5 aminosalicylic acid 5 asa

Male Sprague–Dawley rats (Orient Bio Inc., Seongnam, Korea) weighing at 250 ± 10 g at the time of arrival were acclimated to individual cages for one week. In their cages, the animals had free access to standard rat chow and water. The cages were maintained in a temperature-controlled room at 22°C ± 2°C with a 12 h light/dark cycle. The rats were assigned to various treatment groups (n = 10 per treatment). Mild/moderate colitis was induced by giving rats 5% DSS (MP Bio, Santa Ana, CA, USA) dissolved in drinking water for 7 days. The treatments were initiated 4 days after the first dose of DSS and continued for 10 days. The treatments were once-a-day oral administrations of AD-lico™ at 25/50/100 mg/kg body weight (BW), 5-aminosalicylic acid (5-ASA; Sigma-Aldrich, St. Louis, MO, USA) at 50 mg/kg BW, AD-lico™/5-ASA combination at 25 and 50 mg/kg BW, respectively, and vehicle (water). The rats were checked daily for colitis development by monitoring body weight, gross rectal bleeding, stool consistency and survival. Rats were sacrificed after the final DSS administration. The protocols on treatment of the laboratory animals were approved by the Institutional Animal Care and Use Committee (IACUC) in the laboratory.

All experimental protocols (CBNU2015-089) were approved by the Committee on the Care of Laboratory Animal Resources, Chonbuk National University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Acute colitis was induced by administering drinking water containing 5 % (w/v) DSS (MP Bio, Santa Ana, CA, USA) to mice for 10 days. Thirty mice were weighted before the experiment, and were divided into five groups with six mice per group. Group 1 comprised untreated mice. Mice in group 2 received DSS. In group 3 and group 4, mice with induced colitis were treated with GR 100 mg/kg or 50 mg/kg, respectively. In group 5, mice with induced colitis were treated with 50 mg/kg 5-aminosalicylic acid (5-ASA; Sigma-Aldrich, St. Louis, MO, USA) as the reference drug. Mice were checked daily for body weight, stool consistency and the presence of gross bleeding. GR was diluted with purified water and orally administered once a day during the 10 days of DSS treatment, after which the mice were sacrificed.

GL-V9 (C₂₄H₂₇NO₅, MW: 409.47), prepared from Dr. Zhiyu Li (China Pharmaceutical University, China), was dissolved in dimethylsulfoxide (DMSO) to 100 mM and stored at −20°C, and freshly diluted with Dulbecco's Modified Eagle Medium (DMEM, GIBCO, Carlsbad, CA) to the final concentration in vitro study. In vivo study, GL-V9 was prepared as intragastric administration (0.5% sodium carboxyl methyl cellulose (CMC)) by Dr. Xue Ke from college of pharmacy, China pharmaceutical university. The DSS-treated group of mice were administered 0.5% CMC as vehicle.
LPS (E. coli: Serotype O55:B5) and 5-Aminosalicylic acid (5-ASA) were purchased from Sigma-Aldrich (St. Louis, MO). Dextran sulfate sodium (DSS, molecular weight 36–50 kDa) was brought from MP Biomedicals Inc. (Irvine, CA, USA). Diamidino-phenyl-indole (DAPI) were from Sigma (St. Louis, Missouri). Bovine serum albumin (BSA) was purchased from Roche (Mannheim, Germany).

Sulfasalazine, 5-aminosalicylic acid (5-ASA, mesalamine), sulfapyridine, and capsaicin were purchased from Sigma (St. Louis, MO). 5′-Iodoresiniferatoxin (I-RTX) was purchased from Tocris Cookson (Ellisville, MO). Clostridium difficile toxin A was purchased from TechLab, Inc. (Blacksburg, VA).

DSS (molecular weight 36–50 kDa) was purchased from MP Biomedicals Inc. (Irvine, CA, USA). The 5-aminosalicylic acid (5-ASA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Quercetin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for mouse interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were purchased from BioLegend (San Diego, CA, USA). Hematoxylin and eosin (H&E) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The supplier of diaminobenzidine was Solarbio Science & Technology Co., Ltd. (Beijing, China). The bicinchoninic acid protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against PI3K p85, IL-6 and β-actin were obtained from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against IL-1β and TNF-α were purchased from Bioworld Technology (St. Louis Park, MN, USA). Antibodies against phospho-Akt (Ser473) and Akt (C67E7) were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-occludin antibody was obtained from Proteintech Group (Wuhan, China). Transwell inserts (pore size of 0.4 μm) were purchased from Corning Inc. (Kennebunk, ME, USA).

XLP was purchased from the Hubei Xianglian Pharmaceutical Co., Ltd. (Z42020398, Hubei, China, the compositions were shown in Table 1). Standard solutions of berberine, jatrorrhizine, costunolide, coptisine, and palmatine were purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China, cat# B21449, B21451, B20891, B21438, B21433). DSS (36,000–50,000 MW) was purchased from MP Biomedicals (Santa Ana, CA, United States, cat# 160110). 5-aminosalicylic acid (5-ASA) was purchased from Sigma-Aldrich (St. Louis, MO, United States, cat#A79809). 3-methyladenine (3-MA) was purchased from Selleck (Huston, TX, United States, cat#S2767).

The animals were divided into seven groups (five mice/group) according to the treatment: NOR group (normal control), DSS group [5% DSS (MP Biomedicals, Irvine, CA, USA)], PC group [positive control; 5% DSS + 50 mg/kg/day of 5-aminosalicylic acid (5-ASA; Sigma-Aldrich, St. Louis, MO, USA)], S1 group (5% DSS + 500 mg/kg/day of S1 sample), S2 group (5% DSS + 500 mg/kg/day of S2 sample), S3 group (5% DSS + 500 mg/kg/day of S3 sample), and S4 group (5% DSS + 500 mg/kg/day of S4 sample). The NOR and DSS groups were administered distilled water. Cheonggukjang samples (S1–S4) were orally administered at a dose of 500 mg/kg once a day for 15 days. To induce colitis, mice were administered 5% DSS in drinking water for seven days, and then sacrificed after one day.