Male adult BALB/c mice, aged 8–10 weeks and weighing 20-24g (Aoyide Co., Tianjin, China), were caged in a comfortable experimental condition in the Animal Care Facility, Tianjin General Surgery Institute (Tianjin, China). Mice were provided with 1 week to adapt the new surroundings and free access to ample tap water and mouse food constantly. Total experiments were fulfilled based on the protocols approved by the Animal Care and Use Committee of Tianjin Medical University (Tianjin, China), according to the Chinese Council on Animal Care guidelines.
Primary human ERCs were isolated from menstrual blood by a density gradient centrifugation method in accord with previous study. In brief, the mononuclear cells were firstly separated from menstrual blood and then suspended in the Dulbecco’s modified Eagle’s medium (DMEM) high glucose which was supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Then cells were seeded in 10 cm dishes and cultured at the 37℃ 5% CO2 condition incubator. ERCs would adhere to the bottom of dishes after overnight incubation and the culture medium was changed every two days to wash away the non-adhered cells. Two weeks later, when cells expanded to 80-90% area of dishes and displayed a spindle-shaped morphology, we split and passaged down the ERCs as the rate of 1:3. Typical cell surface markers of ERCs were detected by a flow cytometry as previous study described .
In vitro, we harvested the 3rd to 7th generation of ERCs, divided each generation of cells into 3 groups (unmodified ERCs, GC-treated ERCs, and IL-1β-treated ERCs), and inoculated them at a concentration of 3.5×105/ml with 2.5ml culture media. In an attempt to alter DKK1 secretion, we added glucocorticoid (GC, 100 nM, as the stimulator) or IL-1β (10ng/ml, as the inhibitor) into respective groups. After cultured for 48 hours, supernatants in each group were collected to implement ELISA analysis. Fifth generation of ERCs, the most suitable candidate for treatments, were prepared for RT-PCR test to further analysis the DKK1 secretion changes.
For in vivo use, we selected the 5th generation of ERCs, and pre-treated these candidate cells with GC (100nM) or IL-1β (10ng/ml) for 48 hours respectively. Then ERCs were harvested, washed and suspended at a concentration of 1×106/ml for the following therapy.
The experimental colitis was induced by supplying the mice with 3% (wt/vol) DSS (MP Biochemicals) dissolved tap-water as previous studies described. In this current study, 24 BALB/c mice were randomly allocated into 4 groups: untreated group, unmodified ERC group, DKK1high-ERC group (GC-treated ERCs), DKK1low-ERC group (IL-1β-treated ERCs) (n=6). All experimental groups were firstly supplied with 3% (wt/vol) DSS (MP Biochemicals) soluted water for seven days, and then replaced with the non-DSS tap water. ERCs or pre-treated ERCs were suspended in phosphate buffered saline (PBS) and injected into experimental mice (1×106 cells/ml/mouse, i.v.) on day 2, 5 and 8, respectively. Untreated group was also given the equal volume of PBS as control. Mice body-weights, general conditions and fecal characters were monitored and kept into records daily, convenient for the Disease Activity Index (DAI) assessment and other statistical calculations. DAI is an indicator for disease activity which can comprehensively reflect the severity of inflammation in mice. Its score was calculated by assessing weight loss, fecal character and stool blood, accord to the scoring system (Min = 0, Max = 4) directed by Murthy et al.
On day 10, all mice were sacrificed after being fasted for 8 hours. Colons were dissected carefully from ileocecal junction verge to anus, and their lengths were measured. Then, samples were washed with PBS to clean away the contents and longitudinally severed into two parts. One part was fixed in 10% formalin buffer preparing for pathology analysis, and the other was reserved at -80 °C for other experiments. Spleen samples were also harvested and split into two parts. One was immediately ground in PBS for FACS; the other was stored at -80°C for ELISA test.
Colons were obtained, cleaned with PBS, and fixed in 10% buffered formaldehyde on day 10 after DSS-induction. Undergoing processes of dehydration and paraffin embedding, samples were sectioned on an ultra-microtome (LEICA, Germany) at a thickness of 5um for haematoxylin and eosin (H&E) staining. Histopathological scores were evaluated and calculated in a double-blinded manner, based on the following criteria: (a) inflammation severity: 0 (physiologic inflammation), 1 (mild inflammation or prominent lymphoid aggregates), 2 (moderate inflammation), 3 (moderate inflammation associated with crypt loss), 4 (severe inflammation with crypt loss and ulceration). (b) crypt damage: 0 (no destruction), 1 (1–33% of crypts destroyed), 2 (34–66% of crypts destroyed), 3 (67– 100% of crypts destroyed). The two respective scores, inflammation severity and crypt damage, were summed together to drive the histopathological scores for evaluating colonic inflammation (maximum score 7).
Flow cytometry analysis
Mouse spleens were respectively ground, filtered with sterilized meshes and suspended in 2 ml precooled PBS. Then we added RBC Lysis Solution (1x) (Biolegend Inc., San Diego, CA, USA) into splenic suspensions to lyse erythrocytes, washed twice and resuspended the splenocytes with PBS to a concentration of 1×107/ml. Fluorescent monoclonal antibodies against mouse CD4, IFN-γ, IL-4, IL-17, CD25, FOXP3, CD68, CD206, CD11C, MHCII, and CD86 were applied to detect the populations of Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+), Th17 (CD4+IL-17+), Treg (CD4+CD25+FOXP3+), macrophage (CD68+CD206+) and DC (CD11c+MHCII+/CD86+) cells by FACS Canto II flow cytometer (BD Biosciences, America), as previously described. The absolute number of immune cells detected in Figure 3 and Figure 4 were at the average level of 5×104 and the percentages of TH1, TH2, TH17 and TREG were showed on CD4+ gated cells. In addition, to accurately identify the subpopulation of Th1, Th2 and Th17 CD4+ T cells, splenocytes were firstly incubated with cell stimulation cocktail (including phorbol-12-myristate-13-acetate (PMA), ionomycin, brefeldin A, and monensin) (ebioscience Inc., San Diego, CA, USA) for 5 hours before being stained with fluorescent antibodies. The statistics of various immunocyte proportions were analyzed by Flowjo X software.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was carried out according to the manufacturer’s instructions (Boster, Wuhan, China). Supernatants in culture media of ERCs were collected and prepared for measuring the DKK1 secretion level. Equal quality (30mg) of same area intestinal tissues in each group were gathered and grounded with high efficiency tissue lysate buffer (RIPA) and phenylmethyl sulfonylfluoride (PMSF) (Solarbio, Beijing, China) for testing the level of IFN-γ, IL-4, IL-10 and β-catenin. Splenic tissues frozen in -80°C were also ground for detecting the β-catenin expression level. The reaction absorbance was determined at 450 nm with the Microplate Reader (Tecan, Mannedorf, Switzerland) and each sample was performed in duplicates to lessen the error.
Real-time polymerase chain reaction (RT-PCR)
To determine the transcriptional changes of inflammatory mediators and β-catenin in colons, colonic total RNA was extracted with an RNAprep Pure Tissue Kit (DP431, Tiangen Biotech Co. Ltd., Beijing, China). The pureness and concentration of RNA were determined with an UV spectrophotometer (SANYO, Japan) at the spectrum of 260 and 280nm. cDNA was generated from the obtained RNA by using a Fastquant RT kit (KR106, Tiangen Biotech Co. Ltd., Beijing, China). Real-time quantitative PCR (RT-PCR) was carried out by using SuperReal Color Premix kit (FP216, Tiangen Biotech Co. Ltd., Beijing, China), according to the recommended protocol. The primer sequences involved were designed as follows:
human-GADPH: forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′,
human-DKK1: forward, 5′-ATAGCACCTTGGATGGGTATTCC-3′,
mouse GAPDH: forward, 5′-AGGTCGGTGTGAACGGATTTG-3′,
mouse β-catenin: forward, 5′-GAGTAGCTGCAGGGGTCCTC-3′,
mouse IFN-γ: forward, 5′- GCCGCGTCTTGGTTTTGCAG-3′,
mouse IL-4: forward, 5′-ACAGGAGAAGGGACGCCAT-3′,
mouse IL-10: forward, 5′-AGAAGCATGGCCCAGAAATCA-3′,
mouse TNF-α: forward, 5′-CCCTCACACTCAGATCATCTTCT-3′,
mouse COX-2: forward, 5′- TGAGCAACTATTCCAAACCAGC-3′,
mouse MPO: forward, 5′-AGTTGTGCTGAGCTGTATGGA-3′,
mouse iNOs: forward, 5′-GTTCTCAGCCCAACAATACAAGA-3′,
mouse SOD： forward, 5′-CAGACCTGCCTTACGACTATGG-3′,
Each sample was performed in triplicates on MJ Research DNA Engine Opticon 2 PCR cycler (BIO-RAD, USA). The expressions of target genes among different groups were calculated with the comparative 2−ΔΔCT method.
Experimental data was presented as mean ± standard deviation (SD) and analyzed by SPSS 19.0. Data variance was evaluated by using one-way analysis of variance (ANOVA) (groups≧3) or unpaired two-tailed student’s t test (groups=2) after normality test. The differences between groups were considered significant with p values ≤ 0.05 in statistics.