Neuroprotective effect of Naomaitong extract following focal cerebral ischemia induced by middle cerebral artery occlusion in rats

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J Tradit Chin Med 2017 June 15; 37(3): 333-340 ISSN 0255-2922 © 2017 JTCM. This is an open access article under the CC BY-NC-ND license.

RESEARCH ARTICLE TOPIC

Neuroprotective effect of Naomaitong extract following focal cerebral ischemia induced by middle cerebral artery occlusion in rats

Yang Yongxia, Chen Xi, Wang Shumei, Wang Zhanhong, Li Jiansheng, Liang Shengwang aa Yang Yongxia, College of Basic courses, Guangdong Pharmaceutical University, Guangzhou 510006, China Chen Xi, Division of Education and Research, Shantou Central Hospital, Shantou 515031, China; College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China Wang Shumei, Wang Zhanhong, Liang Shengwang, College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China Li Jiansheng, Institute of Geriatrics, Henan College of Traditional Chinese Medicine, Zhengzhou 450003, China Supported by National Natural Science Foundation of China (Study on the Material Basis and the Ratio of the Effective Components of Naodesheng Based on the Combination of Fingerprint and Metabolic Network, No. 81274059; Study on the Material Basis of Naomaitong in the Treatment of Ischemic Stroke Based on the in vivo Dynamic Effect and Bioinformatics, No. 81274060; Study on the in vivo Process and Compatibility Rule of Naomaitong Based on the PK-PD of Effective Components and the Multiobjective Optimization, No. 81473413) Correspondence to: Prof. Liang Shengwang, College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China. [email protected] Telephone: +86-20-3935-2172 Accepted: June 17, 2016

three groups: sham-operated group, MCA focal cerebral ischemia reperfusion model group, and active extract of Naomaitong treatment group. The model was established by an improved MCA occlusion (MCAO) method. Sham-operated rats received the same surgical procedure, but without occlusion. The Naomaitong treatment group were treated with active extract from Naomaitong at a dose of 3.0 g·kg－1·d－1. Brain tissues and urine samples were collected from all groups for histopathological assessment and proton nuclear magnetic resonance spectroscopy-based metabonomics, respectively. RESULTS: Hematoxylin-eosin and triphenyl tetrazolium chloride staining of brain tissues showed a significant decrease in cerebral infarction area in the Naomaitong group. In model rats, metabonomic analyses showed increased urinary levels of glutamate, taurine, trimetlylamine oxide, betaine, and glycine, and reduced levels of creatinine and creatine. Naomaitong regulated the metabolic changes by acting on multiple metabolic pathways, including glycine metabolism, glutaminolysis, transmethylation metabolism and creatinine metabolism. CONCLUSION: These data demonstrate that extract from Naomaitong is neuroprotective against focal cerebral ischemia induced by MCAO, and can alleviate biochemical changes in urinary metabolism. Metabonomics may be a useful approach for assessing the biochemical mechanisms underlying the neuroprotective actions of extract from Naomaitong.

Abstract OBJECTIVE: To examine the neuroprotective effect of extract from Naomaitong following focal cerebral ischemia reperfusion induced by occlusion of middle cerebral artery (MCA), and to determine the biochemical alterations in urine using proton nuclear magnetic resonance spectroscopy and principal component analysis.

© 2017 JTCM. This is an open access article under the CC BY-NC-ND license.

Keywords: Cerebrovascular circulation; Reperfu-

METHODS: Wistar rats were randomly assigned to JTCM | www. journaltcm. com

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sion; Middle cerebral artery; Principal component analysis; Metabonomics; Magnetic resonance spectroscopy; Naomaitong

ing a biochemical fingerprint of the organism, as well as potential biomarkers. Based on the combination of 1 H NMR with multivariate data analysis, metabonomics is now a well-established technique for metabonome analysis, including widespread applications in pharmacological studies.19 For example, Huo et al 20 adopted NMR-based metabonomics to successfully determine the essence of blood deficiency syndrome and the active mechanism of Siwutang. 1H NMR-based metabonomics was also applied to study the restorative effect and potential mechanisms of Buzhongyiqi Tang in a spleen-Qi deficiency rat model, providing a useful tool for assessment of the restorative effects of Buzhongyiqi Tang both dynamically and holistically.21 In the present study, we analyzed the neuroprotective action of Naomaitong treatment following cerebral ischemia in MCAO model rats, and assessed the urinary metabolites changes using a 1H NMR-based metabonomics approach.

INTRODUCTION Cerebral apoplexy, the acute occurrence of neurological symptoms induced by a vascular lesion in brain, accounts for approximately 60%-80% of all cerebrovascular accidents.1 Occlusion of the middle cerebral artery (MCAO) is a general cause of stroke in humans,2 and is commonly used in animal models for studying cerebral apoplexy.3 There are numerous mechanisms considered to be involved in ischemic brain damage, including lipid peroxidation, overproduction of free oxygen radicals, increased intracellular calcium, and neuroinflammation.4 Nevertheless, there are limited therapeutic options, largely due to the complications resulting from ischemic/reperfusion injury.5 Western Medicines used for clinical prevention of stroke primarily include aspirin, clopidogrel, and rosuvastatin.6 Aspirin is an essential drug used to prevent and control stroke, and it may also be useful for preventing stroke relapse. However, 47% of stroke patients exhibit a resistance to aspirin. Further, even enteric-coated aspirin can have detrimental effects on the gastrointestinal system.7 Clopidogrel also has a potentially serious side effects associated with hepatotoxicity,8 while rosuvastatin can cause ischemic colitis.9 Traditional Chinese Medicine (TCM) can provide therapeutic benefits for numerous diseases, including Complex Salvia Miltiorrhiza Dripping pills for cardiovascular diseases10 and Liuwei Dihuang pills for kidney lesions.11 In addition, the effects of TCM are usually more prolonged than Western Medicine, with less adverse reactions or rebound phenomena after withdrawal of medication. There are numerous TCM for treatment of stroke, including Qingkailing injection12 and Mailuoning injection.13 Naomaitong, which comprises Renshen (Radix Ginseng), Dahuang (Radix Et Rhizoma Rhei), Gegen (Radix Puerariae Lobatae) and Chuanxiong (Rhizoma Chuanxiong), was reported to be neuroprotective against cerebral ischemia injury, by reducing the degree of cerebral ischemia, water content, and brain infarction area.14,15 Nevertheless, the metabolic mechanisms underlying these actions of Naomaitong remain unclear. Metabolomics/metabonomics is a relatively new approach for monitoring biochemical changes caused by endogenous and exogenous factors.16 Analysis techniques for metabolomics include proton nuclear magnetic resonance spectroscopy (1H NMR), mass spectrometry (MS), and gas/liquid chromatograph-MS (GC/LC-MS).17 Nicholson et al 18 reported that 1H NMR spectroscopy of biofluids allowed the simultaneous measurement of endogenous metabolites, providJTCM | www. journaltcm. com

MATERIALS AND METHODS Materials and reagents Renshen, Dahuang, Gegen and Chuanxiong were purchased from Guangzhou Zhixin Pieces of Chinese Medicine Co., Ltd. (Guangzhou, China), and were identified by Prof. Li Shuyuan (Department of Chinese Medicine of Guangdong Pharmaceutical University). The extract of Naomaitong was prepared in our laboratory. The four herbs were weighed according to the prescription of Naomaitong, and placed into a round bottomed flask and dissolved in 60% ethanol with ten times the amount of the herbs. The mixture was then extracted using the water bath reflux method for 1 h, and the procedure was repeated twice more. A 60% ethanol elution solvent and recycled ethanol were collected by rotating evaporation, and then concentrated to 400 mL. Finally, the extract (0.3 g/mL dose) was prepared for purification of the active extract of Naomaitong. The optimal parameters for purification of the active extract of Naomaitong using D101 macroporous absorption resin included a 1∶6 diameter height ratio, a 0.3 g/mL concentration of the extract fluid, and 1 bed volume (BV)/h flow rate. After 2 h of sample loading, we rinsed the macroporous absorption resin using 2 BV water until the solution became colorless, and discarded the eluent. The macroporous absorption resin was then rinsed with 8 BV 50% alcohol. A 50% alcohol elution solvent and recycled alcohol were collected by rotating evaporation. Finally, the concentrated alcohol elution solvent was diluted with distilled water to a concentration of 0.3 g/mL as the active extract of Naomaitong formula. Sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), and heavy water (D2O) containing sodium 3-trimethylsilyl-(2, 2, 3, 334

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the brain tissues removed and stored at － 20 ℃ for 15 min. The brains were then sectioned coronally (2 mm thick) into five slices. The second slice was embedded in paraffin and then staining with hematoxylin and eosin (HE). Changes in brain tissues and nerve cells were observed by light microscopy (× 400). The third slice was immediately immersed in TTC staining medium at 37 ℃ for 15 min, and then incubated for another 15 min after reversal of the slices. Finally, the TTC stained sections were incubated in 10% formalin solution.

3-2H4)-1-propionate (TSP) were obtained from Qingdao Teng Long Technology Co., Ltd. (Qingdao, China). Sodium carboxymethyl cellulose (CMC-NA), formaldehyde, sodium azide (NaN3), and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Shanghai Yuanfan Zhuji factory (Shanghai, China). Chloral hydrate and heparin ([C26H41NO34S4]n) were purchased from Shanxi Tingtai Biological Technology Co., Ltd. (Shanxi, China). Animal experiments A total of 32 male Wistar rats [(280 ± 20) g, five months of age] were obtained from the Laboratory Animal Institute of Guangzhou University of Chinese Medicine (animal certificate #SPF20120125). This study was reviewed and approved by the Ethics Committee of Guangdong Pharmaceutical University. The rats were kept in an air-conditioned experimental laboratory, with a 12 h light/dark cycle and a constant temperature of (25 ± 1) ℃ . After acclimatization for one week, rats were randomly assigned to three groups using the random number table method (sham-operated group [normal controls], MCA focal cerebral ischemia reperfusion model group, and active extract of Naomaitong group. Before modeling, the control and model rats were gavaged with distilled water once a day for four days, while the Naomaitong group received Naomaitong active extract (3.0 g·kg － 1·d － 1) over the same period. All rats were fasted on the fourth day and night, with only free access to water. The Naomaitong group received final treatment with the active extract at one hour before surgery on the fifth day.

Sample collection and preparation for 1H NMR analysis After 6 h reperfusion, urine samples of each group were collected into eppendorf (EP) tubes, with addition of 100 µL of 1% sodium azide for anti-bacterial treatment. All samples were stored at －80 ℃ for NMR assessment. Urine samples were thawed at room temperature before NMR analysis. For each sample, a total of 300 µL of urine samples was mixed with 200 µL phosphate buffer (0.2 M Na2HPO4/NaH2PO4, pH 7.4) containing 80 µL of 0.05% TSP) as a chemical shift reference (δ 0.00 ppm). After centrifugation at 14000 g for 10 min at 4 ℃, the supernatant was pipetted into 5 mm NMR tubes for NMR analysis. H NMR experiments and data processing All NMR spectra were recorded at 298 K on Avance Ⅲ 500 MHz spectrometer (Bruker Ltd., Karlsruhe, Germany). 1H NMR spectra were obtained using a one-dimensional nuclear Overhauser effect spectroscopy with presaturation (NOESYPR) with water suppression during a relaxation delay of 3 s. In NOESYPR experiments, the mixing time was set to 100 ms with a t1 of 3 µs. Sixty-four transients were collected into 32 k data points using a spectral width of 10 kHz. 1

Surgical procedures MCA focal cerebral ischemia reperfusion was generated in the MCAO group and the Naomaitong treatment group as described previously, with slight modifications.22,23 Following overnight fast, anesthesia was performed with intra-peritoneal administration of 10% chloral hydrate (40 mg/kg). Animals were fixed on an operating table in supine position, and the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) exposed through a neck midline incision. The proximal CCA and ECA were then clamped, and a small incision was made in the branch of the CCA and ECA. A 4-0 monofilament nylon surgical suture with a rounded tip was introduced into the initial MCA through the ICA and CCA at 17.5-18.5 mm distal from the bifurcation, occluding the left MCA and tightening the ICA with a spare line. Sham-operated rats received the same surgical procedure, but without occlusion. Body temperature was maintained at 36.5-37.5 ℃ throughout the procedure. At 2 h after ischemia, the embolus was removed to establish blood reperfusion.

Pattern recognition and statistical analysis 1 H NMR spectra were manually corrected for phase and baseline, and then bucketed and automatically integrated with an automated routine in AMIX (Umetrics, Sweden). The spectral region δ 0.5-9.0 was integrated into regions with equal width of 0.004 ppm. Regions distorted by imperfect water saturation (δ 4.6-5.2) and urea signals (δ 5.2-6.2) were discarded. The total sum of the bucket integrals was normalized as variables for principal component analysis (PCA). All 1H NMR spectra were imported into software Simca-P + v12.0 (Umetrics, Sweden) for PCA analysis. In the scores plot, each point represented an individual sample, and the scores plot highlighted inherent clustering trends of the samples. The loadings plot provided metabolites contributing to the separation. Statistical analyses were performed by analysis of variance (SPSS 19.0). P < 0.05 was considered statistically significant.

Sample preparation for measurement of cerebral infarction area At 6 h after reperfusion, animals were euthanized and JTCM | www. journaltcm. com

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RESULTS

cal cerebral ischemia reperfusion model, and Naomaitong treated groups are shown in Figure 3. The 1H NMR spectra signals were assigned according to previously published data25,26 and confirmed by 2D spectra. The dominant metabolites in urine included a series of amino acids (organic acids such as creatine, succinate, citrate, and lactate), ketone bodies (acetone and acetoacetate), membrane metabolites (e.g., choline, glycerophosphocholine and phosphocholine), glycoproteins, and glucose. There were no obvious visual differences in urinary metabolic profiles between the groups. Therefore, we performed PCA for detailed analysis of the metabolic changes in the groups and to examine the urinary metabolic regulatory mechanism of Naomaitong.

Pathological analysis of ischemia/reperfusion injury Brain tissue slices from the sham-operated group showed typical neurons with a well-preserved cytoplasm and a clear nucleus (Figure 1). By contrast, a large region of swelling and focal softening was observed in the model group. In the cerebral cortex of model rats, there was evidence of dissolved and necrotic Nissl bodies and pyknotic nuclei, as well as sparse degeneration of glial cells in the hippocampus, as we previously reported.24 In the Naomaitong pretreated group, there was a decrease in brain swelling and necrotic areas to levels similar to those in the sham-operated group. In agreement with pathological analyses (Figure 2), TTC staining also showed an increase in cerebral infarction area in the model group compared with the other groups. The cerebral infarction area was markedly decreased in the Naomaitong group compared with the MCAO group. These results suggest that we successfully established the MCA focal cerebral ischemia reperfusion model, and that Naomaitong can reduce cerebral damage from ischemia-reperfusion, alleviating brain ischemia and hypoxia, and maintaining physiological balance in the brain. A1

A2

B1

B2

C1

C2

PCA and statistical analysis The scores plot of PCA represents the distribution of all urine samples in the three groups, and provides the distribution trajectory of the samples (Figure 4). An obvious classification was observed between the model and sham-operated groups, which indicated marked metabolic disorder in the MCA focal cerebral ischemia reperfusion group. Naomaitong-treated animals were located between the sham-operated group and the model group, suggesting that the active extract of Naomaitong can partially alleviate the metabolic disorders in model rats. To emphasize the contribution of small metabolites, PCA models were established for the classification between the model group and the sham-operated group, and between the model group and the Naomaitong-treated group. A distinct classification between control and model rats was shown in the scores plot (Figure 5A). From the loadings plot (Figure 5B), the levels of taurine, trimetlylamine oxide (TMAO), betaine, glutamate, and glycine were elevated, while levels of creatine and creatinine were decreased, in samples from the MCA focal cerebral ischemia reperfusion model group compared with the sham-operated group. These data suggest evidence of a marked metabolic disorder in the urine at 6 h of reperfusion, consistent with the severe injury by HE and TTC staining. There was a clear classification between the Naomaitong-treated group and the model group (Figure 6A). From the loadings plot, the active extract reversed the metabolic changes in model animals (Figure 6B; Figure 5B). A summary of the variations of normalized integrals of urine metabolites screened out in Figures 5 and 6 are shown in Table 1.

Figure 1 Hematoxylin and eosin staining (× 40) of the cerebral cortex and hippocampus A1, A2: the sham-operated group, in which the rats received the same surgical procedure, but without occlusion; B1, B2: the model group, middle cerebral artery focal cerebral ischemia reperfusion was generated in the model group; C1, C2: the active extract of Naomaitong-treated group, in which the rats received Naomaitong active extract (3.0 g·kg－1·d－1) for five days before middle cerebral artery occlusion surgery.

DISCUSSION In the present study, the focal cerebral ischemia-reperfusion model was successfully established using an improved MCAO method,22,23 which was confirmed by histopathological and TTC examination. NMR meta-

H NMR spectroscopy The representative 500 MHz urinary 1H NMR NOESYPR spectra of rats from the sham operation, MCA fo1

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A

C

Figure 2 2,3,5-triphenyltetrazolium chloride staining (× 40) of brain sections A: the sham-operated group, in which the rats received the same surgical procedure, but without occlusion; B: the model group, MCA focal cerebral ischemia reperfusion was generated in the model group; C: the active extract of Naomaitong-treated group, in which the rats received Naomaitong active extract (3.0 g·kg － 1·d － 1) for five days before middle cerebral artery occlusion surgery. ×8

A 30 B

19 16 26

27

29

15 24, 25 20, 21 23 26

28

31

22

12

18 16

15 C

11 9 17 11 10 8 6 7 13 14

5 4 3

2

8.0 7.5 7.0 6.5 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm Figure 3 Representative urine proton nuclear magnetic resonance (1H NMR) spectra A: the active extract of Naomaitong-treated group; B: the model group; C: the sham-operated group. 1: 2-hydroxybutyrate; 2: isoleucine; 3: 3-hydroxybutyrate; 4: lactate; 5: alanine; 6: n-acetylglycoprotein; 7: glutamate; 8: acetone; 9: acetoacetate; 10: succinate; 11: α-ketoglutaric acid; 12: citrate; 13: dimethylamine; 14: dimethylglycine; 15: creatine; 16: creatinine; 17: choline; 18: phosphocholine/glycerophosphocholine; 19: taurine; 20: trimetlylamine oxide; 21: betaine; 22: glycine; 23: glycerol; 24: α-glucose; 25: β-glucose; 26: hippurate; 27: n-methylnicotinic acid; 28: fumarate; 29: tyrosine; 30: phenylacetylglycine; 31: formate.

bonomic analysis demonstrated that MCAO surgery in the model group caused comprehensive metabolic alterations in the urine, including marked elevation in levels of taurine, TMAO, betaine, glutamate, and glycine, and a reduction in creatine and creatinine. Further, treatment with the active extract of Naomaitong was effective in treating the ischemia reperfusion injury and reversing ischemia reperfusion-induced metabolic disorders, including glycine metabolism, glutaminolysis, transmethylation, and creatinine metabolism (Figure 7). Our work demonstrates that Naomaitong has a therapeutic effect on focal cerebral ischemia-reperfusion injury, and suggests potential effects on underlying metabolic regulation using 1H NMR-based metabonomics. In the present study there was a significant increase in

0.04

t［2］

0.02 －0.00 －0.02 －0.04 －0.06 －0.06 －0.04 －0.02 －0.00 0.02 0.04 0.06 t［1］ Figure 4 Principal component analyses of urine 1H NMR spectra data ○: the sham-operated group; ●: the model group; ◇: the active extract of Naomaitong-treated group. 1H NMR: proton nuclear magnetic resonance.

0.04 0.02

－0.00 －0.02 －0.04 －0.06

B

0.30 0.20

A p［2］

t［2］

0.06

0.10 －0.00 －0.10 －0.20 －0.30 －0.10 －0.00

0.10 0.20 0.30 0.40 0.50 －0.08 －0.04 －0.00 0.04 0.08 t［1］ p［1］ Figure 5 Principal component analysis of urine proton nuclear magnetic resonance (1H NMR) spectra data A: scores plot, ○: the sham-operated group, ●: the model group; B: loadings plot. JTCM | www. journaltcm. com

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0.02

0.40 0.30 0.20 0.10 －0.00 －0.10 －0.20 －0.30 －0.40

B

p［2］

t［2］

0.04 －0.00 －0.02 －0.04

－0.00 0.06 －0.40 －0.20 －0.00 0.20 t［1］ p［1］ 1 Figure 6 Principal component analysis of urine proton nuclear magnetic resonance ( H NMR) data A: scores plot, ●: the model group; ◇: the active extract of Naomaitong-treated group. B: loadings plot. －0.06

Table 1 Changes in main metabolites changes in urine Variation Metabolite Chemical shift T2 T1 Glutamate ↓a 2.14 (m) ↑a Creatine/creatinine

3.04 (s)

Creatine

3.93 (s)

Creatinine

4.05 (s)

Taurine

3.27 (t)

Taurine

3.43 (t)

TMAO/Betaine

3.28 (s)

Glycine

3.54 (s)

↓b

↑b

↓b

↑b

↓b ↑a ↑a

↑b ↑a

excitatory and inhibitory roles in the central nervous system. For example, glycine is a primary inhibitory neurotransmitter in the spinal cord and brain stem,27 while glycine plays an important role in catalyzing the actions of glutamate in the cerebrum, through an allosteric site on the N-methyl-D-aspartic acid receptor.2 Glutamate is the primary excitatory neurotransmitter in the brain, and overactivation of glutamate receptors can cause an increase in intracellular calcium and generation of free radicals, resulting in cellular necrosis or apoptosis.28 Further, glutamate can be converted to glutamine through glutaminolysis.29 Therefore, the increased glycine and glutamate in the present study is suggestive of cellular necrosis or apoptosis, as well as disordered glutaminolysis. However, compared with the model group, the Naomaitong treated group showed significantly decreased levels of urine glycine and glutamate. Thus, the therapeutic action of Naomaitong may be related to down regulation of glycine and glutamate, and the associated restoration of glutaminolysis metabolism. Taurine is an inhibitory amino acid predominantly found in the brain. During neurotoxicity and hypoxia, taurine was reported to maintain mitochondrial function, and reduce glutamic acid neurotoxicity and resulting calcium influx.30 Taurine, mediated via the glycine receptor, was also reported to protect nerve cells.30 Further, taurine can interact with γ-aminobutyric (GABA) to activate GABA and glycine receptors, which may

↑b ↓b ↓b ↓b ↓a

Notes: ↑: the metabolite increased; ↓: the metabolite decreased; T1, the model group vs the sham-operated group, the arrows denoted the metabolites changes in the model group; T2, the active extract of Naomaitong treatment group vs the model group, the arrows denoted the metabolites changes in the active extract of Naomaitong-treated group; the rats in sham-operated group received the same surgical procedure, but without occlusion; middle cerebral artery focal cerebral ischemia reperfusion was generated in the model group; the rats in the active extract of Naomaitong-treated group received Naomaitong active extract (3.0 g·kg－1·d－1) for five days before middle cerebral artery occlusion surgery; aP < 0.01, bP < 0.05.

urine glycine and glutamate in the model group, which are closely associated with energy substrate depletion and reductions in regional blood flow. Numerous studies have reported that ischemia can induce excessive release of glutamate and glycine.2 Glycine can play both

betaine guanid noacetate

Creatine

Creatinine creatinine metabolism

ADP

glutamine

ATP

Pcr

glutamate

glutaminolysis

choline methylamine metabolism

DMG NMDA receptor

glycine metabolism

0.40

TAMO serine

glycine

glycine receptor taurine

Figure 7 Overview of the metabolic pathway alterations ADP: adenosine diphosphate; ATP: adenosine triphosphate; TMAO: trimetlylamine oxide; DMG: dimethylglycine; NMDA: N-methyl-D-aspartic acid; Pcr: phosphocreatine. JTCM | www. journaltcm. com

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strengthen the neural control effect,31 assist plasma transshipment of Ca2 + , Mg2 + , Na + , and K + , and maintain normal osmotic pressure inside and outside of cells.32 However, excessive taurine can produce nerve toxicity.33 In our study, we observed a significant increase in taurine in model rats, suggesting a potential for CNS excitotoxic cell death. Importantly, Naomaitong treatment reduced the level of urine taurine, suggesting that the extract may also inhibit CNS excitotoxicity. Urinary excretion of betaine is an emerging marker of pathological processes in vascular disease. Betaine has two important metabolic functions. It is a major osmolyte responsible for cell volume regulation in many tissues, and also counteracts urea cytotoxicity in the kidney.34 TMAO, as a betaine metabolite, was reported to be predictive of cardiovascular events.35 Additionally, excessive TMAO may be cytotoxic in mammals.36 Thus, in the present study the increased levels of betaine and TMAO in model animals suggests disruption of the intracellular environment in the brain, including neuronal cell death. Interestingly, betaine was reported to protect cells under stress and act as a methyl source for transmethylation reactions in biochemical pathways.37 Levels of betaine and TMAO were decreased in the Naomaitong treatment group, likely due to, at least in part, its regulation of disordered transmethylation metabolism. Creatinine is a metabolic end product generated from creatine phosphate and creatine, and has a direct function in cellular energy transport.6, 38 Many studies have reported that ischemia-hypoxia can increase levels of creatinine and creatine in the serum and occipital cortex.6,39 In the present study, levels of urinary creatinine and creatine were significantly decreased in the ischemia reperfusion group compared with sham controls, potentially due to a decreased glomerular filtration rate induced by reperfusion.39 By contrast, the extract of Naomaitong increased urinary creatinine and creatine levels, potentially due to improvement of the glomerular filtration rate and the regulation of creatine metabolism. In conclusion, the extract from Naomaitong significantly reduced brain injury and improved urinary metabolism following focal cerebral ischemia. These data suggest that metabonomics are a useful approach to elucidate the biochemical mechanisms underlying the neuroprotective actions of Naomaitong following focal cerebral ischemia induced by MCA occlusion.

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June 15, 2017 | Volume 37 | Issue 3 |