Ingredients and potential targeted genes of SYD in ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)

The herbal mixture underwent two rounds of decoction, with each round lasting for 1 h. Subsequently, the resultant decoction was concentrated and dried under vacuum to yield a powdered form. Firstly, a small amount of SYD powder was transferred to a vial, diluted with 100-fold methanol (Sigma-Aldrich, MO, USA) and extracted by ultrasonication (ThermoFisher, MA, USA) for 30 min, then the extraction was filtered and 1.5 mL filtrate was used to analyze the ingredients of SYD with an Vanquish UPLC system (ThermoFisher, MA, USA) and Q-Exactive Orbitrap mass spectrometer (ThermoFisher, MA, USA). Sheath gas and auxiliary gas were both using nitrogen. The parameters during the positive ion detection mode were: capillary temperature, 400 ℃; spray voltage, 3.5 kV; sheath gas flow rate, 40 arb; aux gas flow rate, 20 arb; while the parameters during the negative ion detection mode were: capillary temperature, 350 ℃; spray voltage, 3.0 kV; sheath gas flow rate, 35 arb; aux gas flow rate, 10 arb. The full scan mode covered 200-2000 m/z, and the resolution was 70000 (full scan) and 17500 (MS/MS). Collision mode was high energy collision dissociation, with NCE30 and stepped NCE50% as the collision energy. The data were analyzed using Xcalibur software (V4.2, ThermoFisher, MA, USA). Furthermore, we quantified the key ingredients in SYD as the following procedure: the TCM formula powder was diluted with 80% methanol and vortexed by BeyoVortex™ (Beyotime, Beijing, CHN) for 5 min, then extracted by ultrasonication for 20 min. The solution after ultrasonic extraction was centrifuged at 13000 rpm at 4 ℃ for 10 min (ThermoFisher, MA, USA), and the supernatant was aspirated and diluted 100-fold with 50% methanol into a clean vial for UPLC-MS/MS, and the standard curve was prepared with 50% methanol aqueous solution at the same time. Subsequently, the prepared sample was analyzed with an Agilent ultrahigh performance liquid chromatography ABSCIEX Exion LC AD system (SCIEX, MA, USA) with a Waters XSelect HSS T3 Column (2.5 µm, 2.1 × 100 mm, Waters, MA, USA) at 40 ℃. Five µL sample was injected, and the flow rate was set at 0.3 mL/min. The mobile phase consisted of acetonitrile (A) and 0.05% formic acid (Sigma-Aldrich, MO, USA) in water (B). The gradient program was as follows: 0 min, A/B (15:85, v/v); 2 min, A/B (30:70, v/v); 6 min, A/B (60:40, v/v); 8 min, A; 10.1 min, A/B (15:85, v/v). To obtain the mass spectrometry (MS) and MS/MS data, we used an ABSCIEX QTRAP 5500 system (SCIEX, MA, USA). The following parameters were applied: spray gas, 55 psi; auxiliary heating gas, 55 psi; capillary temperature, 500 ℃; ion voltage, 4500 V-5500 V; curtain gas, 30 psi; collision gas, 9 psi. Furthermore, we screened all potential targeted genes associated with the ingredients of SYD in the UPLC-MS/MS analysis with the aid of TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, https://old.tcmsp-e.com/tcmsp.php) [11]. Next, all the targeted genes were inputted into the PANTHER (Protein Analysis Through Evolutionary Relationships, http://www.pantherdb.org/) [12] classification system for the purpose of conducting pathway enrichment analysis. The results were presented in the form of a bubble chart in the Supplementary Figures.

Animals and sampling

Colitis models were induced in male BALb/c mice weighing 17-19 g by administering 5% dextran sulfate sodium (DSS, Wako, Tokyo, Japan). Following the detection of occult blood in over 50% of the mice’s stool, they were then administered with various doses of SYD (low dose of Shaoyao Decoction, abbreviated as SYD-L: 8 g/kg/day; medium dose of Shaoyao Decoction, abbreviated as SYD-M: 16 g/kg/day; high dose of Shaoyao Decoction, abbreviated as SYD-H: 32 g/kg/day) or mesalazine (Mes, 0.5 g/kg/day, Etiasa, Shanghai, CHN). Following 7 days of treatment, serum, digesta in the colon (as fecal samples) and colonic tissue were collected for subsequent experiments.

mRNA sequencing and the data processing

Colonic tissues were used to isolate total RNA through the utilization of Trizol reagent (Invitrogen Life Technologies, CA, USA). Subsequently, Poly(A) + RNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). Qualified RNA samples underwent fragmentation and reverse transcription into cDNA employing the NEBNext Ultra RNA Library Prep kit for Illumina (New England Biolabs, MA, USA). RNA sequencing was performed on the Illumina Novaseq Platform (Illumina, CA, USA). The resulting clean data were aligned to the GRCm38 reference database (https://www.ensembl.org/) using HISAT2 (V2.1.0, Johns Hopkins University, MD, USA), followed by transcript assembly using StringTie (V1.3.3b, Johns Hopkins University, MD, USA). RNA expression levels were quantified using fragments per kilobase of transcript per million fragments mapped (FPKM). Principal coordinate analysis (PCoA) with Adonis analysis was used to exhibit the mRNA profiles. The differentially expressed genes (DEGs) were screened according to |Log2Fold change (FC)|≥ 1 and P ≤ 0.05, and displayed using volcano plot and heatmap. DEGs were further subjected to functional annotation and pathway analyses using the ENSEMBL knowledgebase (http://www.ensembl.org/index.html) and the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp/) /PANTHER classification system [13], which were presented in bubble diagrams.

16S rDNA full-length sequencing and the data processing

Genomic DNA was extracted from the fecal samples using a Tiangen DNA kit (Tiangen, Beijing, CHN). Bacterial 16S rDNA was amplified using unique molecular identifier (UMI) primers and KAPA HiFi HotStart ReadyMix (Roche, Shanghai, CHN) with the universal primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGYTACCTTGTTACGACTT-3’). Following amplification, the amplicons were purified with Beckman Agencourt AMPure XP (Beckmancoulter, TX, USA) and quantified using a ThermoFisher Qubit dsDNA assay kit (ThermoFisher, MA, USA). The sequencing library was constructed by pooling equal amounts of purified amplicons and subjected to full 16S rDNA sequencing on the Illumina platform (Illumina, CA, USA). The paired reads derived from the original DNA fragments were merged using Illumina bcl2fastq conversion (V2.20, Illumina, CA, USA). The sequences were then analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software (V2.10, http://www.qiime.org). Operational taxonomic units (OTUs) were assigned at 99% similarity, and a representative sequence from the SILVA 132/16S rDNA database (V132, http://www.arb-silva.de) for bacteria was assigned to each OTU. Taxonomic data were generated using a Bayesian approach with a 99% cutoff value. Kruskal-Wallis analysis was performed to compare the diversity measures statistically. PCoA with Adonis analysis based on the Bray-Curtis distance was used to demonstrate the beta diversity of the intestinal flora. KEGG pathways were obtained using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt, V1.1.4, https://picrust.github.io/picrust/), and the functional difference among groups was displayed using linear discriminant analysis effect size (LEfSe) analysis with P ≤ 0.05 and LDA ≥ 2 as threshold. Spearman's correlation coefficient was used to assess the relationship between genera and the relative abundance of genes in the faeces.

Targeted metabolomics for short-chain fatty acids (SCFAs)

Prepared stock solutions of acetic acid (Aladdin, Shanghai, CHN), propionic acid (Sigma-Aldrich, MO, USA), butyric acid (Sigma-Aldrich, MO, USA), isobutyric acid (Trc, YTO, CA), pentanoic acid (NU-CHEK, MN, USA), hexanoic acid (Dr.Ehrenstorfer, Augsburg, GER) and isovaleric acid (Yuanye, Shanghai, CHN) with a concentration of 1 mg/mL in distilled water (DW). These stock solutions were then diluted with water to the desired concentrations for use as standard reference metabolites in targeted metabolomics. The solutions were stored at -80 ℃. Added 300 μL of ice-cold DW containing acetonitrile (1:1, v/v) to each freeze-dried fecal/serum sample. The samples were then ground for 3 min at 60 Hz (-20 ℃ pre-cooling, WONBIO, Shanghai, CHN). After that, ultrasonic extraction was performed for 10 min in ice-water (FUYANG, Shenzhen, CHN). The samples were then centrifuged at 4 ℃ for 10 min at 12,000 rpm (Lu Xiangyi, Shanghai, CHN) to obtain the supernatant. The resulting supernatant was diluted with DW containing acetonitrile (1:1, v/v) to a volume that is 5 times the original volume.

The 80 μL sample/standard reference metabolites solution, 40 μL 200 mM 3-nitrophenylhydrazine and 40 μL 120 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro chloride-6% pyridine (both two were prepared with water containing acetonitrile, 1:1, v/v) were mixed into injection vial. The resulting mixture was then subjected to a 30-min reaction at 40 ℃ (KEXI, Shanghai, CHN), followed by a 1-min incubation on ice. Next, 160 μL of the reacted mixture was extracted using a syringe and filtered through a 0.22 μm organic phase pinhole filter for subsequent UPLC-MS/MS analysis.

Liquid chromatography (LC) analysis was conducted using a Nexera UHPLC LC-30A system (Shimadzu, TKY, JPN) equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm, Waters, MA, USA). The injection volume was set at 1 μL, and the flow rate was maintained at 0.4 mL/min. Mobile phase A was composed of 0.1% formic acid (ThermoFisher, MA, USA) in water, while mobile phase B consisted of a mixture of acetonitrile and methanol (2:1, v/v). The following gradient elution program was employed: 0 min, A/B (80:20, v/v); 8 min, A/B (60:40, v/v); 8.1 min, A/B (5:95, v/v); 9.6 min, A/B (80:20, v/v); 10 min, A/B (80:20, v/v). Samples were kept at 4 ℃ throughout the elution process, and the column temperature was maintained at 40 ℃.

MS analysis was performed in the negative ion mode using the ABSCIEX Selex ION Triple Quad™ 5500 System (SCIEX, MA, USA), with the electrospray ionization source set at -4500 V and 450 ℃. Nitrogen was employed as the collision gas, and the column temperature was kept at 40 ℃, the flow rate was set as 30 psi. The targeted SCFAs were analyzed utilizing multiple reaction monitoring mode. Data acquisition and analysis were performed using Analyst software (SCIEX, MA, USA), with quantification of all targeted SCFAs conducted using ABSCIEX OS-MQ (SCIEX, MA, USA).

Cell culture

QGP-1 cells (enterochromaffin cells model) and H9 cells (T cells) were acquired from American Type Culture Collection (ATCC, USA, https://www.atcc.org). Cells were cultured in a 37 ℃ incubator with 5% CO2 using RPMI 1640 culture medium (Sigma-Aldrich, MO, USA), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, MO, USA). L-tryptophan (L-Trp, Yuanye, Shanghai, CHN) was added at the concentration of 40 μmol/L, and sodium butyrate (Sigma-Aldrich, MO, USA) was added at concentrations of 2 and 4 mmol/L. The antagonist of G protein-coupled receptor 43 (GPR43), GLPG0974 (MedChemExpress, NJ, USA), was used at the concentration of 5 μmol/L, while the agonist of GPR43, TUG-1375 (MedChemExpress, NJ, USA) was used at the concentration of 16 μmol/L. Additionally, 5-hydroxytryptamine (5-HT, Sigma-Aldrich, MO, USA) was supplied at the concentration of 100 μmol/L, the antagonist of 5-HT receptor 1A (HTR1A), Way100635 (MedChemExpress, NJ, USA), was used at the concentration of 10 μmol/L, and the antagonist of 5-HT receptor 3 (HTR3), Tropisetron (Selleck, TX, USA), was used at the concentration of 25 μmol/L. All the supernatant and the cells should be collected for western blot (WB) and real time-quantitative polymerase chain reaction (RT-qPCR) after 48 h.

Enzyme linked immunosorbent assay (ELISA) and WB

The faeces and grated colonic tissue diluted with DW and centrifuged at 3000 rpm for 30 min, then the supernatant was collected for ELISA. The serum and supernatant from the cell culture were also collected for ELISA. Proteins were extracted from tissue/cell samples using radio immunoprecipitation assay (RIPA, Servicebio, Beijing, CHN), and the protein content was quantified using the PierceTM BCA Protein Assay kit (ThermoFisher, MA, USA).

ELISA was performed according to the manufacturer's instructions for the following cytokines: interleukin-1 beta (IL-1β, Proteintech, Wuhan, CHN), interleukin-2 (IL-2, Proteintech, Wuhan, CHN), interleukin-6 (IL-6, Proteintech, Wuhan, CHN), tumor necrosis factor-alpha (TNF-α, Proteintech, Wuhan, CHN), interferon-gamma (IFN-γ, Proteintech, Wuhan, CHN), and 5-HT (Cloud-Clone, Wuhan, CHN).

The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Epizyme, Shanghai, CHN) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Servicebio, Wuhan, CHN). The membranes were then incubated with protein-free rapid blocking buffer (1 × , Epizyme, Shanghai, CHN) for 1 h at room temperature, and incubated overnight at 4 ℃ with the relevant primary antibodies: tryptophan hydroxylase 1 (TPH1, ABclonal, Wuhan, CHN), dopa decarboxylase (DDC, Abcam, MA, USA), monoamine oxidase B (MAOB, Proteintech, Wuhan, CHN), aldehyde dehydrogenase 1 family member B1 (ALDH1B1, Proteintech, Wuhan, CHN), sodium-dependent serotonin transporter (SLC6A4, Abcam, MA, USA), GPR43 (Proteintech, Wuhan, CHN), protein kinase C theta (PKCθ, Abcam, MA, USA), p-PKCθ (Bioss, Beijing, CHN), NF-κB (ABclonal, Wuhan, CHN), p-NF-κB (Abcam, MA, USA), HTR1A (Abcam, MA, USA), HTR3 (Abcam, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ABclonal, Wuhan, CHN). The membranes were then washed with tris buffered saline with tween (TBST, Servicebio, Wuhan, CHN), and subsequently incubated with HRP-labeled secondary antibodies (ABclonal, Wuhan, CHN) at room temperature for 1 h. Following the wash step, the membranes were exposed using Tanon 4800 (Tanon, Shanghai, CHN) and analyzed using ImageJ (V1.53, https://imagej.nih.gov/ij/, National Institutes of Health, MD, USA).

RT-qPCR and PCR

Total RNA was extracted from colonic tissue and cell samples using the RNA Extraction kit (Accurate Biology, Changsha, CHN), followed by cDNA synthesis using the Evo M-MLV Reverse Transcription kit (Accurate Biology, Changsha, CHN). Amplification of cDNA was performed using the Maxima SYBR Green qPCR Master Mix kit (ThermoFisher, MA, USA) and RT-qPCR analysis was carried out on the BIO-RAD CFX96 Real-Time PCR System (BIO-RAD, CA, USA) and Applied Biosystems ViiA™ 7 Real-Time PCR System (ThermoFisher, MA, USA), following the manufacturer's protocols. Total DNA was extracted from the digesta in the colon using the QIAamp PowerFecal Pro DNA kit (QIAGEN, Dusseldorf, GER), and PCR analysis was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System (ThermoFisher, MA, USA).

The primers were as follows: Mouse-Gapdh (5’-CCATGGAGAAGGCTGGGG-3’, 5’-CAAAGTTGTCATGGATGACC-3’), Human-GAPDH (5’-GGAGCGAGATCCCTCCAAAAT-3’, 5’-GGCTGTTGTCATACTTCTCATGG-3’), Human-HTR1A (5’-AAGACAGTGAAGACGCTGGG-3’, 5’-AGAAGGGCAGAACAAGAGCC-3’), Human-HTR3 (5’-GCAGGAAAACTTGGCAGCTC-3’, 5’-CTCTGCCAACCTCATGTCCC-3’), Mouse-Tph1 (5’-CAGTGGCTCTGAGGTGAGTG-3’, 5’-AGTTGATCCCGTCCTTGCTG-3’), Human-TPH1 (5’-TCTACCCAACCCATGCTTGC-3’, 5’-AAGTAACCAGCCACAGGACG-3’), Mouse-Ddc (5’-GAGCTGGACAATCCCGACAA-3’, 5’-GATCAGGGGCCGAAGATAGC-3’), Human-DDC (5’-ACTCCCGGCTGCCTTTTTC-3’, 5’-CGTCCCTCAATGCCTTCCAT-3’), Mouse-Maob (5’-CCACATTGACCAGACAGGGG-3’, 5’-TCTTCATGCCCAAAGCAGGT-3’), Human-MAOB (5’-AGCGCCATGAGCAACAAATG-3’, 5’-AGGATCCTCCAAGGTCCACA-3’), Mouse-Aldh1b1 (5’-TGCTGAACTGACCGGAGAAC-3’, 5’-TTTGGGATTGGGTTCGGGAG-3’), Human-ALDH1B1 (5’-AGCCTGCTGCAGAGTGTCAG-3’, 5’-CTTGCTGACTGCATCTTGCC-3’), Mouse-Slc6a4 (5’-CAAAACGTCTGGCAAGGTGG-3’, 5’-ACACCCCTGTCTCCAAGAGT-3’), Human-SLC6A4 (5’-TGCAGAAGCGATAGCCAACA-3’, 5’-GTGGGAACTCATCCAGCACA-3’), Mouse-Gpr43 (5’-TGACAGGCTTCGGCTTCTACAG-3’, 5’-AGAGCAGCGATCACTCCATACAG-3’), Human-GPR43 (5’-GAGCAGGTCAGAAGTGGCAATG-3’, 5’-GAAGAAGAGCACCAGGCACAG-3’), Human-PKCθ (5’-CGAGTTCACTGCCACCTTCTTC-3’, 5’-GCACTGGTAGCCCTGTTTGTTC-3’), Human-NF-κB (5’-GCCCACTCGCTGCCTCTC-3’, 5’-ATGTCTCCACGCCGCTGTC-3’).

Hematoxylin–eosin (HE) staining, immunohistochemistry (IHC) and immunofluorescence (IF)

Following sacrifice and dissection, the colon of each mouse was promptly extracted and perfused with normal saline. The colons of mice from different groups were fixed in 4% paraformaldehyde for 24 h, subjected to dehydration in gradient ethanol, and subsequently embedded in paraffin. The paraffin-embedded tissue was sectioned into slices of 5 μm thickness, followed by HE staining to evaluate histopathology using an inverted microscope (Leica Microsystems, Wetzlar, Germany).

Rabbit primary antibodies targeting 5-HT (Abcam, MA, USA) were selected for IHC. Secondary antibodies, derived from goats, were incubated with the samples after which diaminobenzidine (DAB, ZSGB-BIO, Beijing, CHN) was used for signal visualization, and the sections were counterstained with hematoxylin. Finally, the target proteins were observed using an inverted microscope and analyzed using ImageJ.

Sections were washed 3 times with PBST (phosphate buffered saline with tween, Servicebio, Beijing, CHN) and then blocked in 10% normal goat serum, 5% bovine serum albumin (BSA, Servicebio, Beijing, CHN) and 0.3% Triton X-100 (Servicebio, Beijing, CHN) diluted in PBST at room temperature for 1 h. Firstly, antibodies of CD4 (Abcam, MA, USA) was incubated overnight at 4 ℃, and then antibody of PKCθ was incubated for 1 h at room temperature. Nucleus were stained with 4',6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, CHN). IF images were captured using a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software (V25.0, SPSS Inc., IL, USA). The normal distribution of variables was assessed using the Shapiro-Wilk test. Normally distributed variables were analyzed using the one-way analysis of variance (ANOVA) procedure, while non-normally distributed variables were assessed using the Mann-Whitney U test. Statistical significance was determined at P ≤ 0.05.