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(3000 rpm, 10 min), serum was collected and stored at −80°C for Data Analysis
subsequent measurement. The pancreas was removed and fixed in All the values in the text and figures are expressed as mean
4% phosphate-buffered formaldehyde for histopathology observa- (SD). To determine the difference among groups at the same time
tion. Approximately 5-cm intestinal tissue (10 cm away from the point, data were analyzed by one-way analysis of variance followed
ileocecal junction) was removed and 3 cm of which was immedi- by post hoc LSD test. To compare groups subjected to the same
ately frozen on liquid nitrogen and then stored at −80°C. The treatment at different time point, data were analyzed using
remaining part of intestinal tissue was fixed in 4% phosphate- independent-samples t test. Significance level was set at P < 0.05
buffered formaldehyde for histopathology observation. for all these analyses.
Histological Examination
RESULTS
For histological analysis, formaldehyde-fixed pancreas and
intestine were embedded in paraffin, sectioned at 4 μm, and stained
with hematoxylin-eosin (HE). The tissue specimens were examined Histopathological Changes
under light microscope (Leica Microsystems, Shanghai, China). Pancreas
The histopathological examination of the pancreas in the SO
Cytokines Assay group was normal at 6 hours (Fig. 1A). At 12 hours in SO group,
Serum concentrations of IL-18 and IL-1β were measured by scattered cell edema could be observed. Necrotic cells and
commercially available enzyme-linked immunosorbent assay hemorrhagic foci were occasionally observed. No inflammatory
kits, according to the manufacturer's instructions (R&D Systems, cells appeared (Fig. 1B). The pancreas showed pathological
Minneapolis, Minn). changes in SAP-S group at 6 hours. Interstitial edema, necrosis
of acinar cells, visible bleeding, and inflammatory cell infiltration
could be observed (Fig. 1C). At 12 hours in SAP-S group, there
Quantitative Reverse Transcriptase Polymerase were diffuse interstitial edema, large necrosis, splinter hemorrhage,
Chain Reaction Analysis and more inflammatory cell infiltration (Fig. 1D). The SAP-ICE-I
Intestine tissue was homogenized, and total RNA was ex- group showed a little necrosis, bleeding, inflammatory cell infiltration,
tracted with Trizol RNA extraction reagent (Life Technologies, and edema between small leaflets at both 6 hours (Fig. 1E) and
Pleasanton, Calif ). Complementary DNA (cDNA) was synthesized 12 hours (Fig. 1F).
using reverse transcriptase (Promega, Madison, Wis). q-PCR was
performed using SYBR Premix Ex Taq (Takara Bio Inc, Dalian, Small Intestines
China) in an ABI 7500 Real-Time PCR System (Applied Bio- The intestinal mucosa, intestinal epithelium, and villus were
systems, Waltham, Mass). The PCR procedure was 30 seconds at normal in SO groups (Figs. 2A, B). At 6 hours, the SAP-S group
95°C followed by 45 cycles of 5 seconds at 95°C and 40 seconds showed necrosis and exfoliation of the epithelial cell layer. Part of
at 60°C. Primers for each gene were designed using Primer Premier intestinal villi exfoliated and the lamina propria was exposed.
(PREMIER Biosoft, Palo Alto, Calif ). The NOD1 cDNA was am- Congestion and expansion of capillaries were also observed
plified using primers 5′-GCCCGACAGAAACTCCTTCA-3′ and (Fig. 2C). At 12 hours in SAP-S group, there were a small number
5′-GGCATGGCATGTACCTGGTTA-3′ (155 bp). The NOD2 of necrotic intestinal mucosal epithelial cells. Some villi showed
cDNA was amplified using primers 5′-GGGAGCACTTCCATTC abnormal morphology with exfoliated top. Lamina propria edema
CATC-3′ and 5′-CACCCTGCAAAACGTCAACTT-3′ (176 bp). appeared (Fig. 2D). The SAP-ICE-I groups showed mild
The NLPR3 cDNA was amplified using primers 5′-CCAGTAAT edema in lamina propria. The intestinal epithelium and villus were
ACCATTGGGGACAGA-3′ and 5′-ATTGCTCTGTAGGTCCAG normal. No necrosis was observed (Figs. 2E, F).
GTTCTC-3′ (92 bp). Primers for β-actin were 5′-GGAGATTACTG
CCCTGGCTCCTA-3′ and 5′-GACTCATCGTACTCCTGCTTGC Serum IL-18 and IL-1β Concentrations
TG-3′ (150 bp). The relative amount of target gene was calculated Serum concentration of IL-18 in the SAP-S group was sig-
using the 2-△△CT method. nificantly higher than that in the SO group (P < 0.01 at 6 and at
12 hours) and in the SAP-ICE-I group (P < 0.01 at 6 and at
Western Blotting 12 hours). Serum concentration of IL-18 in the SAP-ICE-I
Whole-cell proteins were extracted using Tissue Protein Extrac- group was significantly higher than that in the SO group only
tion Reagent (Thermo Scientific, Waltham, Mass), following the at 6 hours (P < 0.05). The SAP-S group showed significantly
manufacturer's instruction. Protein sample extract (20 μg) was sub- higher IL-18 concentration at 12 hours compared with that at
jected to SDS-PAGE and then transferred to a PVDF membrane 6 hours (P < 0.05) (Fig. 3A).
(Millipore, Billerica, Mass). The membranes were blocked with The SAP-S group showed significantly higher IL-1β con-
5% skim milk for 1 hour and then incubated with primary antibody, centration than that in the SO group (P < 0.01 for both 6 and
anti-NOD1 rabbit polyclonal antibody, anti-NOD2 rabbit polyclonal 12 hours) and in the SAP-ICE-I group (P < 0.01 for both 6 and
antibody, anti-NLPR3 rabbit polyclonal antibody, anticaspase-1 12 hours). Serum concentration of IL-1β in the SAP-ICE-I group
rabbit polyclonal antibody (all using 1:300, Santa Cruz, Dallas, was significantly higher than that in the SO group at both 6
Texas), and anti–β-actin rabbit polyclonal antibody (1:1,000, (P < 0.05) and 12 hours (P < 0.05). The SAP-S group showed sig-
Santa Cruz) overnight at 4°C. The membranes were washed with nificantly higher IL-1β concentration at 12 hours compared with
tris buffered saline with tween, incubated in secondary antibody that at 6 hours (P < 0.05) (Fig. 3B).
horseradish peroxidase conjugated goat anti rabbit IgG (1:3,000,
Santa Cruz) for 1 hour at room temperature, and then developed Protein Levels of Caspase-1 in the Intestine
using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Caspase-1 protein level in the SAP-S group was significantly
Waltham, Mass). The bands on the film were scanned and analyzed higher than that in the SO group (P < 0.01, for both 6 and
using Image J software. 12 hours). Caspase-1 protein level in the SAP-ICE-I group was
FIGURE 1. Histopathologic changes of pancreatic in different groups (HE staining, 200). A, SO group at 6 hours; B, SO group at 12 hours;
C, SAP-S group at 6 hours; D, SAP-S group at 12 hours; E, SAP-ICE-I group at 6 hours; F, SAP-ICE-I group at 12 hours.
significantly lower than that in the SAP-S group (P < 0.01, for showed significantly higher NOD2 mRNA expression at 12 hours
both 6 and 12 hours). There was no difference between the SO compared with that at 6 hours (P < 0.01) (Fig. 5B).
group and the SAP-ICE-I group. The SAP-S group showed signif- The SAP-S group showed significantly higher NLRP3 mRNA
icantly higher caspase-1 protein level at 6 hours compared with expression than that in the SO group (P < 0.01 for both 6 and
that at 12 hours (P < 0.05). The SAP-ICE-I group showed signif- 12 hours). The NLRP3 mRNA expression in the SAP-ICE-I group
icantly higher caspase-1 protein level at 12 hours compared with was significantly lower than that in the SAP-S group (P < 0.01,
that at 6 hours (P < 0.05) (Fig. 4). for both 6 and 12 hours). There was no difference between the
SO group and the SAP-ICE-I group. The SAP-S group showed
significantly higher NLRP3 mRNA expression at 6 hours com-
mRNA expression of NOD1, NOD2, and NLPR3 pared with that at 12 hours (P < 0.01) (Fig. 5C).
in the Intestine
The NOD1 mRNA expression in the SAP-S group and SAP-
ICE-I group was significantly higher than that in the SO group Protein Levels of NOD1, NOD2, and NLPR3 in
(P < 0.01 and P < 0.05, respectively, for both 6 and 12 hours). the Intestine
The NOD1 mRNA expression in the SAP-ICE-I group was signif- The NOD1 protein level in the SAP-S group and SAP-ICE-I
icantly lower than that in the SAP-S group (P < 0.05, for both group was significantly higher than that in the SO group (P < 0.01
6 and 12 hours). The SAP-S group showed significantly higher and P < 0.01, respectively, for both 6 and 12 hours). The NOD1
NOD1 mRNA expression at 12 hours compared with that at protein level in the SAP-ICE-I group was significantly lower than
6 hours (P < 0.01) (Fig. 5A). that in the SAP-S group (P < 0.01, for both 6 and 12 hours). There
The NOD2 mRNA expression in the SAP-S group was sig- was no difference between different time points in the same treat-
nificantly higher than that in the SO group (P < 0.05 for both 6 ment groups (Fig. 6A).
and 12 hours). The NOD2 mRNA expression in the SAP-ICE-I The SAP-S group and SAP-ICE-I group showed signifi-
group was significantly lower than that in the SAP-S group cantly higher NOD2 protein levels than that in the SO group
(P < 0.05, for both 6 and 12 hours). There was no difference be- (P < 0.01 and P < 0.01, respectively, for both 6 and 12 hours).
tween the SO group and the SAP-ICE-I group. The SAP-S group The NOD2 protein level in the SAP-ICE-I group was significantly
FIGURE 2. Histopathologic changes of intestinal mucosa in different groups (HE staining, 200) (A, SO group at 6 hours; B, SO group
at 12 hours; C, SAP-S group at 6 hours; D, SAP-S group at 12 hours; E, SAP-ICE-I group at 6 hours; F, SAP-ICE-I group at 12 hours).
lower than that in the SAP-S group (P < 0.01, for both 6 and enzyme (ICE). Interleukin 1β stimulates the expression of tumor
12 hours). There was no difference between different time points necrosis factor α (TNF-α). Interleukin 18 has the ability to induce
in the same treatment groups (Fig. 6B). production of IL-1β and TNF-α.10 The combined action of IL-18,
The NLRP3 protein level in the SAP-S group was signifi- IL-IL-1β, and TNF-α induces cytokine and chemokine produc-
cantly higher than that in the SO group (P < 0.01 for both 6 and tion and promotes a cascade of inflammatory responses.11 In
12 hours). The NLRP3 protein level in the SAP-ICE-I group accordance with previous studies,12,13 we found that serum con-
was significantly higher than that in the SO group (P < 0.05 only centration of IL-18 and IL-1β was significantly elevated in SAP
at 12 hours) and significantly lower than that in the SAP-S group models. Production of these cytokines appeared within a few
(P < 0.01 for both 6 and 12 hours). The SAP-S group showed sig- hours after administration of sodium taurocholate. These cyto-
nificantly higher NLRP3 protein level at 6 hours compared with kines could act as markers in SAP and cause distant tissues to re-
that at 12 hours (P < 0.01). The SAP-ICE-I group showed signif- spond to pancreatic inflammation, propagating injury to other
icantly higher NLRP3 protein level at 12 hours compared with organs.13 The present study found pathological changes in pan-
that at 6 hours (P < 0.05) (Fig. 6C). creas and small intestine in the SAP rats. Administration of ICE
inhibitor could ameliorate these changes along with reduction of
IL-18 and IL-1β. These results indicated that IL-18 and IL-1β
DISCUSSION played a critical role in intestine injury in SAP.
The present study confirmed the intestine injury in sodium Nucleotide-binding and oligomerization domain–like recep-
taurocholate–induced SAP rats. Along with this, we found that se- tors were known to play an important role in intestinal function.
rum concentration of IL-18 and IL-1β, mRNA expression and The NOD1, NOD2, and NLRP3 mediate regulation of intestinal
protein levels of NOD1, NOD2, and NLRP3 were significantly homeostasis.8 In NOD1-deficient mice, there were greater disrup-
increased in the SAP models. These changes were ameliorated tion of the intestinal epithelial cell barrier and increased intestinal
by administration of ICE inhibitor. permeability in response to dextran sulfate sodium–induced epi-
Interleukins 18 and 1β are inflammatory cytokines converted thelial injury.14 The NLRP3-deficient mice were also highly sus-
from their inactive precursors (pro–IL-18 and pro–IL-1β, respectively) ceptible to dextran sulfate sodium–induced colitis.15 Research in
by caspase-1, which is also known as interleukin-1β–converting NOD2-deficient mice found an exacerbated intestinal inflammation
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