




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 3 (6), pp. 001-009, June, 2015. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

 

Full Length Research Paper 

 

Protease-activated receptor-2 (PAR-2) regulates 
enterotoxigenic Escherichia coli-induced diarrhea 

during weaning in piglets 

 
Xiao-ying Pei1, Ding-zong Guo1*, Dong-hai Zhou1*, Rui Guo2, Hong-Mei Gao1 and Tian Xia1

 
 

1
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. 

2
Key Laboratory 

of Animal Embryo and Molecular Breeding, Hubei Academy of Agricultural Sciences, Wuhan 430064, China. 
 

Accepted 02 January, 2015 
 

Protease-activated receptor-2 (PAR-2) is a member of the G-protein-coupled receptor family. The 
proteases that activate PAR-2 are released during inflammation and injury, with PAR-2 regulating the 
response to these insults. In the gastrointestinal tract, PAR-2 is known to alter gastrointestinal 
secretion, motility, inflammation, and pain. In many cases, PAR-2 has been reported as pro-
inflammatory and proliferative. Paradoxically, PAR-2 is also suggested to be anti-inflammatory in some 
instances. Weaning piglet diarrhea is severely detrimental to the porcine industry, being responsible for 
11% of total piglet deaths, while those that survive from the disease experience developmental 
problems and fail to grow to the size of their healthy counterparts. Thus, we sought to determine any 
correlation between PAR-2 and weaning diarrhea. We hypothesized that PAR-2 might represent a new 
target in the treatment of weaning diarrhea. The current study measured PAR-2 using 
immunohistochemistry on sections of piglet gastrointestinal tract mucosa and identified changes in 
receptor expression during the development and course of weaning. Moreover, the effect of PAR-2 
stimulation using lipopolysaccharide (LPS) and heat-labile enterotoxin (LT) on IL-6 and IL-8 production 
in pig intestinal epithelial cells (IEC) was determined. This study found that PAR-2 is expressed 
abundantly in the piglet gastrointestinal tract mucosa, and revealed that PAR-2 mRNA and protein 
expression are both correlated with the severity of diarrhea. The generation of IL-6 and IL-8 by IECs was 
significantly increased following stimulation with PAR-2 agonists dose-dependently. Thus, we suggest 
PAR-2 may be involved in the development of diarrhea during weaning in piglets. 

 
Key words: Diarrhea, weaning piglet, gastrointestinal tract, protease activated receptor-2, immunostaining, RT-
PCR, IL-6, IL-8. 

 
 
INTRODUCTION 

 
Protease-activated receptors (PARs) are a family of G-
protein-coupled, seven-transmembrane receptors, con-
sisting of four known members, PAR-1 to PAR-4 (Déry et 
al., 1998; Macfarlane et al., 2001). PARs are activated by 

proteolytic cleavage of their extracellular NH2-terminus. 
Among the four PARs identified to date, PAR-1, PAR-3,  
 
 
 
*Corresponding author. +8627-87286251; E-mail: bigdefoot@ 
163.com 

 
 
 
 

 
and PAR4 are known to be activated by thrombin, while 
trypsin and mast cell-derived tryptase are shown to 
activate PAR-2 (Kanke et al., 2005). PAR-2 was first 
identified in 1944 and reported to be widely expressed in 
many cell types (Dulon et al., 2005; Saifeddine et al., 
1996) such as enterocytes, endothelial cells of the lamina 
propria and the submucosa, fibroblasts, myenteric neu-
rons, and immunological and inflammatory cells 
(including lymphocytes, neutrophils, and mast cells) 
(Kawao et al., 2002; Nystedt et al.,1996).  

The physiological and pathological roles of PAR-2 have 



 
 
 

 

been investigated in animal models in vivo using immuno-

histochemistry, pharmacology (employing synthetic PAR-2 

activating peptide), and PAR-2 deficient (PAR2−/−) mice 

(Kawagoe et al., 2002; Vergnolle, 1999). PAR-2 was found 

to participate in leukocyte rolling, ad-hesion, and infiltration 

of neutrophils, as well as inducing nuclear factor kappa B-

DNA binding (Kanke et al., 2001). With regard to the role of 

PAR-2 in the intestine, Hansen et al. (2005) reported PAR 2 

to augment enteritis from many kinds of bacteria. PAR-2 can 

be activated by proteolytic enzymes secreted from bacteria, 

promoting substance P release, which binds and activates 

the neurokinin A receptor, which in turn induces tissue 

edema and effusion. Acute activation of PAR-2 causes 

colitis characterized by gut wall edema, granulocyte 

recruitment, increased permeability and the release of pro-

inflammatory cytokines such as interleukin-1 and TNF-α 

(Cenac et al., 2002). Other studies have shown activation of 

PAR-2 with chronic, systemic administration of a PAR-2 

agonist to provide protection against a chronic model of 

inflammatory bowel disease (IBD). This protection was 

suggested to be the result of PAR-2-induced gastric mucus 

secretion and mucosal cytoprotection (Fiorucci et al., 2001; 

Kawabata et al., 2001). 

 

Cytokines play a central role in the modulation of the 
intestinal immune system and can stimulate proinflam-
matory (IL-1, TNF, IL-12, IL-6, IL-8) or anti-inflammatory 
(IL-4, IL-10, IL-1ra, IL-11) responses. Levels of both 
proinflammatory and anti-inflammatory cytokines are 
elevated in patients with IBD; however the ratio between 
pro- and anti-inflammatory cytokines becomes 
unbalanced, leading to inflammation. The major source of 
IL-6 within the gut is from macrophages, although the 
primary source of IL-6 in epithelial cells remains to be 
defined (Rogler et al., 1997). IL-6 is elevated in both the 
serum and mucosa of patients with IBD (Murata et al., 
1995).  

Furthermore, IL-8 (a powerful neutrophil chemo-
attractant and activator (Daig et al., 1996) correlates with 
the level of inflammation in the colon of patients with 
ulcerative colitis (UC) (Mazzucchelli et al., 1994) in which 
large numbers of neutrophils are found in crypt 
abscesses. Our previous reports have demonstrated that 
direct activation of PAR-1 and PAR-2 in human endo-
thelial cells leads to the production of IL-6, and both LPS 
and TNF-a markedly enhance PAR-induced activation 
(Chi et al., 2001). PAR-2 activation stimulates the 
proliferation of endometriotic stromal cells (ESC) and the 
secretion of IL-6 and IL-8 from the ESC (Hirota et al., 
2005).  

At weaning, piglets are exposed to marked alterations 
in living conditions, such as shifting from milk to a cereal-
based diet, becoming separated from the sow and mixed 
with other piglets. One of the consequences of weaning is 
the high risk of post weaning diarrhea (Wellock et al., 
2007). It has been reported that following three days of 
weaning, piglets become more susceptible to the 

 
 
 
 

 

detrimental effects of Escherichia coli enterotoxin when 
compared to their unweaned littermates. Further investi-
gations report morbidity and mortality of enterotoxigenic 
Escherichia coli (ETEC)-induced diarrhea to account for 
up to 56.2 and 24.7% of total diarrhea-induced morbidity 
and mortality, respectively (Alexander, 1994; Hampson, 
1994; Jin et al., 1998; Stevens et al., 1972). 
Characteristics of ETEC-induced disease include a rapid 
development and fast spread of infection resulting in 
either high mortality or chronically stunted growth. Both 
outcomes seriously compromise productivity and animal 
yield for the porcine industry.  

Thus, PAR-2 plays a potentially protective or patho-
genic role in gastrointestinal tract mucosa when under 
pathological conditions, possibly activated in response to 
tissue injury or inflammation. Many studies have found 
PAR-2 in humans and mice; however, few studies have 
found PAR-2 in pigs. The current study sought to deter-
mine the role of PAR-2 on diarrhea in weaned piglets. 
The results presented in this report demonstrate that 
PAR-2 is expressed abundantly in the piglet 
gastrointestinal tract mucosa, and reveals that PAR-2 
mRNA and protein expression are increased with the 
severity of diarrhea. Direct activation of PAR-2 by both 
LPS and LT in pig intestinal epithelial cells leads to 
enhanced production of IL-6 and IL-8. Taken together we 
suggest that PAR-2 may be involved in the development 
of diarrhea during weaning in piglets. 
 

 
MATERIALS AND METHODS 

 

Regents and materials 
 
Tryptone and yeast extract were obtained from Oxoid (Basingstoke, 

UK). Goat anti-PAR-2 antibody and anti-goat horseradish secondary 

antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, 

CA, USA). Epithelial cells were cultured in high-glucose formulated 

Dulbecco's Modified Eagle's Medium (DMEM, Gibco). Various 

concentrations of epidermal growth factor (EGF; Sigma), insulin 

(Sigma), fetal bovine serum (Gibco), glutamine (Gibco), penicillin 

(sigma) and streptomycin (Sigma) were also added to the medium to 

induce epithelial proliferation. The enzyme-linked immu-nosorbent 

assay (ELISA) kits for IL-6 were purchased from R and D  
systems (Minneapolis, MN). PAR-2 agonist peptides -Ser-Leu-Ile-
Gly-Arg-Leu-NH2 (SLIGRL-NH2) - and related hexa-peptides with 
an altered consensus sequence (LRGILS-NH2) were synthesized 
by Biotechnology Corp. (Xian, China). Trypsin, the soy bean trypsin 
inhibitor, E. coli LPS and E. coli LT were all obtained from Sigma-
Aldrich. 

 

ETEC challenge strains 
 
The ETEC K88 O149 strain (provided by the China Institute of 

Veterinary Drugs Control, China) was grown in Luria broth (LB) medium 

comprising: 1% tryptone, 0.5% yeast extract, and 1% NaCl, final pH 7.0. 

After an overnight incubation at 37 C with shaking, the bacterial cells 

were then diluted to an optical density at 600 nm (OD600) of 0.1 in 

fresh LB medium and allowed to grow to an OD600 of ≈1.0. The cell 

cultures were then centrifuged at 4,000 G  



 
 
 

 
for 10 min at 4°C. The bacterial pellets were resuspended in 20% 
dextrose and 5% non-fat milk. The challenge dose consisted of an 
equal amount of each strain and was determined by serial dilution 

and plating to provide a total of 2×10
10

 CFU/0.5 ml oral dose, twice 
daily with an 8-h interval for three days. 

 

Animals 
 
Animal care and procedures were in accordance with the National 

Institutes of Health recommendations for the humane use of animals. All 

experimental procedures were reviewed and approved by the 

appropriate Animal Use Committee of Huazhong Agricultural University. 

30 Duroc piglets aged 25 - 28 days and weighing ~7 kg were provided 

by HuBei Academy of Agricultural Sciences China. 18 piglets were 

infected with ECET (Enterotoxigenous E. coli), and 12 piglets suffering 

from diarrhea were selected as a diarrhea group. We killed 3 healthy 

piglets and 3 diarrheal pigs on the first, third, fifth and seventh day, 

respectively. All piglets were anaesthetized using sodium pentobarbital 

prior to opening the gut and dissecting the stomach, dodecadactylon 

jejunum ileum, cecum and colon. All sec-tions were washed 3 times in 

cold 0.9% NaCl. Half of the sections were fixed by immersion in 10% 

neutral-buffered formalin, dehydrated, embedded in paraffin wax, and 

stored at 4 C for immunohistochemistry. Other sections were placed into 

liquid nitrogen for 3 h and maintained at -70 C for real-time RT-PCR.  
 

 
Histopathology 
 
A subsection of samples were subject to histological analysis to 
verify the cellular changes within the gastrointestinal tract by H and 
E staining. 

 

Immunostaining of PAR-2 in the piglet gastrointestinal tract 
mucosa 
 
Immunostaining of PAR-2 was performed using a goat polyclonal 
antibody. Briefly, sections of gut were deparaffinized, rehydrated, 
immersed in phosphate-buffered saline (PBS) for 15 min at 37 C 
and blocked in endogenous peroxidase (3% peroxide) for 5 min. 
After washing in PBS, blocking was performed with a 5% solution of 
normal rabbit serum for 30 min at 37 C. Primary goat polyclonal 
antibody, diluted 1:100 in PBS, was applied to each section and 
incubated for 12 h at 4 C in a humidified chamber. After washing 
three times, the slides were flooded with biotinylated rabbit anti-goat 
linking antibody (1:200) for 20 min at 37 C. Sections were then 
washed twice more in PBS, treated with peroxidase-conjugated 
avidin for 30 min, rewashed twice in PBS, and then incubated with 
3-amino-9-ethylcarbazole for 25 min. Gut sections were stained 
with Mayer’s hematoxylin counterstain for 1 min, dewatered, 
mounted by neutral balsam, and then analyzed under a microscope 
(OLYMPUS, IX71, Japan).  

 

SYBR green real-time RT-PCR 
 
PAR-2 mRNA expression was determined by SYBR green I real-
time quantitative polymerase chain reaction (RT-qPCR) analysis 
using an IQ-5 real-time PCR detection system (Bio-Rad, America). 
Total RNA was extracted from the ileum, cecum and colon mucus 
using the TRIzol reagent (Invitrogen). After verification of its 
integrity, RNA was quantified spectrophotometrically with 1 ug 
processed for complementary DNA (cDNA) synthesis using 
SuperScript II reverse transcriptase (Toyobo, Japan). Specific 
primers for the PAR-2 gene were designed using Primer5 software. 

  
  

 
 

 
The sequences of the primers used were: PAR2, sense: 5’-GCA  
ACA ACT GGG TTT ACG GG -3’; antisense: 5’-GGT GTG ATG 
TGA AGG GCT GG -3’; GAPDH, used as a housekeeping gene, 
sense: 5’-GGT GAA GGT CGG AGT GAA CG-3’; antisense: 5’- 
CTC GCT CCT GGA AGA TGGTG-3’.  

The efficacy of the RT-PCR primer pairs was determined by 
amplifying serial dilutions of cDNA. The RT-PCR was performed 
using SYBR Green Real-time PCR Master Mix (Toyobo). Each 
cycle consisted of three steps: denaturation for 30 s at 95°C, 
annealing for 30 s at 60°C, and 30 s of elongation at 72°C. The 
data acquired from each sample were normalized to those of 
GAPDH. The specificity of the real-time reverse transcriptase was 
further confirmed by a regular RT-PCR followed by agarose gel 
electrophoretic analysis to verify the presence of a single band 
corresponding to the predicted size of the amplicon. Relative Ct 
(cycle time) values were obtained using the Bio-Rad iQ5 Optical 
System Software Data Acquisition Server. 

 

Culture of pig intestinal epithelial cells 
 
Newborn piglets were anaesthetized using sodium pentobarbital 
and killed. The small intestine was opened and sectioned into 2 - 3  
mm lengths. Tissue sections were transferred to a 25 ml flask and 
washed at least 8 times in 50 ml of fresh Hanks’ balanced salt 
solution (HBSS) with vigorous shaking. Tissue was then placed on 
a Petri dish and a sharp scalpel blade used to dice the tissue into 

<1 mm
3
 pieces before being returned to a T25 ml flask with 20 ml 

of collagenase type 1 (Sigma-Aldrich). Tissue was shaken 
vigorously for 2 h at 37 °C. 15 ml of DMEM-S (DMEM + 2.5% FCS 
+2% sorbitol) was then added to the flask and shaken again. The 
sedi-ment was allowed to dissociate from the supernatant under 
gravity for 60 s, and the supernatant carefully removed. This 
procedure was repeated twice. 10 ml of DMEM-S was added to the 
supernatant before being vortexed and centrifuged at 200 - 300 
rpm for 2 min. The supernatant was carefully removed and the 
pellet resuspended in 20 ml DMEM-S. This procedure was 
repeated at least 5 - 6 times until the supernatant became 
completely clear and the pellet well defined. Finally, the pellet was 
resuspended in the appropriate growth medium. 

 

Assay of IL-6 and IL-8 production 
 

Intestinal epithelial cells (1×10
5
) were added to each well of a 12-

well microtiter plate and allowed to adhere for 24 h. Following 
adherence, the medium containing serum was removed and serum-
free medium added. Selected concentrations of PAR-2 agonist 
peptides, trypsin, a soy bean trypsin inhibitor, E. coli LPS, E. coli LT 
or medium were added to the monolayers. All incubations were 
carried out at 37°C in 5% humidified CO2 for 24 h unless otherwise 
indicated. After incubation, the supernatant was collected and IL-6 
and IL-8 levels were quantified by ELISA according to the 
manufacturer’s protocol. 
 
 
Statistical analysis 

 
Data are displayed as the mean + SEM. All statistical analyses were 

performed using SPSS statistical software. The means among different 

groups were compared by one way analysis of variance (ANOVA).The 

PAR-2 positive area was calculated in pictures of 

immunohistochemistry using Image-Pro Plus software (IPP6.0). The 

data from SYBR Green Real-Time RT-PCR was analyzed using the  
formula: -[Mean Ct of specific gene of diarrhea group - Mean Ct of 

house-keeping gene of diarrhea] -2- 
ΔΔCt

 (2 [Mean Ct of specific gene 
of normal group - Mean Ct house-keeping gene of normal group]). 



    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. Representative H and E-stained histological sections of stomach, small intestine and large intestine 
from the diarrhea group (Figure 1A, B and C) with slides D, E and F providing normal comparisons respectively. 
Bar = 100 m.  

 
 

 

RESULTS 

 

Microscopic examination of histopathology 

 

The most striking pathological changes of the diarrhea 
group was desquamation and denaturation of the epithe-
lium mucosa, smooth muscle thinning of the bowels, 
infiltrated neutrophils and increased eosinophils exudated 
from the mucosal layer (Figure 1). 
 

 

Immunolocalization of PAR-2 in the piglet 
gastrointestinal tract mucosa 

 

PAR-2 was found expressed throughout the gastro-

intestinal tract, including in the mucosa of the stomach, 

duodenum, jejunum, ileum, cecum and colon (Figure 

2B1-B6). Further observations revealed elevated staining 

of PAR-2 within the lamina propria and greater staining in 

intestinal mucosa of the diarrhea group compared with 

control (Figure 2A1-A6). Histochemical analysis using the 

 
 
 

 

serum from a non-immunized goat displayed no positive 
immunostaining for PAR-2 in the gastrointestinal tract 
mucosa (Figure 2C1-C6). 
 

 

SYBR green real-time RT-PCR 
 

Using the 2
-ΔΔCt

 method to analyze the mean Ct, our 

results display the ratio of PAR-2 mRNA expression in 
pathogenic versus normal tissue over the development of 
diarrhea. During this time the ratio of PAR-2 expression 
was not found to significantly differ in the stomach 
mucosa (Figure 3a), although it was significantly elevated 
in the ileum and colon. Changes in the PAR-2 ratio were 
apparent from day 3 in the ileum and markedly so in the 
colon (Figure 3b and c). 

 

Effect of PAR-2 agonist peptides on IL-6 and IL-8 
production by naive and LPS+LI-stimulated IEC 

 

To examine the role of PAR-2 on intestinal epithelial cell 



    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Immunolocalization of PAR-2 in piglet gastrointestinal tract mucosa from healthy and 
diarrhea piglets using immunohistochemistry. Strong staining was exhibited in the gastric gland of the 
stomach mucosa (A1), duodenum mucosa (A2), jejunum mucosa (A3), ileum mucosa (A4), cecum 
mucosa (A5) and colon mucosa (A6) compared to control cells (slides C1-C6 respectively) and healthy 
cells (B1-B6 respectively). Arrows indicate positive staining for PAR-2, reference bar = 50 m.  

 

 

production of IL-6 and IL-8, the direct and amplifying 
effects of PAR-2 agonists on baseline and LPS+LT-
activated IEC were studied. The data shown in Figure 4 
demonstrate IL-6 and IL-8 production by IECs during 24-h 
incubation with PAR-2 agonist peptides and the effect of 
agonists in the presence of LPS+LT. Non-activated IECs 
produced negligible amounts of IL-6 and IL-8. Incubation 
of IECs with a PAR-2 agonist 

 
 

 

resulted in a low, but significant increase in IL-6 and IL-8 
expression compared with controls. To examine the 
possible synergy between PAR-2 agonists and LPS+LT 
on IEC activation, IECs were incubated with trypsin and 
SLIGKV-NH2 in the presence concentrations of LPS+LT 
Incubation of IECs with the PAR-2 agonists in the 
presence of LPS+LT resulted in marked potentiation of 
IL-6 and IL-8 production at all concentrations tested. The 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Expression of PAR-2 mRNA in the mucosa of the stomach (b), ileum (c), and 

colon (d). Data are displayed as the ratio of PAR-2 mRNA in diarrheal tissue versus that 

of normal tissue using RT-PCR at four time points over a 1-week period. 
 

 

change in IL-8 expression was more pronounced to that 
of IL-6. 
 

 

DISCUSSION 

 

The present study demonstrates that PAR-2 is expressed 
abundantly in the piglet gastrointestinal tract mucosa, and 
reveals that both PAR-2 mRNA and protein expression 
increases with the severity of diarrhea. In vitro analysis 
using piglet intestinal epithelial cells demonstrates that 

 
 

 

activation of PAR-2 leads to the production of IL-6 and IL-
8. The effects of PAR-2 agonists on the IEC were greatly 
enhanced by concomitant stimulation by LPS and LT. 
The mechanism of amplification of PAR-mediated IL-6 
and IL-8 production when in the presence or absence of 
LPS and LT is currently unknown. At least two possible 
mechanisms could explain the observed LPS+LT-
enhanced IEC activity through PAR-2. The first is that 
LPS +LT induce expression of PAR-2 which, in turn, is 
activated by the agonists. Second, the inflammatory 
activators prime PAR-2, causing amplification of 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 4. Specificity of PAR-2 agonist on intestinal epithelial cell activation. IEC monolayers (1×10
5
/well) 

were incubated with an agonist for 24 h in the presence or absence of LPS+LT. After incubation, IL-8 and 
IL-6 expression in the culture medium was measured by ELISA. Values represent the mean ± SEM of 
quadruplicate determinations. Similar results were obtained in three replicate experiments (*P<0.05, 
**P<0.01). 

 
 

 

independent or common signal transduction pathways 
leading to increased cytokine gene expression. One 
report supports the initial hypothesis that LPS 
independently induced the expression of PAR-2 on 
endothelial cells without affecting the expression of PAR-
1 (Nystedt et al., 1996).  

The pathogenic mechanism of ETEC works by invading 
the intestine of a susceptible host. The pathogens pilus 
combines with the enterocyte microvillus and is accepted 
onto the cell surface where it adheres to the enteric 
mucosa to boycott enterokinesia and stimulate intestinal 
secretion. ETEC amplifies rapidly within the intestine and 
releases enterotoxin. The enterotoxin is highly toxic and 
effective in destroying the regulatory system of 

 
 
 

 

gastrointestinal mucous, pathologically remodeling the 

electrolyte/water balance, severely compromising digestive 

function, as observed by the current study (Madec et al., 

2000; Wellock et al., 2007). Taken together, our results 

suggest PAR-2 may be a regulatory factor in the 

pathogenesis of piglet diarrhea during weaning; one 

potential mechanism being the mediation of inflammatory 

factor production. PAR-2 is reported to be activated by 

multiple proteases such as trypsin, mast cell tryptase, and 

coagulation factors VIIa and Xa. These factors may be 

activated and/or accessible to mucosal tissues including 

chief cells and sensory neurons during inflammation or 

tissue injury (Camerer et al., 2000; Kawabata et al., 1999). 

Exogenous and endogenous PAR-2 activation has 



 
 
 

 

been shown to significantly reduce rat intestinal mucosal 
damage, but did not influence leukocyte infiltrates 
induced by intestinal ischemia/reperfusion injury 
(Cattaruzza et al., 2006). With regard to the role of PAR-2 
in piglet weaning diarrhea, we hypothesize that PAR-2 is 
activated by endogenous agonists and the production of 
inflammatory factors will increase further in the present of 
LPS and LT, mediating the pathologenic processes of 
piglet diarrhea during weaning. The physiological dual 
role of PAR-2 in the gastrointestinal tract mucosa remains 
a topic of debate, with application of a PAR-2 agonist 
shown to facilitate pepsin secretion, as well as produce 
dose-dependent cytoprotection at low doses (Kawao et 
al., 2002). The production of inflammatory factors is 
necessary for physiological function, and PAR-2 may 
have many physiological functions despite the patho-
physiological mediation of diarrhea observed here; so we 
tentatively suggest PAR-2 functions as a double-edged 
sword in the gastrointestinal tract dependent upon the 
progressive state of diarrhea. For example, when 
activated PAR-2 initially promotes the release of mucus, 
providing protection for the mucosa; however, the 
mechanism regulating PAR-2 activity is not expounded in 
the present study.  

To the authors knowledge, this study is the first to exhibit 

that both PAR-2 mRNA and protein expression are present 

throughout the gastrointestinal tract mucosa of weaning 

piglets. We found that PAR-2 mRNA expression was 

significantly elevated in the intestine of piglets with weaning 

diarrhea, and PAR-2 mRNA expression increased with the 

course of diarrheal development. Direct activation PAR-2 in 

pig intestinal epithelial cells leads to the production of IL-6 

and IL-8, and both LPS and LT markedly enhanced PAR-

induced activity. Taken together it is suggested that PAR-2 

may be involved in the development of diarrhea during 

weaning in piglets. Future studies are required in which an 

exogenous or endo-genous agonist of PAR-2 will be 

administered into piglets with weaning diarrhea to further our 

knowledge of the disorder. The current study reveals a 

potentially novel therapeutic target in combating weaning 

piglet diarrhea. 
 

 

ACKNOWLEDGMENTS 

 
This work was supported by the National Natural Science 

Fund of China under the contract number 30700588. We 

would like to thank Mr. Qianglin Duan from Tongji Hospital of 

Tongji University for critical reading of the manuscript. 

 
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