Qing-dai powder promotes recovery of colitis by inhibiting inflammatory responses of colonic macrophages in dextran sulfate sodium-treated mice

DSS (molecular weight: 36,000–50,000 Daltons) was purchased from MP Biologicals (Santa Ana, USA). Dimethylsulphoxide (DMSO), lipopolysaccharide (LPS) (Escherichia coli Serotype 055:B5), sulfasalazine (SASP) (purity ≥ 98 %), hexadecyltrimethylammonium bromide, hematoxylin, eosin, O-dianisidine dihydrochloride, protease inhibitor cocktails and hydrogen peroxide were purchased from Sigma-Aldrich (St, Louis, MO, USA). Fetal bovine serum, l-glutamine, penicillin, streptomycin and RPMI 1640 cell culture medium were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against COX-2, iNOS, IκB α and p65 and β-actin were supplied from Cell Signaling Technology, Inc. (Beverly, MA, USA). TNF-α, IL-1β, and IL-6 ELISA kits were purchased from eBioscience (San Diego, CA, USA). Cy5.5 PerCP anti-mouse CD11b, FITC anti-mouse F4/80 were purchased from BD Pharmingen (San Diego, CA, USA). FITC-conjugated donkey anti-rabbit IgG antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). WesternBright™ ECL was supplied from Advansta (Menio Park, CA, USA).

The ratio of I. naturalis and dried alum in QDP is 2:1. Two Chinese medicinal raw herbs were obtained from the dispensary of Traditional Chinese Medicine, Clinical Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong. These two Chinese medicinal materials were authenticated by Dr. Hu-Biao Chen (School of Chinese Medicine, Hong Kong Baptist University, Hong Kong) according to the Chinese Pharmacopoeia (version 2010) and other references [23‐25]. Voucher specimens (nos. TCM-0110-Q01, TCM-0110-Q02) are stored in our Research Laboratory, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong. Two herbs were mixed, powdered to homogeneous size in a mill and sieved through a 120-mesh filter. Each sample was exactly weighed, ultrasonically extracted with DMSO in a KQ-2200DB ultrasonic cleaner bath (Kunshan Ultrasound Instrument Co., Ltd., Jiangsu, China), and filtered through a syringe filter for subsequent UPLC-QTOF-MS analysis and cell culture treatment. For animal study, QDP was freshly suspended in 0.5 % sodium carboxymethylcellulose (CMC-Na) in distilled water prior to oral feeding to mice.

The components in QDP were identified by UPLC-QTOF-MS. Chromatographic separation was performed by an Agilent 1290 Infinity UPLC system (Santa Clara, CA, USA), equipped with a binary solvent delivery system, a standard auto-sampler and photodiode array detectors (DAD). A 100 mm × 2.1 mm ACQUITY BEH C₁₈ 1.7-µm column (Waters Corp., Milford, MA, USA) was used to separate the components of QDP. The mobile phase consisted of (A) 0.1 % formic acid in water and (B) 0.1 % formic acid in acetonitrile. A linear gradient was optimized as follows (flow rate, 0.40 mL/min): 0–2.5 min, 2–5 % B; 2.5–10 min, 5–35 % B; 10–20 min, 35–75 % B; 20–23 min, 75–100 % B; 23–26 min, 100 % B; 26–26.1 min, 100–2 % B; 26.1–30 min, 2 % B. The injection volume was 2 μL and the column temperature was maintained at 40 °C in each run. Mass spectrometry was performed by an Agilent 6540 ultra-high definition (UHD) QTOF mass spectrometer, equipped with a Jet Stream electrospray ionization (ESI) source. Parameters for the Jet Stream technology were set with the superheated nitrogen sheath gas temperature at 350 °C and with a flow rate at 10 L/min. ESI conditions were set as follows: negative ion mode, capillary 4500 V, nebulizer 1.85685 × 10⁶ kPa, drying gas 8 L/min, gas temperature 300 °C, nozzle voltage 300 V, skimmer voltage 65 V; octapole RF peak 600 V, fragmentor 175 V. Mass spectra were recorded across the range m/z 100–1700 with accurate mass measurement of all mass peaks. A sprayer with a reference solution was used for continuous calibration in negative ion mode with reference masses at m/z 112.9856 and 966.0007. The full-scan and MS/MS data were processed with Agilent Mass Hunter Workstation software (version B.02.00) (Santa Clara, CA, USA).

RAW264.7 murine macrophage cells were obtained from the American Type Culture Collection (ATCC No. TIB-71). The cell line was cultured in RPMI 1640 cell culture medium supplemented with 10 % (v/v) fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin. The cells were incubated in a humidified 5 % CO₂ incubator at 37 °C.

Seven to eight-week-old male C57BL/6 mice weighing 20–24 g were purchased from the Laboratory Animal Services Center, The Chinese University of Hong Kong. The animals were fed a standard rodent diet with free access to water, and were kept in rooms maintained at 21–23 °C with a 12 h light/dark cycle following international recommendations. All experimental protocols were approved by the Animal Ethics Committees of Hong Kong Baptist University, in accordance with “Institutional Guidelines and Animal Ordinance” (Department of Health, Hong Kong Special Administrative Region).

Acute colitis was induced by oral administration of 2.0 % (w/v) DSS dissolved in drinking water, for 5 days according to Wirtz et al. [26]. Mice of each experimental group were monitored every day to confirm that they consumed equal volumes of DSS-containing water.

Two sets of experiments were performed. For the first one, 50 colitic mice were arbitrarily allocated into 5 groups: DSS model group, sulfasalazine (SASP, positive reference agent)-treated group, and three QDP-treated groups (n = 10). A vehicle control group with nine normal mice received drinking water without DSS throughout the entire experimental period. Consistent with clinical treatment, QDP was administrated orally to colitic mice at doses of 0.77, 1.54 or 3.08 g/kg/day, comparable with the clinical dosages used in human UC patients. SASP was used as a positive reference agent and it was given at 0.20 g/kg/day according to Kim et al. [27]. The gavage volume was 0.4 mL.

For the second set of experiments to immunophenotype colonic macrophages in colonic lamina propria, twelve colitic mice were arbitrarily allocated into 2 groups: DSS model group (n = 6) and 1.54 g/kg QDP-treated group (n = 6). A vehicle control group of 5 normal mice received drinking water only during the entire experimental period. Both SASP and QDP were dissolved in 0.5 % sodium carboxymethylcellulose (CMC-Na) solution and administrated orally to the mice for 7 days after the onset of colitis. The vehicle control group and DSS model group were fed with 0.4 mL of 0.5 % CMC-Na solution instead of SASP or QDP.

Body weight, stool consistency and rectal bleeding were recorded daily. The DAI was determined by combining the scores of (1) body weight, (2) stool consistency and (3) rectal bleeding [28].

Colon tissues were harvested and fixed in 4.0 % paraformaldehyde. Tissue sections were prepared by conventional tissue processing methods, stained with hematoxylin and eosin (H&E), and examined under the light microscope. Colonic damage was assessed as described previously [28].

MPO activity was measured as described in our previous study [28]. One unit of MPO activity was defined as the amount of enzyme present that produced a change in optical density of 1.0 U/min at 25 °C in the final reaction volume. The results were normalized to equal protein levels and quantified as units/mg protein.

Colonic tissues were fixed in 4.0 % buffered paraformaldehyde, embedded in paraffin and sectioned into 5-μm-thick slices. Sectioned samples were deparaffinized in xylene, rehydrated in a series of graded alcohol, and subjected to antigen retrieval. Antigens were retrieved by incubation with protease K solution (1: 100) for 20 min.

Endogenous peroxidase was quenched with 3.0 % hydrogen peroxide in methanol for 30 min. Sections were further blocked with 3.0 % bovine serum albumin (BSA) in PBS, exposed to 0.5 % Triton X-100 for 1 h for reducing nonspecific antibody binding and incubated with mouse F4/80 antibody (AbD serotec, Raleigh, North Carolina, USA) at 4 °C overnight. The sections were washed with PBS three times, incubated with biotinylated anti-rabbit immunoglobulins, followed by peroxidase-labeled streptavidin, and 3, 3′-diaminobenzidine chromogen substrate added to make antibody binding visible according to the protocol of the LSAB kit (LSAB-DAKO, Copenhagen, Denmark). Sections were then washed with PBS and counterstained with hematoxylin. After dehydration with a series of increasingly concentrated ethanol, sections were mounted with neutral gum. Five random fields at 400× magnification were counted in each sectioned sample by a researcher blinded to the treatment. The number of macrophages per μm² of mucosa was quantified by the Image J software (National Institutes of Health, Bethesda, Maryland, USA).

Colonic lamina propria cells were isolated as previously described with slight modifications [29, 30]. The colons were removed from mice immediately after euthanasia. Subsequently, the colonic tissues were immersed in DMEM containing 100 U/mL of penicillin and 100 μg/mL of streptomycin, dissected longitudinally and cut into 0.5–1-cm pieces. Then, intestinal epithelial cells were dissociated by incubation with Ca²⁺ and Mg²⁺-free HBSS containing 5 mM EDTA and 1 mM DTT for 20 min at 37 °C, twice. After thorough washing with PBS, colonic tissues were then incubated with digestion buffer (RPMI 1640 containing 3 mg/mL dispase II, 0.5 mg/mL collagenase D, and 0.5 mg/mL DNase I) for 30 min at 37 °C, twice. Finally, the cells were labeled with anti-CD11b and anti-F4/80 antibodies, and analyzed by flow cytometry. Appropriate isotype-matched IgGs were used as negative controls.

The cytokines TNF-α, IL-1β and IL-6 in the culture supernatants of RAW264.7 cells and colonic tissues and the chemokine monocyte chemoattractant protein-1 (MCP-1) in serum samples were measured with TNF-α, IL-1β, IL-6 and MCP ELISA kits, respectively (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

After treatment with a range of concentrations of QDP (0.3–3 μg/mL) in the presence or absence of 1 μg/mL LPS, RAW264.7 cells were analyzed by immunoblotting as described previously [31]. Primary antibodies that recognize iNOS, COX-2 and IκB-α were used, and specific proteins were detected by WesternBright™ ECL. Equal loading was assessed by a β-actin antibody, and the amount of total protein present was normalized to the level of β-actin.

RAW264.7 cells were cultured directly on glass cover slips in a 35-mm dish for 24 h and pretreated with QDP for 1 h in the presence or absence of 1 μg/mL LPS. Cells were fixed with 4 % paraformaldehyde, treated with 0.2 % Triton X-100, and blocked with 3 % BSA. Subsequently, cells were incubated with a rabbit anti-NF-κB p65 polyclonal antibody (1:100) at 4 °C overnight. After extensive washing with PBS, cells were further incubated with a secondary FITC-conjugated donkey anti-rabbit IgG antibody (1:100) for 1 h at room temperature. Nuclei were counterstained with DAPI solution (1 μg/mL), and cells were analyzed by a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Under the optimized conditions [32], the major components in QDP were well separated and detected within 30 min (Table 1). Taking retention time (tR), m/z, ultraviolet (UV) absorption characteristics (lambda max), and relevant references into consideration, 27 peaks representing individual chemical components were identified. The representative chromatograms, monitored by DAD and MS, are shown in Fig. 1.

Oral administration of 2.0 % DSS in drinking water resulted in high mortality; up to 30.0 % of the total mice in the DSS model group died. QDP treatment could reduce the mortality rate of DSS-treated mice, to 20.0, 10.0 and 10.0 % for low-, medium- and high-dose QDP-treated groups, respectively (Fig. 2a). Starting from day 5, DSS resulted in rapid loss of body weight and serious clinical disease symptoms (diarrhea and occult fecal blood) in C57BL/6 J mice, and lasted 3–4 days. After that, mice began to recover. QDP treatment improved body weight recovery and DAI of DSS-treated mice, particularly in the medium- and high-dose QDP treatment groups when compared with the DSS model group (both P < 0.001) (Fig. 2b, c). In addition, the DSS-induced model of colitis is associated with a marked decrease in colon length [28]. Treatment with QDP significantly prevented colon shortening in a dose-dependent manner (P = 0.020, P = 0.012, P = 0.001, respectively) (Fig. 2d).

As shown in Fig. 3, 2.0 % DSS in drinking water caused extensive colonic tissue damage, including inflammatory cell infiltration, lesion formation and crypt destruction. Mice receiving QDP treatment showed less colonic damage. Scores of the histological changes are displayed in Fig. 3g. The score was significantly higher for the DSS model group than that for the vehicle-treated control group, i.e., reducing inflammation and mucosal and crypt damage in the colon, in particular in those mice treated with medium and high doses of QDP (both P < 0.001). Consistent with the histological scores, colonic MPO activity was greatly increased in the DSS model group (P < 0.001), whereas the MPO activities in the QDP-treated groups were significantly suppressed in a dose-dependent manner (Fig. 3h).

The infiltration of macrophages in the colon was analyzed with the macrophage marker F4/80. The number of macrophages in the colons of model group mice was significantly higher than in the normal group (P < 0.001) (Fig. 4A). Compared with the model group, there were considerably fewer macrophages in the colons of QDP-treated mice (P = 0.003, P < 0.001 and P < 0.001 for 0.77, 1.54 and 3.08 g/kg QDP, respectively). Similar to the results of the immunohistochemical study, the number of macrophages was increased after DSS treatment (P < 0.001), and QDP at 1.54 g/kg significantly decreased the recruitment of macrophages to the colon lamina propria of the DSS-treated mice (P = 0.004) in flow cytometry (Fig. 4B).

Levels of macrophage-associated proinflammatory cytokines such as TNF-α, IL-1β and IL-6 were measured in the colonic tissues to ascertain the suppressive effects of QDP on colonic macrophages. The levels of colonic TNF-α, IL-1β and IL-6 were significantly higher in the DSS model group (all P < 0.001), and QDP treatment significantly lowered the production of TNF-α, IL-1β and IL-6 in colonic tissue of DSS-treated mice (Fig. 5a–c). Oral administration of 2.0 % DSS in drinking water resulted in a sharp increase of MCP-1 in the serum of DSS-treated mice. Treatment with QDP greatly reduced serum MCP-1 levels in a dose-dependent manner (Fig. 5d).

Macrophages are a major source of proinflammatory cytokines, including TNF-α, IL-1β and IL-6. We therefore evaluated the anti-inflammatory effect of QDP on LPS-stimulated RAW264.7 cells (murine macrophage cell line). Treatment with LPS resulted in significantly increased production of proinflammatory cytokines (by 2.0-fold for TNF-α; by 14.3-fold for IL-6) (both P < 0.001) in culture supernatants of RAW264.7 cells, while QDP at a concentration of 1 μg/mL significantly inhibited LPS-induced production of TNF-α and IL-6 (both P < 0.001) (Fig. 6a).

The activities of iNOS and COX-2 are the immediate modulators of NO and PGE₂, respectively, produced by macrophages for the initiation and progression of IBD [33]. We investigated the effects of QDP on the expression of iNOS and COX-2 by Western blot analysis. Protein levels of iNOS and COX-2 were upregulated in response to LPS (Both P < 0.001), and treatment with QDP resulted in dose-dependent inhibition of iNOS and COX-2 protein expression (Fig. 6b).

We examined the effect of QDP on LPS-induced degradation of IκB-α by Western blotting. The degradation of IκB-α after LPS treatment was inhibited by QDP in a dose-dependent manner (Fig. 7a).

Furthermore, we investigated the effect of QDP on LPS-induced p65 nuclear translocation by immunofluorescence. p65 was normally sequestered in the cytoplasm, while LPS induced p65 accumulation in the nucleus. However, pre-treatment with QDP abolished LPS-induced p65 translocation to the nucleus (Fig. 7b).