Composition and preparation of HQD

Dried Chinese herbal medicines were selected according to the 2020 edition of the Chinese Pharmacopoeia and purchased by the Pharmacy Department of Changzhou Hospital of Traditional Chinese Medicine in March 2022 from Bozhou Traditional Chinese and Western Medicine Pharmaceutical Co. (Bozhou, Anhui, China). The herbs were identified by the Pharmacy Department and all voucher specimens (CF-202203-5) were deposited at Changzhou Hospital of Traditional Chinese Medicine. Scutellaria baicalensis (9 g), Paeonia lactiflora (6 g), Glycyrrhiza uralensis (6 g), and Ziziphus jujuba (49 g) were soaked in 10 volumes the volume of distilled water for 30 min, and boiled at 100 °C for 30 min. The liquid was filtered, and the residue was extracted using 8 times the volume of water. The two filtrates were combined and concentrated into a 1 g/mL herbal decoction after filtration through a membrane and stored at 4 °C for subsequent analysis (Mo et al. 2022).

Network pharmacology

BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm/index.php/Home/Index/index): an enhanced integrative database for known and predicted linkages between TCM ingredients and target proteins (Kong et al. 2024), was used to analyze the ingredient-target-pathway/disease network for HQD. The possible target genes of its main components (Scutellaria baicalensis, Paeonia lactiflora, Glycyrrhiza uralensis, and Ziziphus jujuba) were subsequently analyzed using the Chinese herbal medicine database HERB 2.0: A high-throughput experiment- and reference-guided database of TCM (http://herb.ac.cn/). The targets common to both BATMAN-TCM and HERB 2.0 were obtained through the Jvenn website (https://jvenn.toulouse.inrae.fr/app/example.html).

Animals and experimental design

C57BL/6J wild-type (WT) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and SLC6A4-knockout (SLC6A4-KO) mice with a C57BL/6J genetic background were purchased from Cyagen Biosciences (Guangzhou, Guangdong, China). Mice were maintained under specific pathogen-free (SPF) conditions (The SPF environment complies with the national standard of the People’s Republic of China GB14922-2022: Laboratory animal-microbiological and parasitical standards and monitoring and all mice do not carry pathogens that pose a significant health risk to animals and/or interfere with scientific research) for one week before the experiment. All the procedures were in strict accordance with the P.R. China legislation on the use and care of laboratory animals. The experimental protocols were approved by the Animal Ethics Committee of Changzhou Hospital of Traditional Chinese Medicine (Approval No.: 2022-017).

C57BL/6J mice were divided into 6 groups (n = 6): the control, AOM/DSS, AOM/DSS + Mesalazine, AOM/DSS + low (L)-HQD (2.275 g/kg), AOM/DSS + medium (M)-HQD (4.55 g/kg), AOM/DSS + high (H)-HQD (9.1 g/kg) groups. Except for those in the control group, the mice in the other groups were injected intraperitoneally with 10 mg/kg of AOM (one time, A885948, Shanghai Macklin Biochemical Co., Ltd., Shanghai, China). One week later, 2.5% DSS (D806297, Macklin) was added to the drinking water for one week. This was followed by a return to normal drinking water treatment for two weeks. One cycle of treatment was administrated for three weeks, and the cycle was repeated three times. The body weights of the mice were measured weekly, and the disease activity index (DAI) was evaluated at the beginning of the first cycle of DSS. Euthanasia by intraperitoneal injection of sodium pentobarbital (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was conducted at the end of the third cycle or when the mice developed anal prolapse and/or lost ≥ 20% of their body weight. For the control group, the mice were injected intraperitoneally or gavaged with the corresponding volume of saline and normal drinking water, respectively. For treatment with mesalazine and HQD, mesalazine (200 mg/kg; National Drug Code H19980148; Lulingpharm, Jiamusi, Heilongjiang) (Liang et al. 2019) and HQD (2.275, 4.55, and 9.1 g/kg) (Li et al. 2019) were administered by gavage daily after one week of AOM treatment. The dose of mesalazine was converted from a mouse dose to a human equivalent dose by the body surface area method as = (200 mg/kg × 20 g)/(70 kg × 0.0026) = 4 mg/0.182 kg = 22.0 mg/kg, which follows the recommended daily dose of UC in humans (70 kg) established by the Chinese Pharmacopoeia Commission (1.5 – 4 g = 21.4 mg/kg − 57.1 mg/kg).

For comparison of WT mice and SLC6A4-KO mice (n = 6/group), both groups of mice were induced with AOM/DSS. Considering that a high concentration of HQD had the greatest therapeutic effect on AOM/DSS mice, a high concentration of HQD (9.1 g/kg) was used for the following assays.

Evaluation of colitis

The DAI was used to assess the severity of colitis. The DAI was assessed by a combination of weight loss (percentage), stool consistency, and blood in the stool. The scoring criteria were as follows: (1) body weight loss (0: < 1%, 1: 1–5%, 2: 5–10%, 3: 10–15%, 4: > 15%); stool consistency (0: normal, 2: loose stool, 4: diarrhea); blood in the stool (0: no bleeding, 2: slight bleeding, 4: gross bleeding) (Tajasuwan et al. 2022).

Histological staining

Mouse colon tissues were fixed overnight in Bouin’s fixative, embedded in paraffin, and sectioned. Paraffin-embedded sections were dewaxed with xylene, hydrated with gradient alcohol, and stained with hematoxylin-eosin (HE) staining solution (E607318, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). After the staining was completed, the sections were dehydrated and sealed with neutral resin after being cleared in xylene. Finally, the pathological morphology of mouse colon tissues was observed under a microscope (Wang et al. 2023).

Enzyme-linked immunosorbent assay (ELISA)

The levels of 5-hydroxytryptamine (5-HT), IL-1β, and IL-18 in mouse colon tissues were measured with 5-HT (AD3266Mo, Beijing Andy Huatai Technology Co., Ltd., Beijing, China), IL-1β (JYM0531Mo, Jiyinmei, Wuhan, Hubei, China), and IL-18 (JYM0543Mo, Jiyinmei) kits. Briefly, the optical density (OD) value at 450 nm in each well was measured according to the instructions, and the corresponding concentration in each well was calculated from the standard curve.

RT-quantitative polymerase chain reaction (RT-qPCR) analysis

TRIzol (15,596,026, Invitrogen Inc., Carlsbad, CA, USA) was used for the extraction of mRNA from mouse colon tissues and cells. RNA was subsequently reverse transcribed using a cDNA synthesis kit (11141ES10; Yeasen Biotechnology Co., Ltd., Shanghai, China). The mRNA expression was subsequently detected using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (11184ES03, Yeasen). The data shown are the relative abundances of SLC6A4 normalized to the abundance of β-actin. qPCR was performed using the following primers: SLC6A4: 5’-GTTGATGCTGCGGCTCAGATCT-3′ (forward) and 5′-GAAGCTCGTCATGCAGTTCACC-3′ (reverse); β-actin: 5′-CATTGCTGACAGGATGCAGAAGG-3′ (forward) and 5′-TGCTGGAAGGTGGACAGTGAGG-3′ (reverse).

Western blot

RIPA lysis buffer (89,901; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for the extraction of total protein from tissue and cells. Total protein was quantified using a BCA quantification kit (23,227, Thermo Fisher). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes by the wet transfer method. After being blocked with skim milk for 45 min at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against SLC6A4 (1:1000, H00006532-D01P, Novus Biological Inc., Littleton, CO, USA), p-NFκB p65 (1:1000, #3033, Cell Signaling Technologies, Beverly, MA, USA), NFκB p65 (1:1000, #8242, Cell Signaling Technologies), NLRP3 (1:1000, #15,101, Cell Signaling Technologies), Cleaved-Caspase-1 P20 (1:1000, PA5-99390, Invitrogen), Cleaved-GSDMD (1:1000, #50,928, Cell Signaling Technologies), GSDMD (1:1000, ab225867, Abcam, Cambridge, UK), Caspase1 (1:1000, ab138483, Abcam), ASC (1:1000, #67,824, Cell Signaling Technologies), and β-actin (1:200, ab115777, Abcam). The following day, the PVDF membranes were washed and incubated with an HRP-conjugated secondary antibody (1:5000, ab205718, Abcam) for 1 h at room temperature. Finally, immunoreactive protein bands were detected by the high sensitive ECL luminescence reagent (C500044, Sangon), and β-actin was used as an internal reference to analyze the relative protein expression.

Cell culture and lentivirus infection

Murine intestinal epithelial MODE-K cells were purchased from BLUEFBIO (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. The cells were incubated in a 5% CO2 incubator at 37 °C. The sh-NC and sh-SLC6A4 lentiviruses used for infection were purchased from Obio (Shanghai, China). Lentivirus infection was conducted by seeding the cells into culture plates and adding the lentivirus solution when the cell density reached 60%. Antibiotics were added 48 h after infection to screen for lentivirus-infected cells.

DS preparation

Sprague Dawley rats (from the Vital River) were divided into the control and DS groups via gavage of saline or HQD (9.1 g/kg) for 7 consecutive days. Blood was collected from the rats 3 h after the last gavage. The sera were separated by centrifugation at 3000 rpm for 15 min and inactivated at 56 °C for 30 min, followed by filtration through 0.22-µm filter membranes. The collected serum was stored at -20 °C for cell treatment (Zhu et al. 2019).

Development of an in vitro pyroptosis model and treatment with DS

MODE-K cells were treated with 100 ng/mL LPS (AC11974, Shanghai Acmec Biochemical Co., Ltd., Shanghai, China) for 4 h and then stimulated with 5 mM ATP (A832633, Macklin) for 30 min to induce pyroptosis (Chen et al. 2019). For pretreatment with DS, MODE-K cells were cultured in DMEM supplemented with 10% DS for 24 h before the LPS/ATP induction. The control cells were cultured with control serum.

Cell viability assay

MODE-K cells were assayed for cell viability with a cell counting kit-8 (CCK-8, HY-K0301, MedChemExpress, Monmouth Junction, NJ, USA). Briefly, 10 µL of CCK-8 solution was added to each well, and the cells were incubated for 2 h in an incubator according to the manufacturer’s instructions. Finally, the optical density (OD) values at 450 nm were measured by a microplate reader.

Lactate dehydrogenase (LDH) release assay

LDH kits (C0016, Beyotime Biotechnology Co., Ltd., Shanghai, China) were used to measure the release of LDH from cells. The sample maximum enzyme activity control wells were added to the LDH release reagent provided in the kit as a positive control. The cells were centrifuged at 500 ×g for 6 min for precipitation, after which the cell supernatant of cells was collected. The configured LDH assay solution was added for a 20-min incubation in the dark, and the OD490 values of each group were measured using a microplate reader. The release of LDH was calculated according to the following formula: (OD value of treated samples - OD value of sample control wells) / (OD value of positive control wells – OD value of sample control wells) × 100.

Immunofluorescence

The cells were fixed with paraformaldehyde, permeabilized in Triton X-100 for 20 min, blocked with goat serum for 45 min, and incubated with a diluted primary antibody against ASC (1:500, #67,824; Cell Signaling Technologies) overnight at 4 °C. After the cells were incubated with an Alexa Fluor 488-conjugated secondary antibody (1:1000; #4412; Cell Signaling Technologies) for 2 h in the dark, the cell slides were stained with DAPI, mounted, and then imaged under a fluorescence microscope. The fluorescence intensity of ASC in the cells was observed (Qu et al. 2022).

Statistics

Statistical analysis in this study was performed with Prism 8.0 (GraphPad, San Diego, CA, USA), and the data are presented as mean ± SDs. The unpaired t-test was used to compare the two groups. For multigroup comparisons, the data were analyzed by one-way or two-way ANOVA and Tukey’s multiple comparison tests. Each experiment was conducted with biological and technical replicates with three replications of the entire experiment unless otherwise specified. Differences were considered to be significant at a p-value of less than 0.05.