Animals and reagents
Healthy male C57BL/6 mice aged 7–8 weeks were purchased from the Experimental Animal Center of Nanjing Medical University; they weighed 22–25 g at the time of the experiment. In the center, the mice were kept in an specific-pathogen-free environment at 18℃–23℃ with good ventilation and a 12-h light and dark cycle. Five mice were kept in one cage. The mice were provided food and water ad libitum, and their bedding was replaced regularly. All mice had adapted to these conditions at least 7 days before the experiment began. All animal treatments were approved by the Animal Ethics Committee of Nanjing Medical University. Investigations were accomplished in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Every animal care and protocols of experiments were all approved by the Nanjing Medical University Committee on Animal Care (Permit Number: IACUC 2004006).Mice were sacrificed through cervical dislocation. BTW was provided by Nanjing Hospital of Chinese Medicine, which is affiliated with Nanjing University of Chinese Medicine. Berberine (BBR) was purchased from MedChemExpress (Stockholm, Sweden), and 5-aminosalicylic acid (5-ASA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
BTW decoction preparation
The four medicinal materials were accurately weighed and boiled twice in boiling water at a volume ratio of 1:10 for 1 h. The aqueous solution was passed through five layers of medical gauze, and the concentrated solution was combined into 1 g/ml liquid containing crude drugs followed by storage at 4 ◦C.(Table1)
High-performance liquid chromatography–mass spectrometry analysis
Esculin, aesculetin, jatrorrhizine, coptisine, palmatine hydrochloride, BBR, pulchinenoside A3, and pulchinenoside B4 were obtained from Shanghai Yuanye Biological Co. Ltd. (Shanghai, China). Because of the detection limit, two instruments were used for content determination. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS, Agilent Technologies, Santa Clara CA, USA) was performed to detect the amounts of esculin, aesculetin, jatrorrhizine, coptisine, palmatine hydrochloride, and BBR. Furthermore, a Thermo Fisher UHPLC-Q-Orbitrap HRMS was used to detect the amounts of pulchinenoside A3 and pulchinenoside B4.
The sample (1 mL) was precisely measured in a 10-mL volumetric flask, and methanol was added to 10mL. The flask was plugged, and ultrasonic dispersion was conducted for 30 min. It was then cooled and shaken well. Subsequently, 1.0 mL of the sample–methanol solution was extracted and added to 1.0 mL of water, mixed well, and filtered through a 0.22-μm microporous filter membrane. The filtrate was collected and used as the test product solution for the study. The top layer was used for UPLC-MS/MS analysis. Separation was achieved using an Agilent ZORBAX Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm). The column temperature was set to 30°C. The mobile phase was composed of methanol (phase A) and 0.1% formic acid aqueous solution (phase B), with the flow rate of 0.4 mL/min. The injection volume was 5 μL. The raw MS and tandem MS (MS/MS) data were collected using Agilent 6460 triple quadrupole mass spectrometer. The analytes were ionized using electrospray ionization (ESI) at positive and negative modes. The drying temperature was set to 350℃, the drying gas flow rate was 10 L•min–1, and the capillary voltages were 4000 V(+) and 3500 V(−) (Tables 2, and 3).
A sufficient amount of standard substance was accurately weighed, and pure methanol that can completely dissolve into 2.00 mg/mL reserve solution was added and reserved for use. Pulchinenoside A3 and B4 reserve solutions were diluted with pure methanol, and a series of standard solutions with concentration gradients were prepared for injection analysis. A T3 column (2.1 mm × 150 mm, 3 μm) was used. The mobile phases of 0.10% formic acid in acetonitrile and 0.10% formic acid in water were used for gradient elution (0–0.5 min, 2% A; 0.5–4 min, 2% A; 0.5–4 min, 98% A; 4–9 min, 98% A; 9–9.3 min, 2% A; and 9.3–10 min, 2% A); flow rate: 0.300 mL/min; injection volume: 5.0 μL; and the temperature of the Ultra-High Performance Liquid Chromatography(UHPLC) column was fixed at 45°C. The ion source was ESI, the carrier gas was nitrogen, the spray voltage was 3.2 kV(+), the capillary temperature was 300℃, the auxiliary heating temperature was 300℃, the resolution was 17 500, and the scanning range was 50.0–500.0 m/z (Tables 2 and 3).
Establishment of the dextran sulfate sodium–induced UC mouse model
Acute UC was induced in the mice through the administration of dextran sulfate sodium (DSS) in drinking water (3%, w/v) after 1 week of living in the Experimental Animal Center.. The mice were divided into the following five groups: control, DSS, 5-ASA (800 mg/kg), BBR (100 mg/kg), and BTW (5, 10, and 20 g/kg),6 mice/group . Mice in the treatment groups were administered 5-ASA and BTW through gavage starting from the day of DSS treatment. The control and DSS-treated groups were provided water and 3% DSS, respectively. Mice were sacrificed after 12 days of DSS administration. After the mice were killed, colon segments from the anus to the cecum were removed, and the colon lengths were measured. Colons were rinsed with phosphate buffered saline (PBS), placed in cryopreservation tubes, rapidly frozen in liquid nitrogen for 24 h, and then stored at −80°C until analysis.
Histological analysis
Colon sections (approximately 1 cm long) containing the lesion site were fixed with 4% neutral formalin. Then, conventional paraffin embedding, sectioning, and hematoxylin and eosin staining were performed. Histological changes in pathological sections were observed under a light microscope. The evaluation criteria were previously described[8].
Enzyme-linked immunosorbent assay
Serum was obtained from mice. The concentrations of inflammatory cytokines (IL-6 and IL-1β) were quantified using enzyme-linked immunosorbent assay (ELISA) kits from Elabscience (China). Furthermore, HMGB1 was quantified using ELISA kits from IBL (USA).
Immunohistochemistry
Colon tissue sections were dewaxed in water and washed thrice with 0.01 M PBS (5 min each time). Sections were treated with a 0.3% H2O2 and 0.5% Triton X-100 mixture at room temperature for 30 min. For antigen repair, the sections were treated with 0.05 M PBS solution, cooled to room temperature, and heated to 100°C for 20 min in a microwave oven. Nonspecific antigens were blocked with normal sheep serum and kept in a 37°C water bath for 30 min. The sections were incubated with primary antibody incubated at 4°C overnight. They were then washed thrice with a 0.01 M PBS solution (5 min each) and subsequently incubated with secondary antibody for 1 h in at 25°C, washed thrice with a 0.01 M PBS solution (5 min each), and subjected to diaminobezidin(DAB) color rendering. They were restained with hematoxylin for 3–5 min. Then, they were examined under a microscope, and images were acquired and analyzed. Nuclei stained with hematoxylin were blue, and the positive expression of DAB was indicated in brown{Xuan-Qing, 2020 #2591}.
Malondialdehyde and superoxide dismutase analysis
Colon tissues from five mice in each group were collected and placed in an ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The colon tissue were then homogenized in a Polytron homogenizer (Retech GmbH mm400, Germany) followed by centrifugation at 3000 rpm/min for 15 min; supernatant was collected for superoxide dismutase (SOD) and malondialdehyde (MDA) measurement.
An MDA detection kit (A003; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was selected to determine the MDA level as a marker of lipid peroxidation. The assay was conducted according to the manufacturer’s instructions[9]. An SOD detection kit (A001; Nanjing Jiancheng Bioengineering Institute) was used for SOD measurement. The assay was conducted according to the manufacturer’s instructions.
Western blot analysis
Colon tissues were prepared using RIPA lysis buffer. The protein concentration was determined using a bicinchoninic acid protein assay, and 20 μg of protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Subsequently, membranes were blocked with 5% BSA in tris-buffered saline with 0.1% Tween 20 detergent and incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary antibodies for 90 min. Finally, the membranes were chemically exposed and semiquantitatively analyzed with Quantity One software (Bio Rad){Xuan-Qing, 2020 #2591}.
Statistical analysis
All data are expressed as means and standard deviations. GraphPad Prism 5 software was used to analyze all data. Differences between each group were analyzed using one-way analysis of variance. p values < 0.05 were considered significant (#p < 0.05 and ##p < 0.01 vs the control group; *p < 0.05 and **p < 0.01 vs the DSS group).