Experiments on AnimalsAnimals

Albino male Sprague–Dawley rats, 200–225 g in body weight, were employed throughout the study. The animals were fed with standard laboratory chow and tap water ad libitum and were given at least 1 week to acclimatize after their delivery to the animal facility. They have been housed, three in a cage, in temperature-controlled rooms at 22–24 °C and 50–60% humidity with a 12-h light cycle. Animal care and handling were in accordance with the provisions of the European Community Council Directive 210/63/UE, recognized and adopted by the Italian Government. The experiments have been approved by the Ethical Committee for Animal Experimentation of the University of Pisa and by the Italian Ministry of Health (authorization n 674/2016-PR).

Induction of Colitis and Drug Treatments

Rats were weighed and randomly distributed into separate experimental groups of six rats in each. Colitis was induced in accordance with the method described previously by Pellegrini et al., in 2018 [17]. Briefly, during a short anesthesia with isoflurane (Abbott Labs, Pomezia, Italy), 15 mg of 2,4-dinitrobenzenesulfonic acid (DNBS) in 0.25 ml of 50% ethanol has been administered intrarectally via a polyethylene PE-60 catheter inserted 8 cm proximal to the anus. Control rats received 0.25 ml of 50% ethanol. Following the DNBS administration, rats developed full scale colonic inflammation that became evident 6 days later.

Immediately after the DNBS treatment, test drugs (NC-2600 3, 10, 30 mg/kg; NP-1815-PX 3, 10, 30 mg/kg, and Dexamethasone (DEX) 1 mg/kg) were orally administered to the animals for 6 days. DNBS-untreated animals (control group) and DNBS-treated rats (DNBS group) received only methocel (drug vehicle). When these treatments were initiated, at the same time, we started the daily monitoring of body weight of the rats.

The doses of NC-2600 (3, 10, 30 mg/kg) and NP-1815-PX (3, 10, 30 mg/kg) were selected based on our preliminary experiments designed to measure the effects of increasing doses of these compounds on food intake, body weight, spleen weight, and macroscopic score in the model of DNBS-induced colitis. Effective dosing of NC-2600 (10 mg/kg) and NP-1815-PX (10 mg/kg) was then chosen to further study the effects of these compounds on inflammasome pathway and tight junction expression. The dose of DEX was selected according to a previous study using a rat model of colitis [18]. Macroscopic and histological scores were assessed on the entire length of colon, while biochemical assays were performed on colonic segments collected from inflamed regions adjacent and distal to grossly necrotic tissue areas.

Assessment of Colitis

Colonic tissue samples were graded for macroscopic damage, as reported previously [17]. The criteria for macroscopic grading of colonic damage were as follows: (1) consistency of colonic fecal material (0, formed; 1, loose; 2, liquid stools); (2) presence of abdominal adhesions between colonic tissue and other organs (0, none; 1, minor; 2, major adhesions); and (3) presence of mucosal ulceration (0, none; 1, hyperemia; 2, ulceration without hyperemia; 3, ulceration with inflammation at one side; 4, ≥ 2 sites of ulceration and inflammation; 5, major sites of damage; and 6, major sites of damage extending > 2 cm). The score was then increased by one unit for each millimeter of colonic wall thickness. All parameters of macroscopic damage were recorded and scored for each rat by two observers blinded to the treatment. At the time of experiment, the weight of each spleen was also measured.

Microscopic Tissue Injury Scores

Microscopic tissue injury and inflammation were assessed by light microscopy on hematoxylin/eosin-stained histological sections obtained from whole gut specimens. In detail, upon removal, colonic tissue samples were cut longitudinally, rinsed with cold PBS, and immediately placed in 10% formalin solution for 24 h. Afterward, colon samples were gradually dehydrated with ethanol, embedded into paraffin blocks, and sliced longitudinally into three consecutive serial 7–8 μm sections. Each slice was placed on a glass slide and routinely processed for staining with hematoxylin and eosin. The slices were cut at two different points of the block, starting from the upper towards lower levels. The histological criteria included mucosal architecture loss (0–3), cellular infiltration (0–3), muscle layer thickening (0–3), crypt abscess (0, absent; 1, present), and goblet cell depletion (0, absent; 1, present). All parameters of histological damages were recorded and scored for each rat by two observers blinded to the treatment.

Cytokine Assays

Tumor necrosis factor (TNF) and interleukin (IL)-1β were measured with enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, Camarillo, CA), as described previously [19, 20]. For this purpose, colonic tissue samples (40 mg), stored previously at −80 °C, were weighed, thawed, and homogenized in 0.4 ml of PBS, pH 7.2 and centrifuged at 13,400 g for 20 min. Aliquots (100 μL) of the supernatants were then used for assay. The concentrations of TNF and IL-1β were expressed as picograms per milligram of tissue protein.

Protein Extraction and Western Blot Analysis

The colon was collected from rats and flushed of fecal content with ice-cold phosphate-buffered saline (PBS), as described previously [21, 22]. Tissues were minced and homogenized using a Potter–Elvehjem Grinder homogenizer on ice in 20% (w/v) TNE lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 1% NP-40, 1% SDS, 0.1% DOC) with various protease and phosphatase inhibitors. Samples were then sonicated and boiled for 5 min at 95 °C. Protein concentrations were measured with the Bradford assay. Total lysates were run on 4–20% Criterion™ TGX™ Precast Midi Protein Gels (Bio-Rad, Hercules, CA, USA) and then transferred to PVDF membranes (Trans-Blot TurboTM PVDF Transfer packs, Biorad). Then, membranes were blocked with 3% bovine serum albumin (BSA) diluted in Tris-buffered saline (TBS, 20 mM Tris–HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against caspase-1 (ab1872, Abcam) and occludin (ab167161, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by the IBright Analysis software.

Caspase-1 Activity Assay

Caspase-1 activity in colonic tissues was measured by using the colorimetric protease assay kit (BioVision Research Products, Mountain View, CA), following the protocol provided by the manufacturer. For this purpose, colonic tissue samples (40 mg), stored previously at −80 °C, were weighed, thawed, and homogenized in 0.4 ml of PBS, pH 7.2 and centrifuged at 13,400 g for 20 min. Aliquots (100 μL) of these supernatants were then used for the assay.

Experiments on Cell CulturesCell Culture

Human monocytic THP-1 cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 100 unit/ml penicillin–streptomycin at 37 °C in a humidified atmosphere of 5% CO2.

Cell Treatments

THP-1 cells were seeded at density of 5 × 105 cells/well in 24-well plates and treated with phorbol 12-myristate 13-acetate (PMA, 0.5 µM). After 3 h, the medium was removed, fresh media was added, and cells were incubated overnight (37 °C, 5% CO2).

In the first series of experiments, lipopolysaccharide (LPS)-primed (1 µg/mL, 3 h) cells were treated for 1 h with vehicle (0.03% dimethyl sulfoxide, DMSO) before the addition of ATP (5 mM, 1 h).

In the second series of experiments, LPS-primed (1 µg/ml, 3 h) cells were treated for 1 h with NC-2600 (0.03–0.3–3 µM, 1 h) before the addition of ATP (5 mM, 1 h).

In the third series of experiments, LPS-primed (1 µg/ml, 3 h) cells were treated for 1 h with NP-1815-PX (0.1–1–10 µM, 1 h) before the addition of ATP (5 mM, 1 h). Controls were run in parallel.

A schematic representation of all treatments is shown in Fig. 1.

Fig. 1

Schematic representation of the design of experiments on human monocytic THP-1 cells. THP-1 cells were treated for 3 h with lipopolysaccharide (LPS, 1 µg/mL). Then, cells were incubated for 1 h with vehicle (0.03% dimethyl sulfoxide, DMSO), NC-2600 (0.03–0.3–3 µM) or NP-1815-PX (0.1–1–10 µM) before the addition of ATP (5 mM, 1 h). At the end of experimental protocol, cells were lysed for analysis of inflammasome subunits and caspase-1/-4/-5 and -8 expression and the culture media were collected for analysis of IL-1β release.

After LPS stimulation, THP-1cells were lysed, and the medium were collected and centrifuged for 5 min at 800 rpm to obtain cell-free supernatants. Cell supernatants were used to evaluate IL-1β release, while cells were lysed for analysis of inflammasome subunits and caspase-1/-4/-5 and -8 expression.

Western Blot

Cells were lysed as previously described [23, 24]. Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris–HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (ab8227, Abcam), nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (D2W8U, Cell Signaling), caspase-1 (ab1872, Abcam), caspase-4 (#4450, Cell Signaling), caspase-5 (#46680, Cell Signaling), and caspase-8 (ab227430, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by the IBright Analysis software.

Assessment of IL-1β Release in THP-1 Cells

The release of IL-1β was measured by ELISA kit (R&D system), as previously described [25]. After cell stimulation, the medium was collected and centrifuged for 5 min at 800 rpm to obtain cell-free supernatants. Aliquots of 100 μL were used for the test. IL-1β concentration was expressed as percentage versus THP-1 control cells (Ctrl).

Drugs and Reagents

ATP, DEX, DMSO, DNBS, FBS, LPS, PBS, and PMA were purchased from Sigma Aldrich (St. Louis, MO, USA). The synthesis of NC-2600 and NP-1815-PX were performed by Nippon Chemiphar Co., Ltd. For cell culture experiments, we dissolved the compound NC-2600 in sterile DMSO and subsequently diluted it in culture medium. For the experiments on animals, solid dispersion form of NC-2600 was used, as NC-2600 is hardly dissolved.

Statistical Analysis

Data are presented as mean ± SEM and analyzed by the GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significances were determined by one-way ANOVA followed by Tukey’s post hoc test. A P value < 0.05 was considered statistically significant.