Journal of Ethnopharmacology 154 (2014) 65–75

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Research Paper 1

H NMR-based metabonomic analysis of the effect of optimized rhubarb aglycone on the plasma and urine metabolic fingerprints of focal cerebral ischemia–reperfusion rats

Qinxiao Guan a, Shengwang Liang a, Zhanhong Wang a, Yongxia Yang b,n, Shumei Wang a,nn a b

Department of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, PR China School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 12 July 2013 Received in revised form 19 January 2014 Accepted 1 March 2014 Available online 29 March 2014

Ethnopharmacological relevance: The ischemia cerebrovascular disease is one of leading causes of death and long-term disability in modern society. Rhubarb is one of the common traditional Chinese medicine with many effects, and the main pharmacodynamic ingredients are aloe-emodin, rhein, emodin, chrysophanol and physcion. The five components are also known as rhubarb aglycone. Rhubarb aglycone has been confirmed to play a remarkable curative effect on cerebral ischemia, but the mechanism is not clear. In this study, 1H NMR-based metabonomics approach has been used to investigate the protective effect of the optimized rhubarb aglycone on rats of cerebral ischemia–reperfusion. Materials and methods: Male Wistar rats were divided into four groups: sham operation group, model group, Nimodipine group and the optimized rhubarb aglycone group. Based on 1H-NMR spectra of plasma and urine, principal component analyses were performed to identify different metabolic markers and explore the changes of associated biochemical pathways. Behavior research and brain histopathology examinations were also performed. Results: It was showed that the optimized rhubarb aglycone treatment improved neurological deficits, cerebral infarction and neuronal apoptosis. Principal component analysis scores plots demonstrated that the cluster of model rats was separated from those of sham operation group; rats of the optimized rhubarb aglycone group were classified from model group, but the optimized rhubarb aglycone group closed to the sham operation group. Optimized rhubarb aglycone regulated the associated amino acid, energy and lipid metabolisms disturbed in model rats. Conclusion: Our results suggested that the optimized rhubarb aglycone had protective effect on rats of cerebral ischemia–reperfusion and explored the metabolic regulation mechanism. This work showed that the NMR-based metabonomics approach might be a promising approach to study mechanisms of traditional Chinese medicines. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Metabonomics Cerebral ischemic reperfusion Rhubarb aglycone 1 H NMR Principal component analysis

1. Introduction Nowadays, stroke has become one of the common cerebrovascular diseases, with high incidence and complications rate, which is a serious threat to over 16 million human health worldwide every year (Strong et al., 2007). With the rise of aging population, the burden will also be increased. Almost 80% of all strokes are ischemic cerebral events in both developed and developing countries (Palm et al., 2010). The majority of the surviving patients

n

Corresponding author. Tel.: þ 86 20 3935 2197; fax: þ 86 20 3935 2186. Corresponding author. Tel.: þ 86 20 3935 2559; fax: þ86 20 3935 2174. E-mail addresses: [email protected] (Y. Yang), [email protected] (S. Wang).

nn

http://dx.doi.org/10.1016/j.jep.2014.03.002 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

left sequelae in different degrees, which severely affected their health and quality of life. The treatment of traditional Chinese medicine (TCM) to stroke has significant efficacy and advantages. Researches demonstrated that some TCM preparations, such as Qingkailing (Hua et al., 2008), baicalin and jasminoidin (Zhang et al., 2006) showed effectiveness for the treatment of stroke by anti-oxidation, antiinflammation, protecting against ischemic reperfusion injury, and enhancing the tolerance of ischemic tissue to hypoxia. Rhubarb is one of the common traditional Chinese medicine, with widely effects, of which the main pharmacodynamic ingredients are rhubarb aglycone (aloe-emodin, rhein, emodin, chrysophanol and physcion) and rhubarb glycosides (anthraquinone glycosides and double anthrone glycoside), of which the effective ingredient for treatment of stroke was rhubarb aglycone. Rhubarb,

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rhubarb extracts and rhubarb aglycone have been confirmed to play a remarkable curative effect on ischemic stroke. Rhubarb aglycone could clear a large number of free radicals caused by brain ischemia–reperfusion (Li et al., 2004), effectively reduce NO-mediated cytotoxicity (Li et al., 2003), inhibit inflammatory cascade reaction with cerebral ischemia–reperfusion injury (Li et al., 2005a, 2007), inhibit the thrombosis, the aggregation and adhesion of platelet (Li et al., 2005b), and have protective effects on rats with cerebral ischemia (Liu et al., 2004). Our previous study found that the different ratios of five aglycones had different treatment efficacy on ischemic cerebral. We had performed pharmacological experiments and used the Support Vector Machine regression method to search out the optimized rhubarb aglycone (aloe emodin: rhein: emodin: chrysophanol: physcion¼50:76:38:105:68). But the mechanism of the optimized rhubarb aglycone on stroke is not clear. Metabonomics, a new subject that develops following genomics, transcriptomics and proteomics, is an important part of system biology. Metabonomics is a new scientific platform for investigation of the metabolic response of living systems to any environmental stimuli (Nicholson et al., 1999). Metabonomics was widely used in the study of traditional Chinese medicine. Metabonomics technology contributes to clarify the material basis and mechanism of new drugs (Agnolet et al., 2010; Zhou et al., 2011). Meanwhile, metabonomics also can evaluate the safety of TCM compounds and has been successfully applied in estimating the drug toxicity (Ebbels et al., 2003; Wang et al., 2013). Besides, the metabolic researches indicate that different diseases do not elicit the same biological characteristics, even though conventional biomarker properties may appear similar (Zira et al., 2010; Shi et al., 2013). The application of nuclear magnetic resonance (NMR) spectroscopy to the analysis of biological fluids such as serum, urine, plasma and bile is specified. 1H NMR spectroscopic analysis of biofluids has successfully showed lots of novel metabolic biomarkers for drug development and disease diagnosis (Lindon et al., 2004). Therefore, we investigated the treatment effect of rhubarb aglycone with optimum proportion on cerebral ischemia– reperfusion rat model and explored the metabolic regulation mechanism by using 1H NMR spectroscopy and principal component analysis.

2. Materials and methods 2.1. Chemicals products The purity of aloe-emodin, rhein, emodin, chrysophanol, physcion reached more than 98%, which were purchased from the Plant Chemistry Institute of the Huaihai City, Jiangsu Province. Nimodipine (100903) was purchased from Guangdong Huanan Pharmaceutical Co. Ltd. 2,3,5-Triphenyltetrazolium chloride (TTC), analysis purity, was purchased from Shanghai Yuanfan Auxiliaries Factory for brain tissue staining in order to evaluate the infarct area. Heparin sodium was purchased from Shanxi Xintai Biological Technology Co. Ltd. Sodium chloride injection was purchased from Zhengzhou Yonghe Pharmaceutical Co. Ltd. D2O containing 0.05% sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate (TSP) were purchased from Beijing Dimma Technology Co. Ltd. 2.2. Animal grouping and drug administration 24 male Wistar rats (280720 g) were purchased from the Laboratory Animal Institute of Hebei Medical University. This study was reviewed and approved by the Ethics Committee of Guangdong Pharmaceutical University (No. SPF20120001). 24 rats were randomly divided into four groups (6 rats in each group), i.e. sham operation

group (as placebo group), model group, nimodipine treatment group (as positive group) and the optimized rhubarb aglycone treatment group. Rats in sham operation group and model group were daily given 0.5% CMC-Na suspension. Rats in nimodipine group were daily given nimodipine (6 mg/kg) 0.5% CMC-Na suspension, and rats in the optimized rhubarb aglycone group were daily given rhubarb aglycone with optimal proportion (aloe emodin 50 mg/kg, rhein 76 mg/kg, emodin 38 mg/kg, chrysophanol 105 mg/kg, physcion 68 mg/kg) in 0.5% CMC-Na suspension. They were fed in an environmentally controlled breeding room during the whole period, with standard laboratory food and water. After consecutive administration for 4 days, all the rats were fasting. On the fifth day, they were administered once again an hour before surgery.

2.3. Model preparation The model of focal cerebral ischemia–reperfusion is established with the suture-occluded method by Longa et al. (1989). After 12 h fasting, the rats were injected intraperitoneally with 10% chloral hydrate (0.3 ml/100 g) to be anesthetized. Briefly, the rats in model, nimodipine and the optimized rhubarb aglycone treatment group were fixed on the operating table with supine position. The surgery conducted routine disinfection. After a midline incision was made at the ventral surface of the neck skin, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The CCA and ECA were clamped, leaving a small cutting in crotch of the CCA and ECA. A 4-0 monofilament nylon surgical suture with a rounded tip (Beijing Sunbio Biotech Co. Ltd, Beijing, China) was introduced through the right CCA to the ICA until resistance was felt. After occlusion for two hours, the suture was withdrawn, allowing reperfusion. The rats in the sham group underwent the same surgical procedures, but without arterial occlusion. The environment temperature sustains 25–26 1C during or following surgery.

2.4. Observation and evaluation of neurologic impairment According to the method described by Longa et al. (1989), the neurologic assessments were scored on a five-point scale: a score of 0 indicated no neurologic deficit; a score of 1 (failure to extend left forepaw fully) indicated a mild focal neurologic deficit; a score of 2 (circling to the left) indicated a moderate focal neurologic deficit, and a score of 3 (falling to the left) indicated a severe focal deficit; rats with a score of 4 did not walk spontaneously and had a depressed level of consciousness.

2.5. Plasma, urine collection and sample processing After 6 h reperfusion, 2 ml blood was taken from the tail vein into heparin sodium EP tube (1% heparin sodium infiltration), centrifuged for 10 min (4000 r/min, 4 1C). The supernatant was pipetted into EP tube and refrigerated at  80 1C. Urine was collected within 0–6 h after reperfusion using metabolic cages with the addition of 50 μL sodium azide solutions (0.1% w/w) in each tube. Prior to data acquisition, the plasma samples were thawed at room temperature. 200 μL of phosphate buffer (0.2 M, Na2HPO4/ NaH2PO4, pH7.4) was added into 300 μL plasma sample, centrifuged (4000 rpm) at 4 1C for 5 min. The supernatant was transferred to the 5 mm NMR tube with 80 mL of D2O containing 0.05% sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate (TSP) placed in refrigerator at 4 1C. The urine processing was same to the blood preparation.

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2.6. Brain tissue TTC staining and measurement of infarction area

3. Results

After blood was collected, the rats were decapitated. The brain was rapidly excised and placed in a refrigerator at  20 1C for 15 min. The brain was sliced coronally into 4 pieces (2 mm thick) beginning from optic chiasma. The second piece of brain slices incubated for 20 min in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 1C for vital staining (Bederson et al., 1986). The stained brain slices were washed by saline three times and fixed in 10% formaldehyde solution for being photographed after 24 h. The infarct volumes of brain slices were measured by computer image processing system.

3.1. Evaluation of neurologic impairment

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Two hours after occlusion, rats which undergone cerebral ischemia–reperfusion surgery showed sluggish, piloerection, irritability, and did not fully stretch the right forepaw. Neurological symptoms score results are shown in Table 1. It was demonstrated that the neurological symptoms have been alleviated in the rats of optimized rhubarb aglycone group and nimodipine group in comparison with those of model group, showing the curative effects of optimized rhubarb aglycone on cerebral ischemia model. 3.2. Brain tissue TTC staining and measurement of infarct area

2.7. Pathological examination of brain tissue The third piece of brain slices were fixed with 10% formaldehyde solution overnight at 4 1C. The thickness of paraffin-embedded sections stained with HE was 4 μm. Pathological change of brain tissue was observed under light microscope (  400). Slice of each rat was observed in 10 horizons, and calculated the number of normal neuronal cell under each view. Then the mean values were used to be performed statistical analysis.

Brain slices of TTC staining are shown in Fig. 1, and the results of the cerebral infarction area are shown in Table 1. The ischemic tissue was white and the normal brain tissue looks reddish-purple. The brain slice of the sham operation group showed no infarction. In model group, the left half of the brain slice was of most infarction area. Compared to the model group, infarction areas of brain slices of the optimized rhubarb aglycone group reduced significantly. It was showed that the optimized rhubarb aglycone is effective for the prevention and treatment of ischemic stroke.

2.8. Data acquisition of NMR

3.3. Brain pathological examination

1 H NMR spectra of plasma and urine were collected at 298 K on a Bruker Avance 500 MHz spectrometer. The NMR spectrum was recorded using the water-presaturated standard onedimensional Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence (recycle delay  901 (τ 1801  τ)n  acquisition), which can eliminate interference from macromolecules. For plasma samples, 128 transients were collected into 32 k data points using a spectral width of 10 kHz with a relaxation delay of 3 s, and total echo time (2nτ) of 100 ms. For urine samples, 64 transients were collected into 32 k data points using a spectral width of 10 kHz with a relaxation delay of 3 s, and total echo time (2nτ) of 60 ms. Prior to Fourier transformation, the free-induction decays (FIDs) were multiplied by an exponential function with a line-broadening factor of 0.3 Hz.

The pathologic microscopic examination is shown in Fig. 2. In the sham operation group, the nerve cells at the region of the cerebral cortex were normal (Fig. 2A1), while minority apoptosis of nerve cells in the hippocampus were visible (Fig. 2A2). In the model group, most apoptosis of nerve cells in the cerebral cortex and hippocampus were visible (Figs. 2B1 and 2B2). Meanwhile, the nerve cells in the cerebral cortex shrank slightly as well as those in hippocampus in the nimodipine treatment group (Figs. 2C1 and 2C2). However, the nerve cells in the cerebral cortex shrank slightly and only minority apoptosis of hippocampus nerve cells were visible in the optimized rhubarb aglycone treatment group (Figs. 2D1 and 2D2). The number of normal neuronal cell are shown in Table 1. Compared to the sham operation group, the number of normal cells had a significant reduction in the model group. However, the number of normal cells in the nimodipine group and the optimized rhubarb aglycone group increased in comparison with those in the model group, showing that the optimized rhubarb aglycone could reduce the number of neuronal apoptosis caused by cerebral ischemia–reperfusion.

2.9. Data processing All the spectra were corrected for phase and baseline manually, and then bucketed and automatically integrated with an automation routine in XWINNMR. All NMR spectra were referenced to the TSP at δ0.00. The region of δ4.6–6.2 was deleted to eliminate the effects of water suppression and urea signals. Therefore, the spectra over the ranges δ0.5–4.6 and δ6.2–9.5 were selected. Prior to pattern recognition analysis, each bucketed region was normalized to the total sum of the spectral integrals to compensate for the effects of variation in concentration. Data were further subjected to principal component analysis (PCA) using the software Simca-P þ 12.0 (Umetrics, Sweden). PCA is a multivariate projection method useful in classifying samples according to their common spectral characteristics. Data were visualized with the PCA scores plots, where each point represents an individual spectrum of a sample, and loadings plots, where each point represents a single NMR spectral region or chemical shifts. From the score and loading plots, classification of samples and the biochemical components responsible for the classification respectively can be shown. The normalized integral areas of the selected metabolites form PCA results were further statistically analyzed using SPSS17.0 using One-Way ANOVA analysis.

3.4.

1

H-NMR analysis of plasma

3.4.1. Metabolites identification of 1H CPMG spectra of plasma Fig. 3 shows representative 500 MHz 1H NMR CPMG spectra of plasma from sham operation group, model group, nimodipine and the optimized rhubarb aglycone treatment group. Assignments of endogenous metabolites involved in 1H-NMR spectra were based on the literature (Yap et al., 2006; Chen et al., 2011, 2012; Peng et al., 2011; Shi et al., 2013) and confirmed by 2D spectroscopy. The plasma NMR spectra were dominated by leucine (δ0.95,0.97), valine (δ1.03), 3-hydroxybutyrate (δ1.18), LDL/VLDL (δ0.86, δ1.26), lactate (δ1.33, δ4.12), alanine (δ1.48), acetate (δ1.92), N-acetyl aspartate (NAA) (δ2.02), glutamate (δ2.14), acetoacetate (δ2.24), pyruvate (δ2.38), succinate (δ2.41), citrate (δ2.54, δ2.68), phosphocreatine/creatine (δ3.02), cholestyramine (δ3.18), choline (δ3.22), taurine (δ3.26, δ3.42), glycine (δ3.54), citrulline (δ3.71), methionine (δ3.82), tyrosine (δ3.94), glucose (δ3.2–4.0, 4.66, 5.23) and unsaturated lipids (δ5.30). Visually, the resonance signals of lactate and glutamate were higher in the model group, while the

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Table 1 The results of nervous symptom score, cerebral infarction area and the pathological sections of cerebral issue (n¼ 6, X 7 s). Group

Evaluation of neurologic impairment (score)

Infarct area(%)

Number of normal neuronal cell

Sham operation group Model group Nimodipine group Optimized rhubarb aglycone group

0.00 70.00 2.83 70.87nn 2.2470.81n△ 1.86 70.72n△

0.007 0.00 35.647 10.63nn 22.58 7 8.47nn△ 10.517 3.66n△△

45.98 7 3.45 14.78 7 3.21nn 28.69 7 2.64n△ 36.81 7 3.82n△△

Note:

n

as compared with the sham operation group, np o0.05, np o 0.01; △ as compared with the model group, Δp o 0.05,

△△

p o 0.01.

Fig. 1. The TTC colorable graphs of cerebral tissue from (A) sham operation group; (B) model group; (C) nimodipine group; and (D) optimized rhubarb aglycone group.

resonance signal of phosphocreatine/creatine was lower compared with the sham operation group. The 1H spectra profiles of the optimized rhubarb aglycone group were similar with those of nimodipine group.

3.4.2. PCA and statistical analysis of plasma spectra data To emphasize the contribution of the small metabolites, PCA was done on the CPMG spectral data. Firstly, a tentative PCA model has been performed for all the samples (Fig. 4A1). Samples of model group were separated from the other samples along PC1, and the optimized rhubarb aglycone and the nimodipine treatment group were close to the sham group, suggesting the similar spectra profiles in these three groups. A PCA model was performed for the model group and the sham operation group (Fig. 4B1, PC1 vs PC2, R2 ¼62.7%) and revealed a satisfactory discrimination between these two groups. The loading plots (Fig. 4B2) revealed that the resonances responsible for the discrimination were lactate, glutamate, glycine, taurine, choline, glucose, methionine, 3-hydroxybutyrate, lipids, phosphocreatine/creatinine. Meanwhile, similar PCA plot was obtained when analysis was performed for the model group and the optimized rhubarb aglycone group (Fig. 4C1, PC1 vs PC2, R2 ¼75.7%), which showed clear clustered separation. The loading plots (Fig. 4C2) revealed that the discrimination attributed to the same variables as mentioned above.

Table 2 summarizes the variation of the integrals of the normalized spectral regions (buckets) accounting for different plasma metabolites and lists the results from the statistical analysis (po0.05) for comparison. The resonances assigned to lactate, taurine, glutamate, glycine, choline, methionine, glucose were significantly increased, but the level of lipids, 3-Hydroxybutyrate and phosphocreatine/creatine were statistically decreased in the model group compared to the sham operation group. The optimized rhubarb aglycone group had higher levels of lipids and phosphocreatine/creatine but lower levels of lactate, taurine, glutamate, glycine, methionine and glucose. However, levels of choline and 3-hydroxybutyrate did not present significant alterations in the optimized rhubarb aglycone group compared to the model group. 3.5.

1

H-NMR analysis of urine

3.5.1. Metabolites identification of 1H CPMG spectra of urine Fig. 5 shows representative 500 MHz 1H NMR CPMG spectra of urine from sham operation group, model group, nimodipine group and the optimized rhubarb aglycone group. Assignments of endogenous metabolites involved in 1H-NMR spectra were based on the literature (Yap et al., 2006; Bollard et al., 2010; Shariff et al., 2010; Zhang et al., 2010; Kinross et al., 2011; Zhou et al., 2012; Wang et al., 2013) and confirmed by 2D spectroscopy. The urine NMR spectra were dominated by valine/leucine/isoleucine (δ0.92– 0.98), lactate (δ1.33, δ4.12), alanine (δ1.48), acetate (δ1.92), acetone (δ2.23), succinate (δ2.41), α-ketoglutaric acid (δ2.46, 3.02),

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Fig. 2. HE colorable graphs of cerebral tissue (  400) A1: Cells of cerebral cortex in sham operation group; A2: Cells of hippocampus in sham operation group; B1: Cells of cerebral cortex in model group; B2: Cells of hippocampus in model group; C1: Cells of cerebral cortex in nimodipine group; C2: Cells of hippocampus in nimodipine group; D1: Cells of cerebral cortex in the optimized rhubarb aglycone group; and D4: Cells of hippocampus in the optimized rhubarb aglycone group Neurons.

citrate(δ2.54, δ2.68), dimethylamine (δ2.73), trimethylamine (δ2.92), choline (δ3.22), taurine (δ3.26, 3.42), proline (δ3.46), glycine (δ3.54), glucose (δ3.74, 3.83), tyrosine (δ3.94), creatinine (δ3.06, 4.06), allantoin (δ5.39) and hippurate (δ7.84, 7.64, 7.56, 3.97). Compared with the other groups, the resonance signals of glycine and proline were higher in the model group, while the resonance signals of α-ketoglutaric acid and creatinine were lower.

The 1H spectra profiles of the optimized rhubarb aglycone group were similar with those of nimodipine group.

3.5.2. PCA and statistical analysis of urine spectra data Fig. 6A shows the PCA result as score plots for the first two principal components from the CPMG spectra of urine from the four

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Fig. 3. Typical 1H NMR cpmg spectra of plasma from four groups: (A) the sham operation group; (B) model group; (C) nimodipine group; and (D) the optimized rhubarb aglycone group. Main metabolites: 1. Leucine; 2. Valine; 3.3-Hydroxybutyrate; 4. LDL/VLDL; 5.Alanine; 6. Acetate; 7. N-Acetyl Aspartate; 8. Glutamate; 9. Acetoacetate; 10. Pyruvate; 11. Succinate; 12. Citrate; 13. Phosphocreatine/creatine; 14. Cholestyramine; 15. Choline; 16. Taurine; 17. Glycine; 18. Citrulline; 19. Methionine; 20. Tyrosine; 21. Lactate; 22. β-Glucose; 23. α-Glucose; and 24. Unsaturated lipids.

groups, namely, sham operation group, model group, nimodipine group and the optimized rhubarb aglycone group. The samples in model group were discriminated from those in other groups in score plot (Fig. 6A1). The samples of optimized rhubarb aglycone group and the Nimodipine drug group were close to those of the sham operation group. The clustering (Fig. 6B1, PC1 vs PC2, R2 ¼67.7%) is observable for the model group and sham operation group. The loading plots (Fig. 6B2) revealed that the resonances responsible for the discrimination were taurine, choline, glycine, proline, glucose, α-ketoglutaric acid, creatinine and tyrosine. Meanwhile, it was clearly showed the samples in model group were separated from those in the optimized rhubarb aglycone group (Fig. 6C1, R2 ¼ 0.878). Table 3 showed the statistical results of signal integrals screened out from pattern recognition analysis (Fig. 6), which contributed to the classifications in model group and optimized rhubarb aglycone group. Compared to sham operation group, the model group had higher levels of taurine, choline, glycine, proline and glucose, but lower levels of tyrosine, α-ketoglutaric acid and creatinine. On the other hand, levels of choline, glycine, proline and glucose were decreased while levels of taurine, tyrosine, α-ketoglutaric acid and creatinine were increased in the optimized rhubarb aglycone group compared to the model group.

4. Discussion In this study, we conducted the experiments of neurologic impairment evaluation, brain tissue TTC staining and pathological examination, demonstrating that the optimized rhubarb aglycone could improve the neurological symptoms and reduce brain infarct area and cell apoptosis. Furthermore, we have performed 1H NMRbased metabonomics studies of plasma and urine combined with pattern recognition techniques to discriminate the cerebral

ischemia model group from the optimized rhubarb aglycone group, suggesting the optimized rhubarb aglycone could regulate the metabolic disorders and promote the regression of metabolic phenotype to the normal range. In plasma, rats in the optimized rhubarb aglycone group had higher levels of lipids and phosphocreatine/creatine, but lower levels of lactate, taurine, glutamate, glycine, methionine and glucose compared to those of the model group. In urine, levels of choline, glycine, proline and glucose were decreased while levels of taurine, tyrosine, α-ketoglutaric acid and creatinine were increased in the optimized rhubarb aglycone group compared to the model group.

4.1. Amino acids metabolism During cerebral ischemia, glutamate is released in superphysiological amounts, which possibly revealed the inhibition of reuptake of glutamate in glial cells and presynaptic neurons, resulting in a large number of glutamate accumulations. This excitotoxicity is mediated by several glutamate receptor subtypes (Akins and Atkinson, 2002), subsequently causing the ion imbalance and calcium overload. Extensive researches have shown that Ca2 þ influx via EAA receptors is important in mediating this neurodegeneration (Aarts and Tymianski, 2004). A large number of glutamate can activate the mGluR, which further activate Gq protein coupled phosphatidylinositol signal transduction pathways (Kimura et al., 2003; Crack and Taylor, 2005), and ultimately change the cell permeability. Compared with the model group, the level of glutamate in plasma decreased obviously in the optimized rhubarb aglycone group, indicating that the optimized rhubarb aglycone could significantly reduce the content of glutamate by possibly reducing the influx of Ca2 þ . With combination of pathological observations of brain tissue slices, it was showed that the optimized rhubarb aglycone reduced the number of nerve cells

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Fig. 4. PCA results of plasma 1H-NMR spectra A: score plot of all samples from four groups; B1, B2: score and loading plots of model group vs sham operation group; C1, C2: score and loading plots of model group vs the optimized rhubarb aglycone group; ● Model group Sham operation group Nimodipine group ■ The optimized rhubarb aglycone group. Table 2 Main metabolite changes of plasma by statistical analysis (n¼6). The main metabolite

Model group vs Sham Optimized rhubarb aglycone operation group group vs Model group

Lactate Taurine Glutamate Glycine Choline Methionine Glucose Lipids 3-Hydroxybutyrate Phosphocreatine/ Creatine

↑△△ ↑△△ ↑△ ↑△ ↑△ ↑△ ↑△△ ↓△△ ↓△ ↓△△

↓nn ↓nn ↓n ↓n ↓ ↓nn ↓nn ↑nn ↑ ↑n

Note: △ as compared with the sham operation group, compared with the model group, nnp o0.01, np o 0.05.

△△

p o 0.01, △p o 0.05;

n

as

apoptosis and glutamate excitotoxicity, presenting a protective effect on nerve cells. In comparison with the sham operation group, the glycine level of plasma and urine increased significantly in model rats. Glycine plays a dual role in the nervous system. On one hand, it is an

inhibitory neurotransmitter, which activates the glycine receptors against excitotoxicity caused by cerebral ischemia. On the other hand, it is the NMDA receptor co-agonist, binding to the NMDA receptor, which plays the role of nervous excitability and excitotoxic neuronal injury (Barth et al., 2005). Whether glycine has a toxic effect or a protective effect on cerebral ischemia remains controversial, and the molecular mechanism is unclear. The level of glycine in the optimized rhubarb aglycone group is lower than that in the model group, but it is close to the level in sham group, which indicated that the optimized rhubarb aglycone inversely regulated the concentration of glycine to the normal level. The increase of taurine possibly activates the taurine transporter which inhibits the Na þ –Ca2 þ channel, reducing the influx of Ca2 þ to prevent Ca2 þ overload and maintaining intracellular Ca2 þ homeostasis (Wang et al., 1992). Suleiman and Chapman (1993) found that the Ca2 þ overload in isolated heart of guinea pig could lead to decreased level of taurine in myocardium. It was also demonstrated that supply of taurine may reduce the cardiac dysfunction caused by Ca2 þ overload and increase ATP level, playing a protective effect on myocardial cells. Taurine also acts on the downstreaming of glutamate receptor activation through the regulation of cytoplasmic and intra-mitochondrial calcium homeostasis, to prevent excitotoxicity and accumulation of lactate in the brain, liver and heart tissue caused by ischemia and hypoxia

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Fig. 5. Typical 1H NMR cpmg spectra of urine from four groups: (A) the sham operation group; (B) model group; (C) nimodipine group; and (D) the optimized rhubarb aglycone group. Main metabolites: 1. Leucine/Isoleucine/valine; 2. Lactate; 3. Alanine; 4. Acetate; 5. Acetone; 6. Succinate; 7. α-ketoglutaric acid; 8. Citrate; 9. Dimethylamine; 10. Trimethylamine; 11. Choline; 12. Taurine; 13. Proline; 14. Glycine; 15. Glucose; 16. Tyrosine; 17. Creatinine; 18. Allantoin; and 19. Hippurate.

(Mankovskaya et al., 2000; EIdrissi et al., 2003). Taurine can not only inhibit the activation of neutrophils, but also reduce the release of oxygen free radicals mediated by cerebral ischemia– reperfusion inflammatory response (Schuller-Levis and Park, 2004). Wang found that rhubarb could improve the activity of SOD and reduce the MDA in the brain, indicating that the rhubarb protected the brain tissue by enhancing free radical scavenging and inhibiting damage from free radical (Wang et al., 2003). Our research also showed that compared with the sham operation group, the concentration of taurine in the plasma and urine increased significantly in the model group, suggesting that cerebral ischemia and hypoxia promoted the increases of taurine, which inhibits excitotoxicity caused by glutamate and plays a vital role in neuroprotection. Compared with the model group, the plasma level of taurine decreased in the optimized rhubarb aglycone treatment group, indicating that the optimized rhubarb aglycone continued to maintain the moderate level of taurine to protect the injury of brain tissue. 4.2. Energy metabolism Once cerebral ischemia, ATP rapidly depletes and anaerobic glycolysis strengthens in brain tissue, which will produce large amounts of lactate. With accumulation of lactate H þ , intracellular Na þ increases as a result of activated Na þ –H þ exchanger, which in turn activates Na þ –Ca2 þ exchanger (NCX). The NCX is an important Ca2 þ transporter that can cause Ca2 þ overload (Sugishita et al., 2001) and neuronal injury in certain pathological conditions. Its accumulation may cause lactic acidosis, which further aggravates the injury of nerve cells. The research found that the concentration of lactate in the plasma decreased in the optimized rhubarb aglycone group compared with the model group, indicating that the optimized rhubarb aglycone could decrease the level of lactate and prevent Ca2 þ overload, and had a protective effect on nerve cell. Compared with the sham operation group, the level of blood glucose increased significantly in the model group. It may be a result of improved expression of glucose transporter factor in the early stage of ischemia and hypoxia (Devaskar et al., 1999). The

level of glucose in the plasma and urine decreased in the optimized rhubarb aglycone group in comparison with the level of glucose in the model group, indicating that the optimized rhubarb aglycone had a regulation effect to the glucose metabolism in cerebral ischemia rats. Phosphocreatine/creatine (PCr/Cr) are energy metabolism molecules, which can reflect the changes of energy metabolism in the brain (Ross and Michaelis, 1994). Balestrino found Cr can effectively restrain hypoxia ischemia injury caused by depolarization, which speculated that PCr might be plays a protective role in brain tissue of ischemia hypoxia (Balestrino et al., 2002). This research showed that the levels of PCr and Cr in the plasma decreased in the model group. Energy metabolism was disturbed after cerebral ischemia and reperfusion. The optimized rhubarb aglycone can effectively improve the concentration of PCr and Cr, in order to increase the energy supply and further reduce the injury of brain. Compared with the sham operation group, the level of α-ketoglutaric acid in urine reduced significantly in the model group. The decrease of the α-ketoglutaric acid indicated that tricarboxylic acid cycle was blocked and cerebral ischemia led to metabolic disorder. Α-ketoglutaric acid may prevent the disturbances of neural cells that usually take place during ischemic pathology (Kovalenko et al., 2011). The level of α-ketoglutaric acid in the optimized rhubarb aglycone group increased significantly, indicating that the optimized rhubarb aglycone had an effect on blocked tricarboxylic acid cycle and regulated the aerobic energy metabolism. 4.3. Lipid metabolism Choline is an important component of the phosphatidylcholine and sphingomyelin (Lakher and Wurtman, 1987; NikolovaKarakashian et al., 1997). However, the energy supply is insufficient in the condition of ischemia and hypoxia, resulting in lower enzyme activity (Peng et al., 2003) and hindered synthesis of glycerophospholipids and sphingomyelin. Namely, cerebral ischemia pathological state could lead to abnormal phospholipid metabolism (Chen et al., 1996). Compared with the model group, the concentration of choline in the plasma and urine decreased

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Fig. 6. PCA results of urine 1H-NMR spectra A: score plot of all samples from four groups; B1, B2: score and loading plots of model group vs sham group; C1, C2: score and loading plots of model group vs the optimized rhubarb aglycone group; ● Model group Sham operation group Positive medicine group ■ The optimized rhubarb aglycone group.

Table 3 Main metabolite changes of urine by statistical analysis (n¼ 6). The main metabolite

Model group vs Sham operation group

Optimized rhubarb aglycone group vs Model group

α-Ketoglutaric acid Taurine Choline Glycine Proline Tyrosine Glucose Creatinine

↓△△△

↑n

↑△△ ↑△ ↑△△△ ↑△ ↓△ ↑△△ ↓△

↑n ↓n ↓nn ↓n ↑nn ↓n ↑n

Note: △ as compared with the sham operation group, △△△p o 0.001, △ p o 0.05; n as compared with the model group, nnpo 0.01, np o 0.05.

△△

po 0.01,

in the optimized rhubarb aglycone group, indicating that the optimized rhubarb aglycone may keep the balance of phospholipid metabolism in focal cerebral ischemia rats. NMR spectroscopy is a powerful tool for generating multivariate metabolic data by measuring hundreds of compounds

simultaneously and is a nondestructive method providing a broad unbiased overview of major intermediary metabolites present in biological samples. However, the sensitivity of NMR is poorer and the assignments of the metabolites are limited, while metabolomics research requires the use of high field NMR instrument over 500 MHz. Since NMR produce large amounts of complex data, the use of pattern recognition techniques to aid the interpretation of the data is common. So the extraction of metabolic information is very dependent on the selected data analytical methods. Therefore, the development of instrument and the new variate analytic method will promote the development of metabonomics.

5. Conclusion In our study, a 1H NMR-based metabonomics method combined with PCA was employed to study the protective effect of the optimized rhubarb aglycone. From the PCA score plots, the samples of optimized rhubarb aglycone treated group were close to those of the sham operation group, while a distinct classification between optimized rhubarb aglycone treated group and model group based on their urine and plasma samples has been

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clearly demonstrated. We found that some endogenous metabolites correlated with focal cerebral ischemia–reperfusion altered by the treatment of the optimized rhubarb aglycone. The optimized rhubarb aglycone regulated the metabolic disorders and promoted the regression of metabolic phenotype close to the normal range. This study could provide evidence that 1H NMRbased metabonomics method is a useful tool in the investigation of metabolic regulation mechanisms of traditional Chinese medicine.

Acknowledgment We acknowledge the financial supports from the National Natural Science Foundation of China (81073024 and 81274060). SWL also acknowledges financial supports from the National Natural Science Foundation of China (81274059).

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