Materials
Betaine and LPS were purchased from Sigma-Aldrich (Saint Louis, USA) and dissolved in PBS to prepare stock solutions with concentrations of 100 mmol/L and 1mg/mL respectively. Occludin (Catalog number: #91131) and Claudin-1 (Catalog number: #13995) were obtained from Cell Signaling Technology (Shanghai, China.). All the reagents were stored at -20℃.
Cell culture
IPEC-J2 cells were obtained from Animal Nutrition & Human Health Laboratory, Hunan Normal University. IPEC-J2 cells were cultured in DMEM with 10% of FBS and 1% of penicillin–streptomycin (Hyclone, Logan, UT, USA) at 37 °C, 5% of CO2. The medium was refreshed every second day.
Western blot
Western blot (WB) was used to evaluate the expression of TJ proteins in IPEC-J2 cells. The experimental steps are consistent with regular procedure. First, polyacrylamide gel was prepared and then electrophoresis after adding the required samples. Next, the PVDF membrane was covered by 5% milk for an hour after western transfered, and then the membranes were washed with PBS for three times and incubated with primary antibodies overnight. Next day, the PVDF membranes were washed for six times and then incubated with second- antibody for an hour. Development was performed by chemiluminescence equipment after washing it again for three times and the pictures were measured by Image J.
Morphological observation of cells
IPEC-J2 cells were inoculated 5×105 per well in 6-well plate and cultured for 2 days (the culture medium was replaced 24 hours after the inoculation), then the cells were treated by ctrl, LPS, LPS+betaine and betaine alone for 24h. After that, the culture medium was outwelled and the cells were washed with PBS for two times. The pictures were taken under the light contrast microscopy (Leica, DFC450C, Wetzlar, Germany) in the end.
Immunofluorescence
IPEC-J2 cell monolayers were incubated and cultured in 6-well plates and each plate has a glass slide where the cells attached to. Then the cells were treated with ctrl, LPS, LPS+betaine and betaine for 24 hours. Cells were fixed with paraformaldehyde (4%) no less than 20 minutes after washing twice with PBS (10mins each). Then cells were blocked for 30 minutes with bovine serum albumin (BSA 1%) after washing it again and then incubated with Claudin-1 overnight. The second day the cells were washed once again and incubated with second- antibody for 2 hours. Cells were washed for another 30 minutes and then covered with the DAPI dye solution for 10 minutes. The inverted fluorescence microscope was used to take the microscopic images of the cells.
RT-PCR
Reverse transcriptase-polymerase chain reaction was used to detect the expression of relative mRNA. First cDNA was extracted by conventional methods, and the PCR system was composed by 5.0μL of Green qPCR Mix, 0.3μL of Forward primer and Reverse primer, 4.2μL of double distilled water, and 0.2μL of cDNA. The final volume was 10μL. Then the housekeeping gene GAPDH was used as the standard control, and each sample was repeated for three times, then Real Master Mix SYBP ROX (5’ Prime) was used for quantitative real-time PCR.
Table 1 Sequences of Primers
Gene
|
Forward primer (5′–3′)
|
Reverse primer (5′–3′)
|
GAPDH
|
GAAGGTCGGAGTGAACGGAT
|
CTGGCATTGACTGGGGTCAT
|
Occluding
|
TTGCCTGGGACGAGGCTATG
|
ATCCCTTTGCTGCTCGTGGA
|
IL-6
|
TGGATAAGCTGCAGTCACAG
|
ATTATCCGAATGGCCCTCAG
|
TEER
The 24-hole transwell plate (Costar, Coring Inc, NY, USA) was infiltrated with DMEM culture medium for 2h or overnight before inoculating IPEC-J2 cells (1 × 105 cells per cm2). After being attached, cells were differentiated in the culture medium without serum for 7–10 days. When the resistance value reaches the desired value, the cells were treated with ctrl, LPS, LPS+betaine, betaine respectively. The way to measure the cell integrity is TEER, which used an epithelial voltage ohmmeter with chopstick electrodes (Millicell ERS-2, EMD Millipore, Billerica, MA). TEER measurements were operated in gnotobasis. The plate was taken out of the 37°C cell incubator and put in the operating floor for at least 30 min, the electrodes were disinfected by 70% ethanol and then dried in the air for 30 s. Then the electrodes were douched with culture medium, immersed the electrodes and make sure the shorter electrode is in the insert of the plate and the longer electrode is located between the outer wall and the filter insert. Ensure all the electrode tips are absolutely immersed by solution and the shorter one does not come into contact with the cells which are growing on the insert. And record the resistance value of 12, 24, 36, 48, 72h.
Statistical analysis
All the statistical data were analyzed by the software GraphPad Prism version6. Data were expressed as mean ± SD and relative gene expressions were transformed into natural logarithm scale. One-way and two-way ANOVAs were used to compare the differences between different treatments. A p-value 0.05 was considered statistically significant.