Investigation of the Mechanism of Action of the Mongolian Medicine Eerdun Wurile Basic Formula in the Treatment of Ischemic Stroke Through Transcriptomics and Metabolomics Integration

Chemicals and Reagents

Pearl, licorice, Saussurea lappa, Aucklandia lappa, agarwood, bezoar, Piper longum, safflower, water buffalo horn, and musk were provided by Inner Mongolia Kulun Mongolian Medicine Co., Ltd.; MCAO suture (Guangzhou Jialing Biotechnology Co., Ltd., Guangzhou, China; Batch No. 20230811); isoflurane (Shenzhen RWD Life Science Co., Ltd.); nimodipine tablets (Shandong Xinhua Pharmaceutical Co., Ltd.); dithiothreitol (DTT), iodoacetamide (IAA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, sodium deoxycholate (SDC), Tris buffer, protease inhibitors, and other reagents were purchased from Sigma-Aldrich (USA); trypsin was purchased from Promega (USA); the BCA protein assay kit was obtained from Shanghai Beyotime Biotechnology Co., Ltd.; purified water was purchased from Watsons (China). The botanical names, voucher numbers, and properties of the medicinal components are summarized in Table 1.

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Table 1.

The botanical name, voucher number, Family, Nature, Taste, Efficacy of the Medicine and Content of Plant Content in EWB

Single drug name	Family	Nature of the medicine	Taste of the medicine	Efficacy of the medicine	Content (g; %)
Margarita	Margaritiferidae	Neutral	Sweet、salty	——	20 g,
					15.94%
Glycyrrhizae radix et rhizoma	Leguminosae	Cooling	Sweet	Thinning、Gentle、Softening	10 g,
					7.97%
Inulae radix	Compositae	Neutral	Sweet、bitter、pungent	Cloying、Penetrating、Drying、Grounding	10 g,
					7.97%
Aucklandiae radix	Compositae	Warm	Pungent、bitter	Cloying、Astringent、Light	10 g,
					7.97%
Aquilariae lignum resinatum	Thymelaeceae	Cooling	Pungent、bitter	Softening、Cloying、Drying、Grounding、Mild	15 g,
					11.95%
Bovis calculus artifactus	Bovidae	Cooling	Bitter、sweet	Grounding、Mild、Gentle、Softening	5 g, 3.98%
Piperis longi fructus	Piperaceae	Warm	Pungent	Cloying、Penetrating、Light、Drying	10 g,
					7.97%
Crocussativus L	Compositae	Cooling	Sweet、slightly bitter	Gentle、Softening、Astringing、Mild、Grounding	15 g,
					11.95%
Powerdered buffalo horn extract	Bovidae	Warm	Astringent、salty	——	30 g,
					23.9%
Moschus	Cervidae	Cooling	Pungent、bitter	Mild、Light、Astringent、Cloying	0.5 g, 0.4%

Preparation of Drugs

Precisely weigh 20g of pearl, 10g of licorice, 10g of Saussurea lappa, 10g of Aucklandia lappa, 15g of agarwood, 5g of bezoar, 10g of Piper longum, 15g of safflower, 30g of concentrated water buffalo horn powder, and 0.5 g of musk. The pearl was ground into fine powder to facilitate dissolution or suspension. Combine the ingredients according to traditional pharmaceutical methods, and dissolve them in distilled water to prepare solutions with concentrations of 0.027 g/mL, 0.054 g/mL, and 0.108 g/mL, representing low, medium, and high doses of the base formula, respectively. Prepare fresh solutions daily. Grind the nimodipine tablets into powder and dissolve in distilled water to prepare a solution with a concentration of 1.08 g/L, which is used as the positive control drug.

Animal Grouping and Model Establishment

The study was conducted from February 2023 to December 2023. This study was conducted in accordance with the ARRIVE guidelines for reporting animal research. Ninety male SPF-grade SD rats, weighing (240 ± 30) g, were provided by SPF (Beijing) Biotechnology Co., Ltd. (Animal License No. SCXK [Beijing] 2019-0010). This study was approved by the Experimental Animal Ethics Committee of Inner Mongolia Medical University, and all procedures were conducted in strict accordance with the regulations governing the management of laboratory animals. After 7 days of adaptive feeding, 90 rats were randomly divided into six groups using a random number table: Sham operation group, Model group, Positive control group (Nimodipine), and EWB low, medium, and high dose groups, with 15 rats in each group. In the Sham operation group, after isolating the carotid artery, the wound was cleaned and sutured without inserting or ligating the suture. For the other groups, the right-sided MCAO/R model was established using the suture method. The surgical procedure was conducted as follows ¹⁴ :

1. Rats were fasted for 12 hours before surgery but allowed free access to water. After inducing anesthesia with isoflurane, the rats were placed in a supine position on a 37°C thermostatic pad, and vital signs were closely monitored. The surgical area was shaved and disinfected with povidone-iodine.

The success of the model was evaluated using the Zea Longa scoring method, where rats scoring 1-3 points were included, and those scoring 0 or 4 points were excluded. The sham operation and model groups were administered distilled water by gavage at the same volume. After model establishment, the sham operation and model groups received distilled water by gavage, while the EWB low, medium, and high dose groups received their respective EWB solutions, and the positive control group received nimodipine solution, administered once daily at the same time each day, with a volume of 10 mL/kg, for 21 consecutive days.

Survival Rate and Zea-Longa Five-point Neurological Deficit Score Assessment

The number of surviving rats was recorded during the experiment, and the survival percentage was calculated by normalizing the number of surviving rats to the total number of rats. Neurological deficits were assessed using the Zea-Longa five-point scoring method on days 1, 3, and 7 after model establishment and drug administration. ¹⁵ The scoring criteria are as follows: 0 points: The rat walks normally with no signs of neurological deficits. 1 point: The rat is unable to walk in a straight line, showing mild forelimb weakness. 2 points: The rat circles toward the paralyzed side, exhibiting severe forelimb weakness and significant motor impairment. 3 points: The rat is unable to stand, with body tilt and falling toward the paralyzed side, indicating severe neurological deficits. 4 points: The rat is unable to walk independently, showing markedly reduced consciousness and severely restricted mobility. 5 points: The rat is deceased.

Open Field Test and Morris Water Maze Test

On the first day after drug administration, the open field test was used to assess the spontaneous locomotor activity and exploratory behavior of rats in each group. The floor of the open field apparatus was divided into 25 squares, with the central 9 squares designated as the central area. Each rat was placed in the open field for 5 minutes of free exploration, and the total distance traveled, time spent in the central area, and number of entries into the central area were recorded. Each rat was tested once, and after the test, the open field apparatus was wiped with 75% ethanol to eliminate any residual odors. Video analysis for the open field test was performed using EthoVision XT software (Noldus). On the 17th day after drug administration, the Morris water maze test was used to assess the spatial learning and memory abilities of the rats in each group. The water in the maze was stained white with non-toxic latex to hide the platform. The circular water tank, with a diameter of 120 cm and a depth of 30 cm, was divided into four quadrants, with the platform placed at the center of the third quadrant. During the experiment, the rats were placed in the water and required to find the platform. The test consisted of two parts: the navigation test and the spatial probe test. Navigation Test: Conducted over 4 days, rats were placed into the water facing the tank wall from four different quadrants, and the time required to find the platform (escape latency) was measured, with a maximum time of 90 seconds. This test assessed spatial learning ability. Spatial Probe Test: Conducted for 1 day, the platform in the third quadrant was removed, and the rats were placed in the water from the first quadrant. The number of times the rats crossed the area where the platform was previously located was recorded over 90 seconds to assess spatial memory retrieval ability. Video analysis for the Morris water maze test was performed using EthoVision XT 15 (Noldus), and probability density plots (Figure 1G) were generated using MATLAB R2023a (MathWorks).OPEN IN VIEWER

HE Staining to Observe Pathological Morphological Changes in the Hippocampal Region of Rats

Three rats from each group were anesthetized and placed supine on a dissection board. The chest cavity was opened to expose the heart, and a perfusion needle was inserted from the left ventricle through the apex into the aortic arch. The abdominal aorta was clamped, and the right atrium was incised. The rats were perfused with 150 mL of sterile saline until the lungs and forelimbs turned white, followed by 150 mL of 4% paraformaldehyde. After decapitation, the brains were extracted, fixed in 4% paraformaldehyde for 24 hours, dehydrated through a graded ethanol series (70%, 80%, 85%, 95%, and 100% for 5 minutes each), and cleared in xylene (two changes, 5 minutes each) before being embedded in paraffin. Sections were stained in hematoxylin for 3-5 minutes, washed with tap water, differentiated with differentiation solution, blued with bluing solution, and rinsed with running water. Sections were dehydrated in 85% and 95% gradient alcohol for 5 minutes each, stained in eosin for 5 minutes, and then placed in anhydrous ethanol I, II, and III for 5 minutes each. Sections were cleared in xylene I and II for 5 minutes each and mounted with neutral resin. Five hippocampal sections (4 μm thick) per animal were examined using a Nikon Eclipse E100 microscope equipped with a Nikon DS-U3 camera and a 20x objective to analyze neuronal morphology and cell counts in the CA3 and DG regions.

TUNEL Assay for Detecting Apoptosis

The rat hippocampal tissue was harvested and fixed in 10% neutral formalin for 24 hours. The tissue was subsequently dehydrated, cleared, embedded, and paraffin sections approximately 4 μm thick were prepared. The sections were deparaffinized in xylene, dehydrated through a graded ethanol series, and washed with water. The sections were then digested with proteinase K (20 μg/mL) at room temperature for 15 minutes. After washing with PBS, TUNEL reaction solution (from the TUNEL kit) was applied, and the sections were incubated at 37°C in the dark for 60 minutes. The sections were washed with PBS and stained with DAPI, incubating in the dark at room temperature for 10 minutes. After a final PBS wash, the slides were mounted and five sections per animal were observed under a Nikon Eclipse E100 fluorescence microscope equipped with a Nikon DS-U3 camera and a 20x objective to count TUNEL-positive cells in the CA3 and DG regions.

ELISA Detection of Serum TNF-α, IL-1β, and IL-6 Levels in Each Group of Rats

At the end of the experiment, after anesthetizing each group of rats with isoflurane, 3 mL of blood was drawn via abdominal aortic cannulation and placed in procoagulant tubes. The samples were allowed to stand for 1 hour, then centrifuged at room temperature for 15 minutes (3000 RPM), and the serum was separated. The serum was frozen in liquid nitrogen and stored at −80°C. The levels of TNF-α, IL-1β, and IL-6 were measured using ELISA kits from Quanzhou Ruixin Biological Technology Co., LTD (Quanzhou, China) with catalog numbers RX302869 R (IL-1β), RX302058 R (TNF-α), and RX302856 R (IL-6). The levels of TNF-α, IL-1β, and IL-6 in the serum were then measured according to the ELISA kit instructions.

Transcriptomic Analysis

Total RNA was extracted from brain tissue using TRIzol reagent. Purified RNA extracted from brain tissue was used to construct a cDNA library using the Illumina TruSeq RNA Library Prep Kit, which was sequenced on the Illumina HiSeq 6000 platform. Raw reads were quality-checked with FastQC v0.11.9 and trimmed for quality using Trimmomatic v0.39, aligned to the rat genome (Rnor_6.0) using STAR v2.7.9a, and aligned reads were counted using featureCounts v2.0.1. Differential expression analysis between the two groups was performed using the “limma” package in R. Genes meeting the following criteria were identified as differentially expressed genes (DEGs) between the groups: |log2(fold change)| > 1 and P-value < 0.05. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs were performed using the DAVID database (https://david.ncifcrf.gov/) and Metascape (https://metascape.org/).

Untargeted Metabolomics Analysis

The brain tissue supernatant was extracted using 80% methanol and 0.1% formic acid, followed by centrifugation at 15,000g for 20 minutes to obtain the samples. LC-MS analysis was performed using an UHPLC system (ThermoFisher, Germany) coupled with an Orbitrap Q Exactive™ HF-X mass spectrometer (ThermoFisher, Germany), and all analyses were conducted using a Hypesil Gold column (100 × 2.1 mm, 1.9 µm). The gradient elution program for mobile phase B was as follows: 2%, 0-1.5 minutes; 2-100%, 1.5-12.0 minutes; 100%, 12.0-14.0 minutes; 100-2%, 14.0-14.1 minutes; 2%, 14.1-17.0 minutes. The conditions in positive/negative ion mode were as follows: spray voltage, 3200 V; capillary temperature, 320°C; sheath gas flow rate, 40 arb; auxiliary gas flow rate, 10 arb. The preprocessed data were analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA), and permutation testing (100 permutations) was performed to prevent overfitting. In the OPLS-DA model, metabolites with a VIP score >1 and P-value <.05 were identified as differential metabolites (DMs). Relevant metabolic pathways were identified using MetaboAnalyst (https://www.metaboanalyst.ca/).

Integrated Transcriptomic and Metabolomic Analysis

The Metscape tool in Cytoscape 3.9.0 was used to integrate the DMs from metabolomics and the DEGs from transcriptomics based on a pathway-focused model. After integrating these two omics datasets, key metabolic pathways were further identified. To better understand the mechanisms involved, the STRING database was used to construct a network of DEGs within the key metabolic pathways, and the interactions between the targets were visualized using Cytoscape 3.9.0.

Western Blotting

The collected brain tissue was ground in liquid nitrogen and suspended in RIPA lysis buffer containing protease and phosphatase inhibitors. The tissue was lysed on ice for 30 minutes. The supernatant was collected as a protein sample after centrifugation at 12 000 rpm for 15 minutes at 4°C, and protein quantification was performed using the BCA protein assay kit. Equal amounts of total protein per sample were mixed with loading buffer and boiled for 5 minutes before separation by SDS-PAGE. The proteins were then transferred onto a PVDF membrane, which was blocked at room temperature with 5% bovine skim milk for 1 hour. After blocking, the membrane was incubated overnight at 4°C with the primary antibody solution, including anti-SLC17A6 (Synaptic Systems, catalog no. 135 403), anti-SLC6A11 (Sigma-Aldrich, catalog no. HPA037981), anti-SLC6A9 (Sigma-Aldrich, catalog no. HPA013977), anti-GNG7 (Sigma-Aldrich, catalog no. HPA057790), and anti-ADORA1 (Abcam, catalog no. ab288377). The membrane was washed three times with TBST buffer, each for 10 minutes, followed by incubation with the secondary antibody at room temperature for 1 hour. After washing, the membrane was treated with enhanced chemiluminescence (ECL) reagents, and the luminescence signal was detected and recorded using an imaging system.

Quantitative Real-Time PCR

The collected brain tissue (n = 6) was ground in liquid nitrogen, and total RNA was extracted from the brain tissue using the FastPure RNA Extraction Kit according to the manufacturer’s instructions. RNA concentration was then measured using a Nanodrop spectrophotometer. cDNA synthesis was performed using the M5 Super plus qPCR RT kit with a protocol of incubation at 42°C for 60 minutes followed by 85°C for 5 minutes, using 1 μg of total RNA per reaction, and the undiluted first-strand cDNA was used for qPCR. mRNA expression levels were measured using SYBR qPCR Master Mix. The gene primers are listed in Table 2. Relative mRNA expression was calculated using the 2-^ΔΔCT method.

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Table 2.

Primers for RT-qPCR

NO.	Gene	Sequence (5’− 3’)
1	Slc17a6	Forward 5′-GGGTGGCAAAGTTATCAAGGAG-3′
		Reverse 5′-TGAGGGTAGAGGTAAGCAGTATGG-3′
2	Slc6a11	Forward 5′-ATTCCTGATTCCTTACGTGGTGTT-3′
		Reverse 5′-CCACGCCAGGATGATGATGTAG-3′
3	Slc6a9	Forward 5′-TGCATCGCCTTCTACTACTTCTTC-3′
		Reverse 5′-GCACTTCTCCAAAATCTCCAATGT-3′
4	Gng7	Forward 5′-TGTCAGGTACTAACAACGTCGCC-3′
		Reverse 5′-CTTGGAGACCTTGATGCGTTC-3′
5	Adora1	Forward 5′-CTTATCAACATTGGGCCACAGAC-3′
		Reverse 5′-GCTGGGTCACCACTGTCTTGTA-3′
6	GAPDH	Forward 5′-CTGGAGAAACCTGCCAAGTATG-3′
		Reverse 5′-GGTGGAAGAATGGGAGTTGCT-3′

Data Analysis

Metabolomic and transcriptomic data were compared between the two groups using the Student’s t-test to assess differences in metabolite or protein expression. Metabolomic data were analyzed using the OPLS-DA model with the first principal component’s VIP > 1, combined with a t-test P-value < .05 and Bonferroni correction for multiple comparisons to screen for biomarkers. Data normality was assessed using the Shapiro–Wilk test. For normally distributed data, one-way ANOVA with Tukey’s post hoc test was used for multiple comparisons. For Morris water maze escape latency data, a repeated-measures ANOVA was performed with Bonferroni correction due to its multi-day design across six groups. If normality was not satisfied, the Kruskal–Wallis test with Dunn’s post hoc test was applied. Data analysis and visualization were performed using GraphPad Prism 9. Statistical analysis was conducted using one-way ANOVA, with data presented as mean ± S.D. Statistical significance was set at P < .05, P < .01, P < .001, and P < .0001.

EWB Ameliorated Brain Injury Induced by MCAO/R in Rats

The schematic diagram of the experimental procedure is shown in Figure 1A. MCAO/R rats were treated with low, medium, and high doses of EWB to evaluate its therapeutic efficacy. Compared with the sham group, the MCAO/R group showed a significant reduction in weight gain (P < .001). However, EWB-M, EWB-H, or nimodipine alleviated the stroke-induced weight loss (P < .05, P < .001). The weight gain in the EWB-H group was significantly higher than that in the EWB-L, EWB-M, and nimodipine groups (P < .05, P < .001) (Figure 1B). Subsequently, the effect of EWB on neurological deficits was assessed using the Zea-Longa scoring method (Figure 1C). During the observation period, the neurological scores of the MCAO/R group remained around 3, indicating severe neurological impairment with no spontaneous recovery, thus confirming the success of the model. However, compared to the MCAO/R group, the EWB-M, EWB-H, and Nimodipine groups showed significant improvement in neurological deficits by day 14, with significantly lower scores (P < .05, P < .001). Notably, neurological recovery in the EWB-H group was significantly better than in the EWB-L and EWB-M groups (P < .05, P < .01). Notably, compared with the MCAO/R group at day 21, the infarct rate was significantly reduced in the EWB-M, EWB-H, and Nimodipine groups (P < .01, P < .001), and the infarct rate in the EWB-H group was significantly lower than in the EWB-L and EWB-M groups (P < .01, P < .001) (Figure 1D). After administration, compared to the MCAO/R group, the number of spontaneous activities in the open field test significantly increased in all EWB-treated groups (P < .05, P < .01, P < .05), while the time spent stationary was significantly reduced. This suggests that EWB improves spontaneous activity and exploratory behavior in rats with ischemic stroke (Figure 1E and F). The learning and memory abilities of each group of rats were evaluated using the Morris water maze test (Figures 1G-L). The results of the navigation test showed that, compared with the MCAO/R group, both the EWB-L and EWB-H groups had shorter escape latencies and significantly more platform crossings (P < 0.01). Additionally, the changes in all indicators were more pronounced in the EWB-H group compared with the EWB-L group (P < 0.05). The results of the spatial probe test showed that, on day 5 after platform removal, the number of platform crossings significantly increased in the EWB-H, EWB-M, and nimodipine groups compared to the model group (P < 0.05). Compared to the MCAO/R group, the EWB-H and EWB-M groups significantly increased the swimming time and distance in the target quadrant (P < .01). Additionally, the nimodipine group also showed a significant increase in swimming time in the target quadrant (P < .05). These results indicate that EWB significantly improves spontaneous behavior and learning and memory deficits in rats with ischemic stroke. Finally, there were no significant differences in the average swimming speed across the groups, suggesting that the experimental procedures did not cause physical impairment in the animals. The observed differences were primarily attributed to cognitive dysfunction. Moreover, we identified the high dose of EWB as the most effective, which was further evaluated in subsequent experiments.

Histopathological Changes and Neuroinflammatory Responses in Ischemic Brain Tissue

H&E staining (Figure 2A) showed no obvious changes in the sham operation group, while the MCAO/R group exhibited varying degrees of neuronal damage, including nuclear pyknosis, irregular nuclear shapes, increased intercellular spaces, interstitial edema, and unclear nuclear-cytoplasmic organization. The EWB-L, EWB-M, and EWB-H groups significantly improved neuronal arrangement and cell numbers in brain tissue. As the concentration of EWB increased and the duration of the intervention extended, the improvements in neuronal morphology became more pronounced, with the EWB-H group showing the most notable effect after 7 days of treatment. Additionally, to further analyze the effect of EWB on neuronal apoptosis in the CA3/DG region of the hippocampus in MCAO/R rats, the experiment used TUNEL staining to assess the neuroprotective effect of EWB at different concentrations on cell apoptosis. As shown in Figure 2B, there were very few TUNEL-positive spots in the sham operation group. After 2 hours of MCAO/R treatment followed by 7 days of reperfusion, a significant accumulation of TUNEL-positive spots was observed around the infarct area of the cortical region in MCAO/R rats, indicating extensive neuronal apoptosis compared to the sham group. However, following EWB treatment, the accumulation of positive staining in MCAO/R rats was markedly reduced. Compared with the EWB-L group, the number of apoptotic cells in the EWB-M and EWB-H groups was significantly reduced (P < .01). These results indicate that EWB effectively improved neurological deficits after reperfusion, repaired neuronal damage, and reduced neuronal apoptosis in the hippocampal CA3/DG region of rats, with the effects being proportional to both concentration and duration of treatment. ELISA was used to measure the serum levels of TNF-α, IL-1β, and IL-6 in each group of rats. The results showed a significant increase in IL-1β, IL-6, and TNF-α levels in the MCAO/R model rats. In contrast, the EWB-L, EWB-M, and EWB-H groups significantly reduced the levels of IL-1β, IL-6, and TNF-α in MCAO/R rats (P < .01) (Figure 2E–G).OPEN IN VIEWER

Transcriptomic Analysis

To uncover the neuroprotective mechanisms of EWB, RNA-seq analysis was used to investigate the gene expression profiles of rats treated with EWB following MCAO/R surgery. As shown in Figure 3A–B, 635 differentially expressed genes (DEGs) were identified between the MCAO/R group and the sham group, with 384 upregulated and 251 downregulated. After EWB pretreatment, 1675 DEGs were identified, of which 822 were upregulated and 853 were downregulated. GO and KEGG enrichment analyses were then performed on the 72 overlapping DEGs between the MCAO/R vs sham group and the EWB-H vs MCAO/R group (Figure 3C). The results showed that the biological processes (BP) enriched by these DEGs were related to immune response and angiogenesis. The molecular functions (MF) were closely associated with calcium ion binding and inflammatory pathways, while the cellular components (CC) enriched by the DEGs were primarily related to the extracellular region/matrix and the outer membrane (Figure 3E–F). Additionally, KEGG pathway analysis revealed that DEGs were primarily enriched in inflammatory pathways and ECM-receptor interaction-related signaling pathways, consistent with the results of GO-BP enrichment analysis (Figure 3G–H). In summary, we preliminarily speculate that EWB may prevent MCAO/R-induced injury by modulating inflammatory responses and ECM-receptor interaction-related processes.OPEN IN VIEWER

Using the STRING database, a protein-protein interaction (PPI) network was constructed for DEGs enriched in the GO and KEGG signaling pathways related to “inflammatory response” and “ECM-receptor interaction.” The network was visualized using Cytoscape (version 3.7.2), as shown in Figure 3D. Based on topological parameters, five key targets—SLC17A6, SLC6A11, SLC6A9, GNG7, and ADORA1—were identified as core targets due to having the highest degree of connectivity within the network.

Metabolomic Analysis

To evaluate the host metabolic changes induced by EWB treatment, metabolomic data from the sham group, MCAO/R group, and EWB-H group were analyzed. OPLS-DA models were established to screen for potential biomarkers with significant differences between the sham group and MCAO/R group, and between the MCAO/R group and EWB-H group (Figure 4A–B). The R2Y and Q2 statistical values indicated that the OPLS-DA score plots had good applicability and high predictability. The metabolic profiles between the MCAO/R group and sham group, as well as between the EWB-H group and MCAO/R group, were clearly separated. The parameters for the sham group vs MCAO/R group in positive ion mode were R2Y = 99%, Q2 = 88%; in negative ion mode, R2Y = 99%, Q2 = 80%. For the EWB-H group vs MCAO/R group, the parameters in positive ion mode were R2Y = 99%, Q2 = 89%, and in negative ion mode, R2Y = 98%, Q2 = 88%. The validation test results demonstrated that the model was effective.OPEN IN VIEWER

A total of 848 differential metabolites (DMs) were identified between the MCAO/R group and the sham group, with 254 upregulated and 594 downregulated DMs. Between the EWB-H group and the MCAO/R group, 832 DMs were identified, of which 282 were upregulated and 550 were downregulated. Notably, 416 DMs were shared between the comparisons of the MCAO/R group vs the sham group and the EWB-H group vs the MCAO/R group (Figure 4C–F). Metabolic pathway analysis of the 416 potential biomarkers was performed using the MetaboAnalyst 5.0 database. In positive ion mode, the identified shared DMs were primarily associated with the following metabolic pathways: alanine, aspartate, and glutamate metabolism; GABAergic synapse; tyrosine metabolism; aminoacyl-tRNA biosynthesis; phosphatidylinositol signaling system; steroid hormone biosynthesis; glutathione metabolism; and glycerophospholipid metabolism. In negative ion mode, the identified shared DMs were primarily enriched in the following metabolic pathways: riboflavin metabolism, purine biosynthesis, aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, inosine 5′-monophosphate and hypoxanthine, cofactor biosynthesis, xanthine, glutathione metabolism, tyrosine metabolism, inositol phosphate metabolism, glycerophospholipid metabolism, GABAergic synapse, and the cGMP-PKG signaling pathway. The pathways enriched in tyrosine metabolism, GABAergic synapse, and glycerophospholipid metabolism were identified as key metabolic pathways involved in EWB’s treatment of MCAO/R (Figure 5A). After obtaining the matched information of differential metabolites, pathway searches and regulatory interaction network analyses were performed using the KEGG database for the corresponding species, and the results were visualized as a network plot (Figure 5B). To further visualize the alterations in specific metabolites, violin plots were generated for a set of representative key metabolites, showing their distribution and variation across groups (Figure 6).OPEN IN VIEWER

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Figure 6.

Violin plots showing changes in key metabolite levels (n = 8). MW0138968: Succinic acid; MW0126169: Adenosine monophosphate; MWO015480: Ketorolac; MW0148485: Acetaminophen glucuronide; MADNO541: Tafluprost acid; MW0009282: Fumagillin; MWO114047: cis-2-Oxohept-3-enedioic acid; MW0139814: N(3)-fumaroyl-(S)-2,3-diaminopropanoic acid; MW0140287: 4′-Apo-beta-carotenal; MW0049210: Myricanol 5-beta-sophoroside; MWO141992: DL-3,4-Dihydroxyphenyl glycol; MWO077568: Glycerol 1-myristate; MW0153176: 1-Octadecyl Lysophosphatidic Acid; MW0114676: 3-O-Feruloylquinic acid; MW0000057: Phosphoserine; MWO059443: 5-Aminoimidazole ribonucleotide; MW0150423: Guanosine diphosphate mannose; MEDNO428: Spirolucidine; MEDN1011: 3beta-Cyclopentyl-5alpha-androstan-17beta-ol

Integrated Transcriptomic and Metabolomic Analysis

By integrating transcriptomic and metabolomic data, a reaction-enzyme-gene interaction network was constructed (Figure 7D). We identified three key metabolic pathways: synaptic vesicle cycle, tyrosine metabolism, and glycerophospholipid metabolism (Figure 7E). Further analysis indicated that the DEGs in these metabolic pathways were closely associated with DEGs enriched in inflammatory responses. Among these DEGs, SLC17A6, SLC6A11, SLC6A9, GNG7, and ADORA1 were identified as core genes (Figure 7A). The related key metabolites include adenosine monophosphate, DL-3,4-dihydroxyphenyl glycol, succinic acid, and cis-2-oxohept-3-enedioic acid (Figure 7B). Pearson analysis showed that the targets SLC17A6, SLC6A11, and SLC6A9 were significantly negatively correlated with the levels of adenosine monophosphate, DL-3,4-dihydroxyphenyl glycol, and succinic acid. In contrast, Gng7 and Adora1 were positively correlated with the level of cis-2-oxohept-3-enedioic acid. These findings suggested that these targets are closely associated with the levels of their respective metabolites (Figure 7C). These genes and metabolites may play an important role in the therapeutic effects of EWB on ischemia-reperfusion injury in the brain.OPEN IN VIEWER

EWB Reduced the Expression of Core Genes in MCAO/R Model Rats

To validate the results of the multi-omics analysis, the expression levels of SLC17A6, SLC6A11, SLC6A9, GNG7, and ADORA1 were measured. The mRNA expression levels of SLC17A6, SLC6A11, SLC6A9, and ADORA1 were elevated in MCAO/R model rats, but significantly decreased after EWB treatment at different concentrations (Figure 8A–D). In contrast, the expression of GNG7 showed the opposite trend, with mRNA expression levels increased in MCAO/R rats and significantly reduced after EWB treatment (Figure 8E). The protein expression levels, as determined by Western blot (WB), showed the same trends (Figure 8F–K).OPEN IN VIEWER