2.2. Experimental design of animal study
The animal protocol to characterize the constipation phenotype was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC) based on the ethical procedures for scientific care (Approval Number PNU-2020-2654). All ICR mice were maintained at the Pusan National University-Laboratory Animal Resources Center, accredited by the Korea Food and Drug Administration (KFDA) (Accredited Unit Number-000231) and The Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (Accredited Unit Number; 001525). All mice were provided ad libitum access to a standard irradiated chow diet (Samtako BioKorea Inc., Osan, Korea) and water. Throughout the experiment, mice were maintained in a specific pathogen-free (SPF) state under a strict light cycle (on at 08:00 h; off at 20:00 h) at 23 ± 2°C and 50 ± 10% relative humidity.
Briefly, 7-week-old ICR mice (n = 24) were assigned to either a 1x PBS treated group (Vehicle, n = 6) or MP treated group (MP, n = 18). The MP treated group was further divided into a low concentration MP treated group (LoMP, n = 6), medium concentration MP treated group (MiMP, n = 6), and high concentration MPs treated group (HiMP, n = 6). The three MP treated groups were orally administrated varying concentrations of dispersed MP solution (10 µg/L, 50 µg/L and 100 µg/L) once daily (0.5 mL/day), while the Vehicle treated group was administered the same volume of 1⋅ PBS solution. The physiological condition of all mice in each group was regularly monitored at 10 a.m. every day during the experimental periods; there were no occurrences of severely ill or dead animals. At 2 weeks after MP administration, total stools, urine, food and water were collected from the metabolic cage of each group for further analyses. All mice were subsequently euthanized using CO2 gas, after which the transverse colon and serum samples were acquired and stored at -70°C in Eppendorf tubes until assay.
2.8. Western blotting analysis
The Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Korea) was used to prepare total proteins from transverse colons and IEC18 cells of Vehicle and LoMP, MiMP, HiMP treated groups, according to the manufacturer’s protocol. Protein homogenates were subsequently centrifuged at 13,000 rpm at 4°C for 5 min, after which total protein concentrations were determined using a SMARTTM Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific Inc., Wilmington, MA, USA). Total proteins (30 µg) were subjected to 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. The membranes were then probed with the following primary antibodies, overnight at 4°C: anti-Gα (Abcam, Cambridge, UK), anti-mAChR M2 (Alomone Labs, Jerusalem, Israel), anti-mAChR M3 (Alomone Labs), anti-PKC (Cell Signaling Technology Inc., Cambridge, MA, USA.), anti-p-PKC (Cell Signaling Technology Inc.), anti-PI-3K (Cell Signaling Technology Inc.), anti-p-PI3K (Cell Signaling Technology Inc.), anti-ERK 1/2 (Cell Signaling Technology Inc.), anti-p-ERK (E-4)(Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-p38 (Cell Signaling Technology Inc.), anti-p-p38 (Cell Signaling Technology Inc.), anti- pNFκB (Boster Bio, CA, USA), anti-IκB-α (Cell Signaling Technology Inc.), anti- p-IκB-α (Cell Signaling Technology Inc.) or anti-β-actin (Sigma-Aldrich Co.). Membranes were subsequently washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20), followed by incubation with 1:1,000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) for 2 h at room temperature, after which the blots were developed using a Chemiluminescence Reagent Plus kit (Pfizer Inc., Gladstone, NJ, USA). Signal images of each protein were subsequently acquired using a digital camera (1.92 MP resolution) of the FluorChem® FC2 Imaging system (Alpha Innotech Corporation, San Leandro, CA, USA). Protein densities were semi-quantified using the AlphaView Program, version 3.2.2 (Cell Biosciences Inc., Santa Clara, CA, USA).
2.11. Quantitative Realtime – Polymerase chain reaction (RT-qPCR) analysis
Frozen transverse colon tissue and IEC18 cells was homogenized in RNA Bee solution (Tet-Test, Friendswood, TX, USA). Total RNA molecules were isolated by centrifugation at 15,000 rpm for 15 min, after which RNA concentration was measured by the Nano Drop Spectrophotometer (Allsheng, Hangzhou, China). About 5 µg of total RNA was annealed with 500 ng of oligo-dT primer (Thermo Fisher Scientific Inc.) at 70°C for 10 min. Complementary DNA (cDNA) was synthesized using the Invitrogen Superscript II reverse transcriptase (Thermo Fisher Scientific Inc.). qPCR was performed with the cDNA template obtained (2 µL) and 2× Power SYBR Green (6 µL; Toyobo Life Science, Osaka, Japan) containing specific primers as follows: AQP3 sense primer 5’-GGTGG TCCTG GTCAT TGGAA-3’ and antisense primer 5’-AGTCA CGGGC AGGGT TGA-3’; AQP8 sense primer 5’-TCGCT GGCAG TCACA GTGA-3’ and antisense primer 5’-TCCAA ATAGC TGGGA GATCC A-3’; MUC1 sense primer 5’-CGCCA GCCTT GAGTT TGTTT-3’ and antisense primer 5’-GAAGA AAGGA GCCCG AATGC-3’; MUC2 sense primer 5’-GCACA TTCCT TCGCA TCTTA AA-3’ and antisense primer 5’-AAAGC AAAGA ATGGA ACAGA AACTC-3’; Klf4 sense primer 5’-GGTGC AGCTT GCAGC AGTAA-3’ and antisense primer 5’-AAGTC TAGGT CCAGG AGGTC GTT-3’; CIC-2 sense primer 5’- CAGCA CATGC AAAAG CTAAG AAAA − 3’ and antisense primer 5’- GCGGA TAGAT GTCTC GGAGC TA -3’; CFTR sense primer 5’- TCTGC CGCGC AGCAA − 3’ and antisense primer 5’- GGTGT GAACG TCATC AGATC CA-3’; β-actin sense and antisense primers 5’-ACGGC CAGGT CATCA CTATT G-3’ and 5’-CAAGA AGGAA GGCTG GAAAA GA-3’, respectively. qPCR was performed for 40 cycles using the following sequence: denaturation at 95°C for 15 sec, followed by annealing and extension at 70°C for 60 sec. Fluorescence intensity was measured at the end of the extension phase of each cycle. Threshold value for fluorescence intensities of all samples was set manually. The reaction cycle at which the PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered as the threshold cycle (Ct). Expression of the target gene was quantified relative to the housekeeping gene β-actin, based on a comparison of the Cts at constant fluorescence intensity, as per the Livak and Schmittgen’s method [22].
2.12. Measurement of GI hormone concentrations
The concentrations of cholecystokinin (CCK) and gastrin were quantified using ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. Briefly, transverse colon tissues (50 mg) were homogenized in ice-cold 1× PBS (pH 7.2–7.4) using a glass homogenizer (Sigma-Aldrich Co.). Resultant tissue lysates were then centrifuged at 1,000 ⋅ g for 5 min at 4°C, after which the supernatant was collected for analysis. Specific antibodies for the two hormones (separately in each well) were added to the supernatant, with subsequent incubation for 1 h at 37°C, after which HRP-Streptavidin solution was added to the mixture and further incubated for 1 h at 37°C. This was followed by addition of the TMP One-Step Substrate Reagent and incubation for 30 min at 37°C. The reaction was terminated by addition of the stop solution. Finally, absorbance of the reaction mixture was read at 450 nm using the VersaMax Plate Reader (Molecular Devices, Sunnyvale, CA, USA).
2.13. Measurement of chloride ion concentration
The concentration of chloride ions in transverse colons was quantified using the chloride assay kit (Abcam Co.), according to the manufacturer’s instructions. Briefly, transverse colon tissue (10 mg) was homogenized in ice-cold 1× PBS (pH 7.2–7.4) using a glass homogenizer (Sigma-Aldrich Co.). Resultant tissue lysates were then centrifuged at 13,000 rpm for 5 min at 4°C, after which the supernatant was collected for analysis. After addition of chloride reagent (separately in each well), the supernatant was incubated for 15 min at room temperature. Finally, absorbance of the reaction mixture was read at 620 nm using the VersaMax Plate Reader (Molecular Devices).
2.14. Cell culture and MP treatment
IEC18 cells, intestinal epithelioid cell line, were purchased from ATCC (Manassas, VA, USA). They were grown in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Gyeongsan-si, Korea) supplemented with 10 % fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2. After reaching 70–80 % confluence, IEC18 cells were classified into four different groups; Vehicle, LoMP, MiMP and HiMP treated group. They were exposed to 10 µg/mL (LoMP), 50 µg/mL (MiMP) and 100 µg/mL (HiMP) for 24 h, while Vehicle treated group was received with dH2O of same volume. The cell morphology was also observed under a microscope (Leica Microsystems.) at 100× and 200× magnification. After then, total cells of each group were harvested for western blot and RT-PCR analyses.
2.15. Statistical analysis
Statistical significance was evaluated using the One-way Analysis of Variance (ANOVA) (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA), followed by Tukey post hoc t-test for multiple comparisons. All values are expressed as the means ± SD, and a p-value (p < 0.05) is considered statistically significant.