Abstract Rhubarb (RR), Chinese name Dahuang, is commonly used in the treatment of ischemic stroke (IS). However, its potential mechanism is not fully elucidated. This study intended to verify the effect of RR on IS and investigate the possible mechanism of RR in preventing IS. IS in male rats was induced by embolic middle cerebral artery occlusion (MCAO) surgery, and drug administration was applied half an hour before surgery. RR dramatically decreased the neurological deficit scores, the cerebral infarct volume, and the cerebral edema rate, and improved the regional cerebral blood flow (rCBF) and histopathological changes in the brain of MCAO rats. The 16S rRNA analysis showed the harmful microbes such as Fournierella and Bilophila were decreased, and the beneficial microbes such as Enterorhabdus, Defluviitaleaceae, Christensenellaceae, and Lachnospira were significantly increased, after RR pretreatment. ^1H-nuclear magnetic resonance (^1H-NMR) was used to detect serum metabolomics, and RR treatment significantly changed the levels of metabolites such as isoleucine, valine, N6-acetyllysine, methionine, 3-aminoisobutyric acid, N, N-dimethylglycine, propylene glycol, trimethylamine N-oxide, myo-inositol, choline, betaine, lactate, glucose, and lipid, and the enrichment analysis of differential metabolites showed that RR may participate in the regulation of amino acid metabolism and energy metabolism. RR exerts the role of anti-IS via regulating gut bacteria and metabolic pathways. Keywords: dahuang (rhubarb, RR); ischemic stroke; gut microbiota; metabolomics; ^1H-NMR 1. Introduction Stroke is a common clinical cerebrovascular disease that has high rates of incidence, recurrence, disability, and mortality, and a high treatment cost, presenting a serious danger to human health [[42]1,[43]2]. Stroke mainly includes ischemic stroke (IS) and hemorrhagic stroke. IS accounts for more than 80% of stroke, and there is still an increasing annual trend with the rapid growth in the aging population [[44]3,[45]4,[46]5]. IS is an acute disease caused by cerebral vascular obstruction, leading to cerebral ischemia, hypoxia, necrosis, and neurological function defects [[47]6,[48]7,[49]8]. The intravenous recombinant tissue plasminogen activator (rt-PA) is the only drug currently approved by the FDA for the therapy of IS [[50]8,[51]9]. However, the use of rt-PA in the clinic is limited due to its strict indications and contraindications [[52]9,[53]10]. More effective strategies for IS are urgently needed. IS belongs to the category of “stroke” in traditional Chinese medicine (TCM). The history of treating stroke in TCM is long-standing. Rhubarb plants (Rheum tanguticum Maxim. ex Balf., Rheum palmatum L., or Rheum officinale Baill., RR), also known as dahuang in Chinese. The medicinal parts of dahuang, as stipulated in the Chinese Pharmacopoeia, are the roots and rhizomes. As first recorded in Shennong Bencao Jing, it was commonly used in the treatment of stroke based on the theory of Tong Fu in TCM. The mechanism of action and clinical efficacy of the Tong Fu method in the treatment of stroke have been reported, the prescriptions for stroke based on the theory of Tong Fu have also been summarized in the literature, and the results all show that RR is a commonly used drug in the treatment of stroke [[54]11,[55]12,[56]13,[57]14]. As shown in the literature, study results from research related to the treatment of IS based on the theory of Tong Fu show that RR is the most frequently used drug, which shows that RR is a key drug in the treatment of IS using the method of Tong Fu [[58]15]. The curative role of RR for IS has been reported in multiple studies, such as reducing cerebral infarct volume and neurological deficit score [[59]16,[60]17,[61]18]. However, because of the complex pathogenesis of IS, the effect and mechanism of RR on IS treatment have still not been entirely elaborated. Previous studies have found that RR as a purgative could improve colon mucosal barrier injury and severe acute pancreatitis by regulating gut microbiota [[62]19,[63]20]. The gut flora is a large and complex microbial community that lives in the gastrointestinal tract. It was reported that the gut flora plays a crucial part in keeping the internal balance of the body, which modulates the occurrence and progression of illness. Previous studies suggested that gut microbes could influence metabolism and cerebral function by way of the microbiome–brain–gut axis [[64]21,[65]22,[66]23]. Lee et al. found that fecal transplantation from young mice improved the outcomes of older mice with stroke, which was related to microbes that generate short-chain fatty acids [[67]24]. Hence, we speculated that RR might regulate gut microbes and metabolites and the internal relationship between them, which may affect the brain’s physiological function through the blood circulation or the gut–brain axis. Metabolites are the downstream products of the regulation of genes and proteins, and therefore more immediately reflect the changes in the physiological or pathological state of the organism, and the mechanism of development of IS is associated with alterations in metabolites [[68]25,[69]26]. A metabolomics study found that the phenylacetylglutamine (PAGIn) in plasma in IS patients was dramatically increased compared with that in healthy controls [[70]27]. Refined Huanglian–Jiedu decoction was revealed to play a neuro-protective effect against IS via modulation of the metabolism of energy, amino acids, and nucleic acids based on metabolomics using ^1H-nuclear magnetic resonance (^1H-NMR) [[71]28]. However, whether the disordered composition of gut microbes and levels of metabolites can be reversed by RR treatment in middle-cerebral-artery-occlusion (MCAO) rats is still uncertain. In our study, we observed the role of RR in MCAO rats and further analyzed its impact on alterations in gut microbiota and serum metabolites using 16S sequencing and ^1H-NMR non-targeted metabolomics, which laid the experimental foundation for further clarification of the mechanism of RR for IS. 2. Materials and Methods 2.1. Chemicals and Reagents The following were used: Hematoxylin and eosin dye liquor (Wuhan Servicebio Technology Co., Ltd., Wuhan, China; No. G1004 and G1002), 2,3,5-triphenyltetrazolium chloride (TTC) stain (Beijing Biodee Biotechnology Co., Ltd., Beijing, China; No. BN20391). The standard substances emodin, chrysophanol, aloe-emodin, physcion, rhein, sennoside A, emodin-8-glucoside, and (+)-catechin (Shanghai Yuanye Biological Co., Ltd., Shanghai, China; No. [72]B20240, No. [73]B20238, No. [74]B20772, No. [75]B20242, No. [76]B20245, No. [77]B20380, No. [78]B20241, No. [79]B21722). Chromatographic grade acetonitrile, methanol, and formic acid (Thermo Fisher Scientific Inc., Santa Clara, CA, USA; NO. A955-4, A456-4 and 28905; Purity > 99%). Extract of Ginkgo Biloba Leaf (EGb761, Dr. Willmar Schwabe GmbH & Co., Karslruhe, Germany; No. 5550918). 2.2. Preparation of RR and Dosage Calculation RR was bought in Beijing Bencao Fangyuan Pharmacy Co., Ltd. (Beijing, China), and identified as Rheum tanguticum Maxim. ex Balf. by Xiangri Li, professor of TCM processing at Beijing University of Chinese Medicine. The voucher sample (accession No. 201009) was located at the Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine. To prepare herbal extracts, 1000 g of RR was crushed into a powder using a pulverizer (Zhongcheng Pharmaceutical Machinery, Changsha, China). The herbal powders were decocted 3 times for 20–30 min each decoction, using 10 volumes (w/v) of distilled H[2]O. Three decoctions were combined and filtered, and the RR dry powder was obtained by concentrating at reduced pressure and freeze-drying. The extract rate of RR was 30%. The equivalent dose for rats was calculated using the conversion ratio between rats and humans based on body surface area (BSA) [[80]25], and the high dose of RR (RR-H) used in rats was calculated using the following formula based on the clinical dosage (see the details in [81]Supplementary Materials): Dosage of RR-H = 15 g/person/day × 6.2/70 kg The medium dose of RR (RR-M) is half the dosage of RR-H, while the low dose of RR (RR-L) is a quarter of the dosage of RR-H. The RR powder was dissolved in double-distilled water (ddH[2]O) to appointed concentrations (10, 20, and 40 mg/mL). Thus, the test doses of RR in rats were administered to RR-L (100 mg/kg/d), RR-M (200 mg/kg/d), and RR-H (400 mg/kg/d, the clinical maximum dose) groups. 2.3. Chemical Characterization of RR Extract The chemical components of the RR extract were analyzed using ultra-high-performance liquid chromatography (UHPLC) combined with Q-Exactive Orbitrap mass spectrometry (MS). RR dry powder (1 g) was dissolved with 50 mL of methanol. The chrysophanol, emodin, (+)-catechin, rhein, physcion, aloe-emodin, sennoside A, and emodin-8-glucoside (purity > 97%) were configured with methanol in a mixed standard solution at a concentration of 125 μg/mL. The filtrates of the RR extract and standard solution were placed in a liquid-phase vial after passing through a microporous membrane of 0.22 μm. UHPLC analysis was carried out using a Dionex Ultimate 3000 System (Thermo Scientific, Santa Clara, CA, USA). The separation of the test solution and standards was carried out on an AQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) at 35 °C. The injection volume and flow rate were 5 μL and 0.3 mL/min, respectively. The mobile phases were made up of acetonitrile (A) and 0.1% formic acid/water (v/v) (B). The gradient elution was performed using the following conditions: 0–50 min (5–90% A), 50–50.1 min (90–5% A), and 50.1–55 min (5–5% A). The Q Exactive Plus Orbitrap MS (Thermo Scientific, Santa Clara, CA, USA) was used to analyze the Electrospray ionization (ESI)-MS. The RR sample was analyzed in both ionization modes. The MS analysis was conducted on the heated electrospray ionization source (HESI) with temperatures of 400 °C and 320 °C for the ion source and the capillary, respectively. The voltages of the ionization source and tube lens were 3.5 KV (+) and 3 KV (−), and 55 V, respectively. Nitrogen (purity > 99.99%) was used as a sheath gas and auxiliary gas at 35 arb and 10 arb flow rates, respectively. The full scan was performed using Fourier transform (FT, resolution 70,000, mass scan range of m/z 120.00–1800.00), and the data-dependent acquisition (ddMS^2) and the higher-energy collision dissociation (HCD) were used for the acquisition of MS spectra. The raw MS data were processed using the software Xcalibur 2.2.0, and the composition was confirmed based on the retention time, high-resolution accurate mass, and MS^n multi-stage fragmentation, combined with the data of the standard, ChemSpider database, and literature reports. 2.4. Animals and Experimental Design 2.4.1. Experimental Animals and Ethics Statement A total of 144 SPF Sprague Dawley (SD) rats (male, body weight 230–260 g) were obtained from SPF Biotechnology Co., Ltd. (Beijing, China, certificate No. SCXK (Beijing) 2019-0010). All animals were administered in a barrier environment animal house for free access to food and water with a 12 h light/dark cycle at 20–25 °C room temperature and 40–70% humidity. Our experimental schemes and all surgical and experimental procedures were endorsed and approved by the Animal Ethics Committee of the Beijing University of Traditional Chinese Medicine (No. BUCM-4-2022111001-4036). All surgical and experimental procedures were designed to avoid or minimize discomfort, distress, and pain to the animals. After 3 days of acclimatization, 144 rats were randomized into the Sham, Model, RR-L, RR-M, RR-H, and EGb761 groups, with each group containing 24 rats. The MCAO models were performed in all the other groups, while anesthesia and vascular dissection were performed in the Sham group. About 30 min after the drug administration, the rats were anesthetized by intraperitoneal injection of 20% urethane (1000 mg/kg), and fixed supine on the operating table, and the surgery was performed with the suture-occluded method, as previously described [[82]29,[83]30,[84]31]. After surgery, all the rats were laid on a heating cushion at 37 °C until they recovered from anesthesia. Medical intervention was conducted in the RR-L, RR-M, RR-H, and EGb761 groups by gavage with RR extracts (100, 200, and 400 mg/kg), and in EGb761 (40 mg/kg), sequentially at 0.5 h before the MCAO surgery, while equal volumes of drinking water were given to the rats in the Sham and Model groups. 2.4.2. Neurobehavioral Assessment About 24 h after MCAO, the neuroethology of all animals was assessed by a blinded investigator using a five-level scale (0–4 points) [[85]31], by which neural function and injury degree could be evaluated. The scoring details are as follows: the behavior was completely normal and there was no damage to neural function, which was recorded as 0 points; when the rat was lifted off the ground and the contralateral forelimb was internally rotated, the rat had slight nerve damage, which was marked as 1 point; observing the walking of the rats, it can be seen that the rats circle to the contralateral side, indicating the rats had moderate nerve injury, which was recorded as 2 points; observing the walking of the rats, it can be seen that the rats fall to the contralateral side, indicating the rats have severe nerve function injury, which was marked as 3 points; the rats could not walk and had no consciousness of spontaneous movement, indicating the rats had severe neurological damage, which was scored as 4 points. 2.4.3. Cerebral Edema Rate After neurological assessment, the rats were anesthetized with 5% isoflurane and then underwent CO[2] euthanasia, and their brains were quickly removed. The brain tissue was divided into the right and left hemispheres, which were weighed separately. Cerebral edema was assessed by measuring the cerebral edema rate, and the calculation formula is as follows: Cerebral edema rate (%) = (Weight[ischemic hemisphere] − Weight[non-ischemic hemisphere])/(Weight[ischemic hemisphere] + Weight[non-ischemic hemisphere]) × 100%. 2.4.4. Evaluation of Cerebral Infarction Volume by TTC At 24 h after the MCAO surgery, the rats were anesthetized with 5% isoflurane and then underwent CO[2] euthanasia, and their brains were quickly removed. The brain tissue was cut into six consecutive coronal sections with a 2 mm thickness after excluding the olfactory bulb, cerebellum, and lower brain stem. The brain sections were soaked and incubated in a 1% TTC solution at 37 °C for 30 min under the condition of protection from light. After the sections were stained and fixed in 10% neutral formalin fixative for 24 h, the fluid on the surface of the tissues was drained, and the tissues were placed in order and photographed. The cerebral infarct volume was calculated by the Image J software (version 1.54g) and the percentage of cerebral infarct volume = (cerebral infarct volume/whole brain volume) × 100%. 2.4.5. Regional Cerebral Blood Flow (rCBF) Measurements The rCBF disorder is a golden index for the onset and progression of IS [[86]32]. The Perimed Laser Speckle Imager (Pericam PSI HR; Sweden) was used to measure rCBF at 24 h after MCAO based on Laser Speckle Contrast Analysis technology. The operating distance of the instrument was set at 13.5 cm, the sampling frequency was 21 images/s, and the PIMSoft software (version 1.11.1) was used for monitoring blood flow and processing data. The area below the coronal sutures and within the linear temporalis side was classified as the area of interest, and the following formula was used to calculate the reduced rate of rCBF: The reduced rate of rCBF (%) = (ROI 2 − ROI 1)/ROI 2 × 100 (%) where ROI 1 is the monitoring value of rCBF in selected areas on the ischemic side, and ROI 2 is the monitoring value of rCBF in selected areas on the non-ischemic side. 2.4.6. Histopathological Staining The brain tissue was removed and immediately fixed in 4% paraformaldehyde for more than 24 h after the rats were perfused with the saline and 4% paraformaldehyde at 24 h after MCAO, and the tissue was dehydrated, paraffin-embedded, and sliced into 3 μm thick sections using a tissue slicer (Shanghai Leica Instrument Co., Ltd. RM2016, Shanghai, China). After the sections were dewaxed to water using xylene and gradient ethanol, they were sequentially subjected to hematoxylin staining for 8 min, color separated in 1% hydrochloric acid of alcohol for 30 s, reversed to blue using ammonia for 5 min, and stained with eosin for 2 min. They were then dehydrated with gradient ethanol, made transparent in xylene, and capped with neutral gum. 2.5. Gut Microbiota Determination and Data Analysis The feces of the colon segment of the rats were collected 24 h after MCAO to analyze the intestinal microbial composition. After the total genomic DNA was extracted and quality checked, it was amplified by PCR with specific primers (designed according to the specified sequencing region (V3–V4 variable region of 16S rDNA)), and the amplified products were quantified using a fluorescent quantitative PCR system. The sequencing library was constructed using the kit from Illumina, and sequencing was carried out with the platform provided by Illumina. PE reads from Illumina sequencing were spliced, controlled, and filtered. Species diversity analysis was performed by sample-based OUT clustering analysis. The 16S rRNA database was analyzed by the RDP classifier Bayesian algorithm, and the bioinformatics analysis was performed via the QIIME platform. Further detailed information is available in [87]Supplementary Materials. 2.6. H-NMR Spectroscopy and NMR Data Analysis The serum of 200 μL obtained from rats 24 h after MCAO was added to the deuterated phosphate buffer of 400 μL, vortexed, mixed, and centrifuged (4 °C, 12,000 r/min) for 20 min, and the supernatant was transferred to an NMR tube of 5 mm for future analysis. A 600 MHZ NMR spectrometer was employed to record the ^1H-NMR spectra at 599.672 MHz and 298 K. The relevant measurement parameters and data processing before principal component analysis (PCA) and orthogonal partial least squares discriminant analysis were carried out by referring to the literature (OPLS-DA) [[88]33]. The chemical shift of the NMR spectra was corrected by the lactate and was referenced at δ 1.33. The online Human Metabolome Database (HMDB) and relevant published references were