Intestinal ischemia/reperfusion
Male Wistar rats (250–300 g) were raised in a temperature-controlled room with
12-hr light-dark cycles, and fed regular rat chow and water. Rats were fasted overnight
with free access to water, and subjected to sham operation or mesenteric I/R challenge.
In I/R rats, the superior mesenteric artery (SMA) was occluded with an atraumatic
microvascular clamp for 20 minutes and then released for up to 60 minutes. Ischemia of
the bowel was verified by the loss of mesenteric pulsations and bluish discoloration of
the jejunum. Sham controls rats received mock manipulation of SMA without occlusion.
All animals were placed on heating pads to maintain body temperature at 37°C during
the operation, and the heart rate was closely monitored. All protocols used in this study
were approved and monitored by the Institutional Animal Care and Use Committee,
National Taiwan University.
Experimental design
The experimental protocols were carried out under aseptic conditions. After
anesthetization with urethane (1.2 g/kg, intraperitoneal injection; Sigma-Aldrich, MO),
all rats were subjected to midline laparotomy and a 10-cm jejunal sac was created by
thread ligature at both ends, beginning 10 cm distal to the ligament of Treitz in each
animal. Care was taken not to occlude or puncture mesenteric vessels during the ligation.
A 1 ml syringe with a PE-10 catheter was intubated to one end of the jejunal sac and 0.5
ml of Krebs buffer with the pancaspase inhibitor ZVAD (EMD Chemicals Inc.,
Darmstadt, Germany) or glucose (Sigma) was carefully injected into the lumen. The
formula for Krebs buffer was 115 mM NaCl, 8 mM KCl, 1.25 mM CaCl2, 1.2 mM
MgCl2, 2.0 mM KHPO4, 25 mM NaHCO3, pH 7.33 - 7.37. Animals were then subjected
to sham operation or I/R challenge as described above.
Rats were randomly assigned to six groups (n = 6-8 / group): Group 1, sham
controls that underwent laparotomy and whose jejunal lumen was instilled with Krebs
buffer before sham operation; Group 2, sham+Z rats that underwent laparotomy and
whose jejunal lumen was instilled with 120 μM ZVAD in Krebs buffer 30 minutes prior
to sham operation; Group 3, sham+G rats that were enterally administered 25 mM
glucose in Krebs buffer immediately before sham operation; Group 4, I/R rats that were
enterally instilled with Krebs buffer before SMA occlusion for 20 minutes and
reperfusion for 60 minutes; Group 5: I/R+Z rats that were enterally instilled with 120
μM ZVAD in Krebs buffer 30 minutes prior to the same I/R procedure; and Group 6:
I/R+G rats that were enterally administered 25 mM glucose in Krebs buffer immediately
before I/R challenge. The concentrations of ZVAD and glucose used here have been
previously shown to inhibit cell apoptosis induced by microbial products in epithelial
cell cultures (Yu et al., 2005; Yu et al., 2008).
In some experiments, phloridzin (a SGLT1 inhibitor; 0.5 - 2.5 mM) and phloretin
(a GLUT2 inhibitor; 1.5 and 2.5 mM) were added to the glucose solution for enteral
instillation prior to the I/R procedure. In addition, to investigate the involvement of
PI3K in the signaling pathways of glucose-mediated rescue mechanism, LY294002 (10
mg/kg) and wortmannin (7.5 μg/kg) were administered intraperitoneally and
intravenously, respectively, 30 minutes before I/R challenge in the presence of luminal
glucose. At the end of the surgical procedures, the jejunal tissues, the liver, and the
spleen were collected for experimental analysis.
Histopathological scoring
Jejunal segments were fixed in 4% paraformaldehyde, and care was taken to ensure
proper orientation of the crypt to villus axis during embedding. Sections of 4 μm
thickness were stained with hematoxylin and eosin (H&E). The degree of intestinal
injury was evaluated using a light microscope and graded by two independent persons
blind to the actual treatment. Briefly, intestinal injury was scored from 0 to 5 according
to the following criteria: grade 0, normal mucosal villous structure; grade 1, presence of
subepithelial space at villous tips; grade 2, scattered epithelial denudation on villous tips;
grade 3, denuded tips with exposed lamina propria and villous blunting; grade 4,
epithelial shedding from both the apex and mid-region of the villi associated with
shortened and widened villous structure; grade 5, complete destruction of villi and
disintegration of lamina propria with ulceration.
TUNEL assay
Paraffin-embedded jejunal sections were deparaffinized and in situ detection of
cells with DNA-strand breaks was performed by the TUNEL labeling method using a
TdT-FragEL™ DNA fragmentation detection kit (Oncogene Research Products, Boston,
MA) following the manufacturer's protocol. Negative controls were performed by
substituting tris-buffered saline (TBS) for the TdT enzyme.
Caspase-3 activity assay
Scraped jejunal mucosa was lysed and the protein concentration of the lysate was
adjusted to 50 μg/ml to test for caspase-3 activity (Anaspec, San Jose, CA). The assay is
based on spectrophotometric detection of the chromophore rhodamine 110 (Rh110) after
cleavage from the labeled substrate DEVD-Rh110. The caspase-3 activity of samples
was measured in relative fluorescence units (RFUs) at Ex/Em = 496 nm/520 nm for 60
minutes in 5-minute intervals. The level of caspase-3 activity was expressed as
RFU/min/mg.
Ussing chamber studies and macromolecular flux assay
Intestinal segments were excised and immediately placed in warm Krebs buffer. The
external muscle layers were stripped off, leaving the submucosal plexus and mucosa
intact. From each rat, two pieces of the muscle-stripped tissues (cut longitudinally into
flat sheets along the mesenteric border) were mounted in Ussing chambers (WPI
Instruments, Sarasota, FL). Care was taken to avoid tissues containing Peyer's patches.
The opening area (2 cm2) of the chamber exposed the tissue to 5 ml of circulating
oxygenated Krebs buffer. The serosal buffer contained 10 mM glucose that was
osmotically balanced with 10 mM mannitol in the mucosal buffer. A circulating water
bath maintained the temperature of the buffer at 37°C. The serosal and mucosal tissue
baths were clamped at 0 V using a voltage clamp feedback amplifier (WPI Instruments).
The short-circuit current (Isc, μA/cm2) of the tissue was recorded continuously on line.
At 5-minute intervals, the voltage between the two baths was stepped to 1 mV for one
second, and the change in the Isc caused by the pulse was used to calculate the tissue
conductance (mS/cm2) according to Ohm's law (Yu et al., 2003).
The intestinal epithelial permeability was determined by the level of
mucosal-to-serosal flux of horseradish peroxidase (HRP type II, MW = 44 kD, Sigma).
Tissues in the Ussing chambers were allowed to equilibrate until the Isc stabilized
before HRP was added to the luminal buffer at a final concentration of 5 × 10-5 M.
Samples (300 μl) of serosal buffer were collected at 0, 30, 60 and 90 minutes after
luminal addition of HRP, and were replaced with Krebs buffer. The concentration of
HRP was determined by a kinetic enzymatic assay. Fluxes were calculated according to
standard formulae and were expressed as pmol/cm2/hr (Yu et al., 2003).
Fluorescein-based gut permeability assay
To assess gut permeability in vivo, a fluorescein-based assay was performed as
described previously with slight modification (Wang et al., 1998). The 4-kDa
fluorescein isothiocyanate-conjugated dextran (FD4, Sigma) dissolved in Krebs buffer
was administered into the lumen of ligated jejunal sac to a final concentration of 0.5
mg/ml immediately after the release of the artery clamp. The jejunal sac was placed
back into the peritoneal cavity, and the open abdomen was covered with a saline-wetted
gauze and foil to prevent evaporation and direct light. Arterial plasma from 0.5 ml of
blood was taken at 60 minute post-reperfusion. Fluorescence intensity in arterial plasma
was measured at Ex/Em = 496/520 nm using a plate reader. The concentration (μg/ml)
of FD4 in plasma was calculated using a standard curve.
Magnetic Resonance Imaging (MRI)-based gut permeability assay
To assess gut permeability in vivo, the contrast agent gadodiamide (Omniscan, GE
Healthcare) was instilled into the lumen of the ligated jejunal sac to a final
concentration of 0.25 M immediately after the release of the artery clamp, and the signal
intensity of this agent in the liver and kidney was quantified using abdominal MRI as
described previously (Hsiao et al., 2009). In sham controls, gadodiamide was injected
into the jejunal sac after mock manipulation. The rats were placed in a home-made
resonance coil with inner diameter of 6 cm, and abdominal MRI was performed at
various time points (0, 5, 10, 15, 30, 45 and 60 minutes) using a clinical 1.5 T MR
System (Signa Excite; GE Healthcare). Two dimensional T1-weighted fast spin echo
MRI pulse sequences were used, with the following parameter set: TR/TE = 140/4.2
msec, FOV = 12 × 8.4 cm2, and NEX = 4. The signal intensity produced by
gadodiamide in the region of interest (ROI), liver and both kidneys, was measured.
Signal-to-noise ratio (SNR) was calculated by dividing the signal intensity of the ROI
by that of the background. To quantify the gadodiamide delivered to the systemic
circulation, samples of plasma from 0.5 ml of blood taken before (t = 0 minutes) and 15,
30, and 60 minutes after injecting gadodiamide were prepared. A known concentration
(0.5 M) of gadodiamide solution was serially diluted with neat plasma to prepare
standard solutions for the calibration curve. Plasma and standard solutions were
subjected to MRI scan, and imaging parameters were: TR/TE = 550/67.50 msec, FOV =
14 × 0.5 cm2, and NEX = 4. The SNRs of the standard solutions of gadodiamide were
plotted against their respective concentrations to establish a standard curve. The plasma
gadodiamide concentrations were calculated from the standard curve (Hsiao et al.,
2009).
Analysis of bacterial translocation
The liver and spleen tissues were homogenized, sonicated and adjusted to a protein
concentration of 0.1 g/ml with sterile PBS. Each homogenate was inoculated onto fresh
blood agar plates (200 μl per plate; Scientific Biotech Corp., Taipei, Taiwan) and the
plates were incubated at 37°C overnight. The number of bacterial colony forming units
(CFU) was normalized per gram of tissue (CFU/g).
Myeloperoxidase (MPO) activity assay
Intestinal samples were homogenized and sonicated in 10 volumes of potassium
phosphate buffer (PPB, 50 mM, pH 6.0) containing 0.5% HTAB (Sigma). Lysates were
centrifuged and supernatants were diluted in PPB containing 0.167 mg/ml of
O-dianisidine dihydrochloride (Sigma) and 0.0005% of H2O2. The enzyme
concentration was determined from the absorbance at 460 nm measured every 30
seconds over a 5 minute period. One unit of MPO activity was defined as the quantity of
enzyme degrading 1 μmol of H2O2 per minute, and MPO activity of the gut was
expressed in U/mg of tissue.
ELISA for TNF α and MIP-1 α
Scraped jejunal mucosa were homogenized and sonicated in PBS and the lysate
was centrifuged. The protein concentration in the supernatant was quantified. The levels
of TNFα and MIP-1α in mucosal samples were measured by using ELISA development
kits (PeproTech, NJ) according to the manufacturer's instructions. To measure cytokine
levels, microplates were coated overnight with capture antibodies. The plates were
blocked with PBS containing 1% BSA for 1 hour and washed. The sample and standard
solutions were added and incubated for 2 hours. The biotinylated antigen-affinity
detection antibodies were incubated for another 2 hours. After washing, avidin-HRP
conjugate was added for 30 minutes followed by incubation with ABTS liquid substrate
for color development. Absorbance was measured at 405 nm with correction set at 650
nm. The cytokine levels in jejunal mucosa were expressed in pg/mg of protein.
Immunohistochemical and immunofluorescence staining for rat jejunal tissue
Tissue sections were incubated with 3 % H2O2 to block endogenous peroxidase for
immunohistochemical staining and were quenched with 50 mM NH4Cl in PBS for
immunofluorescence staining, and then blocked with 2% normal goat serum. Tissue
sections were incubated with anti-cleaved caspase-3 (1:1000 Cell signaling, Danvers,
MA), anti-proliferating cell nuclear antigen (PCNA) (1:100, Lifespan biosciences,
Seattle, WA), anti-SGLT1 (1:200, Millipore, Billerica, MA), anti-Akt (1:100, Cell
signaling), or isotype control antibodies. After washing with PBS, tissues stained for
SGLT1 were incubated with biotin-conjugated goat anti-rabbit IgG (1:1000, Molecular
Probes, Carlsbad, CA) for one hour, followed with a streptavidin-conjugated Alexa
Fluor® 488 fluorescent probe (1:1000, Molecular Probes) for one hour; tissues stained
for Akt were incubated with goat anti-mouse IgG conjugated to Alexa Fluor® 488
fluorescent probe (1:1000, Molecular Probes) for one hour. All tissues were stained with
Hoechst dye to visualize cell nuclei. Tissues stained for cleaved caspase-3 and PCNA
were incubated with HRP-conjugated SignalStain® Boost anti-rabbit IHC detection
reagent (Cell signaling) and developed with a DAB peroxidise substrate followed by
counterstain with hematoxylin. The slides were mounted with aqueous mounting media
and viewed under a Zeiss fluorescence microscope.
Western blotting for jejunal mucosa
Scraped jejunal mucosa was homogenized in ice-cold complete RIPA buffer, and
the lysate was sonicated and centrifuged. The protein concentration of the supernatant
was adjusted to 5 mg/ml and diluted at a 1:1 vol:vol ratio in 2× electrophoresis sample
buffer containing 2% (w/v) SDS, 100 mM DTT, and 62.5 mM Tris/HCl (pH 6.8).
Samples were then heated to 95°C in a heat block for 5 minutes, and stored at –20°C
until used for immunoblotting.
The extracted proteins were separated by SDS-PAGE, and the resolved proteins were
electrotransferred onto membranes. After blocking with 5% non-fat milk in TBS, the
membrane was incubated with anti-occludin (1:1000, Invitrogen), anti-Akt (1:500, Cell
signaling), anti-phospho-Akt (1:1000, Cell signaling), anti-IκBα (1:1000, Santa Cruz,
Santa Cruz, CA), anti-phospho-IκBα (1:1000, Santa Cruz), anti-phospho-Bad (1:500,
Cell signaling), anti-phospho-mTOR (1:500, Cell signaling), anti-phospho-GSK3α/β
(1:1000, Cell signaling), or anti-phospho-FoxO1/3a (1:1000, Cell signaling) at 4°C
overnight. A monoclonal mouse anti-β-actin (1:10000, Sigma) was also used to control
for equal loading in each sample. Membranes were washed with 0.1% Tween 20 in TBS
and incubated with either horseradish peroxidase-conjugated goat anti-rabbit or
anti-mouse IgG (1:1000, Cell signaling). The antigens were revealedand band density
quantified by photoimage analysis.
Akt kinase activity
The kinase activity of Akt was determined using a non-radioactive Akt kinase
assay kit (Cell signaling) according to the manufacturer’s instruction. Briefly, scraped
jejunal mucosa was homogenized in ice-cold lysis buffer, and the lysate was sonicated
and centrifuged. For immunoprecipitation, 20μl of immobilized antibody beads
conjugated to anti-phospho-Akt was added to 200μl of cell lysate (the protein
concentration was 2.25 mg/ml) with gentle rocking overnight at 4°C. After washing
with lysis buffer and kinase buffer, the pellet was incubated with ATP and GSK3 fusion
protein for 30 minutes for kinase reaction. Exogenous GSK3, a downstream target of
Akt, served as the substrate of phospho-Akt in this assay. The reaction was terminated
by addition of 2× SDS sample buffer. The Akt kinase activity was determined by
Western blot using anti-phospho-GSK3α/β antibody.
Cell culture models
Human colonic carcinoma Caco-2 and HT29 cells were grown in Dulbecco's
modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY, USA) containing 5
mM glucose and without pyruvate (Kles et al., 2002; Yu et al., 2005). The media was
supplemented with 10% fetal bovine serum, 15 mM HEPES, 100 U/mL penicillin, and
0.1 mg/mL streptomycin (Sigma, St. Louis, MO, USA). Cells were seeded in 96- well
(105 cells/well) or 24-well (106 cells/well) tissue culture plates (Costar, Corning, NY,
USA). Cells were grown to confluency for one week at 37 °C with 5% CO2 and 96%
humidity. In all studies, cells were used between passages 21 and 27.
Hypoxic challenge and glucose deprivation
Cells were deprived of oxygen and glucose as previously described (Kalda et al.,
1998; Kles et al., 2002; Shahrzad et al., 2005). Hypoxic (Hx) challenge was conducted
using a modular incubator chamber (Billups-Rothenberg, CA, USA) by infusion of 5%
CO2 and 95% N2 at 10 L/min for 5 minutes; normoxic (Nx) controls were kept at 5%
CO2 and 95% air (Kalda et al., 1998; Kles et al., 2002; Shahrzad et al., 2005). In some
groups, cells were pretreated with necrostatin-1 (Nec-1; a specific RIP1 inhibitor), BHA
(200 μM; a free radical scavenger), apocynin (1 mM; an inhibitor to NADPH oxidase),
or vehicle controls prior to hypoxic challenge to examine cell death pathways.
In additional experiments, cells were incubated in glucose-free and pyruvate-free
DMEM media (Invitrogen) supplemented as above plus 0-25 mM glucose. To verify the
role of glycolysis in death resistance, cells were pretreated with inhibitors to the cascade
of glucose metabolic pathways such as IA (1 mM; a glycolytic inhibitor to GPD) and
UK (10 μM; a MPC inhibitor), or with vehicle controls prior to hypoxic challenge in the
presence of glucose. In another set of experiments, equimolar concentrations of
substances were apically instilled in place of glucose to verify cellular metabolic status,
such as 3-O-methyl-glucopyranoside (3-OMG; a non-metabolizable sugar analog taken
up by glucose transporters), mannitol (a non-absorbable and non-metabolizable sugar
used as an osmolarity control), or glutamate (an amino acid used as an oxidative fuel
control) before hypoxic challenge. The anti-necrosis effect of a cell-permeable pyruvate
derivative, ethyl pyruvate (25 mM), was also examined in hypoxic cells. All reagents
were purchased from Sigma.
Lactodehydrogenase (LDH) leakage assay
The leakage of intracellular enzyme LDH into the surrounding environment
indicates rupture of plasma membrane, which is a hallmark of cell necrosis. The cell
culture supernatant was collected after hypoxic challenge for the measurement of LDH
activity. Briefly, a reaction mixture of 0.2 mM NADH and 0.36 mM sodium pyruvate
was dissolved in Krebs-Henseleit (K-H) buffer containing 2% BSA. The K-H buffer is
composed of 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.25 mM CaCl2, 1.2 mM
KH2PO4, 24 mM NaHCO3 (pH7.4). Ten μl of cell supernatant and 190 μl of reaction
mixture were mixed well in 96-well plates prior to spectrophotometric kinetic readings.
Owing to the differences in the absorption spectra of NADH and NAD+, changes in the
NADH concentration can be detected at 340 nm. The decrease in absorbance measured
every minute over a 10-min period represents the activity of LDH. One unit of LDH
activity is defined as the quantity for oxidation of 1 μmole NADH per minute; the LDH
activity of cell supernatant was expressed in Units per liter (Unit/L).
Analysis of mitochondrial functions by time-lapse microscopy
Mitochondrial transmembrane potential changes and ROS production were
measured by using cell-permeant cationic fluorescent dyes, including
5,5’,6,6’-tetrachloro-1.1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1)
reagents and MitoSOX (Invitrogen, CA, USA) . The JC-1 reagent emits red
fluorescence in its aggregated form when it accumulates in the negatively charged
mitochondrial matrix of viable cells. The monomeric form of JC-1 emits green
fluorescence when the dye is dispersed in the cytoplasm due to the loss of mitochondrial
transmembrane potential. MitoSOX Red, which selectively targets functional
mitochondria, exhibits red fluorescence after oxidization by superoxide. Cells grown on
96-well culture plates or 8-well chamberslides (2 x 105 cells/well, Costar) were
incubated with JC-1 (10 μg/ml) or MitoSOX (5 μM) for 20 min, and washed twice prior
to hypoxic challenge, and then subjected to fluoremetric readings. Alternatively, cells
were analyzed by time-lapse microscopy using Application Solution Multi-Dimensional
Workstation (AS MDW) (Leica Microsystems, Mannheim, Germany). Cells were
loaded with JC-1 (10 μg/ml) for 30 min before infusion of 5% CO2 and 95% N2 into
the temperature-controlled moisture chamber of the AS MDW for live cell imaging.
Immunoprecipitation of RIP1-RIP3 complex and in vitro kinase assay
Cells lysates were immunoprecipitated with anti-human RIP1 (BD bioscience,
Franklin Lakes, NJ, USA) overnight, and then incubated with protein G agarose beads
for one hour at 4°C followed by centrifugation. The pellet was dissolved in
electrophoresis sample buffer for heat denaturation. The immune complexes were
subjected to reducing SDS/PAGE and the membranes were incubated with anti-RIP1
(1:1000, BD bioscience) or polyclonal rabbit anti-RIP3 (1:1000, Abcam, Cambridge,
UK) for immunoblotting. For in vitro kinase assays, the bead pellets were incubated in
kinase reaction buffer supplemented with 10 μM cold ATP and 1μCi γ-32P-ATP for 30
min at 30°C. The samples were resolved by SDS/PAGE and exposed to film for
autoradiography as previously described (He et al., 2009).
RNAi-mediated knockdown of RIP1
RIP1 siRNA and negative control were purchased from Dharmacon, Lafayette, CA,
USA. Cells were transfected with 100 nM siRNA oligonucleotides using
DharmaFECT® siRNA transfection reagents as per manufacturer’s protocol.
Knockdown efficiency of transfected cells was confirmed by western blotting 96 hrs
post transfection.
Measurement of transepithelial electrical resistance (TER) and paracellular
permeability
Cells grown to confluency underwent normoxia or hypoxia for the indicated times.
The monolayer TER was measured using an electrovoltohmeter (EVOM; World
Precision Instruments, Sarasota, FL, USA). Paracellular permeability was assessed by
apical-to-basal transport of a dextran probe (MW3000) conjugated to fluorescein
(Invitrogen) as described previously (Yu et al., 2005; Wu et al., 2011).
Immunofluorescent staining of tight junction structures
Cells were exposed to normoxia or hypoxia for 16 hrs, fixed with 4%
paraformaldehyde for one hour on ice, and quenched with 50 mM NH4Cl in PBS for 10
min at room temperature. After blocking with 0.1% bovine serum albumin (BSA) in
PBS for one hour, monolayers were incubated with a polyclonal rabbit anti-human ZO-1
antibody (1:100, Invitrogen) in a permeabilizing buffer (0.05% saponin, and 0.1% BSA
in PBS) for one hour. Cells were then incubated with secondary antibodies of goat
anti-rabbit IgG conjugated to Alexa 488 (1:1000, Invitrogen) for one hour in the dark,
and then stained with a Hoechst dye to visualize cell nuclei. The slides were mounted
with aqueous mounting media and viewed under a Zeiss fluorescence microscope.
Measurement of cell apoptosis
DNA fragmentation, which is a final stage of apoptosis, was measured using a cell
detection ELISA kit (Roche) for oligonucleosome amount as previously described (Yu
et al., 2005). The caspse-3 activity assay (Anaspec) was based on spectrophotometric
detection of chromophore rhodamine 110 (Rh110) after cleavage from the labeled
substrate DEVD- Rh110 according to the manufacturer’s instructions (Huang et al.,
2011).
RNA extraction and polymerase chain reaction (PCR) for GLUT transcripts
Total RNA was isolated using Trizol reagent (Invitrogen) according to the
manufacturer's instructions. For semiquantitative PCR analysis, the RNA (2 µg) was
reverse transcribed (RT) with oligo(dT) using RevertAid First Strant cDNA Synthesis
reverse transcribed (RT) with oligo(dT) using RevertAid First Strant cDNA Synthesis