Rabbit polyclonal anti-occludin, rabbit polyclonal anti-junction adhesion molecule (JAM)-A, rabbit polyclonal anti-claudin-1, mouse monoclonal anti-claudin-4, and rabbit polyclonal anti-phosphoserine antibodies were supplied by Zymed (Invitrogen, Carlsbad, CA, United States). Rabbit polyclonal anti-ZO-1 and rabbit polyclonal anti-PKC were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Fluorescein isothiocyanate (FITC)-phalloidin was obtained from Sigma (St. Louis, United States). FITC-conjugated secondary antibodies were supplied by Zymed (Invitrogen). Biotin-labeled goat anti-rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP)-labeled streptavidin were supplied by DAKO (Glostrup, Denmark). All other reagents of analytical grade were purchased from Sigma (St. Louis, United States).
The L. plantarum
(strain CGMCC No. 1258) used in this study was a gift from Dr. Xiao-Ming Hang (Onlly Institute of Biomedicine, Shanghai Jiao Tong University, Shanghai, China). L. plantarum
cultures were prepared exactly as described previously[11
Forty male albino Wistar rats weighing 250-320 g were purchased from Fudan University Medical Animal Center (Shanghai, China). They were housed in stainless-steel cages, three rats per cage, at controlled temperature (23 °C) and humidity and with a 12 h/12 h dark/light cycle. They were maintained on a standard laboratory diet with tap water ad libitum, except for an overnight fast before surgery. The study was conducted according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and it was approved by the Ethics and Research Committee of Shanghai Sixth People’s Hospital, Shanghai, China.
Animals were randomly divided into five groups of eight rats each as described below.
Group I, sham-operation: A 2.0-cm upper midline abdominal incision was made, and the common bile duct (CBD) was freed from the surrounding tissues without ligation or transection.
Group II, bile duct ligation: The CBD was double ligated in its middle third with a 4-0 silk suture and transected between the two ligatures.
Group III, bile duct ligation + L. plantarum: After bile duct ligation (BDL), a volume of 10 mL live L. plantarum (activity, 2 × 108 CFU/mL) divided into two equal doses was administrated daily to the rats by gavage for 10 d. After 10 d, the animals were sacrificed under ketamine anesthesia.
Group IV, BDL + IBD: The CBDs of animals were ligated and isolated. A polyethylene tube PE-10 (American Health and Medical Supply International Corp. Co., Ltd., Scarsdale, New York, United States) was inserted into the proximal CBD in a cephalad direction and fixed. The drainage end was tied and positioned in the right hepatorenal recess. After 5 d of obstructive jaundice, the abdomen was reopened through the previous incision. After releasing the biliary obstruction by transecting the tube, a distal 3-cm segment of the catheter was inserted into the duodenum for internal biliary drainage. The animals were sacrificed after another 5 d.
Group V, BDL + IBD + L. plantarum: Ten days after BDL, live L. plantarum was infused as described for Group III.
All non-L. plantarum control groups, including sham-operation (SHAM), were gavaged with the same volume of the same vehicle (Dulbecco’s phosphate buffered saline) used for the L. plantarum groups. The animals were sacrificed after 10 d.
All surgical procedures were performed under strict sterile conditions and ketamine anesthesia. At the end of the experiment on day 10, 4-5 mL blood sample was collected from each animal by puncturing the portal vein.
Serum total bilirubin and alanine aminotransferase measurement
The serum total bilirubin and alanine aminotransferase (ALT) levels were determined using a kit (Jiancheng Biological Co., Ltd., Nanjing, China) and a Hitachi Model 7600 series automatic analyzer (Hitachi Co., Tokyo, Japan).
Plasma endotoxin measurement
Endotoxin concentrations were determined using a quantitative chromogenic Limulus Amebocyte Lysate test kit (Shanghai Med. and Chem. Institute, Shanghai, China). Samples were processed according to the manufacturer’s instructions[19
Plasma D-lactate and plasma diamine oxidase measurement
-lactate levels were measured by an enzymatic spectrophotometric assay[20
] using a serum D-lactate quantitative colorimetric detection kit according to the manufacturer’s instructions (GMS19038.6, Genmed, Boston, MA, United States). Results were expressed as mol/mL. Plasma diamine oxidase (DAO) activities were determined with an enzymatic spectrophotometric assay[21
] using a Serum DAO detection kit according to the manufacturer’s instructions (Jiancheng Biological Co., Ltd., Nanjing, China). Results were expressed as U/L.
Plasma reduced glutathione/oxidized glutathione measurement
Plasma glutathione (GSH) and glutathione (GSSG) were determined by an enzymatic spectrophotometric assay[22
] using the GSH and GSSG detection kits according to the manufacturer’s instructions (Jiancheng Biological Co., Ltd.). Results were expressed as mol/mL.
Detection of superoxide dismutase and malondialdehyde in the ileum
Superoxide dismutase (SOD) activity was detected using Sun et al’s[23
] nitroblue tetrazolium method. Malondialdehyde (MDA) levels were measured using the thiobarbituric acid test according to Ohkawa et al[24
]. Intestinal tissue samples were thawed, weighed, and homogenized 1:9 (w/v) in 0.9% saline. The homogenates were centrifuged at 3000 × g
for 10 min at 4 °C, and the supernatant was removed for the measurement of MDA content, SOD activity, and total protein. Total intestinal protein concentration was determined by a Coomassie blue method, with bovine serum albumin (BSA) as a standard. SOD activity and MDA levels were detected with kits according to the manufacturer’s instructions (Jiancheng Bioengineering Ltd., Nanjing, China). Results were expressed as U/mg protein and nmol/mg protein.
Samples 1 cm in length were collected from the terminal ileum. To avoid mucosal damage, the intestinal lumen was carefully cannulated and gently washed with normal saline before sampling. Specimens were fixed by immersion in 10% buffered formaldehyde solution and embedded in paraffin. Sections (5 µm thick) were cut and stained for routine light microscopy using HE.
Transmission electron microscopy
Samples 3-4 mm in length were collected from the terminal ileum. These samples were longitudinally cut and immersed in 2.5% phosphate-buffered glutaraldehyde solution for 24 h at 4 °C. Specimens were then washed with phosphate-buffered solution (PBS), fixed in 1% osmium tetroxide for 2 h at 4 °C, dehydrated in ethanol and propylene oxide, and embedded in Epon 812 for 48 h. Sections (1 µm thick) were cut and stained with methylene blue. Ultrathin sections were then cut with a diamond knife, stained with uranyl acetate and lead citrate, and observed under transmission electron microscopy (TEM).
Terminal deoxyuridine nick-end labeling assay
Four-μm thick sections were collected on poly-L-lysine-coated glass slides. The nuclear DNA fragmentation of apoptotic cells was labeled in situ
by the terminal deoxyuridine nick-end labeling immunohistochemical method[25
] using an ApopTag®
Plus Peroxidase In Situ Apoptosis Detection Kit (CHEMICON, Billerica, MA, United States) according to the manufacturer’s instructions.
Terminal ileum tissues were fixed in 3% paraformaldehyde for 3 h, washed with PBS, and embedded in paraffin. Sections (5 μm thick) were cut and attached to glass slides. After deparaffinization and rehydration, sections were permeabilized with 0.2% Triton X-100 in PBS for 20 min. Slides were washed with PBS extensively and blocked with 5% normal goat serum PBS containing 0.05% Tween-20 and 0.1% BSA for 20 min at room temperature. Primary antibodies were added to the slides and incubated overnight at 4 °C in a humidity chamber. After washing, sections were incubated with FITC-conjugated specific secondary antibody (Sigma) at room temperature for 2 h in the dark. The slides were again washed extensively and then mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, United States). Sections were observed under a confocal laser microscope (LSM 510, Zeiss, Jena, Germany).
Expression of PKC by immunocytochemical staining using labelled streptavidin biotin method
After the rats were sacrificed, terminal ileum tissues were excised and fixed in Bouin’s solution and embedded in paraffin. Immunohistochemistry was performed on 5-µm thick paraffin sections. After deparaffinization and dehydration, endogenous peroxidase was blocked with 30 mL/L H2O2 for 15 min. After blocking of nonspecific binding sites with 5% normal goat serum, slides were incubated with specific primary antibody overnight at 4 °C. Primary antibodies were diluted 1:50 (rabbit polyclonal anti-human PKC, Santa Cruz Biotechnology, Inc., United States) in PBS. Next, the slides were washed three times for 5 min each with PBS and incubated with biotinylated goat anti-rabbit IgG at 37 °C for 30 min, washed as before, and developed with HRP-labeled streptavidin. The incubation and the subsequent washing were exactly the same as done before. Finally, diaminobenzidine chromogen, a peroxidase substrate, was added for color development. The reaction was stopped with a tap water rinse. The sections were counterstained with hematoxylin and mounted for examination.
Western blotting analysis
Terminal ileum samples were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer [150 mmol NaCl, 50 mmol Tris·HCl (pH 7.4), 0.5 mmol phenylmethylsulfonyl fluoride, 2.4 mmol EDTA, and 1 mmol sodium orthovanadate with 1% nonidet-40 (NP-40) and Sigma protease inhibitor cocktail (1:100)] for 30 min at 4 °C. After centrifugation at 10 000 × g for 10 min at 4 °C, the protein concentration of each sample was quantified by the Bradford method. An equal amount of total protein was separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to a nitrocellulose membrane. After blocked overnight in tris-buffered saline (TBS) containing 0.05% tween (TBS-T) and 5% dry powdered milk, membranes were washed three times for 5 min each with TBS-T and incubated for 2 h at room temperature in primary antibody (rabbit anti-claudin-1, rabbit anti-occludin, rabbit anti-JAM-A, or rabbit anti-ZO-1). After three washes with TBS-T, the membranes were incubated for 1 h with HRP-conjugated secondary antibody. Following two washes with TBS-T and one wash with TBS, the membranes were prepared for visualization of protein by the addition of enhanced chemiluminescence (ECL) reagent (Amersham, Princeton, NJ, United States). Densitometric analysis was performed using an Alpha Imager 1220 system (Alphainotech Co., San Leandro, CA, United States).
Real-time reverse transcription-PCR
The levels of occludin, ZO-1, claudin-1, claudin-4, JAM-A and UGT1A mRNA were measured by real-time reverse transcription-PCR (RT-PCR) using SYBR1 green[26
]. Total RNA was isolated from terminal ileum samples with the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Real-time RT-PCR was performed with an ABI prism 7000 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The primers were designed using the Primer Express®
Program (Applied Biosystems). Their sequences are shown in Table . The following procedure used 2 μg of RNA. In a sterile RNase-free microcentrifuge tube, 1 μL of 20 μm oligo (dT) 15 primer was added to a total volume of 15 μL water. The tubes were heated to 70 °C for 5 min, cooled immediately on ice, and spun briefly. The following reagents were added to the annealed primer/template: 5 μL of 5 × M-MLV reaction buffer, 1.25 μL of 10 mmol dNTPs and 25 units of RNasin RNase inhibitor, and 200 units of M-MLVRT RNAse H were added to the reagent to yield a 25 μL total reaction volume. All were mixed gently and then incubated for 60 min at 42 °C before the reaction was terminated at -20 °C.
Sequences of oligonucleotide primers and conditions for real-time polymerase chain reaction
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as a house-keeping gene control. Separate PCR reactions (25 μL) were conducted for each transcript and contained 2.0 μL cDNA, 12.5 μL of 2 × SYBR Premix Ex Taq™ (TaKaRa, Ltd., Shiga, Japan), and 0.5 μL each of 10 μmol/L gene-specific forward and reverse primers. PCR conditions were optimized to 95 °C (30 s), followed by 40 cycles (45 s each) at 95 °C, 60 °C (5 s), and 72 °C (30 s), and the reaction was completed at 37 °C for 30 s. Five serial dilutions of cDNA were analyzed for each target gene and used to construct linear standard curves. To compensate for variations in the RNA input and in the efficiency of the real-time RT-PCR, we used a normalization strategy based on the house-keeping gene GAPDH.
Immunoprecipitation and immunoblotting assays
The terminal ileum tissues were homogenized and extracted with the buffer used for Western blotting assays for 30 min at 4 °C. After centrifugation at 10 000 × g for 10 min at 4 °C, the protein concentration of each sample was quantified by the Bradford method. The supernatant was treated with protein G plus protein A agarose beads (Sigma) and incubated overnight at 4 °C with rabbit anti-occludin antibody (Zymed) and protein G + protein A agarose beads. The beads were washed with PBS and ice-cold RIPA buffer, and immunoprecipitated proteins were separated on 10% SDS-polyacrylamide gel electrophoresis gels and transferred onto nitrocellulose membranes (Invitrogen). The membranes were blocked with 1% BSA in PBS overnight at 4 °C and then incubated with rabbit anti-phosphoserine antibodies (Zymed) for 2 h at room temperature, followed by HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., United States). The reaction was visualized by an enzyme chemiluminescence kit from Pierce (Rockford, IL, United States). Western blotting was performed with an anti-occludin rabbit polyclonal antibody (Zymed) followed by an anti-rabbit secondary antibody coupled with peroxidase (Santa Cruz Biotechnology, Inc.) and ECL. For Western blotting of ZO-1, the same protocol was used with the rabbit polyclonal anti-ZO-1 antibody and a rabbit anti-β-actin antibody (both from Santa Cruz Biotechnology, Inc.).
PKC activity assay
The PKC activity assay was conducted according to the instructions of the PepTag non-radioactive PKC assay kit (Promega, Madison, WI, United States). Briefly, terminal ileum tissues were homogenized and lysed in cold lysis buffer, containing 20 mmol/L tris-HCl, 0.5 mmol/L ethylene glycol tetraacetic acid, 2 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L phenylmethanesulfonyl fluoride, and 10 mg/L leupeptin (pH 7.5). Assays were then performed at 30 °C in a total volume of 25 μL containing 5 μL PKC reaction 5 × buffer, 5 μL PLSRTLSVAAK peptide, 5 μL PKC activator, 1 μL peptide protection solution, and 9 μL sample. Reactions were initiated by the addition of the 9 μL sample and terminated after 30 min by incubation of the reaction mixture at 95 °C for 10 min. After added with 1 μL of 80% glycerol, each sample was separated by 0.8% agarose gel electrophoresis at 100 V for 15 min. The intensity of fluorescence of phosphorylated peptides reflected the activity of PKC. All experiments were carried out in triplicate, with each data point representing the results from a separate culture.
Quantification of the immunohistochemical and immunofluorescence staining was performed on stored images of completely scanned tissue sections. Images were acquired with an AxioCam MRc (Carl Zeiss, Jena, Germany) connected to an Axioplan 2 fluorescence microscope (Carl Zeiss), at × 40 magnification. Each microscopic field was individually autofocused before acquisition. Five fields were selected from each slide and a total of five slides per group were examined. All image acquisition and processing were done using custom-written macros in KS400 image analysis software (version 3.0, Carl Zeiss).
Results were presented as mean ± SD of three experiments. The data were analyzed using GraphPad PRISM (GraphPad Software Inc., San Diego, CA, United States) and SPSS 11.0 (SPSS Inc., Chicago, IL, United States). All data were analyzed using one-way analysis of variance with Bonferroni/Dunnett T3 post-hoc test for multiple comparisons to determine differences between two experimental groups. P values < 0.05 were considered to be significant.