Chemicals and reagents
Human colonic NCM 460 epithelial cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). For in vitro experiments, toxin A (TcdA) from C. difficile was obtained from Sigma-Aldrich (St. Louis, MO; C3977-2UG). Dulbecco’s Modified Eagle’s Medium (DMEM) powder, 1% L-glutamine, 100 g/ml streptomycin, 10 units/ml penicillin, and fetal bovine serum (FBS) were purchased from the Gibco (Grand Island, NY, USA); Lipofectin transfection reagent was purchased from Invitrogen (Burlington, ON, Canada). Dimethyl sulfoxide (DMSO) and Annexin V-FITC/Propidium Iodide (PI) Flow Cytometry Assay Kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Small-interfering RNA (siRNA) were synthesised by Wuhan Genesil Biotechnology Co., Ltd (Wuhan, China). An unrelated gene siRNA was chosen as a negative control. All solvents and chemicals were analytical grade.
The human colonic NCM 460 epithelial cells were maintained in DMEM medium containing 10% FBS, 2 mM of glutamine, 1% nonessential amino acids, and antibiotics (100 units/ml of penicillin and streptomycin). The cells were cultured in a 5 % CO2 incubator at 37°C. The human colonic NCM 460 epithelial cells were maintained to approximately 80 % confluence, then treated with a final concentration of 5 ng/ml, 10 ng/ml or 20 ng/ml TcdA for 24 h in a complete medium. Control group (Mock group) were exposed to media only. Toxin A concentrations were selected based on previous data .
gC1qR siRNA-expressing plasmid construction
The siRNA oligonucleotides were targeted to nucleotides 408 to 426. gC1qR siRNA sequenced as follows: 5′-AAC AAC AGC AUC CCA CCA ACA UU-3′. The pGenesil-1 vector using eGFP as the reporter gene was purchased from Wuhan Genesil Biotechnology Co., Ltd. The NCM 460 cells were transfected with a mixture containing Lipofectamine, optiMem, and siRNA oligonucleotides (50 μM) according to the manufacturer’s instructions. The gC1qR siRNA-Lipofectamine complex was then added to NCM 460 cells in suspension and plated on 96-well plates (18,000 cells / well). After 48 h, the complete medium was added, along with TcdA (10 ng/ml). The cells were incubated for 24 h, and assays were performed to detect cell viability, cell apoptosis and cell mitochondrial function.
Cell Viability Assay
The NCM 460 cells viability was assessed by WST-1 assay. Cells were cultured in 96-well plates (1 × 105/well) and treated with 5 ng/ml, 10 ng/ml or 20 ng/ml TcdA. After 24 h of incubation, 10 µL of WST-1 solution (stock solution of 5 mg/mL in PBS) was added to 96-well plates, and the plates were maintained for an additional 4 h at 37 ◦C. The reducing activity of the cells was examined by treatment with dimethyl sulfoxide (DMSO, 150 µl) prior to reading at an optical density (OD) of 490 nm with an automatic microplate reader (Elx808; BioTek Instruments, Inc., Winooski, VT, USA).
Detection of apoptotic cells
The NCM 460 cells apoptosis were examined by Annexin V-FITC/ propidium iodide with flow cytometry analysis. The NCM 460 cells were washed and resuspended in binding buffer including 10 mM HEPES, 140 mM NaCl , 2.5 mM CaCl2, pH 7.4 for 20 min at room temperature. The cell suspension (1 × 106/well) were mixed with 10 µL of Annexin V-FITC, and incubated for 30 min at room temperature. Then the mix stained with 10 µL of PI solution for additional 10 min on ice. The scatter parameters of the cells were calculated by Coulter EPICS XL ﬂow cytometer (EasyCyte; Guava Technologies).
Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNNEL) Assay
The NCM 460 cells were exposed to the 5 ng/ml, 10 ng/ml or 20 ng/ml TcdA for 24 h in a complete medium, and then fixed with 4% paraformaldehyde for additional 20 min and embedded in paraffin at room temperature. The slices were treated with hydrogen peroxide block for 10 minutes and incubated with 50 microliters of TUNEL reaction mixtures for 60 minutes at 37 °C in the dark. After being washed twice with PBS, 50 µL of converter-peroxidase were added on the slices and incubated for 40 minutes at 37 °C, finally, the slices were added to 50 µL of diaminobenzidine substrates for 15 min at 25 °C. After being washed twice with PBS, cells apoptosis level was assessed under a light microscope. Cells with shrunken brown-stained nuclei were considered positive.
Real-time quantitative polymerase chain reaction (real-time qPCR)
Total RNA was extracted from cultured NCM 460 cells with a Total RNA Extraction Kit (Promega, Beijing, China), 10 µL of RNA was reverse-transcribed into complementary DNA (cDNA) according to protocol provided by the manufacturer (Invitrogen, Carlsbad, CA). The following sequences were used to detect gC1qR level: Primer-F (5′-AAT CAC ACG GTA GAC ACT GAA ATG CC-3′) and Primer-R (5′-CAT CAT CCC ATC TAA AAT GTC CCC TG-3′) and β-actin: Primer-F (5′-TCA CCC AVA CTG TGC CCA TCT ATG A-3′) and Primer-R (5′-CAT CGG AAC CGC TCA TTG CCG ATA G-3′). Real-time qPCR was performed using a SYBR Green PCR Kit (Invitrogen) on an ABI PRISM 7500 real-time PCR system (Applied Biosystems). The gC1qR mRNA level was determined using the threshold cycle (2-ΔΔCT) method . The relative amounts of target gene were normalized to the average of the endogenous control.
Western blot analysis
The NCM 460 cells were treated with various treatments, and total protein was harvested and lysed in buffer containing 1 mM of EDTA, 0.5% NP-40, 50 mM of Tris-HCl (pH 7.4), 50 mM of NaF, 1% Triton X-100, 1 mM of PMSF, 10% glycerol, 150 mM of NaCl, 1 mM of Na3VO4, and 1% protease inhibitor cocktail. The equal amount of concentrated proteins were separated using an 10% gradient SDS--polyacrylamide gel running at 100 V for 2 h and transferred onto a polyvinylidene fluoride (PVDF) membrane at 300 mA for 90 min. Non-specific membrane binding sites were blocked in 5% non-fat milk in PBST I for 1 h, and then incubated with primary antibodies specific to gC1qR (1: 1000 dilution, a recombinant rabbit monoclonal antibody, Abcam: ab131284) and actin (1: 2000; ab8227, Abcam) in blocking solution at 37 °C for 2 h. After being washed twice with TBST, the membrane was incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 4000; Santa Cruz). The protein band visualization was detected using enhanced chemiluminescence from Cell Signaling Technology (Beverly, MA, USA). The values used for the histogram were normalized to the endogenous control.
The cultured NCM 460 cells were trypsinized and pelleted at 1000 X for 20 min at 4 °C. Removing the supernatant, the cells mass were embedded in paraffin and cut into 5 µm and dried at 70 °C for 2 h. According to the standard procedures, the slice was treated with 3 % H2O2 for 25 min in the dark. After being washed twice with PBS, the section were underwent epitope retrieval (5 min 750 W, 15 min 350 W in a microwave) in 0.01 mol/L citrate buffer (pH 6.0). The section was incubated with primary antibodies specific to gC1qR (1: 100 dilution, Abcam: ab131284). After being washed twice with PBS, the section was incubated for 30 min with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 4000; Santa Cruz). After being washed twice with PBS, each section was treated with streptavidin-peroxidase complex, and images were recorded under a confocal laser-scanning microscope (Leica, Germany). Since gC1qR localizes to the mitochondrial matrix, which is expressed in the cytoplasm, cells staining brown in their cytoplasm were considered positive.
The cultured NCM 460 cells were digested by trypsin and pelleted at 1000 X for 20 min at 4 °C. After the supernatant was removed, cells were fixed with 2 % glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.4), the cells mass were post-fixed in 1 % OsO4 for 1 h and stained with 1 % uranyl acetate for 2 h. Then the cell mass was dehydrated at an acetone series concentration of 50 % for 15 min, 70 % for 15 min, 80 % for 15 min, 90 % for 15 min and 100 % for 15 min respectively. The mass embedded in Durcupan and sectioned to 60‑70 nm thickness. The ultrastructure of NCM 460 cells was examined at 3700 X magniﬁcation, and photographs were observed under a Zeiss 109 electron microscope (Carl Zeiss, Oberkochen, Germany).
Assay of intracellular ROS
Intracellular ROS production was quantified by a ROS assay kit (Beyotime, Shanghai, China). Briefly, at least 1 × 105 NCM 460 cells were incubated with 10 µM final concentration of H2DCFDA, followed by incubation for 40 min at 37 °C. After being washed twice with PBS, cells were harvested. ROS level were analyzed with flow cytometry (BD FACSCalibur, San Jose, CA, USA) using 488 nm excitation and 530 nm emission wavelength.
Measurement of mitochondrial membrane potential (ΔΨm)
Loss of mitochondrial membrane potential (ΔΨm) was examined in NCM 460 cells using the membrane-permeable JC-1 dye (Beyotime). According to the manufacturer’s instructions, NCM 460 cells were loaded with 10 µM JC-1 for 20 min at room temperature. Depolarization of ΔΨm was analyzed by monitoring the ﬂuorescence intensities at the excitation wavelength 485 nm and the emission wavelength 530 nm using fluorescence microscopy.
Measurement of Intracellular ATP Levels
The ATP content in NCM 460 cells lysates was detected using an ATP Bioluminescent Cell Assay Kit (S0026, Beyotime, Shanghai, China) according to the manufacturer’s instruction, NCM 460 cells were washed twice in cold PBS buffer, 1 mL of 2 % trichloroacetic acid was added into 0.1 mol/L Tris and 2 mmol/L EDTA to stop the luciferase reaction. Cells were removed with a scraper and collected in 1.8-mL Eppendorf centrifuge tubes for 10 min at 15,000 rpm at 4°C. Supernatants were diluted at 0.1 mol/L Tris–2 mmol/L EDTA (Tris-EDTA) (1: 50), and then ATP was determined and protein was analyzed by pellets. The absorbance of samples was examined using a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). A standard curve of ATP concentrations ranging from 0 to 200 nmol/mL was used in this experiment.
All data are displayed as the mean ± standard deviation (SD). Student’s t test was used to compare the means of two groups. p-values less than 0.05 were considered significant (*p < 0.05; ** p < 0.01; # p > 0.05). Statistical analysis of the data was performed using SPSS18.0. All experiments were performed in triplicate.