2.1. Drugs and reagents
HQD consists of the following component herbs: Paeonia lactiflora Pall., Glycyrrhiza uralensis Fisch., Ziziphus jujuba Mill. (purchased from Kangmei Pharmaceutical Co., Ltd.), and Scutellaria baicalensis Georgi (purchased from Guangdong He Xiang Pharmaceutical Co., Ltd.). Paeoniflorin and baicalin were purchased from Guangzhou Whiga Technology Co., Ltd. (Guangzhou, China). Liquiritin (≥ 98% purity), glycyrrhizic acid (≥ 98% purity), and wogonoside (≥ 98% purity) were purchased from Chengdu Feipude Biological Technology Co., Ltd. The cytometric bead array (CBA) assay kit was purchased from BD Biosciences Pharmingen (San Jose, CA, USA). DSS was obtained from MP Biomedicals (Santa Ana, CA, USA). Sulfasalazine tablets were purchased from Shanxi Tongda Pharmaceutical Co., Ltd. (Shanxi, China). The TRIzol reagent, NF-κB p65 gene primers, and receptor-interacting protein-2 (RIP2) gene primers were purchased from Invitrogen (Carlsbad, CA, USA). SYBR® Premix Ex Taq™ (Tli RNase H Plus), DNase/RNase-free deionized water, NOD2 gene primers, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reference gene primers were obtained from Dalian Bioengineering Co., Ltd. (Dalian, China). Anti-TLR4 antibody, anti-MyD88 antibody, anti-Rip2 antibody and anti-NF-κB p65 antibody were purchased from Cell Signaling Technology (Boston, MA, USA). An anti-NOD2 antibody was purchased from Abcam (Cambs, UK). Protein markers were purchased from Fermentas (Burlington, ON, Canada). The enhanced chemiluminescence (ECL) substrate was purchased from Beyotime Institute of Biotechnology (Shanghai, China).
2.2. Preparation of HQD
According to the original proportion (9:6:6:6; g), we weighed and mixed S. baicalensis, P. lactiflora, G. uralensis, and Z. jujuba and prepared 10 batches of a conventional decoction. Each was mixed with eight times the amount of water, soaked for 30 min, then subjected twice to the first fire to boil for 25 min and simmering for about 35 min, and filtered. The two filtrates were combined and subjected to rotary evaporation to obtain a solution equivalent to 2 g/mL crude drug. The solution was diluted with ultrapure water to 0.5 g/mL and further diluted with an equal volume of methanol to a final concentration of 0.25 g/mL. A test sample was obtained after centrifugation for 1 min and filtration through a 0.22-µm membrane.
To prepare reference substances, 5 mg of baicalin, paeoniflorin, glycyrrhizin, and glycyrrhizic acid, was dissolved in 10 mL of methanol and filtered through a 0.45-µm microporous membrane. Paeoniflorin was diluted to a final concentration of 40 µg/mL with 50% methanol, and the other reference substances were diluted to 20 µg/mL. All compounds were prepared as a single standard and mixed standard, followed by centrifugation for 1 min and filtration through a 0.22-µm membrane.
The prepared HQD and reference substance solutions were analyzed by high-performance liquid chromatography (HPLC). The results of an HPLC chromatogram were imported into software (Chinese medicine chromatographic fingerprint similarity evaluation system 2004A) to analyze its fingerprint in an AIA (Analytical Instrument Association) format. The fingerprint of HQD sample S1 was used as a reference spectrum, and control maps were generated using average data. Fingerprint matching was performed by a multi-point calibration method to generate a control map. The following chromatographic conditions were used: a Kromasil C18 column (250 mm × 4.6 mm, 5 m); column temperature: 30℃; mobile phase A: acetonitrile; mobile phase B: water containing 0.1% phosphoric acid; linear elution gradient: 0–10 min, 10% A–11% A; 10–35 min, 11% A–65% A; flow rate: 1.0 mL·min− 1; scanning wavelengths: 220–500 nm; sample size: 10 µL.
2.3. Animals
C57BL/6 mice (male, 6–8-week-old, weight 18–22 g) were obtained from the Laboratory Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China) and acclimated for at least 3 days before experiments. The mice were housed in groups under specific pathogen-free conditions at a temperature of 24 ± 1℃, humidity of 40–80%, and a 12-h light/12-h dark cycle. All experiments were executed according to the guidelines approved by the Ethics Committee of Guangzhou University of Chinese Medicine.
2.4. Induction of colitis and treatments
The C57BL/6 mice were randomly divided into six groups (n = 12 per group): control, model (DSS), sulfasalazine (500 mg/kg), low dose of HQD (250 mg/kg), medium dose of HQD (500 mg/kg), and high dose of HQD (1,000 mg/kg) groups. The mice in the control group had free access to sterile water, while those in the other groups drank 3% DSS freely for 5 days and then drank sterile water. Meanwhile, the mice in the control and model groups were administered with sterile water, while the other four groups were given the corresponding medicines. The drugs were administered for a total of 10 days according to the mouse weight (0.2 mL/10 g), and the experiment was carried out on day 11. The grouping and treatment with HQD of the DSS-induced colitis mice are shown in Fig. 1.
2.5. Assessment of colitis severity
The disease activity index (DAI) score of colitis was determined as Cooper previously described[17]. For the assessment of the severity of colitis in mice, eating, water drinking, the hair color, feces, blood in the stool, and the body weight were carefully observed and recorded daily.
At the end of administration, peripheral blood was collected from eyes after anesthesia, then 20 µL was fully mixed in an EDTA-2K anticoagulant tube, and used to count blood cells with an automatic blood cell analyzer. In addition, we cut colonic segments from the sacrificed mice and measured and recorded their natural length without stretching. The distal colon of 2.0 cm was fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The tissue was cut into about 4 µm slices and stained with hematoxylin and eosin (HE). Then, the stained tissue was observed under an optical microscope, and a histological score was determined according to the standard shown in Supplementary Table 1(Table S1).
2.6. 16S ribosomal DNA (16S rDNA) identification
The contents of the cecum were taken aseptically and were detected by Shenzhen Huada Genomics Technology Service Co., Ltd. using 16S rDNA sequencing technology.
2.7. Cytokine detection by cytometric bead array (CBA) and Flow CytoMetry (FCM)
A colonic segment was mixed with 0.9% NaCl saline at a mass ratio of 1:10 and then automatically homogenized with a homogenizer. The homogenate was centrifuged to obtain a supernatant (3,000 × g, 4 °C, 10 min). The levels of interleukin (IL)-1β, IL-17, IL-6, monocyte chemotactic protein-1 (MCP-1), TNF-α, and interferon-γ (IFN-γ) were determined by CBA following the manufacturer's instructions using a FACS Canto II flow cytometer.
IECs were extracted from small intestine with digestive juice, and incubated with Alexa Fluor 488-CD324 and PE-RegⅢγ flow antibody for 30 min. Intestinal epithelial cells were labeled with CD324 and antibacterial proteins were labeled with RegIIIγ. Then they were detected by flow cytometry.
2.8. Fluorescence in situ hybridization (FISH)
4 µm sections were prepared from paraffin embedded colon tissue for FISH. According to the manufacturer's instructions, bacterial infiltration of the colonic mucosa was detected using the EUB338I FISH probe with FITC labeled Kit (Guangzhou Exon Biotechnology Co., Ltd., China).
2.9. Western blot analysis
Colonic tissue was homogenized and centrifuged (4 °C, 2,500 × g, and 5 min). Cytoplasmic and nuclear proteins were extracted from the collected precipitate using cytoplasmic and nuclear protein extraction kits, respectively. The protein concentration was calculated by measuring optical density at 562 nm with a microplate reader. Proteins (20 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. The membrane was soaked in 5% skim milk at room temperature for 1 h to block non-specific binding sites and then incubated with a primary antibody (TLR4, TLR5, NOD2, MyD88, RIP2 or NF-κB p65), followed by incubation with an appropriate secondary antibody. Positive immunoreactions were directly imaged using a chemiluminescence image analysis system with a Super ECL Plus ultra-sensitive luminescent solution.
2.10. Semi-quantitative reverse transcription and polymerase chain reaction
Total RNA was extracted from the colon with the TRIzol reagent. The purity and concentration of RNA were determined using an ultra-micro ultraviolet spectrophotometer, and the concentration was adjusted to 500–700 ng/µL. DNA was removed from the total RNA using DNase with incubation at 42 °C for 5 min. cDNA was synthesized from 1 µg of total RNA in a total volume of 20 µL at the following conditions: 37 °C for 15 min, followed by 85 °C for 5 s with a first-strand cDNA synthesis kit. SYBR® Premix Ex Taq™ (Tli RNase H Plus) was used for amplification of cDNA. Reactions were performed in triplicate. We analyzed the relative expression of the Nod2, Rip2, and NF-κB p65 genes by comparing transcript levels of the target genes with that of the internal reference Gapdh. The sequences of the primers were as follows: Nod2: 5′-ACCATGTAGAAGCCATGCTGGAG-3′ (forward) and 5′-CTTCACCGCAGCGAGATCAA-3′ (reverse); Rip2: 5′-GCCATTGTGAGCCAGATGA-3′ (forward) and 5′-ATTTGAAGGCGGTGCTTTG-3′ (reverse); RelA (encoding NF-κB p65): 5′-ATGTGCATCGGCAAGTGG-3′ (forward) and 5′-CAGAAGTTGAGTTTCGGGTAG-3′ (reverse); Gapdh: 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse). The amplification conditions were as follows: 95 ℃ for 30 s, 40 cycles at 95 ℃ for 5 s and 60℃ for 30 s, followed by 95℃ for 10 s and generation of a dissociation curve from 65 °C to 95 °C at an increment of 0.5 °C per 5 s.
2.11. Immunohistochemistry analysis
Antigens were retrieved from frozen sections with citrate buffer, and the sections were then fixed with acetone and washed with phosphate-buffered saline. After that, H2O2 (20 µL/sample) was added to inhibit the endogenous enzyme, followed by a blocking solution. The sections were exposed to anti-NOD2, anti-RIP2, or anti-NF-κB p65 at 4 °C overnight and then to the SignalStain® Boost IHC detection reagent at room temperature for 30 min. Images were visualized and captured using an Olympus microscope after staining with hematoxylin, dehydration, and sealing.
2.12. Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Differences between two groups were analyzed by the Student’s t-test, and those among several groups were analyzed by one-way analysis of variance using SPSS (version 17.0). A P-value of < 0.05 was considered statistically significant.