cells. Following incubation at 37°C for 5 min, the cells were mildly washed twice using phosphate-buffered saline (PBS) to eliminate any interference. The NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 100 μmol/l) and acetylcholine (30 μmol/l) were used to validate the measurements. Fluorescence was detected using an Axiovert 200 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

To further assess the impact of HEPM on NO signaling, the phosphorylation of Akt at Ser473, AMPK at Thr172 and eNOS at Ser1177 in HBMEC was assessed by western blotting. Following treatment, HBMECs were washed in cold PBS buffer and then homogenized in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 5 mM NaF, 1 mM PMSF, 1% Triton X-100, 10% glucose]. Proteins were isolated according to standard techniques, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). Immunoblot analysis was performed with anti-eNOS and anti-phospho-eNOS (Ser1177) antibodies (BD Biosciences, San Jose, CA, USA), anti-Akt and anti-phospho-Akt (Ser473) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-AMPKα and anti-phospho-AMPKα (Thr172) antibodies (Cell Signaling Technology, Inc.) followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The intensity of chemiluminescence was measured using an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden). The membrane was reprobed with anti-β-actin antibody (Sigma-Aldrich) as an internal control.

Acetylcholine chloride and L-NAME were purchased from Sigma-Aldrich. LY-294002 and Compound C were purchased from Calbiochem Inc. (San Diego, CA, USA). n-Hexane, ethyl acetate and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals were reagent grade.

All data are expressed as the mean ± SEM. Statistical comparisons were performed using paired or unpaired Student’s t-tests, and one-way analysis of variance (ANOVA) or two-way ANOVA for repeated measures followed by Fisher’s protected least significant difference test. A P<0.05 was considered to indicate a statistically significant result.

We investigated the protective effects of EAEPM, MEPM and HEPM using a focal cerebral ischemic mouse model. EAEPM, MEPM and HEPM (100 mg/kg, intraperitoneally) were separately administered to the mice 30 min prior to ischemic insults. As summarized in Fig. 1, EAEPM, MEPM and HEPM all significantly decreased the cerebral infarct volume (34.9±4.9 mm³, 28.9±9.8 mm³ and 20.5±2.3 mm³, respectively) relative to the vehicle (Veh) treatment (60.6±5.1 mm³). Among these, HEPM exhibited the greatest protective effects against ischemic brain injury. Furthermore, only HEPM significantly improved neurological deficits. Therefore, HEPM was selected for further study.

When administered intraperitoneally at 50 mg/kg and 100 mg/kg 30 min prior to ischemic insult, HEPM decreased the infarct volume and improved the neurological and motor functions in a concentration-dependent manner (Fig. 2). To examine the contribution of eNOS signaling to the cerebroprotective action of HEPM, the impact of HEPM on ischemic brain injury was tested in eNOS KO mice. HEPM (100 mg/kg, intraperitoneally) did not reduce infarct volume or improve neurological and motor function in eNOS KO mice, suggesting that the cerebroprotective effects of HEPM are dependent on eNOS (Fig. 3).

When HBMECs were incubated with 10 μg/ml HEPM, NO production was increased in vitro, as determined by the intensity of fluorescent DAF-FM (Fig. 4A). Previous studies have demonstrated that specific protein kinases, Akt and AMPK, are involved in eNOS phosphorylation and NO production (4,17). To investigate the involvement of Akt and AMPK-dependent pathways in HEPM-induced NO production, we examined NO production in HBMECs co-treated with HEPM and L-NAME (a NOS inhibitor), LY-294002 (an inhibitor of PI3K/Akt) or Compound C (an AMPK inhibitor). HEPM-induced increases in NO production were effectively inhibited by L-NAME and LY-294002, but not by Compound C in HBMEC (Fig. 4A), indicating that HEPM-induced NO production was due to PI3K/Akt-dependent eNOS activation. Following this, we examined the effect of HEPM on the activation of these kinases and the phosphorylation of eNOS. HEPM treatment resulted in an increase in phosphorylation-dependent activation of Akt and eNOS in HBMEC (Fig. 4B). These results suggest that HEPM increased NO production via phosphorylation-dependent activation of Akt and eNOS.