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Amelioration of Experimental Acute Pancreatitis with
Dachengqi Decoction via Regulation of Necrosis-
Apoptosis Switch in the Pancreatic Acinar Cell
Jia Wang
1
, Guangyuan Chen
1
, Hanlin Gong
1
, Wei Huang
2
, Dan Long
3
, Wenfu Tang
1
*
1Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, PR China, 2Physiological Laboratory, University
of Liverpool, Liverpool, United Kingdom, 3Department of Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, PR
China
Abstract
Severity of acute pancreatitis contributes to the modality of cell death. Pervious studies have demonstrated that the herb
medicine formula ‘‘Dachengqi Decoction’’ (DCQD) could ameliorate the severity of acute pancreatitis. However, the
biological mechanisms governing its action of most remain unclear. The role of apoptosis/necrosis switch within acute
pancreatitis has attracted much interest, because the induction of apoptosis within injured cells might suppress
inflammation and ameliorate the disease. In this study, we used cerulein (10
28
M)-stimulated AR42J cells as an in vitro model
of acute pancreatitis and retrograde perfusion into the biliopancreatic duct of 3.5% sodium taurocholate as an in vivo rat
model. After the treatment of DCQD, cell viability, levels of apoptosis and necrosis, reactive oxygen species positive cells,
serum amylase, concentration of nitric oxide and inducible nitric oxide syntheses, pancreatic tissue pathological score and
inflammatory cell infiltration were tested. Pretreatment with DCQD increased cell viability, induced apoptosis, decreased
necrosis and reduced the severity of pancreatitis tissue. Moreover, treatment with DCQD reduced the generation of reactive
oxygen species in AR42J cells but increased the concentration of nitric oxide of pancreatitis tissues. Therefore, the
regulation of apoptosis/necrosis switch by DCQD might contribute to ameliorating the pancreatic inflammation and
pathological damage. Further, the different effect on reactive oxygen species and nitric oxide may play an important role in
DCQD-regulated apoptosis/necrosis switch in acute pancreatitis.
Citation: Wang J, Chen G, Gong H, Huang W, Long D, et al. (2012) Amelioration of Experimental Acute Pancreatitis with Dachengqi Decoction via Regulation of
Necrosis-Apoptosis Switch in the Pancreatic Acinar Cell. PLoS ONE 7(7): e40160. doi:10.1371/journal.pone.0040160
Editor: Juan Sastre, University of Valencia, Spain
Received January 6, 2012; Accepted June 1, 2012; Published July 2, 2012
Copyright: ß2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by the grants of Natural Science Foundation of China (No: 30400576, 30973711 and 30672588). The funder had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: wftang900@gmail.com
Introduction
Acute pancreatitis (AP) is an inflammatory condition that its
severe form involves systemic inflammatory response syndrome
(SIRS) and multiple organ dysfunction syndromes (MODS) [1,2].
The severity of AP depends largely upon the balance between two
forms of cell death-apoptosis and necrosis, the former presumed to
be predominantly protective with mild or no inflammatory
response, while necrosis, cell membrane integrity is lost, associated
with the release of the digestive enzymes and inflammatory
mediators, which can ultimately escalate local and systemic
damage [3,4]. A therapeutic agent that could induce apoptosis
of injured pancreatic acinar cells by regulating the apoptosis/
necrosis switch is likely to reduce necrosis and provide a new
effective treatment [3,5].
Free radicals are molecules produced continuously in cells by
several mechanisms and responsible for a wide variety of diseases
or conditions. It has been shown that reactive oxygen and nitrogen
species (ROS and RNS) contribute to the acinar cell damage
during the early phases of pancreatitis [6]. ROS can act as
a molecular trigger to activate oxidant-sensitive nuclear transcrip-
tion factor kappa B (NF-kB) and thus induces cytokine expression,
participating in various inflammatory processes. Moreover, an
important link between ROS generation and apoptosis has been
shown in both human and experimental pancreatitis. Numerous
studies have shown many anti-oxidant treatments significantly
reduce pancreatic injury and inflammation [7].
In AP, cytokines and other mediators derived from the inflamed
pancreas activate the production of the inducible nitric oxide
synthase (iNOS). An enhanced formation of nitric oxide (NO) due
to the induction of iNOS may be an important factor in the
systemic and local haemodynamic disturbances and regulation of
pancreatic exocrine secretion associated with AP. Excess of NO
cause hypotension and decrease blood perfusion of various organs,
including the pancreas and lung, correlating with apoptotic
changes [8]. Therefore, treatments that could regulate free
radicals ROS or RNS may directly contribute to the modality of
acinar cell death and the degree of inflammation.
Dachengqi Decoction (DCQD) composed of Radix et Rhizoma
Rhei (Dahuang), Cortex Magnoliae Officinalis (Houpu), Fructus Aurantii
Immaturus (Zhishi) and Natrii Sulphas (Mangxiao) is traditionally
used as the representative prescription purgative for the treatment
of constipation and for clearing internal heat in the stomach and
intestine [9]. In China, DCQD has been used to treat AP for over
30 years [10]. Recent studies have shown that DCQD can
PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40160
promote gastrointestinal motility, inhibit cytokine activity and
immune inflammatory response in AP [11–13]. However, most of
its biological activities have been studied individually on its
ingredients. Studies designed to test the molecular mechanisms of
the compound herb formula DCQD in the modality of pancreatic
acinar cell death have not been elucidated to date.
Thus, in our present work, we studied the effect of DCQD in
regulating the inflammatory response via selective induction of
pancreatic acinar cell apoptosis and explored the regulation
mechanism of apoptosis/necrosis switch through its opposite effect
in regulating ROS and NO in vitro and in vivo.
Materials and Methods
1. Materials
1.1. Drugs and reagents. The spray-dried Radix et Rhizoma
Rhei,Cortex Magnoliae Officinalis,Fructus Aurantii Immaturus and Natrii
Sulphas powder were purchased from Chengdu Green Herbal
Pharmaceutical Co. Ltd (Chengdu, China). The spray-dried
powder was mixed of an equal amount and reconstituted with
sterile distilled water at concentrations for the crude drug of 2 g/
mL DCQD in vivo study [14]. In vitro study, the mixed powder was
reconstituted with PBS to prepare a 50 mg/mL stock solution in
dimethylsulfoxide (DMSO) and kept in 220uC. Before being
added to cells, the DCQD stock solution was diluted with PBS to
prepare the working solutions. The final DMSO concentrations
were all less than 0.1% when DCQD was added to cells. The dose
of DCQD was calculated and diluted according to its contents
quantitatively analyzed by HPLC system. Fetal bovine serum
(FBS) was obtained from HyClone (Logan, UT). DMSO,
Cerulein, F12K medium and DCFH-DA were obtained from
Sigma (St. Louis, MO, USA).
1.2. Rats. Sprague-Dawley rats (243618 g) were purchased
from the Experimental Animal Center of West China Center of
Medical Sciences of Sichuan University. All animal studies were
performed according to the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Committee on the Ethics of Animal
Experiments of the Sichuan University.
2. Methods
2.1. Cell culture. Rat pancreatic acinar AR42J cells (ATCC,
Rockville, MD, USA) were cultured in F12K medium containing
20% FBS and 100 U/mL penicillin, 100 mg/mL streptomycin in
standard condition (37uC and 5%CO
2
). All experiments were
carried out 24 h after cells were seeded. To investigate the
protective effects of DCQD against AP, AR42J cells were treated
with or without DCQD prior for 30 min, then further co-
incubated with cerulein (10
28
M) for another 24 h.
2.2. Cell viability assay. Cell survival was assessed by WST
viability assay kit containing WST-8(2-(2-methoxy-4-nitrophenyl)-
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monoso-
dium salt) according to the manufacturer’s protocol (Roche, Basel,
Switzerland). AR42J cells were plated in 96-well plates at 2610
4
cells/well. After 24 h incubation, cells were pretreated with or
without DCQD at different concentrations (0–0.004 g/mL) and
then were further co-incubated with cerulein for 24 h, WST-8
solution (0.5 mg/mL) was added to each well and incubated at 5%
CO
2
37uC for 2 h. The cell viability was determined by the
differences absorbance at wavelengths of 450 nm and 630 nm.
The relative cell viability rate was calculated according to the
following formula: Cell viability rate (%) = 100%6mean absor-
bance of cells in sample groups/mean absorbance for cells in
control group.
2.3. LDH assay. Necrotic cell death was assessed by the
release of lactate dehydrogenase (LDH) from the cytosol of the
damaged cells into the supernatant [15], using the LDH
cytotoxicity detection kit (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China) for various time point 0–24 h according
to the manufacturer’s instructions. Values for LDH release are
presented as the percentage of total cellular LDH from the
following equation [16]: LDH release (%) = total extracellular
LDH activity at the time point6100/total LDH activity.
2.4. Apoptosis assay. Cells were stained with Annexin V-
FITC Apoptosis detection kit (Nanjing Kaiji, Nanjing, China)
following the manufacturer’s instructions to detect early apoptotic
cells (Annexin V+PI2events) and necrotic or late apoptotic cells
(Annexin V+PI+) by flow cytometry. Briefly, AR42J cells were
treated or not with DCQD prior for 30 min and then stimulated
with cerulein (10
28
M) for 24 h. Then cells were collected and
resuspended in the culture medium at a density of 1610
6
cells/
mL, stained with 5 mL of Annexin V-FITC and 5 mL propidium
iodide (PI) in 300 mL binding buffer (10 mM HEPES, pH 7.4,
140 mM NaOH, and 2.5 mM CaCl
2
) according to the manu-
facturer’s instructions for 15 min at room temperature in the dark.
Quantification of apoptotic cells was analysis by flow cytometry
(FACScan, Becton Dickinson, USA).
2.5. Measurement of ROS generation. The generation of
ROS in cells was determined using a FACScan flow cytometry
following the manufacturer’s instructions. Briefly, AR42J cells
were pretreated with DCQD 30 min before stimulated with
cerulein for 24 h. Cells were collected and incubated with 10 mM/
L DCFH-DA 30 min in the dark and then washed twice with PBS.
Intracellular low-molecular-weight peroxides oxidize DCFH-DA
to the highly fluorescent compound dichlorofluorescein (DCF).
Then the cells were harvested and the pellets were suspended in
300 ml PBS at an excitation wavelength of 488 nm and an
emission wavelength of 525 nm.
2.6. Animal models and treatment with DCQD. Sprague-
Dawley rats were divided randomly into sham-operated group, AP
group and DCQD- treated group (n = 6). While the rats were
under ether anesthesia and laparotomy, pancreatitis was induced
by retrograde perfusion into the biliopancreatic duct of 3.5%
sodium taurocholate (Sigma, St. Louis, MO, USA) (1 mL/kg body
weight) at a rate of 0.2 mL/min with a microinfusion pump [17].
The entire procedure from induction of anesthesia to closure of the
incisions requires ,30 min for each animal. The same procedure
was applied to sham-operated group but receiving an intraductal
perfusion of saline (NaCl 0.9%) instead of sodium taurocholate. In
DCQD- treated group, the rats recovered from anesthesia and
were administered intragastrically DCQD 20 g/kg body weight
(equivalent to 2 g/mL crude herbs) 2 h after operation. In the
sham-operated group and AP group, rats were given equal volume
of saline. After 48 h, blood were obtained from the vena caudalis
and centrifuged to obtain serum for amylase examination. The
animals were sacrificed by exsanguination while under ether
anesthesia and the pancreatic tissues were rapidly collected for
pathological and apoptotic examinations. Tissue homogenate was
collected for NO and iNOS concentration measurement.
2.7. Amylase and NO and iNOS Assay. Serum was
collected from the rats for amylase activity (U/L) measurement
by an enzymatic assay kit from Sigma (St. Louis, MO, USA)
according to manufacturer’s instructions. Tissue homogenate was
collected for NO and iNOS concentration measurement, using
nitric oxide and inducible nitric oxide synthetase assay kits (Nanjin
Jiancheng Biological engineering Company, Nanjin, China)
according to manufacturer’s instructions.
Dachengqi Decoction Regulates Cell Death Pathways
PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40160
2.8. TUNEL assay. The apoptotic cells in tissue samples
were detected using an In Situ Cell Death Detection kit (Roche,
Switzerland) according to the manufacturer’s instructions. Briefly,
paraffin embedded specimens were cut into 4–5 mm thickness
sections. After deparaffinization and washed in PBS, The sections
were treated with proteinase K, then incubated with TUNEL
reaction mixture at 37uC for 1 h. Slides were washed with PBS
and then treated with HRP and DAB terminated until the color
was developed. Apoptotic index was determined by counting the
number of TUNEL-positive cells. Eight slides per block were
evaluated. For each slide, 8 fields were randomly chosen, and
`100
cells per field were counted and calculated the apoptosis index.
2.9. Histopathologic analysis of pancreas. At the end of
experiment, pancreatic tissues were promptly collected, fixed in
10% neutral formalin and embedded in paraffin. The paraffin-
embedded tissue blocks were cut into 5 mm thick sections and
stained with hematoxylin and eosin. Specimens were graded by
two independent pathologists blinded to the experimental setup
using a scoring system for the extent and severity of pancreatitis
(0–4, normal to severe, respectively), the degree of interstitial
edema, hemorrhage, hyperemia, necrosis, leukocyte infiltration of
the pancreatic tissue as previously described [18,19].
2.10. Leukocyte infiltration assay. For evaluation of the
infiltration number of leukocyte during pancreatitis we randomly
chose 8 to 10 consecutive high-power fields for each rat (n = 6) on
a scale of 0–4 by two researchers in a blinded manner as
previously described [18,19].
3. Statistical analysis
Statistical analysis was carried out using the PEMS3.1 statistical
program. All data represent at least three independent experi-
ments and are expressed as the mean 6standard errors of mean
(S.E.M.). One-way repeated-measures ANOVA (followed by
multiple pair-wise comparisons using Student-Neuman-Keuls
procedure) were used for the analysis of differences between the
experimental and control groups. Values of P,0.05 were regarded
as statistically significant.
Results
1. DCQD enhanced cerulein-induced AR42J cell viability
In order to examine the effect of DCQD on cell viability,
pancreatic acinar AR42J cells were treated with increasing
concentrations of DCQD (0.00025 g/mL, 0.0005 g/mL,
0.001 g/mL, 0.002 g/mL and 0.004 g/mL) for 0–24 hours based
on the components’ concentrations of DCQD in blood of our
previous study [20,21]. As shown in Figure 1A, there were few
dead cells present in the control group, and the cell viability
significantly decreased after cerulein was added. Treatment of
AR42J cells with DCQD for 24 hours caused a concentration-
dependent protective effect, and the maximum effect was obtained
at the dose of 0.004 g/mL. The viability of cells pretreated with
0.004 g/mL DCQD for 24 h increased significantly compared to
the cerulein stimulated group. One characteristic of pancreatic
acinar cell stimulated with supramaximal doses of cerulein is the
induction of necrosis [16]. The process of necrosis damages the
plasma membranes, and release LDH into the extracellular
medium. To evaluate necrosis in the present study, we measured
the release of LDH from the damaged AR42J cells following 24 h
treatment with cerulein. The release of LDH in the control group
was at relatively lower levels, and the levels of LDH significantly
increased after the addition of cerulein and different concentra-
tions of DCQD. The level of necrotic cells was decreased after the
pretreatment of DCQD with increased concentration. In our
study, supramaximal cerulein treatment significantly increased
LDH release from pancreatic acinar cells. However, pretreatment
with 0.004 g/mL DCQD significantly diminished LDH release
compared to the cerulein-stimulated cell AP model group at 24 h
(Figure 1B).
2. DCQD induced pancreatitis AR42J cells apoptosis
To determine the effects of inducing apoptosis by DCQD on
AR42J cells, we further analyze apoptosis using Annexin V/PI
Figure 1. Effects of DCQD on the reduction of cerulein-induced
necrosis of AR42J cells. The cells were pre-treated with increasing
concentrations of DCQD (0.00025 g/mL, 0.0005 g/mL, 0.001 g/mL,
0.002 g/mL and 0.004 g/mL) for 30 min and then co-incubated with
or without 10
28
M cerulein for another 0–24 h. (A) Cell viability rate was
examined using WST-8 assay. (B) Necrotic cell death rate was assessed
by the release rate of LDH. The results are mean 6SE (n = 5) for three
independent experiments. *P,0.05 versus control group; +P,0.05
versus DCQD 0.004 g/mL group;ˆP,0.05 versus cerulein group.
doi:10.1371/journal.pone.0040160.g001
Dachengqi Decoction Regulates Cell Death Pathways
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staining. The Annexin V2/PI2population was regarded as
normal healthy cells, while Annexin V+/PI2cells were taken as
a measure of early apoptosis and Annexin V+/PI+as necrosis/late
apoptosis. Our results showed that there was a very low level of cell
death in the control group, 24 h treatment with cerulein
significantly increased cell death (Figure 2A). In the AP group,
there were fewer apoptotic cells but more necrotic cells (Figure 2B).
After pretreated with DCQD, the number of apoptotic cells
increased and the number of necrotic cells decreased significantly
comparing with AP group cells at 24 h (Figure 2C).
3. DCQD reduced ROS in cerulein-induced AR42J cells
Acinar cellular damage induced by supramaximal cerulein
could originate from premature intracellular enzyme activation,
but also from injurious levels of ROS. We explored whether
DCQD could diminish the supramaximal cerulein-induced
necrosis by interfering with ROS production. The ROS positive
cells pretreated with or without DCQD before stimulated with
cerulein for 24 h were analyzed (Figure 2D). A very low level of
ROS positive cells were detected in the control group, but in AP
group ROS positive cells significantly increased. DCQD pre-
treated pancreatic acinar cells before cerulein stimulating signif-
icantly decreased ROS positive cells remarkably (Figure 2E).
4. DCQD reduced the release of serum amylase in the
rats’ model of AP
Sodium taurocholate stimulation caused a statistically significant
increase of serum amylase at 48 h in the AP group compared with
the sham-operated group in vivo. In DCQD-treated group, the
serum amylase values were significantly lower than that in the AP
group (Figure 3A).
5. DCQD induced apoptosis of pancreatitis acinar cells
determined by TUNEL staining
TUNEL-stained slides revealed that pancreas tissue from sham-
operated rats exhibited very low levels of apoptosis. In contrast,
a significant number of TUNEL-positive cells were detected in
pancreas tissue within the AP group and DCQD- treated group at
48 h. Treatment with DCQD significantly increase the percentage
of TUNEL-positive cells compared with the AP group (Figure 3B).
This result indicates that DCQD may preferentially induce
apoptosis within injured cells, which is consistent with our in vitro
study.
6. DCQD alleviated the severity of experimental AP
In the sham-operated group, the pancreas was few edematous,
with the infiltration of a few inflammatory cells but without
obvious hemorrhage, necrosis of acinar cells or the adjacent fat
tissues. However, the AP group showed the features of a severe
form of AP characterized by expansion of interlobular and
interlobular spaces caused by moderate to severe interstitial
edema, extensive infiltration with inflammatory cells, obvious
pancreatic acinar cells vacuolization, necrosis and hemorrhage
(Figure 3C). The rats treated with DCQD showed a significant
reduction of inflammatory cells infiltration, hemorrhage, necrosis
and interstitial edema compared to the AP group. The standard
pathological scores in both AP and DCQD-treated groups
significantly exceeded the sham-operation group at 48 h. The
scores of DCQD-treated group were significantly lower than those
of the AP group at 48 hours (Figure 3D).
Figure 2. DCQD regulated cerulein-induced AR42J necrosis-apoptosis switch through ROS. The cells were pre-treated with 0.004 g/mL
DCQD for 30 min and then co-incubated with or without 1028 M cerulein for another 24 h. (A) Flow cytometry analysis of the apoptotic and necrotic
cells among the AR42J cells. Four different regions can be found in each panel in the figure for flow cytometry detection: Viable cells (Annexin V2/
PI2) are located in the lower left, early apoptotic cells (Annexin V+/PI2) in the lower right, late apoptotic and necrotic cells (Annexin V+/PI+) in the
upper right and primary necrotic cells (Annexin V2/PI+) in the upper left quadrants, respectively. (B) The percentages of apoptotic cells and (C)
necrotic cells were compared. (D) Generation of ROS in AR42J cells were detected by DCF using flow cytometry (E) and ROS positive cells were
compared. The results are mean 6SE for three independent experiments.
#
P,0.05 and
##
P,0.01 versus control cells;
*
P,0.05 and
**
P,0.01 versus
AP group.
doi:10.1371/journal.pone.0040160.g002
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7. DCQD reduced the leukocyte infiltration of pancreatitis
tissues
In AP group, there was a significant increase of leukocyte
infiltration of pancreas tissue at 48 h after sodium taurocholate
stimulation compared with the sham-operated group. In DCQD-
treated group, leukocyte infiltration was significantly lower than
that in the AP group (Figure 3E).
8. DCQD enhanced the acute pancreatitis-associated
tissue concentration of NO and iNOS
There were significant differences in the concentration of NO
and iNOS among the groups. The results showed that the
concentration of NO and iNOS in the AP group were significantly
lower than that in the sham-operated group, whereas the
concentration of NO and iNOS in the DCQD-treated group
were significantly higher than that in the sham-operated group at
48 h (Figure 4). These results suggest that treatment with DCQD
Figure 3. DCQD alleviated acute pancreatitis-associated tissue damage. Rats (n = 6 per group) were given DCQD (20 g/kg body weight) 2 h
after operation. After 48 h, the blood was obtained for amylase examination. The pancreatic tissues were collected for examination of pathological
and apoptotic examinations (A) Serum amylase activity assay. (B) TUNEL-positive cells percentage in the pancreatic tissue. (C) Pathological changes of
pancreas in different groups observed by HE staining (Light microscopy, 6400). Sham-operated group rats showed slightly edematous. Compared
with sham-operated rats, AP group rats showed a severe degree of pancreatic damage with edema, hemorrhage, necrosis, pancreatic acinar cell
vacuolization and infiltration of inflammatory cells. In DCQD-treated group, rats showed a reduction of edema, hemorrhage, necrosis, and
inflammatory cells infiltration. (D) Pathological scores of pancreatic injury (E) and leukocyte infiltration counted in 8 to 10 consecutive high-power
fields per slide of Sham-operated group, AP group and DCQD-treated group. The results are mean 6SE for three independent experiments.
#
P,0.05
and
##
P,0.01 versus sham-operated group;
*
P,0.05 and
**
P,0.01 versus AP group.
doi:10.1371/journal.pone.0040160.g003
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could increase the production of NO in pancreatic tissues, which is
a major factor correlated with the appearance of apoptosis.
Discussion
AP is a multifactorial disease associated with the excessive
inflammatory response, which can ultimately lead to devastating
consequences. The form of cell death determines the severity of
AP. Recent studies prove the apoptosis could be a beneficial
reaction to AP, because apoptotic cells maintain membrane
integrity, therefore, unlike necrotic cells, the leakage of in-
tracellular components containing proinflammatory and immu-
nogenic materials could be prevented actively to avoid in-
flammatory response [3,22]. Crucially the two kinds of cell
death, necrosis and apoptosis, may be interchangeable under
certain conditions [23]. Therefore, if the therapeutic agent could
regulate the apoptosis/necrosis switch, it might inhibit the
inflammatory response, protecting against progression toward AP.
In some in vivo studies, the modified formula of DCQD and its
components exhibited potencies in inducing apoptosis of pancre-
atitis acinar cell [24,25]. This study shows that DCQD promotes
acinar cell apoptosis both in vitro and in vivo models of AP with
a reduction in necrosis, suggesting a switch from deleterious
necrotic cell death to a milder apoptotic form. Additionally,
pancreatic tissue necrosis, hemorrhage and inflammatory cell
infiltration were significantly alleviated in DCQD-treated group in
vivo. The concentration of LDH in the supernatant of the culture
solution can indirectly reflect cell membrane damage resulting in
necrosis [26]. It was seen that both the number of necrotic cell and
the level of LDH were lower in DCQD-treated group than in the
AP group in vitro. The plasma concentration of amylase is another
parameter for the severity of AP. In this study, its levels were
significantly lower in DCQD-treated group than in the AP group
in vivo. The treatment mechanism of DCQD may occur via
inducing apoptosis of injured acinar cells, decreasing necrosis,
which help to avoid the release of digestive enzyme and various
inflammatory mediators, significantly attenuating the progression
of pancreatic injury.
Oxidative stress is regarded as an important determinant of the
severity of acute pancreatitis [27]. Large amounts of ROS can
directly activate the oxidant-sensitive transcription factor of NF-kB
and generate an inflammatory response, which attracts more
oxidative stress-generating neutrophils to worsen local tissue
destruction and to cause distant organ injury [28,29]. Moreover,
it has been demonstrated that the level of ROS involved in
apoptosis/necrosis switch [30,31]. In our study, there was a lower
level of ROS and necrosis but a higher level apoptosis in DCQD-
treated group than in AP group in vitro, indicating that DCQD
have the antioxidant effect to reduce the production of ROS and
might switch acinar cells death from necrosis to apoptosis,
alleviating subsequent inflammatory response.
The role of NO in the pathogenesis of AP remains controversial.
Some studies showed that the proinflammatory cytokines activate
the production of the iNOS, resulting in overproduction of NO,
which could promote pancreatic injury [32,33]. Whereas others
reported that NO acts as a biological scavenger and inactivates
ROS, which protects pancreatic acinar cells [34] and has also
beneficial effects by inhibition of neutrophil accumulation and
improvement of microcirculation [35,36]. Our data showed that
after treated with DCQD, the production of NO in pancreatic
tissues was increased, accompanying the increase of apoptosis with
the decrease of inflammatory cells infiltration and pathological
scores in pancreatic tissues. This indicated that the increase of NO
and iNOS was not the reason of pancreatic tissue damage, but
a protective factor might be involved in inducing apoptosis and
reduce pancreatic tissue pathological severity.
ROS and nitrogen oxide species play important and different
roles in various physiological and pathological states. In our study,
DCQD exerting an opposite regulation on NO and ROS may
come from its scavenging effects which remain to be understood.
These findings provided evidence that treated with DCQD reduce
the generation of ROS and increase the NO pancreatitis acinar
cells, therefore regulate apoptosis/necrosis switch, induce apopto-
sis of injured acinar cells and inhibit the subsequent amplifying
inflammatory response, which in turn protects against AP.
In the present study, we introduce the ideas and methods of
translating research into traditional Chinese medicine, and this is
the first time to research the way of compound Chinese herb
formula DCQD regulating apoptosis/necrosis switch on AP in vitro
and in vivo. Here we conclude that DCQD could inhibit the local
and systematic inflammatory response and alleviate the pancreatic
damage via regulating the pancreatic cell necrosis/apoptosis
switch. Future study should be directed at signaling pathways of
ROS and NO in regulating injured pancreatitis acinar cell
apoptosis by the treatment of DCQD.
Author Contributions
Conceived and designed the experiments: WFT JW. Performed the
experiments: JW GYC HLG WH. Analyzed the data: DL. Contributed
reagents/materials/analysis tools: GYC HLG WH. Wrote the paper: JW.
Figure 4. DCQD enhanced the acute pancreatitis-associated
tissue concentration of NO and iNOS. Tissue homogenates were
collected for NO and iNOS concentration measurement after 48 h
treatment. (A) NO concentration (B) and iNOS concentration of sham-
operated group, AP group and DCQD-treated group. The results are
mean 6SE (n = 6 per group) for three independent experiments.
#
P,0.01 versus sham-operated group;
*
P,0.01 versus sham-operated
group and AP group.
doi:10.1371/journal.pone.0040160.g004
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Dachengqi Decoction Regulates Cell Death Pathways
PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40160
