A total of one hundred male sprague-dawley (SD) rats (weighing 180–220 g) were purchased from Ji’nan Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, China). The production license number is SCXK (LU) 2022-0006. They were fed adaptively under the conditions of temperature (20°C–25°C), relative humidity 40%–50%, and alternating light (12 h light/dark cycle) with unrestricted free access to sufficient food and water. The animals were allowed to habituate to the housing facilities for at least 1 week before the experiment began. All animal experiments were approved by the Animal Ethics Committee of the 940th Hospital (No. 2024KYLL145) and performed according to the Guidelines for the Care and Use of Laboratory Animals.

8-OaS(purity ≥ 98%; batch number: AZCC2903) was purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China); highly active dry yeast was purchased from Angel yeast Co., Ltd. (Yichang, China). Aspirin enteric-coated tablets was purchased from Hunan Xinhui Pharmacy Co., Ltd. (Changsha, China); prostaglandin E₂ (PGE₂) rat assay kit, tumour necrosis factor-α (TNF-α) assay kit, NF-κB test Kit, interleukin-6 (IL-6) assay kit, interleukin-1β (IL-1β) assay kit, arginine vasopressin (AVP) assay kit, 5-HT test kit, macrophage inflammatory protein-1α (MIP-1α) assay kit, interferon-γ (IFN-γ) assay kit, corticotropin-releasing hormone (CRH/CRF) and substance P (SP) assay kit were purchased from Nanjing Jiancheng Bioengineering institute (Nanjing, China). Anti-TLR4 rabbit pAb, anti-phospho-NF-κB p65 (S536) rabbit pAb, anti- TNF-α rabbit pAb, and anti-COX2/Cyclooxygenase 2 rabbit pAb were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). HSP70 antibody was purchased from Affinity Biosciences (Liyang, China).

Three days prior to the formal experiment, one hundred SD male rats underwent adaptive measurements three times daily, as well as two additional measurements conducted before the establishment of the fever model, with a 1-h interval between each measurement. The rats were excluded if they exhibited a single temperature higher than 38°C or a temperature difference greater than 0.5°C between the two temperatures, and the average temperature of the two temperatures was taken as the basal body temperature of the rat.

The body temperatures of rats were measured using a calibrated medical thermometer. Prior to measurement, the temperature probe was prepared by establishing a physical limit marker at 3 cm from its tip to standardize insertion depth. A thin layer of medical-grade Vaseline was uniformly applied to the distal 3 cm of the probe to ensure adequate lubrication and prevent rectal tissue damage. During the procedure, each rat was humanely restrained using a dedicated rodent immobilization device. The lubricated probe was then gently inserted rectally until the pre-marked 3 cm depth was reached. The thermometer was activated, and temperature readings were automatically recorded upon completion of the device’s audible notification signal. Three consecutive measurements were obtained at 2-min intervals for each subject, with the probe thoroughly disinfected between animals using 70% ethanol.

Following the screening of body temperature, the seventy-two rats were randomly divided into six groups (n = 12): normal control group (NG, sterilized water for injection, 10 mL/kg), fever model group (MG, sterilized water for injection, 10 mL/kg); 8-OaS high dose group (8-OAS-H, 200 mg/kg); 8-OaS medium dose group (8-OAS-M, 150 mg/kg); 8-OaS low-dose group (8-OaS-L, 100 mg/kg); positive control group (PG, aspirin, 100 mg/kg). The dosage of 8-OaS is obtained based on preliminary experiments. Except for the NG, all other groups were injected subcutaneously with 20% dry yeast suspension to establish the rat fever model (Li et al., 2024; Sherif et al., 2024). After a subcutaneous injection of dry yeast for 5 h, the rats were screened out and subsequently administered corresponding drugs; the body temperature was measured continuously for 7 h following drug administration, with an interval of 1 h between each measurement. At the end of the temperature measurements, the maximum temperature rise △t at each time point was calculated (△t = body temperature at each time point - basal body temperature).

At the conclusion of temperature measurement, blood was collected from the abdominal aorta using a negative pressure tube. A volume of 5 mL was extracted from each rat and subsequently centrifuged at 2,200 g for 10 min. The supernatant was aspirated, aliquoted and stored in a refrigerator at −80°C. Additionally, the hypothalamic tissues and colonic contents were removed and stored at −80°C.

ELISA was conducted to assess the levels of IL-1β, IL-6, TNF-α, IFN-γ, PGE₂, MIP-1α, and other corresponding indicators in blood serum, according to the manufacturer’s protocol (Zhang et al., 2019). The sample (50 μL) or standard (50 μL) was added to the enzyme-labeled plates that have been pre-coated with antibodies, then biotin-labeled recognition antigen was added and incubated at 37°C for 30 min. After washing five times by PBST, 50 μL of HRP-conjugated avidin was added and incubated at 37°C for 30 min. After washing the plate, the plate was developed with 50 μL of TMB substrate for 15 min in the dark. The reaction was stopped with 2 M H₂SO₄ and optical density was measured at a wavelength of 450 nm using a spectrophotometer.

The RNA of hypothalamic tissue was extracted by the trizol method and reverse transcribed to cDNA using a reverse transcription kit. Primers were designed according to gene sequences (Ping et al., 2020), NF-κB: upstream GCT​GAT​GGA​GTA​CCC​TGA​AGC, downstream ATG​TCC​GCA​ATG​GAG​GAG​AAG; TNF-α: upstream: CCA​CGC​TCT​TCT​GTC​TAC​TG, downstream GCT​ACG​GGG​CTT​GTC​ACT​C; IL-6: upstream GTT​GCC​TTC​TTG​GGG​ACT​GAT​GT, downstream ATA​CTG​GGT​CTG​TTG​GGG​TGG​GT,; IL-1β: upstream GCT​TCA​AAT​CTC​ACA​GCA​GCA​T, downstream TAG​CAG​GTC​GTC​ATC​ATC​CCA​C; HSP70: Upstream CGA​GGA​GGT​GGA​TTA​GAG​GC, downstream GCT​GAG​GTG​TTC​GCA​GGA​A; TRPV4: Upstream AAG​TGG​CGT​AAG​TTC​GGG, downstream CGTGGTACGGTAAGGGT; GAPDH: Upstream ACA​GCA​ACA​GGG​TGG​TGG​AC, downstream TTT​GAG​GGT​GCA​GCG​AAC​TT. The amplification reaction of the GAPDH and the target gene was performed simultaneously for each batch of samples.

The total protein in the hypothalamic tissue was extracted utilizing a high-efficiency RIPA lysis buffer, which was supplemented with protease inhibitor and phosphatase inhibitor. The protein concentration was determined by the BCA protein assay kit. The protein samples were separated by 10% SDS-PAGE, run at 80 V for 40 min and then at 110 V for 80 min, before being transferred to a polyvinylidene fluoride (PVDF) membrane. After the transmembrane process, the PVDF membrane was removed and immersed in 1×TBST containing 5% skim milk powder. This mixture was sealed at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4°C. The primary antibodies used in this study were against TLR4 (1:1,000), TNF-α (1:1,000), p-NF-κB p65 (1:1,000), p-IκB-α (1:1,000), HSP70 (1:1,000), COX2 (1:1,000), and GAPDH (1:1,000). After primary antibody incubation, wash with 1×TBST at room temperature, allowing for 8 min of washing each time. Subsequently, the PVDF membrane was immersed into the secondary antibody solution (1:5,000) and incubated at room temperature for 2 h. ImageJ software was utilized for the purpose of quantitative analysis (Wang et al., 2024; Hou et al., 2024).

Hypothalamic tissues were paraffin sectioned and deparaffinized for hydration, antigen repair, and closure. Then it was incubated with 3% endogenous H₂O₂ for 10 min. Then, the tissue sections were incubated with primary antibodies against IL-1β (1:500) and IL-6 (1:500) overnight. On the following day, secondary antibody was added and reacted for another 10 min, and stained with diaminobenzidine for 1 min. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and sealed for observation using a microscope. The whole positive rate was analyzed using ImageJ software.

Microbial DNA was extracted utilizing the HiPure Soil DNA Extraction Kit (Magen, China) in accordance with the provided instructions. Subsequently, PCR sequencing was conducted. The amplification of the target region of the 16 S rDNA gene via PCR was performed under the following conditions: 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and finally 72°C for 7 min using the primers (sequences upstream: CCTACGGGGNGGCWGCAG; downstream: GGACTACHVGGGTATCTAAT). Amplification system: 50 μL mixture containing 10 µL 5×Q5@ Reaction Buffer, 10 µL 5×Q5@ High GC Enhancer, 1.5 µL 2.5 mM dNTPs, 1.5 µL upstream and downstream primers (10 µM), 0.2 µL Q5@ High-Fidelity DNA Polymerase and 50 ng of template DNA. Finally, Illumina sequencing was applied. Amplification product quality was assessed using a 2% agarose gel, PCR product purification was performed using AMPure XP Beads (Beckman, United States) and quantification was conducted using Qubit 3.0. Sequencing libraries were prepared using the Illumina DNA Prep Kit (Illumina, United States). Library quality assessment was performed utilizing the ABI StepOnePlus Real-Time PCR System (Life Technologies, United States). Qualified libraries were up-sequenced using PE250 mode pooling on Novaseq 6000.

After sequencing to obtain raw reads, the initial step involves filtering out low-quality reads, followed by their subsequent assembly. The double-ended reads are spliced into tags. The tags are subsequently filtered and the resulting dataset is referred to as a clean tag. Subsequently, clustering is performed based on a clean tag to eliminate the chimeric tags detected during the cluster comparison process. The final output obtained from this procedure is referred to the as effective tag. After obtaining OTUs, OTU abundance statistics were performed based on the effective tag. The following analyses were performed: species annotation, alpha diversity analysis, beta diversity analysis, species composition analysis, and differential species screening.

Appropriate samples of hypothalamic tissue and colon contents were carefully placed in 2 mL centrifuge tubes. A total of 500 μL of an 80% methanol-water solution (containing 0.1% formic acid) was added to the tube. The mixture was vortexed for 1 min and subsequently ground for 5 min; after centrifugation, the supernatant was collected for LC-MS/MS analysis.

The liquid chromatography system utilized was a Waters Acquity UPLC, coupled with a mass spectrometer from AB SCIEX 5500 QQQ-MS. The chromatographic columns employed in this study were Acquity UPLC BEH C18 (1.7 µm, 2.1 mm × 100 mm) and Acquity UPLC HSS T3 (1.8 µm, 2.1 mm × 100 mm). The chromatographic separation was conducted under conditions that included a column temperature of 40°C and a flow rate of 0.20 mL/min. The composition of the mobile phase comprised two components: component A, which was water containing 0.01% formic acid, and component B, which was acetonitrile (0.01% formic acid). The total run time was 10 min, with an injection volume of 6 µL. The gradient eluting conditions for samples were as follows: 0–3 min, 95%–55% A; 3–13 min, 55%–5% A; 13–14 min, 5% A; the UHPLC elution conditions for urine samples were as follows: 0–1 min, 99% A; 1–7 min, 99%–60% A; 7–8 min, 60% A; 8–9 min, 60%–99% A; 9–10 min, 99%A. The mass spectrometric conditions were as follows: ion source, ESI; curtain gas, 35 arb; collision gas, 9 arb; ion spray voltage, 4,500 V; ion source temperature, 550°C; ion source gases, 55 arb each for Ion Source Gas1 and IonSource Gas2. The parameters for the MRM acquisition were established based on the aforementioned chromatographic and mass spectrometric conditions. Subsequently, standard solution preparations were injected into the sample vials for analysis.

Preparation of standard solutions is outlined as follows: a 100 μg/mL standard solution was prepared using 80% methanol containing 0.1% formic acid. Subsequently, a series of standard solutions with appropriate concentrations (0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 ng/mL) was subsequently prepared through further dilution.

The standard curve was plotted according to the peak areas of the standards at various concentrations and their corresponding concentrations. The R ² was exceeded 0.99, which indicated that the standard curve had a superior linear relationship and could be used for the calculating the concentrations of the neurotransmitters in the samples. Finally, the integration was carried out using MultiQuant software and quantitative analyses were performed using standard curves.

Statistical analyses were performed using SPSS 25.0 and GraphPad Prism 8.0 software. All data was presented as mean ± standard deviation ( x ¯ ±s). Unless otherwise stated, statistical comparisons were performed using a single-factor analysis of variance (ANOVA) followed by the LSD test. The p-value < 0.05 indicated statistical significance. The pearson correlation coefficient was utilized in the correlation analysis between the intestinal flora and metabolites. Metabolomic profiling was performed in collaboration with Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China) including mass spectrometry analyses and/or bioinformatics analysis.

The variations in temperature observed within 7 h post-administration are illustrated in Figure 1a. Compared with the control group, the remaining five groups exhibited a significant increase in body temperature after 5 h of modeling (P < 0.01), indicating that the yeast-induced pyrexia model in rats was successfully established. Compared with the fever model group, various dosage groups of 8-OaS demonstrated an antipyretic effect.

Serum cytokines, thermoregulatory mediators, neurotransmitters were closely associated to the degree of fever (Figures 1b–l). ELISA was employed to assess the alterations in cytokine levels within serum. The concentrations of IL-1β, IL-6, TNF-α, MIP-1α, IFN-γ, PGE₂, CRH, SP, and NF-κB in the MG were found to be elevated. Conversely, the serum AVP, 5-HT of the MG was decreased. The above results indicated that the yeast-induced pyrexia model in rats was successfully established. After the administration of 8-OaS, the above indicators tended to recover to the normal levels.

The mRNA expression of the hypothalamus was detected by PCR. The findings are presented in Figures 2a–f. Compared with the NG, the mRNA expressions of TNF-α, IL-6, IL-1β, NF-κB, TRPV4, and HSP70 in the MG were significantly upregulated (P < 0.01), indicating that the expression of mRNA was enhanced under fever conditions. Compared with the MG, the mRNA expressions of TNF-α, IL-6, IL-1β, NF-κB, TRPV4, and HSP70 in the 8-OaS dose groups and the PG were significantly downregulated (P < 0.01).

Western blotting method was used to detect the expression of proteins in the rat hypothalamus. The results are shown in Figures 2g–l. Compared with the NG, the protein expressions of TLR4 (P < 0.05), TNF-α, p-NF-κB p65 (P < 0.05), HSP70 (P < 0.01), COX2 (P < 0.05) were upregulated and the protein expression of p-IκBα was downregulated in the MG. Compared with the MG, the protein expressions of TLR4 (P < 0.05), TNF-α (P < 0.05), p-NF-κB p65 (P < 0.05), HSP70 (P < 0.01), and COX2 (P < 0.05) were significantly decreased in the 8-OaS-L, while the protein expression of p-IκBα was increased. The protein expressions of TLR4 (P < 0.01), TNF-α (P < 0.01), p-NF-κB p65 (P < 0.05), HSP70 (P < 0.01), and COX2 (P < 0.05) in the 8-OaS-M were significantly elevated, but the protein expression of p-IκBα was reduced. The protein expressions of TLR4 (P < 0.01), TNF-α (P < 0.01), p-NF-κB p65 (P < 0.01), HSP70 (P < 0.01), and COX2 (P < 0.05) were significantly downregulated and the protein expression of p-IκBα was upregulated in the 8-OaS-H. In the PG, the protein expression of TLR4 (P < 0.01), TNF-α, p-NF-κB p65 (P < 0.05), COX2 (P < 0.05) declined and the protein expression of p-IκBα significantly augmented (P < 0.05).

Immunohistochemical staining of IL-1β and IL-6 expressions in the hypothalamus revealed a significant increase in both IL-1β and IL-6 significantly increased in the MG (Figures 2m, n). Meanwhile, a marked decreased in the expressions of IL-1β and IL-6 were observed in the 8-OaS dose groups.

The Veen diagram presented in Figure 3a illustrates that there were 999 OTUs in the NG, with 193 of these being unique. Additionally, there were 1,026 OTUs in the MG, with 198 of these being unique, and 910 OTUs in the 8-OaS-H, with 187 of these being unique. The taxonomic richness and homogeneity of a sample are visualized by rank abundance curves (Figure 3b). As the number of test samples increases, the curves gradually flatten, evenly distributed and concentrated. With sufficient sampling, it can be used for subsequent data analysis. The NMDS analysis revealed significant structural differences among the three groups (Figure 3c). As shown by the β diversity Adonis grouping test (Table 1) and the Anosim grouping test (Table 2), the composition of the rat intestinal flora in the MG differed significantly from that of the NG and 8-OaS-H group.

The top ten species from each group of rat intestinal flora objects, ranked according to their constitutive abundance at the taxonomic phylum, class, order, family, genus, species level, are shown in Figure 3d. After conducting fever modeling in rats and administering drug, the proportions of the top ten species exhibited distinct changes.

The analysis of indicators calculates the indicator value of every species for each subgroup, based on the abundance and frequency of occurrence of the species in the sample. A higher value indicates that the species is more likely to serve as an indicator species for that subgroup. At the phylum level (Figure 3e), Bacteroidota, Proteobacteria, and Cyanobacteria in the three comparison groups were screened as indicator species. Welch’s t-test found that compared with the NG, the relative abundance of Cyanobacteria in the MG was significantly higher (P < 0.05), while the relative abundance of Cyanobacteria in the 8-OaS group was significantly lower (P < 0.05). At the genus level (Figure 3e), Escherichia-Shigella, Prevotellaceae_NK3B31_group, Alistipes, Rodentibacter, Christensenellaceae_R-7_group, Enterococcus, Acinetobacter, Allobaculum, Fournierella, Clostridium_sensu_stricto_12, Odoribacter, Muribaculum, Enterorhabdus, Kosakonia, Clostridioides, Rothia were screened as indicator species. Welch’s t-test showed that compared with the NG, the relative abundances of Alistipes and Odoribacter in the MG were significantly increased (P < 0.01). Compared with the MG, the relative abundances of Alistipes (P < 0.01) and Odoribacter (P < 0.05) in the 8-OaS-H were significantly decreased. Bacteroides_thetaiotaomicron, Pasteurellaceae_bacterium, Bacteroides_sartorii, bacterium_YE57, Acinetobacter_sp_ ACNIH1, Clostridium_tyrobutyricum, Alistipes_finegoldii, Lachnospiraceae_bacterium_COE1 as indicator species screened at species level. Welch’s t-test showed that the relative abundance of Alistipes_finegoldii in the MG was significantly higher than that in the NG (P < 0.05). Compared with the MG, the relative abundance of Alistipes_finegoldii in the 8-OaS-H was significantly decreased (P < 0.05).

The diversity of intestinal flora was examined using the multi-level species hierarchy of LEfSe at genus level. The cladogram and LDA histogram (Figures 4a, b) indicated that 80 species exhibited differences in their relative abundances among the three groups, including 17, 41, and 22 species belonging to the NG, MG, and 8-OaS-H, respectively. The relative abundances of Clostridia_UCG_014, Eubacterium_coprostanoligenes_group, UCG_005, Acidaminococcaceae, Negativicutes, Acidaminococcales, Phascolarctobacterium, Prevotella and Allobaculum were abundant in the NG, while those of Bacteroidota, Bacteroidia, Bacteroidales, Muribaculaceae, Prevotellaceae, Prevotellaceae_NK3B31_group, Rikenellaceae, Alistipes, Erysipelatoclostridiaceae, Erysipelatoclostridium, NK4A214_group, Christensenellaceae, Christensenellales, Christensenellaceae_R_7_group, Mitochondria, Rickettsiales, RF39, Marinifilaceae, Clostridia_vadinBB60_group, and Butyricimonas were abundant in the MG. Moreover, the relative abundances of Lactobacillales, Pasteurellaceae, Pasteurellales, Rodentibacter, Enterococcus, Enterococcaceae, Clostridium_sensu_stricto_12 and Nesterenkonia were enriched in the 8-OaS-H. To explore the potential effects of 8-OaS intervention on metabolic pathways of intestinal microflora in rats induced with yeast, we conducted PICRUSt 2 analysis based on the KEGG database (Figure 4c). In contrast to rats of the MG, multiple microbial metabolic pathways were significantly changed in 8-OaS-fed rats (8-OaS-H), which were mainly characterized by the regulation of carbohydrate metabolism, metabolism of cofactors and vitamins, amino acid metabolism, metabolism of terpenoids and polyketides, metabolism of other amino acids and so on.

Neurotransmitter metabolites and its variation trends in hypothalamic tissues were showed in Figure 5; Table 3. In hypothalamic tissue, neurotransmitters of the phenylalanine metabolic pathway are impacted. Compared with NG, the contents of phenylalanine and DL-adrenaline were found to be reduced in MG; however, these differences did not reach statistical significance. The contents of tyramine, dopamine and homovanillic acid were significantly decreased (P < 0.01). The levels of tyrosine and NE were significantly increased (P < 0.01), and the levels of DL-metanephrine were increased but the differences were not statistically significant. Compared with MG, the contents of phenylalanine, tyrosine, tyramine, DL-adrenaline and NE in 8-OaS-H were significantly decreased (P < 0.01), the contents of dopamine and homovanillic acid were significantly increased (P < 0.01), and the content of DL-metanephrine was increased but the difference was not statistically significant. In the neurotransmitters of L-tryptophan metabolic pathway, compared with the NG, the levels of L-tryptophan, KYN and 5-HT were significantly increased in the MG (P < 0.01). In contrast, while the level of 5-HTP was increased, the differences observed were not statistically significant. The contents of 5-HIAA (P < 0.01) and tryptamine were decreased. Compared with MG, the contents of L-tryptophan, KYN, 5-HT and tryptamine in 8-OaS-H were significantly decreased (P < 0.01), while the contents of 5-HTP were decreased but the difference was not statistically significant. The contents of 5-HIAA was significantly increased. In the neurotransmitters of glutamate metabolic pathway, the contents of glutamate, GABA and glutamine in MG were increased compared with NG, and the difference of GABA content was statistically significant (P < 0.01). Compared with the MG, the contents of glutamic acid (P < 0.01), GABA and glutamine in the 8-OaS-H were decreased. In the neurotransmitters of histidine metabolic pathway, the contents of histidine (P < 0.01) and histamine (P < 0.01) in the MG were significantly elevated compared with NG. Compared with MG, the content of histidine (P < 0.01) and histamine decreased in the 8-OaS-H. In the neurotransmitter of arginine metabolic pathway, the level of arginine in MG was significantly increased compared with NG (P < 0.01). Compared with MG, the level of arginine in 8-OaS-H was significantly decreased (P < 0.01). Among the neurotransmitters of the choline metabolic pathway, choline in the fever model group compared with NG, and the content of acetylcholine increased (P < 0.01). Compared with MG, the contents of choline and acetylcholine increased in the 8-OaS-H, but there was no statistical significance.

The pearman correlation analysis of intestinal flora and metabolomics between 8-OaS-H and the MG at the genus level (Figure 5d). It was worth noting that Alistipes have significant negative associated with 5-HIAA and homovanillic acid, and a significant positively associated with DL-adrenaline, NE, 5-HT, histidine, KYN, tyramine, tryptamine, glutamic acid, tyrosine, arginine, L-tryptophan and phenylalanine.

The pearman correlation analysis of intestinal flora and metabolomics between 8-OaS-H and the MG at the species level (Figure 5f). It is worth noting that Alistipes_finegoldii showed a significant negative correlation with 5-HIAA, DL-METANEPHRINE and homovanillic acid and a significant positive correlation with NE, 5-HT, arginine, L-tryptophan and phenylalanine.

In colon contents (Figure 6; Table 4), the neurotransmitters associated with the phenylalanine metabolic pathway are affected. Compared with NG, the contents of phenylalanine, tyrosine, tyramine, dopamine, NE and 3-methoxytyramine(3-MT) in MG were significantly decreased (P < 0.01), while the content of DL-metanephrine was significantly increased (P < 0.01). The content of DL-adrenaline was increased. Compared with MG, the contents of phenylalanine, tyrosine, NE, DL-adrenaline, DL-metanephrine and 3-MT in 8-OaS-H were significantly increased (P < 0.01), while the content of tyramine was increased and dopamine content decreased, but the differences were not statistically significant. In the neurotransmitters of L-tryptophan metabolic pathway, compared with NG, the contents of 5-HT and 5-HIAA acid were significantly increased in MG (P < 0.01), while the content of L-tryptophan was increased but the difference was not statistically significant. The contents of KYN, 5-HTP and tryptamine were significantly decreased (P < 0.01). Compared with MG, the levels of 5-HT and 5-HIAA acid in 8-OaS-H were significantly decreased (P < 0.01), while the contents of L-tryptophan and KYN were significantly increased (P < 0.01). The content of 5-HTP and tryptamine (P < 0.01) was decreased, but the difference of 5-HTP was not statistically significant. In the neurotransmitter of glutamate metabolic pathway, compared with NG, the content of GABA acid in MG was significantly increased (P < 0.01), and the contents of glutamate and glutamine were significantly decreased (P < 0.01). Compared with the MG, the content of GABA in 8-OaS-H was significantly decreased (P < 0.01), and the contents of glutamate and glutamine were significantly increased (P < 0.01). In the neurotransmitters of the histidine metabolic pathway, the content of histidine in MG was significantly decreased (P < 0.01) and the content of histamine was significantly increased (P < 0.01) compared with the NG. Compared with MG, the histamine and histidine contents of 8-OaS-H were significantly increased (P < 0.01). In the neurotransmitter of arginine metabolic pathway, the content of arginine in MG decreased compared with NG. Compared with MG, the content of arginine in 8-OaS-H was significantly increased (P < 0.01). In the neurotransmitters of choline metabolic pathway, the contents of choline and Ach in MG were significantly increased compared with NG (P < 0.01). Compared with MG, the choline content in 8-OaS-H was decreased without statistical significance, but the Ach content was significantly increased (P < 0.01).

The pearman correlation analysis of intestinal flora and metabolomics were performed between 8-OaS-H and the MG at the genus level (Figure 6d). Alistipes showed a significant negative correlation with histamine, DL-adrenaline, KYN, 3-methoxytyramine, Ach, DL-metanephrine, glutamic acid, NE, glutamine, histidine, tyrosine, arginine, L-tryptophan and phenylalanine and a significant positive correlation with 5-HIAA, GABA, 5-HT and tryptamine. At the species level (Figure 6f), It is worth noting that Alistipes_finegoldii showed a significant negative correlation with histamine, KYN, 3-methoxytyramine, Ach, DL-metanephrine, glutamic acid, glutamine, histidine, tyrosine, arginine, L-tryptophan and phenylalanine and a significant positive correlation with 5-HIAA, GABA, 5-HT and tryptamine between 8-OaS-H and the MG.