Huangqi Guizhi Wuwu Decoction Ameliorates Focal Cerebral Ischemia-Reperfusion Injury in Rats: Pharmacological and Metabolomics Evidences

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(1. The Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, China; 2. Nanjing Dorra Pharmaceutical Co., Ltd., Nanjing, 210012, China; 3. School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, China)

ABSTRACT: OBJECTIVE To investigate the neuroprotective effect of Huangqi Guizhi Wuwu Decoction (HGWD) on cerebral ischemia injury, and to explore the underlying mechanism.METHODS Fifty rats were randomly divided into 5 groups: sham operation group (sham), middle cerebral artery occlusion (MCAO) model group, and MCAO rats treated with HGWD at 5, 10, 20 g·kg-1. The first two groups were administered intragastrically (i.g.) with saline for 7 days, while HGWD groups were treated with HGWD at 5, 10, and 20 g·kg-1, correspondingly. The neurological deficits scores, cerebral infarct size, lipid peroxidation and inflammatory cytokines levels were used to evaluate the brain ischemic injury. The possible mechanism of HGWD on cerebral ischemia-perfusion injury was investigated by metabolomics. RESULTS HGWD treatment significantly attenuated middle cerebral artery occlusion (MCAO)-induced brain ischemic injury of rats as evidenced by decreasing the neurological deficits scores, cerebral infarct size, as well as lipid peroxidation and inflammatory cytokines levels. Further metabolomics study indicated that great metabolic disorders in serum were induced following cerebral ischemia/reperfusion injury. Totally 23 metabolites associated with ischemic stroke were proposed as potential biomarkers. The metabolic pathway analysis revealed that MCAO induced brain ischemic injuries mainly by targeting glyoxylate and dicarboxylate metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. Correspondingly, HGWD could restore most of the imbalanced endogenous metabolites. CONCLUSION HGWD plays a protective role in focal cerebral ischemia-reperfusion injury in rats, which may be associated with the inhibition of inflammatory effect and improvement of multiple metabolic pathways.

KEYWORDS:Huangqi Guizhi Wuwu Decoction; cerebral ischemic-reperfusion; middle cerebral artery occlusion; metabolomics; UPLC/MS

1 Introduction

Stroke is considered the leading cause of death or adult disability worldwide, among which 87% is ischemic[1]. The pathophysiology and mechanisms involved in ischemic stroke are varied and complex, including excitotoxic amino acids (AAs), inflammation, oxidative stress, apoptosis, angiogenesis, ionic imbalances and so on. Anticoagulation and antiplatelet agents are commonly used to restore cerebral blood flow for stroke treatment. However, the efficiency of these therapy are poor because of high risk of bleeding and ischemic-reperfusion (I/R) injury itself[2]. Currently, there is no universally accepted therapeutic regimen available. Thus, it is an urgent need to identify safe and effective strategy for stroke therapy. Numerous studies have demonstrated that multiple drugs combination regimens applied in treating complex illness can amplify the therapeutic efficacy and reduce adverse effects. Therefore, combination regimens have been considered as a promising choice for ischemic stroke treatment[3].

Traditional Chinese Medicine (TCM) using multiple components has been practiced for 2 500 years to prevent and treat complex, refractory illnesses. There is no doubt that TCM could act on multiple targets simultaneously and result in synergistic therapeutic efficacies. First recorded inSynopsisoftheGoldenChamber(JinguiYaolüein Chinese) in the Eastern Han Dynasty, Huangqi Guizhi Wuwu decoction (HGWD) has long been used for treating "blood impediment" characterized by numbness, pain and rough pulse[4]. HGWD is composed of five Chinese medicinal herbs:Astragali Radix (Haungqi, HQ, 18 g), Cinnamomi Ramulus (Guizhi, GZ, 9 g), Paeoniae Radix Alba (Baishao, BS, 9 g), Jujubae Fructus (Dazao, DZ, 9 g), and Zingiberis Rhizoma Recens (Shengjiang, SJ, 9 g). All the component herbs are reported to improve qi, promote the flow of yang, and invigorate the blood to promote coronary circulation[5]. Chemically, various phytochemical constituents including saponins, flavonoids and phenolic acids, have been identified in HGWD or its single herbs[6]. The chemical profile of HGWD was also previously established by our group using HPLC-MS/MS analysis, and a total of 36 compounds were unambiguously or tentatively characterized[6]. These constituents possess series of biological functions, such as anti-oxidation, anti-inflammatory and neuroprotection[7]. Previous studies have also found that HGWD could prevent diabetic peripheral neuropathy and improve neurological function[8]. However, the therapeutic effects of HGWD against cerebral ischemia have yet been unknown.

Due to the double complexity of stroke and chemical compositions of HGWD, it is difficult to assess the efficacy of HGWD by conventional pharmacological methods. Metabolomics, an approach for monitoring the changes in endogenous metabolites, has widely been applied as versatile tool for discovery of disease-related biomarkers and drug therapeutic evaluation[9-11]. Metabolomics could comprehensively reveal the metabolite perturbations in complex biological samples[12-13]. The concept of metabolomics is consistent with the holistic theory of TCM on the treatment of disease. Therefore, it is reasonable to explore the holistic and synergistic effects of TCM using metabolomics technology. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) is an ideal tool for metabolomics studies due to its fast-scanning capabilities, high mass accuracy and widespread availability[14]. In this work, we aimed to investigate the protective effects of HGWD on focal cerebral ischemia-reperfusion injury in rats via pharmacological study combined with a metabolomics approach. Potential plasma biomarkers closely related with the perturbed metabolic pathways would be identified for better understanding the intervention effect and responsible molecular mechanism of HGWD.

2 Materials and methods

2.1 Materials and animals

Crude drugs including HQ, BS, GZ, SJ and DZ obtained from Jiangsu Hospital of Integrated Traditional Chinese and Western Medicine, were identified, and authenticated based on the instructions recorded in Chinese Pharmacopoeia (2015 edition). The voucher specimens were deposited in Jiangsu Province Academy of Traditional Chinese Medicine.

Adult male SD rats weighing (260±10)g were purchased from Experimental Animal Center of QingLongShan Forest Farm (Nanjing, China). All animals were reared at (25±2)℃ (12∶12 dark∶light) with a relative humidity of (50±10)%. The animals were regularly fed during the experiment period. The animal experiments were approved by the Animal Care and Use Committee of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, and performed in accordance with the guidelines for Care and Use of Laboratory Animals.

2.2 HGWD preparation

HGWD was prepared as follows: HQ, BS, GZ, SJ and DZ were mixed in the ratio of 2∶1∶1∶1∶1, and immersed with 10-times the volume of distilled water for 0.5 h. The crude drugs were then subjected to reflux extraction twice, 1 h each time. The extracts were mixed and concentrated to 2 g·mL-1under reduced pressure, and stored at -80 ℃ for subsequent experiments.

2.3 Drug administration

Fifty rats were randomly divided into 5 groups (Fig.1): sham operation group (sham), middle cerebral artery occlusion (MCAO) model group, and HGWD 5, 10, 20 g·kg-1groups. The first two groups were administered intragastrically (i.g.) with saline for 7 days, while HGWD groups were treated with HGWD at 5, 10, 20 g·kg-1, correspondingly. The drugs were administered at 8: 30 am. The experiment process was schemed in Fig.1.

Fig.1 Scheme of experimental design of the study

2.4 MCAO model establishment

On the 8th day, transient focal ischemia was induced in rats by MCAO as previously described with slight modification[15]. Concisely, a midline incision in the neck was made on rats anesthetized by 10% chloral hydrate to expose and separate the external carotid artery, left common carotid artery as well as internal carotid artery (ICA). A nylon monofilament with round tip was inserted into the right ICA, and slowly advanced to obstruct the origin of the middle cerebral artery (MCA). After occluding the MCA for 2 h, the filament was removed for reperfusion. Rats in the sham group only underwent the same operation without MCAO.

2.5 Neurological deficit evaluation

Neurobehavioral dysfunction of rats was evaluated by an observer-blind study using Zea-Longa five-point scale[15].

2.6 Cerebral infract area measurement

After 24 h reperfusion, the brain tissue was excised and frozen at -20℃for 20 min. The brain tissue was sectioned to obtain uniform 2 mm sections, which were then co-incubated at 37℃for 30 min with 2,3,5-triphenyltetrazolium chloride (TTC). The infarct size expressed as infarct area percentage (%) was calculated using Image-Pro Plus 5.1.

2.7 Histopathological assessment

Rat brains were fixed with 10% formalin overnight, and then embedded in paraffin. Sections with 5 μm thick were cut in the coronal plane, and then stained with hematoxylin and eosin (HE). The histopathological abnormalities were examined by light microscopy.

2.8 Biochemistry evaluation

Changes in the levels of lactate dehydrogenase (LDH),malondialdehyde (MDA),glutathione (GSH) in the ischemic brain hemispheres were determined using the biochemical assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.9 Serum samples preparation

The blood of rats was collected and centrifuged (3 500 r·min-1at 4℃for 15 min) to obtain serum samples. The supernatant was stored at -80℃for analysis. Prior to metabolomics profiling analysis, serum sample (100 μL) was mixed with cold acetonitrile (300 μL),and vortexed for 30 s. The resulting mixture was centrifuged (12 000 r·min-1at 4℃for 10 min),and the supernatant was transferred to an UPLC analysis.

2.10 Inflammatory cytokines measurement

Cerebral cortex was gently rinsed, homogenized with PBS and stored at -20℃overnight. After two brief freeze-thaw cycles, the tissue homogenates were centrifuged, and the obtained supernatant was assayed by the bicinchoninic acid (BCA) method for protein content. TNF-α, IL-6, and IL-1β levels in ischemic cerebral cortex were determined using commercially available ELISA kits[MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China].

2.11 UPLC-MS analysis

Chromatographic separation was accomplished using an Acquity UPLC system and ACQUITY UPLC®HSS T3column (150 mm×2.1 mm, 1.8 μm, Waters). The gradient elution of serum samples was performed with 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B) at flow rate of 0.25 mL·min-1. The gradient elution program was applied as follows:0-1 min, 2%B;1-11 min, 2%-50%B;11-17 min, 50%-98%B;17-18 min, 98%B;18-18.5 min, 98%-2%B;18.5-21 min, 2%B.

The ESI-MSnexperiments were performed using a Thermo LTQ-Orbitrap XL mass spectrometer. The ionization conditions were as follows:capillary temperature, 325℃;sheath gas flow, 40 L·min-1;auxiliary gas flow, 15 L·min-1;nebulizer pressure, 35 psi (1 psi=6.895 kPa);capillary temperature, 325℃. The Orbitrap mass analyzer scanned over range fromm/z50 tom/z1 000 at a resolution power of 60 000.

2.12 Statistical analysis

The acquired mass data were aligned based on the retention time andm/z. The detected ion signals were normalized to the sum of the peak area of the chromatogram.

Data from negative and positive modes were merged, and multivariate analysis was conducted using the SIMCA-P program (version13.0). Non-supervised principal components analysis (PCA) was applied for data visualization and outliers identification. Then supervised regression modeling was analyzed with a partial least squares discriminant analysis (PLS-DA). The quality of the fitting PLS-DA models can be evaluated byR2YandQ2Yvalues. HigherQ2Yvalues indicated more significant differences between study groups. The biomarkers were screened and confirmed based on the variable importance in projection (VIP) values (VIP>1) combined with one-wayANOVAtest (P<0.05).

All the data in this study are expressed as mean±standard error of the mean (SEM).ANOVAfollowed byTukeymultiple comparison was applied for statistical significance evaluation. The difference was considered statistically significant ifP<0.05.

3 Results

3.1 HGWD improved neurological deficits scores in MCAO rats

Neurobehavioral abnormality scores of each group were exhibited in Fig.2A. It was found that the mean neurological deficit score in the MCAO group (2.33±0.52) was significantly higher than that in the sham group, suggesting a successful MCAO model. However, the neurological symptoms were improved noticeably when the MCAO rats were treated with HGWD at dose of 10 or 20 g·kg-1(compared to the MCAO model group,P<0.05 andP<0.01, respectively).

3.2 HGWD attenuated infarction volume rate in MCAO rats

HGWD could obviously reduce the infarct area of the MCAO rats. The TTC graphs of brain tissue and the corresponding cerebral infarct area were presented in Fig.2B-C. Compared with the sham group, MCAO induced a significant infarct area in the brain tissue. However, for rats pretreated with HGWD at dose of 10 and 20 g·kg-1, the cerebral infarct area was reduced from (38.51±4.36)% to (25.55±7.11)% and (13.09±5.70)%, respectively. There was no statistically significant difference between the HGWD at low dose (5 g·kg-1) and the model group (P>0.05). These results showed that HGWD pretreatment could protect against cerebral infarction in ischemic rats. Based on the data, rats were treated with HGWD at 20 g·kg-1(with best efficacy) were chosen for further metabolomics analysis.

Note: (A) Neurological deficit scores were determined according to Zea-Longa 5-point scheme. (B) TTC staining photograph in Sham, MCAO, HGWD groups. (C) infarct volume. *P<0.05, **P<0.01, compared with MCAO group. ##P<0.01, compared with sham group.Fig.2 The effect of HGWD on scores of neurological deficit and cerebral infarction size in rats

3.3 HGWD ameliorated histopathological alteration in the brain of MCAO rats

As seen in Fig.3, severe cellular edema, infiltration of inflammatory cells and shrunken neuronal bodies were observed in the cortical region of rats in MCAO model group. HGWD at 10, 20 g·kg-1could relieve the abnormalities in morphology. No significant difference was found among the sham, the medium and high dose HGWD treatment groups.

Fig.3 Degeneration of brain tissues and neurons in MCAO group, ameliorated by HGWD pretreatment

3.4 HGWD inhibited lipid peroxidation in the brain of MCAO rats

Compared with the sham group, MDA was found to be significantly elevated in MCAO model group (P<0.01, seen in Fig.4A),and was decreased dose-dependently by HGWD. GSH, another marker of lipid peroxidation, decreased after MCAO/reperfusion (Fig.4B) . HGWD (10, 20 g·kg-1) inhibited the GSH reduction induced by ischemia/reperfusion. In addition, LDH activity increased in rats with MCAO when compared with the sham-operated group. HGWD (20 g·kg-1) significantly reversed the increase of LDH levels (Fig.4C).

3.5 HGWD reduced inflammatory biomarkers in MCAO rats

To explore the anti-inflammatory effects of HGWD in cerebral ischemia injury, TNF-α, IL-1β and IL-6 levels in the cortex were measured by using ELISA kits. In comparison with the sham group, the contents of TNF-α, IL-1β and IL-6 elevated significantly in model group rats at 24 h after MCAO/reperfusion (P<0.05 orP<0.01, Fig.4D-F). However, HGWD at the dose of 20.0 g·kg-1could dramatically inhibit the release of IL-1β, IL-6 and TNF-α (P<0.05 orP<0.01).

Note: ##P<0.01, compared with the control group; *P<0.05, **P<0.01, compared with the MCAO group.Fig.4 Restoring effects of HGWD on biological parameters and expression of inflammatory factors in brain of rats

3.6 Metabolic disturbances of MCAO-induced focal cerebral ischemia

PCA manifested MCAO induced obvious metabolic disturbances. The system stability was evaluated using QCs. The QC samples were analyzed in positive as well as negative modes, and theRSDs of the retention times and peak areas of chromatograph peaks were calculated. The results showed that over 90%RSDs of QC samples were less than 30%, indicating acceptable repeatability and stability for experimental performances. The cluster of QC samples observed in the PCA plot also suggested good repeatability and stability of the method for metabolic profiling.

To identify the significant metabolism variations, PLS-DA models of MCAO and sham control groups were further constructed. The score plots demonstrated obvious cluster between the two groups (Fig.5A, positive:R2X=0.791,Q2=0.653;Fig.5B, negative:R2X=0.557,Q2=0.575). TheVIPvalues of variables in MCAO model group were ranked based on their contribution to the classification. The identified differential metabolites were further validated by Student's t-test and selected as the potential biomarkers. The variables were considered significantly different with criticalP-value≤0.05. Following the above criteria, a total of 23 differential endogenous metabolites were detected and identified (Table 1). Among these metabolites, most of them were amino acid (L-Tyrosine, L-Arginine, L-Tryptophan) or organic acid (2-ketovaleric acid, 2-ketohexanoic acid, citric acid, hippuric acid, et al) . According to metabolite concentrations, a heatmap was drawn to present the variation trend of the identified significant metabolites (Fig.6A). As shown in Fig.6A, 14 metabolites were down-regulated, and 9 metabolites were up-regulated in MCAO group. Some of the serum metabolite biomarkers have been considered to be closely associated with ischemic stroke progression, such as uric acid, L-glutamine, L-tryptophan, L-arginine, glucose, acetylcarnitine and 1-linoleoylglycerophosphocholine[16].

To further reveal the metabolism pathways affected by MCAO/reperfusion, the significant metabolites (Table 1) were input into Metaboanalyst for the metabolic networks construction (Fig.6B). The results showed that MCAO/reperfusion primarily disturbed the glyoxylate and dicarboxylate metabolism (impact 0.5) and phenylalanine, tyrosine and tryptophan biosynthesis (impact 0.296 3),as were consistent with prior literature[17]. With significant impact score, glyoxylate and dicarboxylate metabolism was considered as an important target pathway of MCAO.

Note: The PLS-DA plots showed clear separation between Control and MCAO. (A) Positive: R2X=0.791, Q2=0.653. (B) Negative: R2X=0.557, Q2=0.575.Fig.5 PLS-DA Plots of metabolic disturbances of MCAO-induced focal cerebral ischemia

Table 1 Significantly differential metabolites

3.7 HGWD rebalanced the metabolic disorders

HGWD administration rebalanced most disturbed metabolites in MCAO rats. The alteration of 19 metabolites appeared to be rebalanced after HGWD treatment (Fig.7). Especially, indoxylsulfuric acid, citric acid, 3-hydroxydodecanoic acid, 3-methyl-2-butenoic acid, malic acid, butanal, and uric acid were rebalanced by HGWD. In addition, 2-ketovaleric acid, LysoPC(20∶4(5Z, 8Z,11Z,14Z)), L-tyrosine, L-tryptophan, threonate, 3-methyl-2-butenoic acid, andα-D-glucose were also regulated by HGWD treatment.

To explore the underlying molecular mechanism of HGWD, metabolite-protein interaction networks were constructed. Using HMDB database, a total of 18 enzymes correlated to the regulated metabolites were identified (Table 2). Using STRING analysis, the interaction networks between the enzymes and proteins were constructed. As shown in Fig.8A, acly, ido1, mdh2 and tph1 with high confidence scores (score=0.9) were 4 most interactive enzymes. The interaction networks of these enzymes and their proteins were then further analyzed (Fig.8B-E). As presented in Table 3, a total of 28 proteins with interaction score more than 0.9 were assumed to be possible targets for the disturbed metabolites. Besides, for exploring the metabolite-related signal pathways, the identified enzymes and proteins were mapped to the biological pathways in DAVID 6.8. Two important biological pathways including citrate cycle (TCA cycle) and tryptophan metabolism were identified. Other metabolism pathways such as glyoxylate and dicarboxylate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and tyrosine metabolism, cysteine and methionine metabolism, pyruvate metabolism pathways were also regulated by HGWD treatment.

Table 2 Key enzymes of HGWD treatment

Table 3 Key proteins of HGWD

4 Discussion

The disease process after ischemic stroke is complex and lack of understanding. Significant effort has been focused on elucidating the mechanisms involved. However, it still remains a big challenge to understand ischemic stroke from a metabolomics perspective. At present, there are no drugs available that can effectively cure ischemic stroke. Previous studies have shown that HGWD, a topical traditional Chinese formula, was promising in alleviating neurological damage. In this work, neuroprotective effects of HGWD in ischemic stroke and the mechanisms behind it were studied for the first time using combined pharmacological and metabolomics approaches. Our results showed that HGWD could ameliorate neurological deficits scores, cerebral infarct size, lipid peroxidation as well as inflammatory response, indicating the neuroprotective property of HGWD on ischemic stroke. Based on metabolomics study, 23 remarked metabolites in serum of MCAO rats were identified, including amino acid, lipids (LysoPCs or PCs),and organic acids. Further metabolism pathway analysis showed that MCAO induced ischemic stroke mainly by targeting glyoxylate and dicarboxylate metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. Correspondingly, HGWD attenuated symptoms of ischemic stroke by primarily rebalancing citrate cycle (TCA cycle) and tryptophan metabolism. The identified biological pathways and potential targets would help shed light on further molecular mechanism research of HGWD.

Note: (A) Functional interaction networks of the key enzymes were collected and input into the STRING database to analyze interactions between them(interaction score=0.9). Acly, ido1, mdh2 and tph1 were 4 most interactive enzymes. Functional interaction networks of acly (B), ido1 (C), mdh2 (D) and tph1 (E). Only proteins with highest confidence (interaction score=0.9) were considered as possible targets network for the metabolites.Fig.8 Interaction networks of enzymes and proteins

4.1 Amino acid metabolism

The results of this study suggested that 4 amino acids including glutamine, tyrosine, tryptophan, and arginine significantly increased after cerebral ischemia. Glutamine is synthesized from glutamate and ammonia through the action of glutamine synthetase, which is an enzyme sensitive to oxidative damage and closely linked to cerebral ischemia[18]. As previous reports have detailed, glutamine and glutamate in the brain may serve as important markers for monitoring cerebrovascular risk factors[19]. In our work, the increased level of glutamine in the MCAO-treated rats further confirmed that glutamine may play a crucial role in the pathophysiological process of cerebral ischemia.

Alanine, an essential amino acid for the human, is commonly synthesized by the reductive amination of pyruvate involving alanine transaminase. Because pyruvate is ubiquitous, and transamine reactions are easily reversed, alanine is easily formed and closely related to glycolysis, gluconeogenesis and citric acid cycle. Previous studies have shown that alanine levels of cerebrospinal fluid increased significantly after 8 h of cerebral ischemia[20]. Similarly, an obvious increase in serum alanine level at 24 h after MCAO in rats was observed in our study.

4.2 Energy metabolism disturbance

The perturbed metabolites found in this study are also closely related to energy metabolism. Disturbance of energy metabolism is one of the main pathological mechanisms of brain injury after cerebral ischemia. In MCAO-treated rats, creatine significantly elevated. It is well known that creatine is phosphorylated to form phosphocreatine, an energy store in the brain[21]. During ischemia, energy stores in the brain is depleted, so high energy must be transferred from ADP to ATP through phosphocreatine. This phenomenon may explain why a remarkable increase in creatine level was observed in the MCAO model group.

Another metabolite glucose, the main source of energy is also involved in energy metabolism. Tight regulation of glucose metabolism has been recognized as essential for brain physiology. Glucose transported by transporters crosses the blood-brain barrier and enters the brain. It is actively transported across the cell membrane, produces pyruvate in the cytoplasm, and then enters the mitochondria where glucose enters into the tricarboxylic acid cycle and undergoes oxidative decarboxylation. Studies have shown that glucose metabolism disorder is the starting point of neuronal damage after ischemia[22]. The significant decrease in glucose observed in the model group suggests that energy metabolism pathways may be disrupted under local hypoxia during stroke.

4.3 Lipid metabolism

The biosynthesis and degradation of lipids are closely related to the mechanism of nerve cell injury and repair during cerebral ischemia. In this study, lipids such as PE (16∶0),LysoPC (16∶1) and LysoPC (20∶3) were found to be decreased significantly in the MCAO model group compared with the sham group. After treatment with HGWD, the levels of PE (16∶0),LysoPC (16∶1) and LysoPC (20∶3) presented a trend toward normalcy, suggesting that the protection of HGWD against cerebral ischemia may be partially mediated by lipid metabolism regulation.

4.4 Other metabolism pathways

Previously, uric acid was found to be a predictor for stroke[23]. In this study, compared with the sham control group, serum uric acid levels increased significantly in the model group after 24 h cerebral ischemia, whereas HGWD could reduce the elevated serum levels of uric acid.

5 Conclusion

In this study, combined pharmacological and metabolomics studies were carried out to explore the neuroprotective effects of HGWD against focal cerebral ischemia-reperfusion injury in rats. After HGWD treatment, we observed decrease in the neurological deficits scores, cerebral infarct size, as well as lipid peroxidation and inflammatory cytokines levels in MCAO rats. Metabolomics analysis showed that the serum metabolic profiles of sham, MCAO model and HGWD-treated group were different, and HGWD could rebalance the metabolic disorders in focal cerebral ischemic rats. Based on univariate and multivariate statistical analysis, 23 significant changed serum metabolites at 24 h after cerebral ischemia/reperfusion were identified. These markers were mainly involved in amino acids, lipids and energy metabolism. Further study demonstrated that HGWD attenuated MCAO-induced neurological deficits by regulating these pathways in a multi-target manner. Based on the evidences collected from metabolomics and pharmacological studies, HGWD may be a potential therapeutic option for cerebral infarction treatment.