Boswellic acid and myrrh sesquiterpene alleviate hypoxic-ischemic brain injury via regulating TLR4/NF-κB and Ang (cid:0) /Ang (cid:0) /Tie signaling pathway on MCAO rats

Background: The frankincense and myrrh can be used in traditional Chinese medicine (TCM) clinic with the treatment of stroke, while its mechanism is still unclear. The aim of this study was to investigate the effects of boswellic acid (BA) and myrrh sesquiterpene (MS) compositions on ischemic stroke in rats. Methods: The ischemic stroke rat model induced by ischemia-reperfusion (I/R) after transient middle cerebral artery occlusion (MCAO) was adopted and treated with boswellic acid and myrrh sesquiterpenes with proportions compatibility (BA-MS) of 10:1, 5:1 and 20:1. Biochemical indexes, histopathological analysis, metabolomics analysis of plasma and urine, as well as 16S rDNA sequencing of gut microbiota and short chain fatty acids were investigated. Results: BA and MS could obviously signicantly ameliorated neurological decits scores, reduced cerebral infarction area in I/R, and improve the damage of brains and lungs of MCAO rats. Additionally, it could signicantly reduce the expression of neuroinammatory factors (IL-1β, nNOS, BDNF, NGF) and angiogenic growth factors (ET-1, PAI-1, PDGF, VEGF, VWF), and signicantly increase the expression of nerve indicators (TDP-43, NeuN) and angiogenic growth factors (Ang (cid:0) , TGF-β1, bFGF). After the administration of BA-MS, the metabolites of ischemic stroke in plasma and urine of ischemic stroke of rats were reversed, mainly correlated with the metabolic pathway of linoleic acid metabolism (impact value =1), and the gut microbiota and SCFAs metabolism of cecal contents were also improved. Conclusions: BA-MS can improve brain injury of MCAO rats via Ang/Tie and TLR4 signaling pathway, improve metabolism disorder by regulating linoleic acid metabolism pathway, and ameliorate gut microbes and their metabolic disorders. The internal close the middle cerebral artery (MCA). The wire from the external carotid artery to the internal carotid artery lumen 17 20 mm until resistance was ensure occlusion at the origin of the MCA. The line was allowed stay there 120 minutes before gently retracting. The rats of sham group were performed by the method of MCAO without inserting the line. All the animals were scored 1–5 for neurological function 3 h after by people who did not know the experimental group, until specimens were taken. 1–4 points can be classied as successful modeling. Scoring criteria see Supplementary.


Background
In 2017, stroke became the leading cause of death in China [1]. It is clinically divided into ischemic stroke and hemorrhagic stroke, among which, ischemic stroke accounts approximately 80%. Ischemic stroke is devastating and caused by thromboembolic occlusion of the cerebral aorta or its branches [2]. The victim may suddenly experience paralysis, speech problems or vision loss [3]. The risk factors of ischemic stroke are various. Some are unchangeable, while some can be adjusted to reduce the risk, like hypertension [4], diabetes [5], dyslipidemia [6], etc. Cerebral ischemia reperfusion (I/R) injury is mediated by multiplex mechanisms. Vascular obstruction not only leads to the loss of oxygen and energy, which followed by the formation of reactive oxygen species, the release of glutamic acid, the accumulation of intracellular calcium, but also induces neuroin ammation [7]. The series of reactions eventually leads to brain injury or even brain death, and infections may occur, especially in the lungs [8]. Therefore, inhibiting the development of neuroin ammation [9] and stimulating angiogenesis [10,11] are the key to recovering the brain's focal I/R response.
Currently, thrombolytic therapy is a powerful clinical treatment for ischemic stroke [12]. The standard method is to use of an enzyme tissue plasminogen activator (t-PA), which lyses plasminogen and converts it into active thrombolytic plasminogen [13]. However, using t-PA has limitations, including complications of intracranial hemorrhage. Traditional Chinese medicine has been used in the clinical treatment of cerebrovascular diseases for a long time known as little side effects and multiple targets [14].
Frankincense is a gelatinous resin exuded from Boswellia carterii Birdw. or Boswellia bhaw-dajiana Birdw. Myrrh is an oily gelatinous substance exuded from the bark of Commiphora myrrh Engl. or Commiphora molmol Engl. Frankincense and myrrh (FM) are typical Chinese medicine pair, which have the effects of activating blood and removing stasis, astringent wound, and generating muscle. Boswellic acids (BA) and myrrh sesquiterpenes (MS) are respectively the main bioactive compound in frankincens and myrrh.
Previous studies found that the most common ratios of FM were 1:1, 1:2, and 2:1, and then converted them to the ratio of 10:1, 5:1, 20:1 of BA-MS according to their respective extraction rates [15]. In addition, modern studies have shown that adding frankincense when treating of stroke patients can effectively improve muscle weakness and non-dominant muscle strength in patients with acute neurological dysfunction [16]. The oil extracted by mixing white pepper, long pepper, myrrh, cinnamon and saffron in a conventional ratio of 1:2:2:2:2 (w/w) protected PC12 cells from oxygen glucose deprivation [17]. So BA-MS has the potential to treat ischemic stroke, while the unclear mechanism limits its clinical application.
In this study, we examined the effects of different proportions of BA-MS on I/R in MCAO rats through pathological sections, biochemical indicators, metabolomics analysis, intestinal ora diversity analysis, short chain fatty acids (SCFAs) analysis, and comprehensive correlation analysis between biochemical indicators and metabolites, intestinal ora and SCFAs to understand the potential mechanism of oral BA-MS in the treatment of ischemic stroke.

Animal experiment
Animal protocol was approved by the Institutional Animal Ethics Committee of Nanjing University of Chinese Medicine. SD rats were fed conventional feeding and water freely. Temperature and humidity were maintained at 23 ± 2 °C and 60 ± 2%, respectively, and the light cycle was 12 h.
After adaptive feeding period, the fasted (12 h) rats were anesthetized with 10% chloral hydrate via intraperitoneally injection (400 mg/kg) and placed on warm pads to maintain the body temperature at 37 ± 1 °C throughout the whole procedure. The left common carotid artery, external carotid artery and internal carotid artery were exposed and then a line with rounded ends was used to close the middle cerebral artery (MCA). The wire passed from the external carotid artery to the internal carotid artery lumen for 17 to 20 mm until resistance was felt to ensure occlusion at the origin of the MCA. The line was allowed to stay there for 120 minutes before gently retracting. The rats of sham group were performed by the method of MCAO without inserting the line. All the animals were scored 1-5 for neurological function 3 h after surgery by people who did not know the experimental group, until specimens were taken. 1-4 points can be classi ed as successful modeling. Scoring criteria see Supplementary.
Successful modeling rats were randomly divided into nine groups: sham group (n = 7), model group (M, n = 12), Nimodipine group (YXY, 21.6 mg/kg/d, n = 9), the ratio of BA and MS was 10:1 with high dose and low dose groups  11.88 mg/kg/d, n = 9). Then twice per day, 10 mL/kg was given by gavage each time. All rats were administrated daily at scheduled time for 3 consecutive days. For sham and model groups rats were given physiological saline undergoing the same procedure.
At the end of treatment, 12-hour urine was collected with metabolic cage and all rats were anaesthetized (10% chloral hydrate) after overnight-fasted. Plasma was collected and centrifuged (3000 r/min, 10 min).
The left ventricle was perfused with saline until the e uent from the right ventricular ear became colorless and the liver color became white. The brains, lungs, thymus and spleens were quickly taken, weighed, and viscera index was calculated (viscera index = viscera weight (g)/rat weight (g) × 100%). A portion of the brains and lungs were placed in formalin and the rest in liquid nitrogen and then stored at -80 °C for further study. The collected cecum contents were stored at -80 °C for 16 s rDNA sequencing and short-chain fatty acids analysis.

Longa neurological scores and weight changes
Longa neurological scores were performed at 24 h, 48 h, and 72 h after I/R. Daily and 24 h weight after surgery was recorded, change in body weight was calculated, and neurological function was scored 3 h after surgery by people who did not know the experimental grouping during until the whole experiment.
Change in body weight (%) = (body weight at each time point -body weight before surgery)/ body weight before surgery × 100%.
2.5 Measure infarct size and general histology 2.5.1 TTC staining Fresh brain was removed and then cut into 2 mm coronal sections and immediately incubated with 2% 2,3, 5-triphenyl-tetrazolium chloride (TTC, Solarbio, lot. G3005) at 37 °C for 15 minutes. The tissue was gently shaken at intervals of 5-8 min to evenly contact the staining solution, then the reaction was terminated by adding a stationary solution (Solarbio, lot. p1110). Image J software was used to quantitatively analyze the infarct area.

HE staining
Brains and lungs tissue were removed and dehydrated with 4% paraformaldehyde solution xed with tissue, embedded in para n and cut into 4 µm. And observed under 200 × by optical microscope after the sections were stained with hematoxylin and eosin (H&E).

Metabolomics study on plasma and urine
The plasma and urine were thawed at room temperature before preparation, and were extracted with three times of acetonitrile to precipitate protein, respectively. Then, the mixture was swirled for 30 seconds and centrifuged at 13,000 rpm for 15 minutes at 4 °C. The samples was redissolved using 50% acetonitrile (2:1) after centrifugally concentrated, and then 2 µL was injected into UPLC-MS for metabolic analysis.
Quality control sample, liquid phase conditions and mass spectrometry conditions see Supplementary. 2.10 Intestinal microecology analysis 2.10.1 Gut ora diversity analysis Extract total DNA from cecum contents samples according to E.Z.N.A. soil kit instructions. The concentration and purity of DNA were determined by NanoDrop2000, and the quality of DNA was determined by 1% agarose gel electrophoresis. The variable region of V3-V4 was ampli ed by 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') primers. PCR products were recovered by 2% agarose gel, puri ed by AxyPrep DNA gel extraction kit (Axygen, USA), eluded by Tris-HCl, and detected by 2% agarose gel electrophoresis. QuantiFluor™ -ST (Promega, USA) was used for quantitative testing. PE*2 × 300 library was constructed according to Illumina MiSeq platform (Illumina, USA) standard operating procedure. The original sequence was controlled by Trimmomatic software and assembled by FLASH software. According to the similarity of 97%, will not repeat sequence (not including single sequence) according to operating classi cation unit (OTU) clustering, get OTU the representation of the sequence using UPARSE (vsesion 7.0 http://drive5.com/uparse/) software. The abundance and diversity of the single-sample microbial community were re ected by Alpha dilution curve analysis. In addition, at the phylum level to genus level, linear discriminant analysis (LDA) was conducted and Linear discrimination analysis effect size (LEfSe) software was used to identify the species with signi cant differences on sham group and model group. Finally, the function of the 16S amplicon was predicted by PICRUSt.

Metabolites of intestinal ora analysis
Sample treatment: 100 mg of cecum contents thawed on ice were added to 4 × methanol homogenate, centrifuged at 13 000 rpm for 10 min. Supernatant redissolved and transferred to a liquid bottle after centrifuged and concentrated, and 2 µL of sample was injected for GC-FID analysis.

Statistical analysis
All data were statistically analyzed by Graphpad 7, The results were expressed as mean ± SD. The oneway ANOVA was used for comparison between groups. A P value < 0.05 was regarded as signi cant.

Effects of nerve function, body weight and visceral indexes
There was a signi cant decrease in body weight between M group and the sham group at 24 h, 48 h and 72 h after I/R after operation. At each time point, it showed no signi cant difference, but a decreasing trend can be seen (Fig. 1a). The neurological function score showed there were different degrees of improvement after 72 h of administration (Fig. 1b). Viscera index (Fig. 1c) showed that brain, lung and spleen indexes increased signi cantly in M group compared with the sham group, while thymus index decreased signi cantly.
3.2 Effects of BA-MS on cerebral infarct area and general histology As shown in Fig. 2a, viable tissue was deep red by TTC staining, whereas area of infarction in the injured side cerebral hemisphere was white. The brain infarction area in M group was signi cantly increased in comparison to the sham group (P < 0.001). Compared with M group, 20:1 group showed the smallest infarction area (P < 0.001), followed by 5:1 group (P < 0.01) and H10:1 group (P < 0.01). In addition, the brains of the rats in M group exhibited an edematous morphology with neuron loss and pyknotic nuclei in H&E staining, and this condition was effectively ameliorated in each treatment group (Fig. 2b). The lungs of the rats in M group appeared pulmonary bullae with alveolar structure damaged. Alveolar capillary wall thickened and in ltrated the alveolar space and peribronchi in H&E staining, which was consistent with the development of pulmonary infection in rats after stroke [18,19], and it was effectively ameliorated in treatment groups (Fig. 2c).

BA-MS improved biochemical indexes
The results of biochemical indexes were shown in Fig. 2d. M group showed signi cant increase in IL-1β, nNOS, BDNF, NGF, ET-1, PAI-1, PDGF-BB and VEGF-A levels, as well as a signi cant decrease in Ang level in comparison with sham group. After the administration, the above indicators all tended to be normal. H5:1 group and H20:1 group were generally better than other administration groups.
As shown in Fig. 3a  Western blotting results (Fig. 3c) showed that the expression of NF-κB, p-NF-κB and TLR4 protein in cerebral I/R region after MCAO were signi cantly increased, and Ang , Ang and Tie2 protein were signi cantly decreased compared with the sham group (P < 0.05). In general, the H20:1, H10:1 and H5:1 had signi cant effects on the expression of NF-κB, p-NF-κB, TLR4, Ang , Ang and Tie2, and H5:1 showed the best regulatory effect.

Metabolism analysis
The OPLS-DA score plots ( Figure S1a) showed that sham and M group could be clearly separated, indicating that metabolic disorders occurred in the model group. PCA score ( Figure S1b) showed that the plasma and urine samples of sham and M group had signi cant clustering. The administration groups deviated from M group and was closer to the sham operation group, indicating the drug administration groups could improve the metabolic disorder of I/R in MCAO rats. In the S-plot plot ( Figure S1c), metabolites that deviate from the main ion cluster and VIP ≥ 1 was extracted as differential metabolites. Then, 21 potential biomarkers (Table S1, among them 9 from plasma and 12 from urine.) and 16 potential metabolism pathways (impact ≥ 0.1) were identi ed by HMDB (www.hmdb.ca) and Metabo Analyst (http://www.metaboanalyst.ca/) database. As shown in Figure S1d, the occurrence of I/R in MCAO rats may be associated with retinol metabolism, pentose and glucuronate interconversions, linoleic acid metabolism, glycerophospholipid metabolism, biotin metabolism, ascorbate and aldarate metabolism, phenylalanine metabolism, terpenoid backbone biosynthesis, pantothenate and CoA biosynthesis, ether lipid metabolism, sphingolipid metabolism, pyruvate metabolism, galactose metabolism, cysteine and methionine metabolism, arachidonic acid metabolism and steroid hormone biosynthesis. Among them, linoleic acid metabolism had an impact-value of 1, which suggested the administration of BA-MS could modify this metabolic pathways and be related biomarkers to exert the curative effect for I/R after MCAO rats. The results also showed BA-MS could improve metabolic disorders in I/R after MCAO by regulating metabolites. Other details see Figure S2.

Gut microbiota analysis
In order to investigate whether the therapeutic effect of the bioactive parts of BA-MS in different proportions on I/R after MCAO was related to intestinal ora, we analyzed the cecum contents ora of rats after 72 h of treatment and the species diversity of the sample at OTU level. Figure S3 showed the sample length distribution was in the range of 400 to 440 bp. Sobs index was to measure colony richness, Heip index was to measure colony evenness, and Shannon index was to evaluate species diversity of the samples at OUT level, as shown in Figure S4a. The results showed that there were high richness, diversity and uniformity of the micro ora in each group, and there was no signi cant difference in the diversity index. Dilution curve ( Figure S4b), showed that the current sequencing depth was enough to re ect the diversity contained in the current sample, and a large number of undiscovered new OTU cannot be detected by increasing the sequencing depth. In addition, the number of each sample at the level of phylum and genus was counted. At the phylum level (Fig. 4a), rmicutes, proteobacteria, bacteroidetes and verruciformis were found after the combination of species with abundance less than 0.1%, and Firmicutes accounted for the largest proportion. At the genus level (Fig. 4a), escherichiashigella, allobaculum, norank_f_muribaculaceae, akkermansia, lactobacillus, prevotella_9 were found after the combination of species with abundance less than 0.1%. According to the LEfSe multi-species hierarchy tree diagram (Fig. 4b), compared with the normal group, the intestinal ora of the model group was different in phylum, class, order, family and genus. The Linear discriminant analysis (LDA) discriminant bar graph (Fig. 4b) was made from phylum level to genus level. By comparing the sham group with M group and Welch's t test, signi cant differences were found in 3 phylums and 27 genus. It was found that H5:1 signi cantly increased the relative abundance of actinobacteria and tenericutes at the phylum level (Fig. 4c). The administration groups could regulate 10 species of bacteria at the genus level, among which H5:1 had the best effect, followed by H10:1, H20:1, L10:1 and YXY. Further studies showed that the intestinal ora of I/R in MCAO rats were involved in 24 related functions ( Figure S5) and metabolic pathways of the top 20 abundance values (Table S2). Among them, amino acid transport and metabolism, cell wall/membrane/envelope biogenesis, replication, recombination and repair, ribosomal structure and biogenesis, energy production and conversion have high functional abundance, and carbohydrate metabolism, amino acid metabolism, replication and repair, energy metabolism, nucleotide metabolism and lipid metabolism pathways disorder contribute directly or indirectly to the development of ischemia stroke.

Short-chain fatty acids analysis
In order to investigate the effects of different proportions of BA-MS bioactive ingredients on intestinal ora metabolites in I/R rats, we analyzed SCFAs in cecum contents of rats after 72 h of I/R. The chromatogram of the mixed reference substances, sham and M groups was shown in Figure S6, and the precision, stability, repeatability, average recovery and linear relation of six SCFAs components were shown in Table S3. It can be seen in Fig. 4d, compared with the sham group, the contents of acetic acid and propionic acid in the caecum of M group were signi cantly decreased, and the contents of each administration group were increased to different degrees. H5:1 had a signi cant effect on the contents of acetic acid and propionic acid in the contents of the I/R rats' cecum after MCAO, suggesting that H5:1 could improve the effect of ischemic stroke by increasing the content of SCFAs in the contents of the cecum of MCAO rats.

Discussion
Currently, a variety of animal stroke models have been developed to identify the mechanisms of cerebral ischemia and developing new treatments for stroke [2]. Middle cerebral artery (MCA) and its branches are the most common cerebral blood vessels in human ischemic stroke, thus, the technique to block the artery is closest to a human ischemic stroke [2]. In occlusive MCA stroke models, intra-arterial suture blockade of MCA (MCAO) is the most common method in rodents [20]. Therefore, in this study, the MCAO rat model of I/R was established to evaluate the therapeutic effect of BA-MS with different compatibility ratios on ischemic stroke, and to clarify the mechanism.
When cerebral ischemia occurs, a decrease in blood ow due to arterial occlusion will lead to hypoxia, which quickly induce neurological de cits or death. And it not only changes the ultrastructure and function of the brain [10], but also affects the function of the lung [21]. Post stroke pulmonary infection was found to be the result of bacterial ora displacement due to the destruction of the intestinal barrier after stroke [22]. In addition, after ischemic stroke, microglia and astrocytes are activated within a few hours, producing cytokines and chemokines [23] and causing the in ltration of white blood cells [24], suggesting that secondary neuroin ammation further promotes brain damage [25]. In our study, the administration groups were shown to improve neurological function, cerebral infarction area, and brain and lung morphology; as well as modulate anti-in ammatory factors (IL-1β, nNOS), neurotrophic factors (BDNF, NGF), and angiogenic growth factors (ET-1, VEGF, PDGF, Ang , PAI-1).
The sequelaes of ischemic stroke include language barrier, limb muscle atrophy and so on. Recent studies suggest that TDP-43 may be pathological marker proteins for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) [26,27] and frontotemporal lobar degeneration (FTLD) [28]. In the pathological case, TDP-43 accumulates from the nucleus to the cytoplasm, causing a decrease of TDP-43 in the nucleus [29]. This explains the signi cant reduction of TDP-43 in M group compared to the sham group, and the signi cant callback in the administration groups (L5:1, YXY). NeuN is a soluble nuclear protein, a marker for mature neurons [30]. Our results showed that NeuN positive expression was signi cantly decreased in M group, and NeuN positive cell expression was signi cantly increased in administration groups (20:1, H10:1, YXY) compared with the sham group. This suggests that the administration groups improved ischemic stroke by inducing NeuN expression. TGF-β1, VWF, and bFGF are important regulators of angiogenesis. Our results also demonstrated that BA-MS can promote angiogenic growth by improving TGF-β1, VWF and bFGF. In addition, western blot results showed that the TLR4/NF-κB and Ang , Ang /Tie2 signaling pathways were activated after I/R. TLR4 is a member of the toll-like receptor family and can activate downstream NF-κB and participates in in ammatory responses [31]. Ang , Ang and their receptors Tie2 are related to a new angiogenesis signal pathway discovered in recent years, except for vascular endothelial growth factor (VEGF) [32]. Studies have shown that the combination of Ang and the receptor activates Tie2, and Tie2-activated endothelial cells form a complete vascular wall, thus promoting angiogenesis and maintaining the stability of vascular structure [33,34]. BA-MS may improve damaged brains by inhibiting in ammatory responses and promoting angiogenic growth.
Metabolomics is a systematic approach to the study of metabolic changes. By analyzing the composition and changes of endogenous metabolites in biological samples, the physiological and pathological state of the subjects was explained to re ect the overall metabolic law under the in uence of internal and external factors [35]. Metabolomics results showed that the impact value of linoleic acid metabolic pathway was 1, indicating that BA-MS interfered with the occurrence of ischemic stroke by regulating linoleic acid metabolism. Moreover, the correlation analysis of metabolic markers and biochemical indicators also showed that linoleic acid was strongly negatively correlated with IL-1β (r= -0.641) and VWF (r=-0.606). It also found that the content of linoleic acid, which is essentially a polyunsaturated fatty acid, decreased in the M group. Therefore, we speculated that the decrease of linoleic acid was closely related to the increase of IL-1β and VWF after stroke. Studies have found that substituting linoleic acid for saturated fats, glycemic carbohydrates, or monounsaturated fats may reduce the risk of ischemic stroke [36], possibly because linoleic acid lowers plasma low density lipoprotein cholesterol levels [37].
The balance of intestinal ora is critical to the normal development of the immune system [38]. Intestinal microorganisms interact with the host, providing energy, nutrition and immune protection to the host against disease, as well as producing toxins that affect the health of the host [39]. Many diseases associated with abnormal microbes have an "in ammatory" basis, from autoimmune diseases to atherosclerotic heart disease, high blood pressure and even stroke [40]. SCFAs are metabolites of intestinal microorganisms, mainly including acetic acid, propionic acid and butyric acid. And it may have important associations with diet, gut ora, and in ammatory responses [41]. The correlation between gut microbiota and SCFAs showed that isovaleric acid was positively correlated with norank_f_Lachnospiraceae, Clostridium_sensu_surico_1 (r ≥ 0.4); acetic acid was positively correlated with Lactobacillus (r ≥ 0.4), and acetic acid, propionic acid and butyric acid were negatively correlated with Parasutterella (r ≤ -0.4). This was consistent with callback the gut microbial in I/R rats after administration groups, suggesting that the decrease of those ora disrupts the balance of the intestinal environment, thus exacerbating the progression of ischemic stroke, which was effectively improved in the administration groups.
Hyperglycemia is one of the important causes of ischemic stroke [5], report found that the advancedstage diabetic rats have the characteristics of cognitive impairment, its relative abundance of gut microbes showed Lachnospiraceae_NK4A136_group increased, while the relative abundance of Clostridium_sensu_stricto_1 reduced [42]. Lachnospiraceae is a butyrate producing bacteria in rmicutes [43], and butyrate is an anti-in ammatory short-chain fatty acid salt that can provide nutrition for intestinal mucosa and enhance intestinal mucosal immunity [44]. Lactobacillus can reduce plasma endotoxin [45] and inhibit in ammatory response [46]. Parasutterella is related to irritable bowel syndrome and chronic intestinal in ammation [47]. Compared with the sham group, the relative abundance of Parasutterella increased signi cantly in M group, while Lactobacillus and norank_f_Lachnospiraceae decreased. In addition, there has been evidence that the imbalance of intestinal ora after stroke can aggravate the cerebral infarction area [48,49]. So we suspected that after brain injury, the increase in Parasatella and the decrease in Lactobacillus and Lachnospiraceae leads to intestinal in ammatory response, which in turn exacerbates brain injury. After administration, it was found that administration groups signi cantly increased the relative abundance of Lactobacillus and norank_f_Lachnospiraceae, and signi cantly reduced the relative abundance of Parasatella, suggesting that BA-MS may reduce the incidence of ischemic stroke by improving the in ammatory response of intestinal microorganisms through the "brain-gut axis".

Conclusion
In general, BA-MS can improve brain and lung injury of MCAO rats, which may alleviate nerve in ammation and promote angiogenesis growth by regulating TLR4/NF-κB signaling pathways and Ang /Ang /Tie signaling pathway, improve metabolism disorder by regulating linoleic acid metabolism pathway, and ameliorate gut microbes and their metabolic disorders. In terms of overall treatment effect, H5:1 has the best effect, followed by H10:1 and H20:1 (Fig. 6). The results of the study can provide bene cial inspiration for the treatment of ischemic stroke.

Declarations
Availability of supporting data The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
All the experiments involving animals were approved by the Institutional Animal Ethics Committee of Nanjing University of Chinese Medicine.