Influence of endurance exercise training on antioxidant enzymes, tight junction proteins, and inflammatory markers in the rat ileum

Animals

Adult female Sprague–Dawley rats (4 months old) were assigned to a sedentary (SED)
or endurance exercise-training (EXE) group (n = 8 per group). The animals were housed
on a 12-h: 12-h light–dark cycle (20–22 °C) and provided food and water ad libitum
throughout the experiment. The University of Florida Institutional Animal Care and
Use Committee approved the use of animals in this experiment.

Experimental design

The EXE animals were familiarized to treadmill running for five consecutive days (10,
20, 30, 40, and 50 min of exercise/day, respectively). Following 2 days of rest, EXE
animals were trained on the treadmill for 10 days at a speed of 30 m/min at 0° incline,
estimated work rate of 70 % maximum oxygen consumption 9], for 60 min per day. SED and EXE animals were sacrificed 24 h after the final training
bout and the ileum (last part of the small intestine and vital organ for nutrient
absorption) was stored and used for analyses.

Western blot analysis

Western blot analysis determined protein abundance in the ileum tissue. Briefly, ileum
tissue samples were homogenized 1:10 (wt/vol) in 5 mM Tris (pH 7.5) and 5 mM EDTA
(pH 8) with a protease inhibitor cocktail (Sigma, St. Louis, MO) and centrifuged at
1500×g for 10 min at 4 ?. The supernatant was collected and ileum protein content was assessed
by the Bradford method. Proteins from the supernatant fraction of the ileum homogenates
were separated via polyacrylamide gel electrophoresis (C.B.S. Scientific Company,
San Diego, CA). After electrophoresis, the proteins were transferred to polyvinylidene
difluoride membranes (Ameresco, Solon, OH) via the C.B.S. Scientific Company system
for 2 h at 200 mA. Non-specific sites were blocked for 1 h at room temperature in
PBS solution containing 0.05 % Tween and 5 % non-fat milk. Membranes were then incubated
for 1 h with primary antibodies directed against the proteins of interest. The primary
antibodies used were: superoxide dismutase 2 (SOD2; # GTX116093; GeneTex, Irvine,
CA), catalase (GeneTex, # GTX110704), 4-hydroxynonenal conjugated proteins (4-HNE,
# ab46545, Abcam, Cambridge, MA), p-p65 (Cell Signaling, Danvers, MA, # 3033) and
p65 (Cell Signaling, # 8242). Following incubation with primary antibodies, membranes
were washed extensively with PBS-Tween and then incubated with secondary antibodies.
Membranes were then developed using an enhanced chemiluminescent reagent (Amersham,
Pittsburgh, PA), and band densitometry was performed through the use of a gel documentation
system and associated densitometry software (UVP, LLC, Upland, CA). Alpha tubulin
(# 12G10, Developmental Studies Hybridoma Bank, Iowa City, IA) was used as the normalizing
control for SOD2 and catalase. For 4-HNE, the whole lane was quantified and normalized
to Ponceau S.

RT PCR for ileum mRNA expression

RNA was isolated from ileum using the Ribozol method (Ameresco) according to the manufacturer’s
instructions. Concentration and purity of the extracted RNA were measured spectrophotometrically
at 260 and at 280 nm using the NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific,
Waltham, MA). Following isolation, 1 ?g of RNA was reverse transcribed into cDNA using
a cDNA synthesis kit (Quanta, Gaithersburg, MD) per manufacturer’s recommendations.
Real-time PCR was performed by utilizing the CFX Connect instrument (Hercules, CA)
and SYBR green chemistry (Quanta) with the gene-specific primers listed in Table 1. Tested genes were: occludin (OCLN), claudin 1 (CLDN1), zonula occluden-1 (ZO1),
haptoglobin/zonulin (Zonulin), nuclear factor of kappa light polypeptide gene enhancer
in b-cells (NF
_K
B), tumor necrosis factor (TNF?), gamma interferon (IFN?), interleukin 6 (IL6), chemokine
(C–C motif) ligand 2 (CCL2), toll-like receptor 4 (TLR4), interleukin 10 (IL10), solute
carrier family 15 member 1 oligopeptide transporter (SLC15A1), solute carrier family
6 member 19 neutral amino acid transporter (SLC6A19), solute carrier family 7 member
6 amino acid transporter light chain, y + L system (SLC7A6), solute carrier family
7 member 7 amino acid transporter light chain, y + L system (SLC7A7), solute carrier
family 16 member 10 aromatic amino acid transporter (SLC16A10), solute carrier family
5 member 8 sodium/monocarboxylate co-transporter (SLC5A8), solute carrier family 27
member 2 fatty acid transporter (SLC27A2), and solute carrier family 6 member 4 sertonin
neurotransmitter transporter (SLC6A4). Beta-glucuronidase (GUSB) expression was not
different between the two groups and was used as the reference gene. Relative quantification
of gene expression was performed using the 2??CT method whereby ?CT [CT(reference
gene) ? CT(gene of interest)].

Table 1. Gene-specific primers used for RT-PCR

Statistical analysis

Dependent variable comparisons between groups were made by independent t tests with
the significance set at p  0.05. Data are presented as mean ± SE.