# The Differences of Metabolites in Different Parts of the Brain Induced by Shuxuetong Injection against Cerebral Ischemia-Reperfusion and Its Corresponding Mechanism

Authors: Tingyue Jiang; Jiakang Jiao; Jinfeng Shang; Lei Bi; Huanhuan Wang; Chao Zhang; Hongwei Wu; Yiran Cui; Peng Wang; Xin Liu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Ischemic stroke is often associated with a large disease burden. The existence of ischemia-reperfusion injury brings great challenges to the treatment of ischemic stroke. The purpose of this study was to explore the differences of metabolites in different parts of the brain induced by Shuxuetong injection against cerebral ischemia-reperfusion and to extend the corresponding mechanism. The rats were modeled by transient middle cerebral artery occlusion (t-MCAO) operation, and the success of modeling was determined by neurological function score and TTC staining. UPLC-Q/TOF-MS metabolomics technique and multivariate statistical analysis were used to analyze the changes and differences of metabolites in the cortex and hippocampus of cerebral ischemia-reperfusion rats. Compared with the model group, the neurological function score and cerebral infarction volume of the Shuxuetong treatment group were significantly different. There were differences and changes in the metabolic distribution of the cortex and hippocampus in each group, the distribution within the group was relatively concentrated. The separation trend between the groups was obvious, and the distribution of the Shuxuetong treatment group was similar to that of the sham operation group. We identified 13 metabolites that were differentially expressed in the cortex, including glutamine, dihydroorotic acid, and glyceric acid. We also found five differentially expressed metabolites in the hippocampus, including glutamic acid and fumaric acid. The common metabolic pathways of Shuxuetong on the cortex and hippocampus were D-glutamine and D-glutamate metabolism and nitrogen metabolism, which showed inhibition of cortical glutamine and promotion of hippocampal glutamic acid. Specific pathways of Shuxuetong enriched in the cortex included glyoxylate and dicarboxylate metabolism and pyrimidine metabolism, which showed inhibition of glyceric acid and dihydroorotic acid. Specific pathways of Shuxuetong enriched in the hippocampus include arginine biosynthesis and citrate cycle (TCA cycle), which promotes fumaric acid. Shuxuetong injection can restore and adjust the metabolic disorder of the cortex and hippocampus in cerebral ischemia-reperfusion rats. The expression of Shuxuetong in different parts of the brain is different and correlated.


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## Body

## 1. Introduction

Ischemic stroke (IS) is one of the most common types of stroke. It is an acute cerebrovascular disease that can seriously harm human health. IS is characterized by poor prognosis and easy coexistence with other chronic diseases, bringing severe psychological and economic burden to patients around the world [1]. The core treatment of IS is to unclog blocked blood vessels, but even after the unclog is completed, there are still risks like cerebral ischemic-reperfusion (CIR) injury [2]. The brain pathological mechanism of the CIR injury process is complex, usually involving energy metabolism disorder, inflammatory response, oxidative stress injury, cell necrosis and apoptosis, excitatory toxicity, and other processes [3]. The coexistence of brain injury and recovery during reperfusion makes the treatment of IS more difficult [4]. Therefore, it is important to study the mechanism of anti-CIR injury for the treatment of IS to improve the quality of life of patients with cerebral ishemia and reduce the mortality of the disease.After an acute stroke, the cascade of brain remodeling is activated, and there are vital regional differences in the survival, migration, and maturation of brain cells, which have different effects on subsequent functions [5]. New neuronal precursors in the subventricular zone migrate to the cortex after stroke and are closely related to metabolic regulation [6]. New neurons in the subgranular zone of the hippocampus with abnormal function or morphology after stroke are associated with hippocampal dependence defects, affecting cognitive functions such as memory and pattern separation [7]. It has also been shown that neurovascular coupling and oxygenation are lower in the hippocampus than in the cortex under pathological conditions such as stroke. It is suggested that the hippocampus is more susceptible to hypoxic injury in the presence of neurovascular coupling damage [8]. The pathological features of the hippocampus and cortex are different and correlated during ischemic injury. The balance of excitatory and inhibitory pathways in the hippocampus is disrupted after middle cerebral artery distal occlusion (MCAO), when the oscillatory activity of the hippocampal EEG curve is more pronounced and the brain connections between the cortex and the hippocampus are disrupted. Impairment is closely related to changes in cognitive function after stroke [9]. By performing MCAO in the somatosensory cortex and recording the functional dynamics between the cortex and hippocampus, increased cortical activity was found to modulate hippocampal activity [10].Shuxuetong injection is a standardized product extracted from traditional Chinese medicine Shuizhi (Hirudo) and Dilong (Pheretima). Each ampoule SXT (2 mL) is extracted from 0.5 g Shuizhi and 0.5 g Dilong. TCM believes that blood stasis block is the key to the onset of IS, and SXT has the effects of promoting blood circulation and removing stasis and activating channels (Huo Xue Hua Yu & Tong Jing Huo Luo), which has been widely used in the treatment of cardiovascular and cerebrovascular diseases, with precise curative effect and high safety for stroke diseases [11, 12]. SXT has a protective effect on brain microvascular endothelial cells after oxygen-glucose deprivation reperfusion (OGD/R), which is related to its effects on platelet aggregation, antithrombosis, reduction of mitochondrial superoxide production, inhibition of inflammation, and inhibition of vascular endothelial cells growth factors [13]. The main active components of SXT include hirudin and lumbrokinase. Hirudin has anticoagulant, antithrombotic, antifibrosis, antiapoptosis, and other pharmacological effects [14]. Lumbrokinase has antibacterial and anti-inflammatory effects and promotes the repair of the nervous system [15]. Researchers analyzed and predicted bioactive peptides in SXT and screened out YLKTT peptides that had protective effects on OGD/R injury of astrocytes [16].Over time, metabolomics has evolved from an exploratory tool for qualitative studies to a more mature and complete biochemical technique for quantitative studies [17]. The identification of different metabolomic profiles helps to identify and improve clinical treatment strategies for diseases [18]. The brain has a high level and specific metabolic activity that is sensitive to ischemic injury [19]. Therefore, metabolomic studies of IS have a certain research base. Wang et al. described the metabolic characteristics of each subtype of IS, and the differential metabolites in patients with large atherosclerosis and small arterial occlusions were mainly related to glycerophospholipid metabolism and histidine metabolism [20]. The metabolomic analysis of edaravone on CIR injury in mice by Ma et al. screened cysteine sulfonate decarboxylase as a possible therapeutic target [21]. Other researchers have subdivided them by gender, identifying 49 metabolites that are significantly altered in female patients with acute IS, mainly glycerophosphocholine, and 39 metabolic pathways that are significantly altered in male patients with acute IS, including 5-α-Androstan-3-α,17-β-diol and threonate [22].In the early stage of this project, the pharmacological effects of SXT injection on cerebral ischemia-reperfusion model rats were evaluated from the perspectives of transcriptomics and proteomics. In order to further study the pharmacological effects, this study aims to explore SXT's role in the process of the injection against CIR injury and its influence based on different parts of the brain metabolism, to explore the different parts of the brain function and metabolic material basis.


## 2. Materials and Methods

### 2.1. Animals

In total, 88 SPF male SD rats aged 7–9 weeks with an average body weight of 240–270 g were purchased from Beijing Weitong Lihua Co., Ltd. The certificate number of the experimental rats was SCXK (Beijing) 2019-0010. All animals were fed under the conditions of temperature (22 ± 2)°C, relative humidity (50 ± 10) % and a 12-hour dark/light cycle. All the procedures in the experiment were approved by the Medical Ethics Committee of China Academy of Chinese Medical Sciences.


### 2.2. Drugs and Reagents

Shuxuetong injection freeze-dried powder (Manufacturer: Youbo Pharmaceutical Co., Ltd., No.20042201, freeze-dried powder content: 22.38 mg· mL−1), Edaravone Injection (manufacturer: Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd., No. 80–151211), TTC (2,3,5-Triphenylte-Trazoliumchloride, Factory: McLean, No. C12221739), 4% paraformaldehyde phosphate buffer (Factory: Solarboi, No. P1110), methanol (chromatopure, factory: McLean), formic acid (chromatographic pure, manufacturer: McLean), acetonitrile (chromatographic pure, manufacturer: McLean), and rat MCAO nylon plug (thread diameter: 0.26 mm, tip diameter: (0.36 ± 0.02) mm, manufacturer: Beijing Shadong Company, No. 2636-100).


### 2.3. Surgery and Drug Administration

The cerebral ischemia-reperfusion model of rats was prepared after 3 days of adaptive feeding. According to Zea Longa method, a little improvement was made [23, 24]. t-MCAO was performed on rats, with specific operations as follows: we weigh and anesthetize the rats, make an incision at the middle part of the neck, and separate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The distal ECA was then ligated. The CCA and ICA was temporarily clamped. Prepare at the intersection of ECA and ICA. At the ECA cut, insert the marked thread from ECA and pull back to insert the thread in the ICA direction about 18∼19 mm. At this point, ICA’s temporary clamp has been opened. After fixation of the bolus, the ECA was burned and timed. The surgical incision was sutured and disinfected, and the animals were placed at a constant temperature of 37°C. After 15 min of ischemia, the groups were administered by tail vein injection. After 2 h of ischemia, the insertion line was removed and the temporarily clamped CCA was opened to ensure the establishment of reperfusion. For the sham-operated group, the steps were the same, except that the line was not inserted. The experimental animals were randomly divided into six groups: sham group (sham), model group (model), SXT high-dose group, medium-dose group, and low-dose group (SXT-H, 1.08 mg·kg−1; SXT -M, 0.54 mg·kg−1; SXT-L, 0.27 mg·kg−1), positive drug group (edaravone injection, ED. 1.35 mL·kg−1). Saline was given in the sham-operated and model groups.


### 2.4. Neurological Function Score

Six hours after modeling, the neural function of experimental rats was evaluated in the form of scoring, and Logna scoring method was adopted [25]. The rats were divided into five grades according to their behaviors: 0 point, no symptoms of nerve damage; 1 point, left front paw cannot be fully extended; 2 points, when walking, the rats turned to the left side (paralyzed side); 3 points, when walking, the rat body tipped to the left side (paralyzed side); 4 points, cannot walk spontaneously, conscious loss.


### 2.5. The Volume of Cerebral Infarction Measured by TTC

After neurological function evaluation, the rats were euthanized, and their brains were taken for staining with TTC [26]. The extent of cerebral infarction was calculated as a percentage of the total volume of the infarcted portion. Taking the position 3 mm away from the forehead as the starting point, slices were carried out in the order from front to back, and the thickness of a single slice was about 2 mm. The slices were stained with 2% TTC buffer solution and placed in a constant temperature light-proof box at 37°C for 10 min during the dyeing process. Uninfected brain tissue appears rose-red, while infarcts appear white. After staining, 4% paraformaldehyde buffer was used for fixation. After 30 minutes of fixation, the slices were sequentially arranged in the correct order and then electronically scanned and recorded. Image J was used to determine the area of different color brain regions, and the infarct rate was calculated according to the area:(1)Infarction rate=Vc−ViVc×100%,Vc=d×∑AcAc−Single slice area,Vi=d×∑AiAi−Red area of single brain slice.


### 2.6. Sample Pretreatment

The rats were decapitated, and their brains were taken out and dissected on ice. The cortex and hippocampus of the affected side were taken out and frozen at −80°C for later use. The cortex and hippocampus of 3 groups were extracted with 1 mL methanol as the extraction solvent. The cortex and hippocampus were homogenized, and the cortex and hippocampus were ground for 45 s and 60 s, respectively, with a 70 Hz high-throughput tissue grinder. The tissue grinding machine was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. The model is ScientZ-192. Methanol 800µL was added to 200 µL cortex abrasive solution, which was vortically mixed for 1 min, then centrifuged at 12 000 r·min−1 for 5 min at 4°C, and 5 µL supernatant was injected. The centrifuge (GT16-3) was purchased from Beijing Times Beili Centrifuge Co., Ltd. The abrasive solution of the hippocampus was centrifuged at 12 000 r·min−1 at 4°C for 5 min, and 5 µL supernatant was used for sample injection.


### 2.7. UPLC-Q/TOF-MS Analysis

The separation was performed by UPLC (1290 binary liquid chromatography, Agilent Technologies, USA) and screened by ESI-MS (Targeted MS/MS mode). The LC system consisted of Agilent ZORBAX Eclipse XDB-C18 (RRHD) 1.8μm column and an American Pheromone guard column. The mobile phase consisted of solvent A (0.1% formic acid-water) and solvent B (0.1% formic acid-acetonitrile), gradient elution (0-1 min, 95% A; 1–6 min, 95–70% A; 6–20 min, 70–5% A), and the mobile phase velocity was 0.5 mL·min−1. Column temperature is 45°C and sample manager temperature is 4°C. The mass spectrometry was performed by Q/TOF-MS with a dual Agilent jet (AJS) ESI source. The scanning mass-charge ratio ranges from 50 to 1500, and the scanning rate is 1.00 spectra·s−1. The capillary voltage is 4000 V and 3500 V (positive and negative mode, respectively), and the voltage is 175 V. Atomizer pressure is 35 psi, gas temperature 325°C, and continuous gas flow 5 L·min1. Instrument mode is set to an extended dynamic range. Quality control (QC) samples from normal rat cortex or hippocampus were performed at the beginning of the sequence to avoid small changes in chromatographic signal strength.


### 2.8. Data Analysis

SPSS 25.0 software (SPSS, Chicago, USA) was used to perform the Kruskal–Wallis test for neurological function scores. Under the condition of normal distribution and homogeneity of variance, a one-way ANOVA test was used for cerebral infarction volume data. The results of the cerebral infarction area were presented by means ± standard error of the mean (SEM).P<0.05 indicates that the difference was statistically significant. Raw data are collected and calibrated using the MassHunter Workstation (B0.06.00, Agilent) based on the M/Z value and retention time of the ion signal. The results of ion ESI− and ESI+ were imported into SIMCA-P (11.5) for multivariate analysis. Principal component analysis (PCA) is used as an unsupervised method to visualize data and identify outliers. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used for supervised regression modeling of datasets. Screen for metabolites with significant differences between the model group and the sham group and between the dosed group and the model group. Metabolites with P<0.05 were used as differential metabolites. Metabolic pathway analysis was performed using MetaboAnalyst 5.0 and the KEGG database.


## 2.1. Animals

In total, 88 SPF male SD rats aged 7–9 weeks with an average body weight of 240–270 g were purchased from Beijing Weitong Lihua Co., Ltd. The certificate number of the experimental rats was SCXK (Beijing) 2019-0010. All animals were fed under the conditions of temperature (22 ± 2)°C, relative humidity (50 ± 10) % and a 12-hour dark/light cycle. All the procedures in the experiment were approved by the Medical Ethics Committee of China Academy of Chinese Medical Sciences.


## 2.2. Drugs and Reagents

Shuxuetong injection freeze-dried powder (Manufacturer: Youbo Pharmaceutical Co., Ltd., No.20042201, freeze-dried powder content: 22.38 mg· mL−1), Edaravone Injection (manufacturer: Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd., No. 80–151211), TTC (2,3,5-Triphenylte-Trazoliumchloride, Factory: McLean, No. C12221739), 4% paraformaldehyde phosphate buffer (Factory: Solarboi, No. P1110), methanol (chromatopure, factory: McLean), formic acid (chromatographic pure, manufacturer: McLean), acetonitrile (chromatographic pure, manufacturer: McLean), and rat MCAO nylon plug (thread diameter: 0.26 mm, tip diameter: (0.36 ± 0.02) mm, manufacturer: Beijing Shadong Company, No. 2636-100).


## 2.3. Surgery and Drug Administration

The cerebral ischemia-reperfusion model of rats was prepared after 3 days of adaptive feeding. According to Zea Longa method, a little improvement was made [23, 24]. t-MCAO was performed on rats, with specific operations as follows: we weigh and anesthetize the rats, make an incision at the middle part of the neck, and separate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The distal ECA was then ligated. The CCA and ICA was temporarily clamped. Prepare at the intersection of ECA and ICA. At the ECA cut, insert the marked thread from ECA and pull back to insert the thread in the ICA direction about 18∼19 mm. At this point, ICA’s temporary clamp has been opened. After fixation of the bolus, the ECA was burned and timed. The surgical incision was sutured and disinfected, and the animals were placed at a constant temperature of 37°C. After 15 min of ischemia, the groups were administered by tail vein injection. After 2 h of ischemia, the insertion line was removed and the temporarily clamped CCA was opened to ensure the establishment of reperfusion. For the sham-operated group, the steps were the same, except that the line was not inserted. The experimental animals were randomly divided into six groups: sham group (sham), model group (model), SXT high-dose group, medium-dose group, and low-dose group (SXT-H, 1.08 mg·kg−1; SXT -M, 0.54 mg·kg−1; SXT-L, 0.27 mg·kg−1), positive drug group (edaravone injection, ED. 1.35 mL·kg−1). Saline was given in the sham-operated and model groups.


## 2.4. Neurological Function Score

Six hours after modeling, the neural function of experimental rats was evaluated in the form of scoring, and Logna scoring method was adopted [25]. The rats were divided into five grades according to their behaviors: 0 point, no symptoms of nerve damage; 1 point, left front paw cannot be fully extended; 2 points, when walking, the rats turned to the left side (paralyzed side); 3 points, when walking, the rat body tipped to the left side (paralyzed side); 4 points, cannot walk spontaneously, conscious loss.


## 2.5. The Volume of Cerebral Infarction Measured by TTC

After neurological function evaluation, the rats were euthanized, and their brains were taken for staining with TTC [26]. The extent of cerebral infarction was calculated as a percentage of the total volume of the infarcted portion. Taking the position 3 mm away from the forehead as the starting point, slices were carried out in the order from front to back, and the thickness of a single slice was about 2 mm. The slices were stained with 2% TTC buffer solution and placed in a constant temperature light-proof box at 37°C for 10 min during the dyeing process. Uninfected brain tissue appears rose-red, while infarcts appear white. After staining, 4% paraformaldehyde buffer was used for fixation. After 30 minutes of fixation, the slices were sequentially arranged in the correct order and then electronically scanned and recorded. Image J was used to determine the area of different color brain regions, and the infarct rate was calculated according to the area:(1)Infarction rate=Vc−ViVc×100%,Vc=d×∑AcAc−Single slice area,Vi=d×∑AiAi−Red area of single brain slice.


## 2.6. Sample Pretreatment

The rats were decapitated, and their brains were taken out and dissected on ice. The cortex and hippocampus of the affected side were taken out and frozen at −80°C for later use. The cortex and hippocampus of 3 groups were extracted with 1 mL methanol as the extraction solvent. The cortex and hippocampus were homogenized, and the cortex and hippocampus were ground for 45 s and 60 s, respectively, with a 70 Hz high-throughput tissue grinder. The tissue grinding machine was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. The model is ScientZ-192. Methanol 800µL was added to 200 µL cortex abrasive solution, which was vortically mixed for 1 min, then centrifuged at 12 000 r·min−1 for 5 min at 4°C, and 5 µL supernatant was injected. The centrifuge (GT16-3) was purchased from Beijing Times Beili Centrifuge Co., Ltd. The abrasive solution of the hippocampus was centrifuged at 12 000 r·min−1 at 4°C for 5 min, and 5 µL supernatant was used for sample injection.


## 2.7. UPLC-Q/TOF-MS Analysis

The separation was performed by UPLC (1290 binary liquid chromatography, Agilent Technologies, USA) and screened by ESI-MS (Targeted MS/MS mode). The LC system consisted of Agilent ZORBAX Eclipse XDB-C18 (RRHD) 1.8μm column and an American Pheromone guard column. The mobile phase consisted of solvent A (0.1% formic acid-water) and solvent B (0.1% formic acid-acetonitrile), gradient elution (0-1 min, 95% A; 1–6 min, 95–70% A; 6–20 min, 70–5% A), and the mobile phase velocity was 0.5 mL·min−1. Column temperature is 45°C and sample manager temperature is 4°C. The mass spectrometry was performed by Q/TOF-MS with a dual Agilent jet (AJS) ESI source. The scanning mass-charge ratio ranges from 50 to 1500, and the scanning rate is 1.00 spectra·s−1. The capillary voltage is 4000 V and 3500 V (positive and negative mode, respectively), and the voltage is 175 V. Atomizer pressure is 35 psi, gas temperature 325°C, and continuous gas flow 5 L·min1. Instrument mode is set to an extended dynamic range. Quality control (QC) samples from normal rat cortex or hippocampus were performed at the beginning of the sequence to avoid small changes in chromatographic signal strength.


## 2.8. Data Analysis

SPSS 25.0 software (SPSS, Chicago, USA) was used to perform the Kruskal–Wallis test for neurological function scores. Under the condition of normal distribution and homogeneity of variance, a one-way ANOVA test was used for cerebral infarction volume data. The results of the cerebral infarction area were presented by means ± standard error of the mean (SEM).P<0.05 indicates that the difference was statistically significant. Raw data are collected and calibrated using the MassHunter Workstation (B0.06.00, Agilent) based on the M/Z value and retention time of the ion signal. The results of ion ESI− and ESI+ were imported into SIMCA-P (11.5) for multivariate analysis. Principal component analysis (PCA) is used as an unsupervised method to visualize data and identify outliers. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used for supervised regression modeling of datasets. Screen for metabolites with significant differences between the model group and the sham group and between the dosed group and the model group. Metabolites with P<0.05 were used as differential metabolites. Metabolic pathway analysis was performed using MetaboAnalyst 5.0 and the KEGG database.


## 3. Results

### 3.1. Pharmacodynamics of SXT Injection against Cerebral Ischemia-Reperfusion

After modeling, the rats in the model group showed neurological impairment and cerebral infarction. Compared with the model group, the neurological function evaluation and cerebral infarction volume of the high-dose, medium-dose, and low-dose SXT groups and the ED group were significantly different and showed a quantitative effect relationship. The higher the concentration of SXT, the more significant the therapeutic effect, as shown in Figure1 and Table S1.Figure 1
Pharmacodynamic results of cerebral ischemia after 6 h in different groups. (a) Map of cerebral infarction after 6 h of cerebral ischemia in different groups. (b) Cerebral infarction rate after cerebral ischemia 6 h in different groups. (c) Neurological function scores of different groups after cerebral ischemia at 6 h. Cerebral ischemia-reperfusion began at 2 hours after cerebral ischemia. Sham represents the sham operation group, model represents the model group, SXT-H represents the Shuxuetong high-dose group, SXT-M represents the Shuxuetong medium-dose group, SXT-L represents the Shuxuetong low-dose group, and ED represents the edaravone positive drug group. Compared to the Nodel group,∗P<0.05, ∗∗P<0.01, AND ∗∗∗P<0.001.
(a)(b)(c)


### 3.2. Metabolomics Results of Cortex

PCA, OPLS-DA, and S-plot[M2] results of cortical tissues are shown in Figure2. Results of cortical positive and negative ions are in Table S2. Each dot in the figure represents a sample of cortical tissue, and the distribution of each group represents the metabolic distribution of the sample. PCA analysis showed (R2 = 0.682, Q2 = 0.413) that the separation trend of metabolites in the three groups was obvious. The distribution of metabolites in each group was different, and the distribution within the group was relatively concentrated. The distribution of the administered group was close to that of the sham-operated group. OPLS-DA analysis showed that the cortical tissue samples in the model and sham-operated groups were distributed on both sides of the space, and the distribution of samples within the group was relatively concentrated, indicating that the metabolites of the posterior cortex were significantly changed. Modeling and the changes were closely related to the pathogenesis. In S-plot[M2] figure, the further the distance from the origin, the more likely the reactive ions are to become differential metabolites between groups.Figure 2
Metabolic analysis of cortical samples at 6 h after cerebral ischemia. (a) PCA-X image of cerebral ischemia cortical samples at 6 h. (b) OPLS-DA diagram of cerebral ischemia cortical samples at 6 h. (c) S-plot[M2] of cerebral ischemia cortical samples at 6 h; color represents the group in (a) and (b). Cerebral ischemia-reperfusion began at 2 hours after cerebral ischemia. Green represents the model group. Blue represents the sham group. Red represents drug, SXT-M group, the same as below.
(a)(b)(c)


### 3.3. Metabolomics Results of Hippocampus

PCA, OPLS-DA, and S-plot[M2] results of hippocampal tissue are shown in Figure3. Results of hippocampal positive and negative ions are shown in Table S3. PCA results (R2 = 0.674, Q2 = 0.204) showed that compared with the sham operation group, the model group had a significant separation trend, and the distribution of the administration group and the sham operation group were closer, revealing the therapeutic effect of the drug from the perspective of metabolic distribution. The OPLS-DA results showed that 6 h after IR, the samples of the model and sham-operated groups were symmetrically distributed on both sides of the space, and the distribution of samples within the groups was relatively concentrated indicating that hippocampal metabolites changed significantly by modeling. In the S-plot [M2] plot, the farther away from the origin they are, the more likely the reactive ions are to be differential metabolites between groups.Figure 3
Metabolic analysis of hippocampus samples at 6 h after cerebral ischemia. (a) PCA-X image of hippocampal samples at 6 h after cerebral ischemia. (b) OPLS-DA of hippocampal samples at 6 h after cerebral ischemia. (c) S-plot[M2] of hippocampal samples at 6 h after cerebral ischemia.
(a)(b)(c)


### 3.4. Differential Metabolite Results

The differential metabolites among different groups are shown in Table1. The differential metabolites obtained by screening were all metabolites with significant differences between the model group and the sham operation group and between the administration group and the model group. All the differential metabolites obtained by screening belong to ESI− ion mode, and all have the performance of callback; that is, drug administration can reverse the injury of the model group to a certain extent, making the expression of the drug administration group more similar to that of the sham group. A total of 13 different metabolites were screened from the cortex, including amino acids (e.g., glutamine, dihydroorotic acid, and threonic acid), alkaloids (e.g., humantenine and yohimbic acid), and other acid products (including benzenesulfonic acid, glyceric acid, and hydroxy acid) which were the main products. Five kinds of differential metabolites were screened from the hippocampus, mainly amino acids (glutamic acid) and benzene-related derivatives (benzoquinone, benzophenone, amd benzoic acid).Table 1
Differential metabolites of Model group, Sham group, and SXT-M group.
PartNo.Metabolite nameRetention time(min)Measured mean mass (Da)Chemical formulaNormalized abundance (×105)ShamModelSXT-MCortex1Orthanilic acid0.85172.0213C6H7NO3S1.151.92▲▲▲1.62∗∗2Glutamine0.87145.0610C5H10N2O321.5030.72▲▲▲24.54∗∗3Humantenine17.73353.1976C21H26N2O34.933.17▲▲▲5.11∗∗∗4Dodecylbenzenesulfonic acid24.89325.1818C18H30O3S53.4064.05▲▲▲51.95∗∗∗5Dodecyl sulfate24.40265.1460C12H26O4S113.22129.05▲▲106.75∗∗∗6Yohimbic acid25.14339.1974C20H24N2O342.1649.02▲▲▲40.19∗∗∗7S-sulforaphene1.38174.0010C6H9NOS23.484.78▲▲4.00∗83-Hydroxypropanoic acid1.3189.0240C3H6O3137.90171.41▲▲141.70∗∗9Dihydroorotic acid0.28157.0108C5H6N2O43.263.54▲3.06∗∗∗10Glyceric acid0.76105.0166C3H6O42.492.69▲2.39∗∗11Embelin24.86293.1771C17H26O481.5693.37▲▲77.97∗∗∗12Etidronate23.68204.9879C2H8O7P21.621.80▲1.45∗∗∗13Threonic acid23.14135.0102C4H8O51.451.59▲▲1.45∗∗Hippocampus1Fumaric acid1.29115.0030C4H4O40.320.28▲0.32∗2Embelin13.24293.1733C17H26O412.016.18▲▲▲9.10∗∗∗3Glutamic acid14.55146.0348C5H9NO45.403.69▲▲5.25∗∗4Butylparaben12.04193.0854C11H14O33.262.04▲▲▲2.90∗∗∗52,4-Dihydroxybenzophenone0.84213.0473C13H10O34.116.39▲▲▲5.01∗The model group was compared with the sham group,▲P<0.05, ▲▲P<0.01, ▲▲▲P<0.001. The SXT-M group was compared with the model group: ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.


### 3.5. Metabolic Pathway Enrichment Results

Metabolic pathway enrichment of differential metabolites was carried out through the KEGG database, and the enriched pathways are shown in Figures4 and 5. There were four metabolic pathways with significant differences in the cortex: D-glutamine and D-glutamate metabolism, glyoxylate and dicarboxylate metabolism, pyrimidine metabolism, and nitrogen metabolism. Different metabolite concentrations in the metabolic pathways of the hippocampus and eight pathways with significant differences, respectively, are D-glutamine and D-glutamate metabolism, arginine biosynthesis, alanine, aspartate, and glutamate metabolism, nitrogen metabolism, butanoate metabolism, histidine metabolism, citrate cycle (TCA cycle), and pyruvate metabolism.Figure 4
Enrichment results of differential metabolite metabolic pathways in the cortex. The abscissa represents −log10P, the ordinate represents the enrichment pathway, and the bubble size represents the enrichment ratio. The darker the bubble color is, the smaller the P value is, as shown in Figure 5.Figure 5
Enrichment results of differential metabolite metabolic pathways in hippocampus.


## 3.1. Pharmacodynamics of SXT Injection against Cerebral Ischemia-Reperfusion

After modeling, the rats in the model group showed neurological impairment and cerebral infarction. Compared with the model group, the neurological function evaluation and cerebral infarction volume of the high-dose, medium-dose, and low-dose SXT groups and the ED group were significantly different and showed a quantitative effect relationship. The higher the concentration of SXT, the more significant the therapeutic effect, as shown in Figure1 and Table S1.Figure 1
Pharmacodynamic results of cerebral ischemia after 6 h in different groups. (a) Map of cerebral infarction after 6 h of cerebral ischemia in different groups. (b) Cerebral infarction rate after cerebral ischemia 6 h in different groups. (c) Neurological function scores of different groups after cerebral ischemia at 6 h. Cerebral ischemia-reperfusion began at 2 hours after cerebral ischemia. Sham represents the sham operation group, model represents the model group, SXT-H represents the Shuxuetong high-dose group, SXT-M represents the Shuxuetong medium-dose group, SXT-L represents the Shuxuetong low-dose group, and ED represents the edaravone positive drug group. Compared to the Nodel group,∗P<0.05, ∗∗P<0.01, AND ∗∗∗P<0.001.
(a)(b)(c)


## 3.2. Metabolomics Results of Cortex

PCA, OPLS-DA, and S-plot[M2] results of cortical tissues are shown in Figure2. Results of cortical positive and negative ions are in Table S2. Each dot in the figure represents a sample of cortical tissue, and the distribution of each group represents the metabolic distribution of the sample. PCA analysis showed (R2 = 0.682, Q2 = 0.413) that the separation trend of metabolites in the three groups was obvious. The distribution of metabolites in each group was different, and the distribution within the group was relatively concentrated. The distribution of the administered group was close to that of the sham-operated group. OPLS-DA analysis showed that the cortical tissue samples in the model and sham-operated groups were distributed on both sides of the space, and the distribution of samples within the group was relatively concentrated, indicating that the metabolites of the posterior cortex were significantly changed. Modeling and the changes were closely related to the pathogenesis. In S-plot[M2] figure, the further the distance from the origin, the more likely the reactive ions are to become differential metabolites between groups.Figure 2
Metabolic analysis of cortical samples at 6 h after cerebral ischemia. (a) PCA-X image of cerebral ischemia cortical samples at 6 h. (b) OPLS-DA diagram of cerebral ischemia cortical samples at 6 h. (c) S-plot[M2] of cerebral ischemia cortical samples at 6 h; color represents the group in (a) and (b). Cerebral ischemia-reperfusion began at 2 hours after cerebral ischemia. Green represents the model group. Blue represents the sham group. Red represents drug, SXT-M group, the same as below.
(a)(b)(c)


## 3.3. Metabolomics Results of Hippocampus

PCA, OPLS-DA, and S-plot[M2] results of hippocampal tissue are shown in Figure3. Results of hippocampal positive and negative ions are shown in Table S3. PCA results (R2 = 0.674, Q2 = 0.204) showed that compared with the sham operation group, the model group had a significant separation trend, and the distribution of the administration group and the sham operation group were closer, revealing the therapeutic effect of the drug from the perspective of metabolic distribution. The OPLS-DA results showed that 6 h after IR, the samples of the model and sham-operated groups were symmetrically distributed on both sides of the space, and the distribution of samples within the groups was relatively concentrated indicating that hippocampal metabolites changed significantly by modeling. In the S-plot [M2] plot, the farther away from the origin they are, the more likely the reactive ions are to be differential metabolites between groups.Figure 3
Metabolic analysis of hippocampus samples at 6 h after cerebral ischemia. (a) PCA-X image of hippocampal samples at 6 h after cerebral ischemia. (b) OPLS-DA of hippocampal samples at 6 h after cerebral ischemia. (c) S-plot[M2] of hippocampal samples at 6 h after cerebral ischemia.
(a)(b)(c)


## 3.4. Differential Metabolite Results

The differential metabolites among different groups are shown in Table1. The differential metabolites obtained by screening were all metabolites with significant differences between the model group and the sham operation group and between the administration group and the model group. All the differential metabolites obtained by screening belong to ESI− ion mode, and all have the performance of callback; that is, drug administration can reverse the injury of the model group to a certain extent, making the expression of the drug administration group more similar to that of the sham group. A total of 13 different metabolites were screened from the cortex, including amino acids (e.g., glutamine, dihydroorotic acid, and threonic acid), alkaloids (e.g., humantenine and yohimbic acid), and other acid products (including benzenesulfonic acid, glyceric acid, and hydroxy acid) which were the main products. Five kinds of differential metabolites were screened from the hippocampus, mainly amino acids (glutamic acid) and benzene-related derivatives (benzoquinone, benzophenone, amd benzoic acid).Table 1
Differential metabolites of Model group, Sham group, and SXT-M group.
PartNo.Metabolite nameRetention time(min)Measured mean mass (Da)Chemical formulaNormalized abundance (×105)ShamModelSXT-MCortex1Orthanilic acid0.85172.0213C6H7NO3S1.151.92▲▲▲1.62∗∗2Glutamine0.87145.0610C5H10N2O321.5030.72▲▲▲24.54∗∗3Humantenine17.73353.1976C21H26N2O34.933.17▲▲▲5.11∗∗∗4Dodecylbenzenesulfonic acid24.89325.1818C18H30O3S53.4064.05▲▲▲51.95∗∗∗5Dodecyl sulfate24.40265.1460C12H26O4S113.22129.05▲▲106.75∗∗∗6Yohimbic acid25.14339.1974C20H24N2O342.1649.02▲▲▲40.19∗∗∗7S-sulforaphene1.38174.0010C6H9NOS23.484.78▲▲4.00∗83-Hydroxypropanoic acid1.3189.0240C3H6O3137.90171.41▲▲141.70∗∗9Dihydroorotic acid0.28157.0108C5H6N2O43.263.54▲3.06∗∗∗10Glyceric acid0.76105.0166C3H6O42.492.69▲2.39∗∗11Embelin24.86293.1771C17H26O481.5693.37▲▲77.97∗∗∗12Etidronate23.68204.9879C2H8O7P21.621.80▲1.45∗∗∗13Threonic acid23.14135.0102C4H8O51.451.59▲▲1.45∗∗Hippocampus1Fumaric acid1.29115.0030C4H4O40.320.28▲0.32∗2Embelin13.24293.1733C17H26O412.016.18▲▲▲9.10∗∗∗3Glutamic acid14.55146.0348C5H9NO45.403.69▲▲5.25∗∗4Butylparaben12.04193.0854C11H14O33.262.04▲▲▲2.90∗∗∗52,4-Dihydroxybenzophenone0.84213.0473C13H10O34.116.39▲▲▲5.01∗The model group was compared with the sham group,▲P<0.05, ▲▲P<0.01, ▲▲▲P<0.001. The SXT-M group was compared with the model group: ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.


## 3.5. Metabolic Pathway Enrichment Results

Metabolic pathway enrichment of differential metabolites was carried out through the KEGG database, and the enriched pathways are shown in Figures4 and 5. There were four metabolic pathways with significant differences in the cortex: D-glutamine and D-glutamate metabolism, glyoxylate and dicarboxylate metabolism, pyrimidine metabolism, and nitrogen metabolism. Different metabolite concentrations in the metabolic pathways of the hippocampus and eight pathways with significant differences, respectively, are D-glutamine and D-glutamate metabolism, arginine biosynthesis, alanine, aspartate, and glutamate metabolism, nitrogen metabolism, butanoate metabolism, histidine metabolism, citrate cycle (TCA cycle), and pyruvate metabolism.Figure 4
Enrichment results of differential metabolite metabolic pathways in the cortex. The abscissa represents −log10P, the ordinate represents the enrichment pathway, and the bubble size represents the enrichment ratio. The darker the bubble color is, the smaller the P value is, as shown in Figure 5.Figure 5
Enrichment results of differential metabolite metabolic pathways in hippocampus.


## 4. Discussion

There are complex pathological mechanisms in the process of CIR injury. It is helpful and significant to extend the study of its mechanism to different regions and scopes to clarify the transmission process of the disease. This study explored the effects of SXT injection on the distribution of metabolites in different parts of the brain of rats with cerebral ischemia-reperfusion in order to extend the relevant mechanism and target from the metabolites and lay a material foundation for further clarifying the pathological mechanism and site difference of CIR injury.In order to determine the appropriate dose and effect of the dosing group, we performed a pharmacodynamic evaluation of SXT injection before metabolic analysis. The results showed a significant therapeutic effect in the high-, medium-, and low-dose groups of SXT. However, in combination with other studies in the group, the risk of bleeding was higher in the SXT high-dose group, provided that both the high-dose and medium-dose groups had significant therapeutic effects [27]. Therefore, the SXT medium-dose group (0.54 mg·kg−1) was selected for the follow-up metabolomics experimental study. The results showed that the disorders of brain metabolism in the model group were closely related to the metabolic synthesis of various amino acids, carbohydrates, nucleotides, and energy. SXT injection can regulate a variety of metabolites, correct the high and low levels of some metabolites, improve metabolic disorders in many aspects, and exert pharmacological effects on the treatment of cerebral ischemia-reperfusion from multiple perspectives, which is consistent with the overall concept.SXT can restore and regulate the metabolic disorders of the cortex and hippocampus caused by CIR, in which D-glutamine and D-glutamate metabolism and nitrogen metabolism are the same differential metabolic pathways of the cortex and hippocampus. The effects of SXT on these two pathways are mainly manifested as inhibition of cortical glutamine and promotion of hippocampal glutamate. Glutamate is the most abundant excitatory neurotransmitter in the brain, most of which is present in cortical synapses. It is involved in learning and memory in the brain and is closely related to the pathological process of stroke disease [28]. In total, 50–60% of the neurotransmitter glutamate is derived from the glutamate-glutamate cycle between neurons and astrocytes. Glutamate is converted to glutamine by glutamate synthase in astrocytes, and glutamine transporter proteins on the plasma membrane of astrocytes and neurons mediate the transfer of glutamine from astrocytes to neurons. Glutamine is converted to glutamate in neurons by glutaminase, which is loaded into synaptic vesicles for further release [29, 30]. Ischemic stroke severely destroys the synaptic network in the hippocampus. Transient ischemic occlusion is responsible for about 30% of synaptic closure in the hippocampus, and glutamate receptor expression in the ischemic hippocampus decreases. The transport of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPA) is inhibited [31]. Glutamine was significantly increased in the model group, possibly due to the increase in the reactivity of astrocytes in response to excitatory toxic injury. Under ischemic conditions, the continuous increase of glutamine can further aggravate the osmotic swelling of astrocytes and neurons, thus aggravating the metabolic disorders in the brain [32]. Therefore, the effect of SXT may be related to the protection of neurons in the cortex, inhibition of the excessive activation of astrocytes in the cortex, and promotion of glutamic acid conversion and uptake in the hippocampus, thereby reducing brain cell swelling and neurological dysfunction after CIR in rats.The effects of SXT on metabolic pathways in different parts of CIR are both different and correlated. Glyoxylate and dicarboxylate metabolism and pyrimidine metabolism are the unique metabolic pathways enriched by cortical differential metabolites. The effects of SXT are mainly shown as inhibition of glyceric acid and inhibition of dihydroorotic acid. The specific pathways enriched in the hippocampus include arginine biosynthesis, alanine, aspartate, and glutamate metabolism, butanoate metabolism, histidine metabolism, citrate cycle (TCA cycle), and pyruvate metabolism. The effect of SXT is mainly manifested as the promotion of fumaric acid. Differential metabolic pathways can be mainly attributed to carbohydrate metabolic pathway, nucleotide metabolic pathway, and amino acid metabolic pathway. Glyceric acid is derived from the oxidation of glycerol, and the metabolic pathways of glyoxylate and dicarboxylate metabolism are closely associated with the TCA cycle. The chain cycle of glyoxylate oxidative decarboxylation can be used as the protometabolic cycles analogue of the TCA cycle, converting glyoxylate into CO2 and producing aspartic acid in the presence of ammonia [33]. Dihydroorotic acid is an amino acid compound and can be oxidized to orotic acid under the action of dihydroorotate dehydrogenases (DHODHs), which plays an important role in pyrimidine metabolism pathway and energy metabolism [34]. DHODH is associated with oxidative phosphorylation process through its electron receptor coenzyme Q (CoQ). Oxidative phosphorylation damage of cells will lead to the decrease of CoQ, resulting in the inhibition of DHODH function and the increase of ROS production [35]. Fumaric acid is a precursor of L-malate in the TCA cycle and is formed by the oxidation of succinic acid by succinate dehydrogenase (SDH). Selective accumulation of succinic acid in the TCA cycle is a common metabolic feature of tissue ischemia and is closely associated with the generation of mitochondrial ROS during reperfusion. The ischemic accumulation of succinic acid is caused by the reversal of SDH, which reduces the amount of fumaric acid and intensification of CIR injury [36].Nitric oxide (NO), a small molecule involved in normal blood flow, vasodilation, and neuronal and glial activity, plays a neuroprotective and neurotoxic role in different stages of cerebral ischemia. It is a key factor in pathological development after stroke [37]. NO is produced by the oxidation of arginine by nitric oxide synthase (NOS) or the reduction of nitrite by various reductases [38]. There are three subtypes of nitric oxide synthase (NOS) involved in NO synthesis: neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), and inductive nitric oxide synthase (iNOS). ENOS and nNOS are known as structural nitric oxide synthases (cNOS) and depend on Ca2+ and calmodulin complexes [39]. In ischemic conditions, the content of superoxide (O2−) and other reactive oxygen molecules (ROS) increases, and NO reacts with O2− to generate active nitrogen (RNS), including highly toxic peroxynitrite (ONOO−), and other nitrogen oxide components such as NO2+ and NO2, leading to a cascade redox reaction that leads to neurotoxicity [40]. Changes in NO induced by ischemia are closely related to the activity of NOS subtypes. In the early stage of ischemia, the release of NO produced by eNOS surges, which may protect neurons from death by inducing vasodilation and inhibiting microvascular aggregation, but in the late stage of stroke, excessive NO produced by nNOS or iNOS can lead to cell apoptosis and neuron death [41]. Therefore, selective nNOS or iNOS inhibitors and eNOS agonists may have positive therapeutic significance for ischemic stroke.In the metabonomic study of the mouse IS model, arginine biosynthesis and alanine, aspartate, and glutamate metabolism have been implicated in the development of disease [42]. During the acute phase of IS, arginine is significantly elevated and may exert a protective effect by improving cerebral microcirculation in the prethrombotic environment induced by complement activation [43]. Arginine administration is associated with clinical and biochemical improvement of stroke-like episodes in patients with mitochondrial disease. During stroke-like episodes in patients with mitochondrial disease, transient changes and fading were observed in brain magnetic resonance imaging (MRI) within 1 week of arginine administration [44]. Bone bridge protein peptides containing arginine, alanine, and aspartate motifs significantly reduced brain infarct volume in the MCAO model [45]. Mitaki et al. showed that histidine-rich glycoprotein was significantly upregulated in future IS patients, which also provided a possible biomarker for predicting or targeting treatment of IS [46]. In addition to therapeutic and predictive effects, some amino acids are often used to modify therapeutic factors to improve therapeutic and targeting effects. Arginine was applied to modify polyamidoamine dendrimer, reduce the cytotoxicity of the compound, and improve its gene delivery ability in animal models of stroke [47]. Unmodified stem cell-derived extracellular vehicles (EVs) are poorly targeted and cannot be transported to ischemic sites. When their domain fuses arginine-glycine-aspartic acid to form the RGD-EV complex, their targeting of ischemic brain lesions is significantly improved and can be used as a potential therapeutic agent for IS [48]. Fatty acid-binding protein inhibitor linked with butanoic acid molecule has a neuroprotective effect on CIR injury in mice and has the potential as a neuroprotective drug [49].The ischemia-reperfusion injury that occurs after cerebral infarction is usually treated by lowering cranial pressure and dehydration [50]. For the ischemia-reperfusion injury of the limb, embolization and revascularization are performed after arterial embolization of the lower or upper extremity, and ischemia-reperfusion injury occurs after surgery, and osteofascial syndrome occurs in severe cases, and osteofascial syndrome dissection and decompression is performed if necessary, and VSD decompression and drainage is now commonly used in clinical practice, and when muscle swelling caused by ischemia-reperfusion injury is completely relieved, second-stage suturing can be performed [51–53]. During the reuse of VSD drainage for ischemia-reperfusion injury, it is important to prevent infection and closely observe the ischemic status of the limb, which may cause limb necrosis in severe cases [54]. In Western medicine, statins are effective in stabilizing atherosclerotic plaques and inhibiting inflammatory responses and are commonly used as clinical lipid-regulating drugs, and sparing blood combined with Western medicine for transient ischemic attacks can significantly improve the therapeutic effect and reduce the risk of cerebral infarction in patients [55, 56].


## 5. Conclusions

In this study, CIR rats were selected as models to analyze the metabolomics of the cortex and hippocampus of the rats after SXT injection, aiming to reveal the characteristics of metabolic molecules and pathways and to understand the key process of SXT repairing different parts of the brain. We demonstrate the feasibility of using metabonomics to analyze regional characteristics of CIR and explore the mechanisms involved. Our data provided not only 13 cortical differential metabolites (glutamine, dihydroorotic acid, glyceric acid, etc.) and 5 hippocampal differential metabolites (glutamic acid, fumaric acid, etc.) but also 4 metabolic pathways that were significantly altered in the cortex and 8 metabolic pathways that were significantly altered in the hippocampus. These findings provide evidence for the prevention and treatment of ischemic stroke by SXT injection and lay a foundation for the follow-up study on the mechanism of cerebral ischemia-reperfusion. However, there are limitations to the experiment, our experimental sample is small, and more clinical trials are needed to find different effects, including dose changes.



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