Pinocembrin protects rats against cerebral ischemic damage through soluble epoxide hydrolase and epoxyeicosatrienoic acids

2013 年 5 月第 11 卷第 3 期 Chin J Nat Med May 2013 Vol. 11 No. 3 207

Chinese Journal of Natural Medicines 2013, 11(3): 0207−0213

ChineseJournal of Natural Medicines

Pinocembrin protects rats against cerebral ischemic damage through soluble epoxide hydrolase and

epoxyeicosatrienoic acids WANG Shou-Bao, PANG Xiao-Bin, GAO Mei, FANG Lian-Hua, DU Guan-Hua*

Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

Available online 20 May 2013

[ABSTRACT] AIM: To investigate the relationship between cerebroprotection of pinocembrin and epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH). METHODS: Rats underwent middle cerebral artery occlusion (MCAO) to mimic permanent focal ischemia, and pinocembrin was administrated via tail vein injection at 10 min, 4 h, 8 h and 23 h after MCAO. After 24 MCAO, rats were re-anesthetized, and the blood and brain were harvested and analyzed. RESULTS: Pinocembrin displayed significant protective effects on MCAO rats indicated by reduced neurological deficits and infarct volume. Importantly, co-administration of 0.2 mg·kg–1 14, 15-EEZE, a putative selective EET antagonist, weakened the beneficial effects of pinocembrin. 14, 15-EET levels in the blood and brain of rats after 24 h MCAO were elevated in the presence of pinocembrin. In an assay for hy-drolase activity, pinocembrin significantly lowered brain sEH activity of MCAO rats and inhibited recombinant human sEH activity in a concentration-dependent manner (IC50, 2.58 μmol·L–1). In addition, Western blot and immunohistochemistry analysis showed that pinocembrin at doses of 10 mg·kg-1 and 30 mg·kg–1 significantly down-regulated sEH protein in rat brain, especially the hippocampus CA1 region of MCAO rats. CONCLUSION: Inhibiting sEH and then increasing the potency of EETs may be one of the mechanisms through which pinocembrin provides cerebral protection. [KEY WORDS] Pinocembrin; Cerebral ischemic injury; Epoxyeicosatrienoic acids (EETs); Soluble epoxide hydrolase (sEH)

[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2013)03-0207-07

1 Introduction

Ischemic stroke is a medical emergency leading to neu-ronal dysfunction and death in the affected brain areas. It remains a frequent cause of death worldwide, and is associ-ated with serious long-term physical and cognitive disabili-ties, especially in elderly patients [1-2]. However, the molecu-lar and biological mechanisms remain unclear. Recent studies suggest that active epoxyeicosatrienoic acids (EETs) which

[Received on] 07-Jun.-2012 [Research funding] This project was supported by the Research Special Fund for Public Welfare Industry of Health (Nos. 200802041, and 200902008) and Major Scientific and Technological Special Project for "Significant New Drugs Creation" of China (Nos. 2009ZX09302- 003, and 2009YZH-LCH07). [*Corresponding author] DU Guan-Hua: Prof., Tel/Fax: 86-10- 63165184, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

are derived from arachidonic acid through cytochrome P450 epoxygenases are involved in the progression of ischemic injury in diverse organs. Soluble epoxide hydrolase (sEH) is considered as the primary enzyme responsible for the degra-dation of EETs to the less potent dihydroxyeicosatrienoic acids (DHETs). Several studies indicate that pharmacological inhi-bition or gene depletion of sEH protects rats against cerebral ischemia via vascular and neural protection [3-5].

Propolis is a resinous substance collected by bees from exudates of different plants and is rich in well-known health- relevant phenolic compounds such as flavonoids and phenolic acids. Pinocembrin (5, 7-dihydroxyflavanone; Fig. 1) is a flavanone found abundantly in honey and propolis. Many

Fig. 1 Structure of pinocembrin

WANG Shou-Bao, et al. /Chinese Journal of Natural Medicines 2013, 11(3): 207−213

208 Chin J Nat Med May 2013 Vol. 11 No. 3 2013 年 5 月第 11 卷第 3 期

studies have established that it possesses multiple activities, including neuroprotection, anti-inflammation, vasorelaxa-tion, antioxidant, antimicrobial, and antiproliferative ef-fects [6-11].

Recent studies have indicated that pinocembrin could protect rat brain against ischemic injury by improving brain blood flow, inhibiting the inflammatory cascade, and allevi-ating blood-brain barrier injury [12-15]. Considering the poten-tial roles of EETs in ischemia, it was hypothesized that the inhibition of sEH and increasing EETs were implicated in the protective effects of pinocembrin against cerebral ischemic injury. In this study, the rat middle cerebral artery occlusion (MCAO) model was used to test the hypothesis.

2 Materials and Methods

2.1 Animals Male Sprague–Dawley rats (220–250 g) were provided

by Vital River Laboratory Animal Center (Beijing, China). The protocol was approved by the institutional animal care and use committee and the local experimental ethics com-mittee. All rats were kept on a 12 h light/12 h dark regime, with free access to food and water. Room temperature (25 ± 2) °C and room humidity (55 ± 10) % were controlled. 2.2 Drugs and reagents

Racemic pinocembrin ((+)-pinocembrin: (–)-pinocembrin = 1 : 1, 99% purity) used in the present study was synthe-sized and processed as a sterile injection powder at the De-partment of New Drug Development, Institute of Materia Medica, Chinese Academy of Medical Sciences. It was dissolved in 0.9% NaCl before use. 14, 15-EEZE and PHOME were purchased from Cayman Chemical Co., MI, USA, and were dissolved and kept below –20 °C. Antibod-ies of sEH (H-215) and β-actin were purchased from Santa Cruz, CA, USA. 2.3 Experimental design in vivo

Rats were generally anesthetized with an intraperitoneal injection of 10% chloral hydrate (Tianjin, China) at a dose of 400 mg·kg–1. A standard intraluminal MCAO, as described previously [12], was performed to provide a permanent focal ischemia. Sham-operated rats received the same procedure except for filament insertion. During the operation, a heating pad was used to keep the body temperature at (37 ± 0.5) °C. Pinocembrin (3, 10 and 30 mg·kg–1 body weight), 14, 15-EEZE (0.2 mg·kg–1 body weight), and vehicle were ad-ministrated via tail vein injection at 10 min, 8 h and 23 h after MCAO or sham operation. MCAO group animals received an equal volume of 0.9% NaCl. Sham group animals received the sham operation and an equal volume of 0.9% NaCl. At the end of the experiments, rats were re-anesthetized and euthanized, and the blood and brain samples were collected promptly. Serum was separated by centrifugation at 3 000 r·min–1, 4 °C, then stored at –20 °C before analysis of the 14,15-EET level. The whole brain was used to measure in-farct size. Other brain samples were divided into two parts: the hippocampus CA1 region was sepa-rated and immersed

in 10% neutral buffered formaldehyde for immunohisto-chemical examinations and the rest was stored at –80 °C for further analysis. 2.4 Neurological deficits

After 24 h MCAO, an experienced experimenter blind to the experimental groups assessed the neurological function of rats in accordance with Longa's method on a 5-point scale: no neurological deficit = 0, failure to extend right paw fully = 1, circling to right = 2, falling to right = 3, being unable to walk spontaneously and depression of consciousness = 4 [16]. 2.5 Measurement of infarct volume

The whole brain was sectioned coronally into 2 mm-thick slices and immediately stained with 2% 2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma, USA) for 10 min at 37 °C. The stained slices were recorded using a digital camera (Canon EOS 450D), and the photographs were quan-tified for infarct volume by ImageJ software (version 1.37c, NIH). 2.6 Measurement of 14, 15-EET by ELISA

The 14,15-EET levels in the blood and brain were meas-ured with a commercial ELISA kit (Detroit R&D, Inc MI, USA). Briefly, ispilateral hemisphere was collected in TPP (triphenylphosphine) with a final concentration of 0.1 mmol·L–1. After acidification with acetic acid to a pH of ap-proximately 3–4, the samples were extracted three times with ethyl acetate. The collected organic phases (ethyl acetate) were pooled and evaporated under nitrogen. The residue was dissolved in 20 μL of ethanol, and 20 μL acetic acid added to ca. pH 3–4, followed by reaction for 12 h at 45 °C. Under the acidic conditions, EET is hydrolyzed to DHET. After the reaction, 1.5 volumes of water were added to the sample and extracted three times with equal volumes of ethyl acetate. After three extractions, the ethyl acetate phases were pooled and evaporated under nitrogen. The residue was dissolved in 20 μL of ethanol for ELISA assay of 14, 15-DHET according to the manufacturer’s instructions. At the same time, meas-urement of the 14, 15-DHET level without hydrolysis of 14, 15-EET was conducted on the same sample. By subtracting that value from the 14, 15-EET + 14, 15-DHET level, and the 14, 15-EET level in the sample is obtained. 2.7 Assay for hydrolase activity of sEH in brain

Hydrolase activity of sEH in brain was measured by in-cubating with 14,15-EET and assaying for 14, 15-DHET. Briefly, the right cerebral hemisphere was homogenized in 4 volumes of ice-cold buffer (20 mmol·L–1 Tris-HCl (pH 7.4), 0.32 mol·L–1 sucrose, 1 mmol·L-1 EDTA), centrifuged at 1 000 × g for 10 min, and the supernatant was further centrifuged at 10 000 × g for 20 min. Hydrolase enzymatic reactions were initiated by adding 1 μmol·L–1 14, 15-EET and incubated in a 37 °C shaking water bath for 1 h. The 14, 15-DHET level was measured by ELISA according to the manufacturer’s instruc-tions. 2.8 Western blotting for sEH

Right cerebral hemisphere was homogenized for 30 min



at 4 °C in 4 volumes of RIPA lysis buffer (50 mmol·L–1 Tris-HCl (pH 7.4), 150 mmol·L–1 NaCl, 1 mmol·L–1 EDTA, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mmol·L–1 PMSF). After centrifugation at 12 000 × g, 4 °C for 30 min, the protein concentration of the supernatant was quantified using a BioRad Dc Protein Assay kit (BioRad, USA). Equal amounts of protein samples (n = 3 in each group) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with Tris buffered saline, 0.1% Tween 20 with 5% W/V nonfat milk, membranes loaded with the protein of interest were incubated with primary antibodies (rabbit polyclonal anti-sEH and anti-actin, both 1 : 500 dilu-tion) at 4 °C overnight. HRP-conjugated secondary antibody (goat anti-rabbit, 1 : 1 000 dilution, Rockland, Gilbertsville, PA, USA) was used to identify primary antibodies. Blots were developed with SuperSignalWest Pico Chemilumines-cent Substrate (Pierce, USA) and visualized using a Chemi-Doc XRS system (BioRad, USA). The intensity of immuno-reactive bands was quantified using Quantity One Software (BioRad) and normalized against the loading control β-actin. Results were expressed in arbitrary units relative to that in the sham group which was set to 1. 2.9 Immunohistochemistry for sEH

Rats were anesthetized, and the brain was perfused from the apex of the heart with PBS, and perfusion-fixed with 4% paraformaldehyde (PFA) in PBS. The right cerebral hemi-sphere was removed and kept for post-fixation in 4% PFA for 24 h. After the brains were fixed and embedded in paraffin, sections at 5 μm were used. Immunohistochemistry was per-formed by using the avidin-biotin-peroxidase complex tech-nique. The primary antibody sEH H-215 (Santa Cruz, CA, USA) was used at a dilution of 1 : 200. Following incubation with the primary antibody, a biotinylated secondary antibody, followed by addition of a complex of avidin and biotinylated peroxidase, was applied, and diamino-benzidine (DAB) was used as a chromogenic substrate for visualization. Images were photographed with an Olympus microscope connected with a digital camera (Canon EOS 450D) at 400 × magnifica-tion. The mean optical density (MOD = integrated optical density (IOD)/area) of the positive immunohistochemical staining in each preparation was acquired using Image-Pro Plus 6.0 software. 2.10 Effect of pinocembrin on the hydrolase activity of re-combinant human sEH in vitro

Recombinant human sEH was produced, purified, ana-lyzed, and quantified in E. coli cells as described previously [17-18]. Its substrate PHOME was used to determine epoxide hydrolase activities of the produced enzymes. Final enzyme concentration was 3 nmol·L–1 and final substrate concentra-tion was 50 μmol·L–1. The appearance of the fluorescent product was monitored kinetically for 10 min with an interval of 2 min by a SpectraMax M5 microplate reader (Molecular Devices, CA, USA) with an excitation wavelength of 340 nm and an emission wavelength of 460 nm. The Vmax (RFU per second) was used to represent enzyme activity.

Each 384 well black microplate contained blanks (no en-zyme) in column 1, controls (enzyme, no compound) in col-umn 2, and SAA concentration gradients (SAA 10–8, 10–7, 10–6, 10–5, 10–4, and 10–3 mol·L–1 in ultrapure water, respec-tively) in columns 3–9 in quadruplicate. The percent inhibi-tion was calculated using the following equation:

% Inhibition = (1– (Vmax sample – Vmax blank)/(Vmax control – Vmax

blank)) ×100 Vmax sample represents the Vmax of samples; Vmax control

represents the Vmax of the controls; Vmax blank represents the Vmax of the blanks.

The IC50 was calculated by sigmoidal fit with Origin Pro 7.0 software.

3 Statistical Analysis

Data were presented as x ± SEM. Neurological deficit scores between groups were analyzed using a non-parametric test. Statistical significance of the quantitative data was deter-mined by Student’s t test or one-way analysis of variance (ANOVA) followed by Dunnett’s test. All calculations were performed with SPSS version 13.0 (SPSS Inc., IL, USA). Dif-ference was considered significant if P < 0.05.

4 Results

4.1 Effects of pinocembrin on neurological deficit and in-farction size

As shown in Fig. 2, sham rats had no neurological deficit and no infarct volume. In contrast, the neurological deficit score 3.41 ± 0.18 and infarct volume (187 ± 8) mm3 of MCAO rats were significantly higher (P < 0.01). Pinocem-brin treatment ameliorated the neurological deficit and infarct volume induced by MCAO. The neurological scores of 3, 10 and 30 mg·kg–1 pinocembrin treated MCAO rats were 2.63 ± 0.19, 2.15 ± 0.17 and 1.82 ± 0.16, respectively. Relatively, the infarct volumes were (123 ± 9) mm3, (95 ± 7) mm3 and (82 ± 6) mm3, respectively. As compared to pinocembrin 30 mg·kg–1 alone treated MCAO rats, the neurological scores 2.97 ± 0.17 and infarct volume (135 ± 8) mm3 of 30 mg·kg–1 pinocembrin plus 14, 15-EEZE co-treated MCAO rats were significantly higher. 4.2 Effects of pinocembrin on 14, 15-EET levels in the blood and brain

There was no statistically significant difference in the 14, 15-EET level in the blood and brain between sham and MCAO rats: (21.9 ± 2.6) pg·mL–1 and (89 ± 6.1) pg·mg–1 protein vs (28.6 ± 2.4) pg·mL–1 and (96 ± 6.2) pg·mg–1 pro-tein (Fig. 3). The 14, 15-EET levels in the blood and brain from 30 mg·kg–1 pinocembrin treated sham rats were (44.7 ± 2.5) pg·mL–1 and (139 ± 7.3) pg·mg–1 protein, showing a significant difference from sham rats (P < 0.05). As com-pared to the MCAO alone rats, 10 and 30 mg·kg–1 pinocem-brin-treated MCAO rats displayed increased blood 14, 15-EET levels [(50.5 ± 3.1) pg·mL–1 and (55.2 ± 2.7) pg·mL–1] and higher brain 14, 15-EET levels [(138 ± 8.1) pg·mg–1 protein and (156 ± 8.2) pg·mg–1 protein].



Fig. 2 Pinocembrin reduced the infarction size and neurological deficits of rats after 24 h MCAO, and 14,15-EEZE attenuated these effects. Panel A shows infarct volumes in rats after 24 h MCAO ( x ± SEM, n = 6). Panel B shows neurological deficit scores in rats after 24 h MCAO ( x ± SEM, n = 15). There were remarkable infarct volumes and neurological deficits in MCAO alone rats. Pinocembrin treatment at the doses of 3, 10 and 30 mg·kg-1 reduced infarct volumes and neurological deficits. The putative selective EET antagonist 14, 15-EEZE (0.2 mg·kg–1) blocked the beneficial effects of pinocembrin. *P < 0.05 vs sham group; #P < 0.05, ##P < 0.01 vs MCAO alone group; $P < 0.05 vs 30 mg·kg-1 pinocembrin treated MCAO group

Fig. 3 Pinocembrin increased 14,15-EET levels in the blood and brain of rats after 24 h MCAO. Panel A showed blood 14,15-EET levels of rats ( x ± SEM, n = 6) and panel B showed brain 14, 15-EET levels of rats ( x ± SEM, n = 4). The 14,15-EET levels in the blood and brain from 30 mg·kg-1 pinocembrin treated sham rats were significant higher than sham rats (*P < 0.05). The doses of 10 and 30 mg·kg–1 of pinocembrin significantly improved the 14, 15-EET levels in the blood and brain compared to MCAO alone rats (#P < 0.05) 4.3 Effects of pinocembrin on brain sEH activity

As shown in Fig. 4, there was no significant difference in the brain sEH activity, as indicated by the content of 14, 15-DHET between the sham and MCAO alone rats [(1 489 ± 67.5) pg·mL–1 vs (1 379 ± 62.4) pg·mL–1]. The brain sEH activity in 30 mg·kg-1 pinocembrin treated sham rats was significantly reduced (984 ± 65.3) pg·mL-1. As compared to the MCAO alone rats, 10 and 30 mg·kg-1 pinocembrin sig-nificantly lowered the 14,15-DHET contents to (1 045 ± 66.5) pg·mL–1 and (904 ± 63.7) pg·mL–1, respectively (P < 0.05). At 3 mg·kg-1 pinocembrin show no significant impact on brain sEH activity [(1 202 ± 68.3) pg·mL–1, P > 0.05]. 4.4 Effects of pinocembrin on protein expression of sEH

Western blot analysis showed that cerebral ischemic in-jury by MCAO had minimal impact on sEH protein expres-sion (0.92 ± 0.04), and there was no significant difference compared to sham rats. The expression level of sEH protein in brain from 30 mg·kg-1 pinocembrin treated sham rats (0.54 ± 0.04) was significantly lower than sham rats. As compared

Fig. 4 Pinocembrin inhibited brain sEH activity in rats after 24 h MCAO. Brain tissue was incubated with 1 μmol·L–1 14, 15-EET for 1 h, and then assaying for 14, 15-DHET was performed using a commercial ELISA kit (Detroit R&D, Inc, Detroit, MI). The results showed that pinocembrin at the doses of 10 and 30 mg·kg-1 significantly decreased brain sEH activity as indicated by the 14, 15-DHET level ( x ± SEM, n = 4, #P < 0.05 vs MCAO alone)



Fig. 5 Pinocembrin decreased sEH protein expression in the brains of rats after 24 h MCAO. Panel A showed representive immunoblotting images of sEH protein. In each lane, 30 μg protein was loaded. The dilution of anti-sEH is 1 : 200. Panel B showed the relative normalized amount of sEH protein in rat brain ( x ± SEM, n = 4). Each immunoreactive band was normalized as a ratio of corresponding β-actin band and expressed in arbitrary units relative to that in the sham group. At 30 mg·kg–1 pinocembrin reduced sEH protein ex-pression in the brain of sham rats. Pinocembrin at doses of 10 and 30 mg·kg–1 significantly decreased sEH protein ex-pression in brain of MCAO rats. *P < 0.05 vs sham group; #P < 0.05 vs MCAO alone group

Fig. 6 Pinocembrin reduced sEH expression in hippocam-pus CA1 of rats after 24 h MCAO (× 400, A: sham group; B: MCAO alone group; C: pinocembrin 3 mg·kg–1 group; D: pinocembrin 10 mg·kg–1 group; E: pinocembrin 30 mg·kg-1 group) . The difference of IOD/area in the CA1 region be-tween groups was presented (F) ( x ± SEM, n = 4). Pinocem-brin at doses of 10 mg·kg–1 and 30 mg·kg-1 reduced sEH im-munoreactivity in hippocampus CA1. *P < 0.05 vs sham group; #P < 0.05 vs MCAO alone group

Fig. 7 Different concentrations of pinocembrin inhibited hydrolase activity of recombinant human sEH in vitro. The result of sigmoidal fit with Origin Pro 7.0 software indicated that pinocembrin had an IC50 of 2.58 μmol·L–1 on recombi-nant human sEH. Each dot was repeated three times

to MCAO rats, expression of sEH protein in 10 and 30 mg·kg–1 pinocembrin treated MCAO rats was significantly reduced to 0.65 ± 0.04 and 0.52 ± 0.03, respectively.

The expression of sEH protein in the hippocampus CA1 region was also investigated by immunohistochemistry (Fig. 6). Compared with sham rats (Fig. 6A), the neurons in hippo-campus CA1 of rats from MCAO alone group were swelling, even fragmenting. The sEH positive neurons scattered with a larger gap (Fig. 6B). Pinocembrin treatment improved the morphology and spatial arrangement of neurons. The sEH expression in hippocampus CA1 decreased significantly in 10 and 30 mg·kg–1 pinocembrin treated MCAO rats (0.55 ± 0.07 and 0.57 ± 0.06) compared to MCAO alone rats (1.11 ± 0.12, P < 0.05) (Figs. 6C-E, F). 4.5 Effect of pinocembrin on the hydrolase activity of re-combinant human sEH in vitro

Enzyme activity measurements showed that the hy-drolase activity of recombinant human sEH decreased gradu-ally with the addition of different concentration of pinocem-brin. At concentrations of 10–8, 10–7, 10–6, 10–5, 10–4, and 10–3 mol·L–1 of pinocembrin, the percent inhibitions against re-combinant human sEH were 1.71%, 8.02%, 31.32%, 74.16%, 97.53%, 103.46%, respectively (Fig. 5). Based on sigmoidal fit, the IC50 value was 2.58 μmol·L–1. The results indicated that pinocembrin could inhibit recombinant human sEH in a concentration-dependent manner.

5 Discussion

In the present study, it was demonstrated that pinocem-brin at doses of 10 and 30 mg·kg–1 significantly ameliorated the reduced infarct volume and improved the neurological recovery in rats subjected to MCAO. There was no signifi-cant improvement in the neurological recovery, but a signifi-cant decrease in infarct volume was shown at the dose of 3 mg·kg–1. These results indicated that pinocembrin could pro-vide potent cerebro- and neuro-protection against cerebral ischemic rats, consistent with previous studies [11, 19-20].



The cytochrome P-450 (CYP) monooxygenase pathway metabolizes arachidonic acid to produce epoxyeicosatrienoic acids (EETs) [21]. There are four epoxyeicosatrienoic acid (EET) regioisomers: 14, 15-, 11, 12-, 8, 9-, and 5, 6-EET. Each regioisomer has specific paracrine and autocrine func-tions, and they have been shown to potently dilate blood vessels, activate potassium channels, promote angiogenesis and attenuate inflammation [22]. EETs are produced in the brain, and perform important biological functions, including vasodilation and neuroprotection [23]. To investigate the rela-tionship between the protective effects of pinocembrin against cerebral ischemic injury and EETs, the level of 14, 15-EET, which has the strongest biological activities among four regioisomers, was determined in this study. The results showed that treatment with 30 mg·kg–1 pinocembrin in-creased the 14, 15- EET levels in the blood and brain of sham rats. Pinocembrin at the doses of 10 and 30 mg·kg–1 signifi-cantly increased 14, 15-EET levels in the blood and brain of MCAO rats. On the other hand, a putative selective EET antagonist 14, 15-EEZE was used to test whether EETs and its downstream pathway were involved in the cerebroprotec-tion of pinocembrin. The results showed that simultaneous treatment with 0.2 mg·kg–1 14,15-EEZE attenuated the re-duced infarction volume and the improved the neurological outcome of MCAO rats compared to 30 mg·kg–1 pinocembrin alone treatment, indicating that the beneficial effects of pino-cembrin were blocked by 14,15-EEZE. The results demon-strated that enhancing EETs level may be one of the mecha-nisms through which pinocembrin provides neuroprotection in rats subjected to MCAO.

As is known, EETs are rapidly hydrolyzed and deacti-vated via sEH. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. The enzyme sEH has recently emerged as a potential therapeutic target in the treatment of ischemic stroke [5, 24]. The effect of pinocembrin on sEH activity was evaluated in the current study. The re-sults indicated that brain sEH activity in 30 mg·kg–1 pino-cembrin treated sham rats was significantly reduced, and that pinocembrin at doses of 10 and 30 mg·kg–1 significantly low-ered brain sEH activity of MCAO rats. A rapid kinetic fluo-rescent assay in vitro using recombinant human sEH protein also showed that pinocembrin could inhibit hydrolase activity of recombinant human sEH in a concentration-dependent manner with an IC50 of 2.58 μmol·L–1. These data indicated that pinocembrin could inhibit sEH activity, and retard the degradation of EETs to DHETs.

There are few studies that examine the changes in expression level of sEH protein in animals subjected to cerebral ischemia. In this study, Western blot analysis showed that pinocembrin at doses of 10 and 30 mg·kg–1 significantly decreased the expression level of sEH protein in the brain. In addition to cortical infarction, MCAO may result in damage to the ipsi-lateral hippocampus that leads to neurodegeneration and de-layed neuronal death [28-29]. The hippocampus is of particular

interest in view of its well-described functions in learning, memory and stress responses, and it is a common target of age-related disease and brain damage. In the CA1 region, the structures of pyramidal neurons and synapse are the morpho-logical bases of learning and memory abilities. Immunohis-tochemical analysis also showed that pinocembrin reduced the expression level of sEH protein in the hippocampus CA1 region of MCAO rats, and improved the structure and mor-phology of pyramidal neurons.

In conclusion, pinocembrin displayed potent protective effects on rats subjected to MCAO. It may be a promising drug for the development of novel, multiple-action therapies in treating ischemic stroke. Inhibiting she, and then enhanc-ing the levels of EETs, may be one of the mechanisms un-derlying the cerebral protection effect of pinocembrin.

Acknowledgments

The authors thank Mr. LAN Bao-Qiang, from Guangxi Institute of Chinese Medicine & Pharmaceutical Science, for his assistance in the conducting animal experiments.

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匹诺塞林通过可溶性环氧化物水解酶和环氧二十碳三烯酸对脑缺血大鼠发挥保护作用

王守宝, 庞晓斌, 高梅, 方莲花, 杜冠华*

“药物靶点研究和新药筛选”北京市重点实验室, 中国医学科学院北京协和医学院药物研究所, 北京 100050

【摘要】目的：探讨匹诺塞林的脑保护作用与环氧二十碳三烯酸(EETs)及其代谢关键酶可溶性环氧化物水解酶(sEH)之间

的关系。方法：大鼠进行大脑中动脉闭塞(MCAO)制备永久性局灶性脑缺血, 术后 10 min, 4 h, 8 h 和 23 h 经尾静脉注射给予匹诺

塞林。手术 24 h 后, 大鼠重新麻醉, 收集血液和脑等样本进行检测。结果：研究表明匹诺塞林对脑缺血大鼠具有明显的保护作

用, 可以减轻神经功能障碍及降低梗死面积。重要的是, 给予 EETs 的选择性拮抗剂 14, 15-EEZE(0.2 mg·kg–1)可以减弱匹诺塞林

的保护作用。手术 24 h 后, 匹诺塞林能明显升高脑缺血大鼠血液和脑组织中 14, 15-EET 水平。匹诺塞林能显著降低缺血大鼠脑

中 sEH 活性并能浓度依赖地抑制重组人 sEH 活性(IC50, 2.58 μmol·L–1)。此外, 免疫印迹和免疫组化结果表明, 10 和 30 mg·kg–1匹

诺塞林显著下调 sEH 蛋白在缺血大鼠大脑, 尤其是在海马 CA1 区的表达。结论：抑制 sEH 而提高 EETs 水平可能是匹诺塞林发

挥脑保护作用的机制之一。【关键词】匹诺塞林；脑缺血；环氧二十碳三烯酸；可溶性环氧化物水解酶

【基金项目】卫生部公益性行业科研专项(Nos. 200802041, 200902008)和“重大新药创制”科技重大专项(Nos. 2009ZX09302-003, 2009YZH-LCH07)

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Pinocembrin protects rats against cerebral ischemic damage through soluble epoxide hydrolase and epoxyeicosatrienoic acids