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ORIGINAL ARTICLE

Involvement of Nucleotide Binding and Oligomerization


Domain-Like Receptors in the Intestinal Injury of Severe
Acute Pancreatitis in Rats
Shan Xu, MS,*† Shuqing Wei, MS,*‡ Yu Guo, PhD,* Donglai Cui, PhD,* and Jinfeng Yao, PhD*

and 2 (NOD2) are the first identified members of the “nodsome”


Objectives: The aim of the study was to observe the role of nucleotide- family, characterized by a caspase activation and recruitment do-
binding and oligomerization domain (NOD)-like receptors (NLR) in intes- main at the N-terminal. Activation of NOD1 and NOD2 activate
tine injury of severe acute pancreatitis (SAP) in rats. NF-κB and MAPK pathway and induce proinflammatory cyto-
Methods: Severe acute pancreatitis was induced by retrograde infusion of kines. It is also reported that NOD1 and NOD2 can directly bind
sodium taurocholate into the biliopancreatic duct. Rats were divided into and activate caspase-1 to trigger interleukin 1β (IL-1β) process-
the following 6 groups: sham operation, SAP treated with saline, and SAP ing through the caspase activation and recruitment domain.5,6
treated with interleukin 1β (IL-1β)-converting enzyme inhibitor, killed at NACHT-LRR-PYD–containing protein 3 (NLRP3) is the best char-
6 or 12 hours after operation. Serum IL-18 and IL-1β concentrations were acterized member of the “inflammasome” family, characterized by
measured. mRNA expression and protein levels of NOD1, NOD2, and a pyrin domain at the N-terminal.7 NLRP3 mediates secretion of
NLRP3 in the intestine were measured. IL-18 and IL-1β via caspase-1 pathway.4 It is well studied that
Results: Severe acute pancreatitis resulted in significantly higher serum NLRs play an important role in shaping the innate immune re-
IL-18 and IL-IL-1β concentration, higher mRNA expression, and protein sponse within the intestine, maintaining homeostasis, inducing
levels of NOD1, NOD2, and NLRP3 in intestine in SAP treated with saline tissue repair after injury, and promoting tumorigenesis.8 However,
groups compared with sham operation groups. This effect was attenuated the role of NLRs in intestine injury in SAP remains unclear.
by administration of IL-1β–converting enzyme inhibitor. The present study induced SAP in rats by sodium taurocholate
Conclusions: The NLRs, including NOD1, NOD2, and NLRP3, were injection. Expression of NLRs in the intestine was observed to de-
involved in the intestine injury in SAP through a caspase-1 pathway. termine the involvement of NLRs in SAP-induced intestine injury.
Key Words: severe acute pancreatitis, intestine injury, Caspase-1 pathway is also studied here to determine possible
oligomerization domain (NOD)-like receptors, caspase-1, mechanism underlying this process.
inflammatory cytokines
(Pancreas 2018;00: 00–00)
MATERIALS AND METHODS

S evere acute pancreatitis (SAP) is a systemic disease with high


mortality, characterized by complications such as multiple or-
gan failure, pancreas hemorrhage, and necrosis. Previous studies
Animals
Adult male Sprague-Dawley rats weighing 220(20)g were
indicated that intestinal tract played an important role in the prog- housed 3 to 4 per cage in a colony room under controlled temperature
ress of SAP. In SAP, the structure and function of intestinal mu- (23[2]°C) and 12-hour light/dark schedule (light on at 7:00 A.M.)
cosa are damaged and lead to gut mucosal barrier dysfunction, with free access to food and water. All animal handling procedures
resulting in increased intestinal permeability.1,2 Increased intestinal were carried out in accordance with the Guide for the Care and
permeability may result in bacterial translocation from the intestinal Use of Laboratory Animals of Hebei Medical University, China.
tract and cause infectious complications.3 However, the mechanism
underlying intestinal injury in SAP is still not fully understood. Experimental Model and Groups
Nucleotide-binding and oligomerization domain (NOD)-like Rats were divided into 6 groups with 10 rats in each group:
receptors (NLRs) are a family of cytosolic proteins that play an im- sham operation (SO), SAP with saline treatment (SAP-S), SAP
portant role in inflammation and immunity. Multiple NLRs function with IL-1β converting enzyme (ICE, which is also known as
as pattern recognition receptors, detecting host-derived damage sig- caspase-1), and inhibitor treatment (SAP-ICE-I), killed at 6 or
nals or conserved molecular motifs present on pathogens and initiat- 12 hours after operation. Acute pancreatitis was induced following
ing the innate immune response. The NLRs can be distinguished into previous study.9 In brief, animals were anesthetized by intraperito-
two broad families: the “nodsome” family and the “inflammasome” neal injection of chloral hydrate (10%, 1 mL/100 g of body weight).
family.4 Nucleotide-binding and oligomerization domain 1 (NOD1) The SAP model was induced by retrograde infusion of a fresh pre-
pared 5% sodium taurocholate solution (Sigma-Aldrich, St. Louis,
From the *Department of Gastroenterology, The Second Hospital of Hebei Mo, 1 mL/kg) into the biliopancreatic duct at a speed of 0.1 mL/min
Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Medical after laparotomy. The SO groups were treated in the same way ex-
University, Hebei Institute of Gastroenterology, Shijiazhuang; †The Central
Hospital of HanDan, Handan; and ‡Hebei Chest Hospital, Shijiazhuang, China. cept that equivalent volume of saline solution was used instead of
Received for publication May 5, 2017; accepted December 1, 2017. sodium taurocholate solution. The ICE inhibitor Ac-YVAD-CHO
Address correspondence to: Jinfeng Yao, PhD, Department of Gastroenterology, (Chinese Peptide Inc, Hangzhou, China, 0.25 mg in 1-mL PBS)
The Second Hospital of Hebei Medical University, Hebei Key Laboratory was intraperitoneally injected in the SAP-ICE-I groups, whereas
of Gastroenterology, Hebei Medical University, Hebei Institute of
Gastroenterology, No. 215 Heping West Rd, Shijiazhuang 050000, Hebei, 1 mL of saline was injected in other groups 2 hours after operation.
China (e‐mail: yaojf1965@outlook.com).
The authors declare no conflict of interest. Blood and Tissue Preparation
S.X. and S.W. are co-first authors.
Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. After 6 or 12 hours after operation, animals were killed and
DOI: 10.1097/MPA.0000000000000977 blood samples were taken via femoral artery. After centrifugation

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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

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Pancreas • Volume 00, Number 00, Month 2018 NLRs in Intestinal Injury in SAP

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

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Xu et al Pancreas • Volume 00, Number 00, Month 2018

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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Pancreas • Volume 00, Number 00, Month 2018 NLRs in Intestinal Injury in SAP

FIGURE 3. IL-18 (A) and IL-1β (B) concentrations in serum. Data


presented as mean (SD). *P < 0.01 vs SO group at the same time
point; #P < 0.05 vs SO group at the same time point; ▲P < 0.01 vs
SAP-S group at the same time point; &P < 0.05 vs 6 hours in the
same treatment group.

FIGURE 5. mRNA expression of NOD1 (A), NOD2 (B), and


NLRP3 (C) in intestinal tissue. Data are presented as mean (SD).
*P < 0.01 vs SO group at the same time point; #P < 0.05 vs SO group
at the same time point; ▲P < 0.01 vs SAP-S group at the same
time point; △P < 0.05 vs SAP-S group at the same time point;
@P < 0.01 vs 6 hours in the same treatment group.

in response to parasites and yeasts.16 These studies indicated a pro-


tective role of these substances in intestinal epithelium. In the pres-
ent study, we found that mRNA expression and protein levels of
NOD1, NOD2, and NLRP3 were significantly elevated in rat intes-
FIGURE 4. The protein level of caspase-1 in intestinal tissue. Data
are presented as mean (SD). *P < 0.01 vs SO group at the same tine within a few hours after SAP. This elevation might be a protec-
time point; ▲P < 0.01 vs SAP-S group at the same time point; tive response to SAP. Because these proteins were shown to
&P < 0.05 vs 6 hours in the same treatment group. enhance IL-18 and IL-1β processing, increased expression of these
(western-blotting bands from left to right: SO-6 h, SAP-S-6 h, proteins may also contribute to the increased production of IL-18
SAP-ICE-I-6 h, SO-12 h, SAP-S-12 h, SAP-ICE-I-12 h). and IL-1β. Interestingly, contrary to NOD1 and NOD2, mRNA

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Xu et al Pancreas • Volume 00, Number 00, Month 2018

expression as well as protein levels of NLRP3 was significantly


lower at 12 hours than that at 6 hours. This result indicated that
these proteins may play different roles in intestine injury in SAP.
The NLRP3 couples with an apoptosis-associated speck-like pro-
tein (ASC) and procaspase-1 to form inflammasomes and convert
caspase-1 to its active form.17 The time course of caspase-1 ex-
pression was similar to that of NLRP3 in the present study, which
indicated an interaction of NLRP3 and caspase-1 in intestine in-
jury in SAP.
To determine possible mechanism underlying the involve-
ment of NLRs in intestine injury in SAP, ICE inhibitor was admin-
istrated in a subgroup of SAP rats. Ac-YVAD-CHO is a specific
ICE inhibitor (caspase-1 inhibitor), which could inhibit caspase-1
from processing pro–IL-18 and pro–IL-1β to mature IL-18 and
IL-1β.18 As expected, serum concentrations of IL-18 and IL-1β
were reduced under the effect of Ac-YVAD-CHO. We also found
that administration of ICE inhibitor could ameliorate intestine in-
jury in SAP rats, along with reduction of expression of NOD1,
NOD2, NLRP3, and caspase-1 levels. Previous in vitro experi-
ments found that NOD1 and NOD2 mRNA expressions were
increased by treatment of IL-1β administration of in vitro.19 In-
terleukin 18 was shown to simulate production of IL-1β and
TNF-α.10,20 Tumor necrosis factor α was reported to induce NLRP3
and caspase-1 expression.21 Because NOD1, NOD2, and NLRP3
could induce production of IL-18 and IL-1β,4 these pathways may
form positive feedback in regulating inflammatory response.
Thus, it could be speculated that the ameliorating effect of ICE in-
hibitor on pathological changes in intestine injury might be a re-
sult from blocking of these positive feedback.
The present study revealed an involvement of NLRs in intes-
tine injury of SAP. The NOD-like receptors participated in this
process through a caspase-1 pathway, by playing a role in positive
feedback formed by NLRs, caspase-1, and inflammatory cytokines.
However, besides caspase-1, many other molecules were known to
mediate NLR-related signal transduction.4 For example, activation
of either NOD1 or NOD2 leads to the recruitment and activation
of RIP2, which results in MAPK pathway and NF-κB activation.22
Acute pancreatitis induced a temporal increase in p38 MARK ex-
pression and activation as well as NF-κB activation.23 NF-κB p65
protein expression in the intestine tissue was significantly in-
creased in SAP rats.24 Thus, NLRs may also play a role in intestine
injury through NF-κB and MAPK pathways. A further systematic
investigation of these signaling pathways will help us better un-
derstand the role of NLRs in intestine injury of SAP.
The mRNA expression and protein levels of NOD1, NOD2,
and NLRP3 in intestine were significantly increased in the SAP
models, along with an increase in serum concentration of IL-18
and IL-1β. Administration of ICE inhibitor could ameliorate patho-
logical changes of intestine tissue and reduce the changes of these
signaling molecules. These results indicated an involvement of
NOD1, NOD2, and NLRP3 in intestine injury in SAP models, and
this involvement may be partially through the caspase-1 pathway.

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Copyright © 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
This paper can be cited using the date of access and the unique DOI number which can be found in the footnotes.
Pancreas • Volume 00, Number 00, Month 2018 NLRs in Intestinal Injury in SAP

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