Sprague–Dawley male rats (age 6–8 wk, weighing 200–220 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). All rats were maintained under specific pathogen-free conditions at Experimental Animal Center, Dalian Medical University (Certificate of Conformity SYXK Liao 2013-0006). The study was conducted following the guidelines of the National Institute of Health Guide for Care and Use of Laboratory Animals (Publication no. 85 − 23, revised 1985), and was proved by the Dalian Medical University Animal Care and Ethics Committee (No: AEE20047). The animals were acclimatized to laboratory conditions of 23°C, 12 h/12 h light/dark, and 50% humidity, with ad libitum access to food and water for 2 weeks prior to experimentation. No animals died before the study was started.
2.2. SYD preparation
SYD was made from nine herbs: Paeonia lactiflora Pall., Angelica sinensis (Oliv.) Diels., Coptis chinensis Franch., Areca catechu L., Aucklandia lappa Decne., Rheum palmatum L., Scutellaria baicalensis Georgi., Cinnamomum cassia Presl., and Glycyrrhiza uralensis Fisch., The raw herbs for SYD were purchased from Beijing TongRenTang Co.Ltd. These raw herbs of Shaoyao Decoction were mixed in the ratio of 4:2:2:1:1:1:2:1:1 (dry weight) and then decocted with 10 times volume of distilled water (v/m) for 2 h. The aqueous extracts of SYD were extracted and centrifuged. Supernatant was collected and subjected to condensation under reduced pressure to obtain the semisolid SYD solution . SYD were suspended again in distilled water at a final concentration of 1.2 g/mL. The solution was stored in aliquots at -20°C.
2.3. Experimental design
Acute colitis was induced according to previously established protocols with slight modifications. Briefly, after a fasting period of 24 h with free access to drinking water, a catheter was inserted through the anus so that its terminus reached approximately to the level of the splenic flexure (8 cm proximal to the anal verge) under urethane anesthesia. Subsequently, the colon was infused with 1 mL TNBS dissolved in ethanol (40% v/v) at a dose of 125 mg/kg. Rats were randomly assigned to five groups of 12 animals each. Control rats in group I were given sterile saline. Group II was a TNBS colitis model control. Groups III, IV, and V were treated by intragastric administration of 150 mg/kg sulfasalazine (SASP), low-dose 4 g/kg SYD, or high-dose 24 g/kg SYD. SASP is an anti-inflammatory drug used to treat IBD, and was a positive control for the effects of SYD on colitis.
2.4. Shaoyao Decoction medicated serum
The SYD-medicated serum was manufactured according to the previous study. Thirty rats were randomly divided into 3 groups, consisting of control group, low dose SYD (4g/kg) group and high dose SYD (24g/kg) group. The rats in the SYD group underwent intragastric administration of SYD two times a day for five consistent days. The rats in the blank serum group received orally administration of physiological saline twice a day for five days. At 0.5 h after the final administration, the rats were anesthetized and sacrificed, blood samples were collected from abdominal aorta and centrifuged at 2500 rad/min at 4°C for 15 min. The serum was isolated and stored for further analysis.
2.5.Monitoring TNBS-induced colitis
Inflammation was evaluated using HE stained colon sections according to previously described morphological criteria. Animal body weights, food intake, rectal bleeding, and diarrhea incidence for each group were recorded daily. The colon was scored for macroscopically visible damage on a 0–10 scale. The disease activity index (DAI) was determined as previously reported.
2.6.Measurement of epithelial barrier function
As is previously reported, epithelial barrier function was measured in vivo and in vitro. Rats were denied access to food, but allowed water for 3 h. Then, 22 mL/kg body weight of PBS (pH 7.4) containing 22 mg/mL FITC-dextran were gavaged and serum was harvested 1 h later. Multi-function microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure serum recovery of fluorescein isothiocyanate-dextran (FD-4) after SYD administration. An increase in serum recovery of FD-4 indicated the loss of epithelial barrier.
Epithelial barrier loss dysfunction model in vitro was established using Caco-2 cells (1×106) monolayer incubated LPS (0.5 ng/mL) for 3 days . Caco-2 monolayers were treated with LPS or Shaoyao Decoction medicated serum. Caco-2 monolayers without drug treatment served as normal control. Epithelial voltohmmeter was used to determine transepithelial electrical resistance (TEER) of Caco-2-plated filters. Decreased level of TEER implicated an increase in monolayer permeability and epithelial barrier loss in vitro.
2.7.Transmission electron microscopy
Rats were harvested and colonic tissue was fixed with 2.5% glutaraldehyde, followed by post-fixation in 2% osmium tetroxide with 1.6% potassium ferrocyanide in 0.1 mol/L sodium cacodylate. Colonic tissues were cut into 5-mm3 samples, stained with 2% uranyl acetate, dehydrated in ethanol, and embedded in eponate. Semi-thin sections (80 nm) were stained with hematoxylin and eosin, 2% uranyl acetate and lead citrate. Images were captured with a Hitachi H7600 TEM in the microscope core.
2.8.Flow cytometry analysis
Caco-2 cells were incubated with BODIPY 581/591 C11 (5µM) at 37°C in cell incubator for 30 min. Cells were subsequently washed, resuspended in PBS and transferred through a cell strainer for flow cytometry (The BD Accuri™ C6 Plus).
2.9. Small interference RNA-target silencing of GPX4
GPX4 siRNAs were introduced into Caco-2 cell line at a concentration of 30 nmol/L by transient transfection with Lipo2000 Transfection Reagent, following guidelines provided by manufacturer. At 48-hour post-transfection, the cells were collected for further studies. The effect of siRNA was evaluated by measuring the expression of GPX4 .
2.10 Luciferase assay
Caco-2 cells were co-transfected at day 2 with GPx4 promoter-driven luciferase fusion construct (pGL3-GPX4) and Renilla luciferase expression plasmid pRL-TK together with Lipo 3000 (Invitrogen) transfection reagent in serum-free medium. The promoter luciferase vector pGL3 basic was co-transfected with pRL-TK in control experiments for the determination of background.
2.11 MS analysis
Wogonoside, Paeoniflorin, Baicalin, Emodin, Liquiritin, Baicalein, Ferulic acid, Cinnamic acid, Gallic acid, Palmatine, Coptisine, Berberine, Dehydrocostus Lactone, Wogonin, Imperatorin, Arecoline, Coumarin were mixed at a concentration of 50 ng/mL. Chromatographic separation with MS was achieved using a programmed gradient mobile phase consisting of (A) 0.1% formic acid in water and (B) acetonitrile. The flow rate used was 0.3 mL/min and the injection volume was 4 µL for all the analyses.
2.12 Macromolecular docking
We molecularly docked five monomers (wogonoside, wogonin, palmatine, paeoniflorin and liquiritin) with GPX4. The small molecule structures were retrieved from the NCBI PubChem database (https://pubchem.ncbi.nlm.nih.gov), and the Compound CIDs were 3084961, 5281703, 19009, 442534, 503737. The 3D structure of GPX4 (PDB code: 5l71) was obtained from The Protein Data Bank (PDB; http://www.rcsb.org/pdb/). Ligand tools in Autodock4 are used to prepare ligands and receptors for AutoGrid, including removing existing ligands and crystal water from the target protein, adding hydrogen bonds and partial charges to small molecules, and identifying rotatable bonds that will be explored during docking. Then run autodock for automated molecular docking. The binding energy between the ligand and receptor is then obtained. The lower the value, the more stable the docking result. Finally, use PyMOL for graphing.
2.13 Statistical analysis
The animal experiments, in vitro experiments, and data analysis were conducted according to a single-blind study design. One-way analysis of variance was used for multiple group comparisons; multiple comparison between the groups was performed using S-N-K method. Two-tailed unpaired Student’s t-test was used to evaluate between two group comparisons. Data were expressed as means ± SD. The data were a normally distributed and the groups had equal variances. All experiments were repeated for at least three times. P-values less than 0.05 were considered statistically significant.