Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Original Article
Protective effect of Dachengqi decoction on the pancreatic
microcirculatory system in severe acute pancreatitis by
down-regulating HMGB-TLR-4-IL-23-IL-17A mediated neutrophil
activation by targeting SIRT1
Jia Wang1#, Yang Zou2#, Dan Chang2, Da-Qing Hong2, Jiong Zhang2
1General Practice Center, Sichuan Provincial People’s Hospital & Sichuan Academy of Sciences, University of Electronic Science and Technology,
Chengdu, China; 2Division of Nephrology, Sichuan Provincial People’s Hospital & Sichuan Academy of Sciences, University of Electronic Science
and Technology, Chengdu, China
Contributions: (I) Conception and design: J Zhang; (II) Administrative support: DQ Hong; (III) Provision of study materials or patients: D Chang; (IV)
Collection and assembly of data: Y Zou; (V) Data analysis and interpretation: J Wang; (VI) Manuscript writing: All authors; (VII) Final approval of
manuscript: All authors.
#These authors contributed equally to this work.
Correspondence to: Jiong Zhang; Da-Qing Hong. Division of Nephrology, Sichuan Provincial People’s Hospital & Sichuan Academy of Sciences,
University of Electronic Science and Technology, Chengdu, China. Email: zhangjiong831224@163.com; hongdaqing11@126.com.
Background: Dachengqi decoction (DCQD), one of classic prescription of Chinese herbal medicine has
been widely used in clinic to treat severe acute pancreatitis (SAP). The damage of pancreatic microcirculation
plays key pathogenesis of SAP. However, little is known about the molecular pharmacological activity of
DCQD on pancreatic microcirculation in SAP.
Methods: Sodium taurodeoxycholate and cerulein were used to establish model of SAP in vitro and in vivo,
respectively. The pancreatic pathological morphology, wet weight ratio, myeloperoxidase (MPO) activity, cell
viability and microcirculatory function of the pancreas, as well as serum lipase and amylase expressions were
evaluated. The expression levels of SIRT1, acety-HMGB1, TLR-4, HMGB1, IL-23, IL-17A, neutrophil
chemokines (KC, LIX, and MIP-2), and inflammation-related factors (IL-6, IL-1β, and TNF-α), the
translocation of HMGB1 and the interaction of SIRT-HMGB1 in the pancreas and serum were determined
by ELISA real-time PCR, western blotting and immunoprecipitation.
Results: In vivo studies showed that DCQD or neutralizing antibody (anti-23p19 or anti-IL-17A) could
all signicantly decrease lipase, amylase activity, down-regulate the expression of CD68, Myeloperoxidase
(MPO), wet/weight, IL-1β, IL-6, TNF-α, and neutrophil chemokines (KC, LIX, MIP-2), alleviate
pathological injury and improve pancreatic microcirculatory function in rats with SAP. Furthermore, DCQD
remarkably increased SIRT1 expression, promoted SIRT1 and HMGB1 combination, reduced HMGB1
translocation from nuclear to cytoplasm, and alleviated the expression of acetyl-HMGB1, HMGB1, IL-17A,
TLR-4, and IL-23 in vitro and vivo with SAP. However, the intervention with EX527 (SIRT1 inhibitor) or
r-HMGB1 (recombinant HMGB1) obliviously reverses the above mentioned influence mentioned above
of DCQD in SAP. In vitro, we conrmed that DCQD could decrease HMGB1 acetylation, migration, and
release, and improve the decline of cell viability, SIRT1 expression and SIRI-HMGB1 combination induced
by cerulean with promoting macrophage to release IL-23 by relying on the HMGB1/TLR-4 way.
Conclusions: DCQD treatment improves SAP-induced pancreatic microcirculatory dysfunction by
inhibiting neutrophil-mediated inammation via inactivating HMGB1-TLR-4-IL-23-IL-17A signaling by
targeting SIRT1.
3044
3031
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Introduction
Severe acute pancreatitis (SAP), one of the acute abdominals
in the clinic, accounts for 10% to 20% of all acute
pancreatitis cases (1). Rapid onset characterizes it, rapid
progression, and poor prognosis (1). In addition, it causes
inammatory cascades and organ failure in its early stages,
resulting in high mortality (1). Therefore, SAP is a serious
threat to human health, life, and the economy (1). Despite
extensive research and efforts to develop pharmacological
tools, there are currently no specic treatments for SAP (1).
Therefore, determining the pathophysiological mechanisms
underlying SAP is crucial to identify effective therapeutic
drugs and preventive measures for SAP.
The pathogenesis of SAP is complex and involves the
abnormal activation of pancreatic inammatory mediators,
microcirculation dysfunction, translocation of intestinal
flora, apoptosis, and oxidative stress. A large number of
latest studies have shown that microcirculation disorder
plays an extremely important role in the progression of
SAP. It is not only the initiating factor of SAP, but also the
factor of sustained and aggravated injury. Acute pancreatitis
can damage the pancreas and systemic microcirculation,
thus forming a vicious circle (1,2). Additionally, the
immune-inflammatory response-mediated pancreatic
microcirculation dysfunction is always active, which is
also an important molecular biomarker to determine the
outcome of pancreatitis (2). In particular, the activation
of the inflammatory immune response relies on the
recruitment and migration of neutrophils in SAP (3).
Therefore, it is an attractive treatment strategy to improve
SAP by suppressing the dysfunction mediated by the
inflammatory response of the pancreatic microcirculation
by promoting neutrophil inactivation.
HMGB1, one of the endogenous molecules secreted
from apoptotic and necrotic cells, has a pivotal influence
on the development of SAP by activating the immune
response (2). Once pancreatic cells are harmfully stimulated,
HMGB1 in the nucleus will be modified by acetylation
and then released to the extracellular space where it pairs
with receptors (TLR-4) on the surface of macrophages to
induce TLR-4-mediated immune responses (2,4). This
further promotes the release of HMGB1 and amplifies
the immune reaction. It is worth noting that HMGB1-
TLR-4 signaling stimulates macrophages to secrete the
pro-inflammatory cytokine IL-23 and IL-23, promoting
Th17, γδ T, NK, and NKT cells to secrete IL-17A to
regulate neutrophil activation (5). In addition, the activity
and nucleocytoplasmic shuttle of HMGB1 depend on the
acetylation degree of HMGB1. SIRT1, one of the key
acetylation modifying enzymes, can suppress HMGB1
transcription by deacetylation of HMGB1. Therefore, it
is an effective strategy to alleviate the activation mediated
by TLR-4 of a neutrophil by triggering SIRT1/HMGB1
signaling in SAP development (6).
Dachengqi decoction (DCQD) is a traditional decoction
widely used for SAP treatment (7,8). Its main ingredients
include Houpu, Da Huang, Mangxiao, and Zhi Shi (7).
DCQD appears positively associated with the improvement
of pancreatic microcirculation in SAP (8,9). However,
it is a pity that the concrete mechanism that DCQD
regulates HMGB1 activation and microcirculation is not
clear to date. Therefore, the purpose of the study was to
confirm that the improvement of DCQD in pancreatic
microcirculation is associated with the suppression of the
neutrophil-mediated immune-inflammatory response.
However, it promotes the inactivation of the HMGB1-
TLR-4-IL-23-IL-17A axis by targeting the SIRT1 signal
pathway in SAP. We present the following article in
accordance with the ARRIVE reporting checklist (available
at https://dx.doi.org/10.21037/gs-21-655).
Methods
Drugs, reagents, instruments, and animals
DCQD consists of Houpu, Da Huang, Mangxiao, and
Zhi Shi. The above herbs were acquired from Lv Yuan
Pharmaceutical Co, Ltd. in Chengdu. The DCQ granules
collected were dissolved in saline to obtain DCQD
Keywords: Severe acute pancreas (SAP); Dachengqi decoction (DCQD); neutrophil; HMGB1-TLR-4-IL-23-IL-
17A; pancreatic microcirculatory
Submitted Sep 05, 2021. Accepted for publication Oct 14, 2021.
doi: 10.21037/gs-21-655
View this article at: https://dx.doi.org/10.21037/gs-21-655
3032 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
(2 g/mL). According to the previous article, the concrete,
specic compatibility, steps, extraction, and use (9).
Reagents were obtained from the following sources:
sodium taurocholate, cerulean (Shanghai Dibao Biology
Technology Co., Ltd.); lipase, and amylase, HMGB1,
TNF-α, IL-23, IL-1β, KC, IL-17A, LIX, IL-6, and MIP-
2 ELISA Kits (Biyuntian); TLR-4, HMGB1 and Acety-
HMGB1 antibodies (Abgent, USA); Myeloperoxidase
(MPO) and CD68 Colorimetric Activity Assay Kit
(BioVision, USA). EX527, r-HMGB1, neutralizing IL-
23p19, and IL-17A (Biolegend, San Diego, CA, USA) were
purchased from Sigma.
The following instruments were purchased: enzyme
labeling instrument, two-dimensional electrophoresis
instrument, PCR (Bio-Rad, USA), parafn embedding and
pathological section machines (Leica, Germany), real-time
PCR (Applied Biosystems, USA), Western blot imager (Pei
Qing, Shang Hai).
Seventy healthy male SD rats (weighing 220±30 g,
6–8 weeks) were obtained from Sichuan University. The rats
were located in a standard SPF environment with humidity
of 60%±4% and a temperature of 22±2 ℃. All animal
procedures were approved by the animal care committee
of the Sichuan Provincial People’s Hospital (No. 2011220),
and the experimental protocols were strictly carried out
based on NIH Guidelines for the care and use of animals. A
protocol was prepared before the study without registration.
Animal grouping, model establishment, and intervention
The 70 rats were randomly separated into seven groups:
Sham, SAP, DCQD, EX527, anti-IL-17, recombinant
HMGB1(r-HMGB1), and anti-IL-23p19 groups (n=10).
Rats were fed ad libitum for 7 days before establishing the
SAP model.
Rats in the SAP, DCQD, EX527, anti-IL-17, r-HMGB1,
and anti-IL-23p19 groups were anesthetized with
pentobarbital sodium (40 mg/kg). An upper abdominal
incision was made by cutting open the skin and muscle
to expose the bile duct, temporarily clamped to the
small artery at the portal site on the liver. The insulin
injection needle was stretched into the biliopancreatic
duct by the nipple, and sodium taurocholate (1 mg/
kg) got into the biliopancreatic duct by injection. After
2h of injecting sodium taurocholate, the animal in the
EX527, DCQD, and r-HMGB1 group were treated with
DCQD using an oral gavage at 20 g/kg dose at 2 h after
operation (9). Animals in the EX527 and r-HMGB1 group
were also treated with EX527 (10 mg/kg) or r-HMGB1
(100 μg/kg) by intraperitoneal injection, according to the
previous article (10,11). Rats in the anti-IL-17A groups and
the anti-IL-23p19 group were treated with neutralizing
antibodies against IL-17A (1 mg/kg) and IL-23p19
(0.2 mg/kg), respectively, by intraperitoneal injection,
according to the previous article (12,13). After 12 h, rats in
all groups were sacrificed with pentobarbital sodium and
serum, and pancreatic tissue was collected for further study.
Cell culture
Rat pancreatic acinar cell lines (AR42J) and RAW264.7
were acquired from ATCC (Rockville, MD, USA). After
30 min of treatment without or with DCQD, AR42J cells
were co-incubated with cerulean for 24 h.
Gene silencing
AR42J cells were transfected with control siRNA or TLR-
4. The RAW 264.7 cells were intervened with rHMGB1.
TLR4 overexpression
RAW 264.7 cells were designated to transfect TLR4 and
control genes using Lipofectamine2000, according to the
instructions. TLR4 and IL-23 expression were examined
after transfection at 24 h.
Assaying cell viability
MTT was used to evaluate cell survival, and the concrete
steps were based on the instruction. AR42J cells were
preconditioned with or without DCQD, EX527, or
r-HMGB1 (0.004 g/mL), successively 24 co-incubation
with cerulean (10 nmol/L).
Microcirculation of the pancreas
Each rat was injected with 1.5 mL of FITC-RBC through
the tail vein. We measured ow velocity, functional blood
vessel number, red blood cell ow, and blood vessel number
to evaluate pancreatic microcirculation using the BI-2000
medical image analysis system.
Histology and wet/weight
After performing hematoxylin and eosin (HE) staining,
3033
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
the excised pancreatic tissue was sectioned and mounted
in paraffin, after performing hematoxylin and eosin
(HE) staining to evaluate the pathological morphology
of the pancreas. The pancreas was assayed under a light
microscope. Pancreatic tissue was classified by evaluating
necrosis + hemorrhage + edema + inammation according
to the modified Kusske scoring standard blinded by
two independent pathologists (9). No inflammatory cell
infiltration, hemorrhage, edema and necrosis were scored
0; necrosis area (1–10%), hemorrhage and edema (0–25%)
were 1 point; necrosis area (11–20%), hemorrhage and
edema (25–50%) were 2 points; necrosis area (21–30%),
hemorrhage and edema (50–75%) were 3 points; necrosis
area (>30%) hemorrhage and edema (50–55%) were 4
points; 0.5 point for 5 inammatory cells and 4 points for
more than 30 inammatory cells. The weighted index of the
pancreas was calculated as the weight of the pancreas (g)/
body weight (g) ×100%.
Amylase and lipase activity assays in serum
The blood collected was centrifuged to obtain the
supernatant. The amylase and lipase activity was assayed by
exploiting colorimetric activity kits for amylase and lipase,
following the manufacturer’s instructions.
Assaying IL-23, HMGB1, IL-1β, IL-17A, MIP-2, TNF-α,
LIX, IL-6, and KC
IL-23, HMGB1, IL-1β, IL-17A, MIP-2, TNF-α, LIX, IL-
6, and KC concentrations in the pancreas and serum were
determined using rat ELISA kits for each protein, following
the manufacturer’s instructions.
Detection of IL-23, IL-1β, IL-17A, MIP-2, TNF-α, LIX,
IL-6, and KC mRNA expression in the pancreas
Total RNA was extracted using the TRIzol method after
homogenizing the frozen pancreas in liquid nitrogen. Real-
time PCR was exploited to detect mRNA expression. The
concrete steps were in line with the instructions. The
primer sequences are displayed in Table 1.
Examining SIRT1 activity
SIRT1 activity was evaluated by the Fluorescent Assay Kit
for SIRT1 activity based on instructions.
Western blotting
Total, nuclear, and cytoplasmic proteins were extracted
from pancreatic tissues by lysis, centrifugation, and
quantication. Western blot analysis was performed using
SIRT1 (1:400), Acey-HMGB1 (1:400), TLR-4 (1:1,000),
HMGB1 (1:400), β-actin (1:4,000) and Histone (1:1,000).
Signals were detected by incubation with a secondary
antibody, followed by exposure to the ECL-Plus reagent.
MPO and CD68 assay
Immunohistochemistry was used to evaluate the expression
of MPO and CD68 in pancreatic tissues.
Statistical methods
All documents are displayed as mean ± SEM. T-test
and one-way ANOVA tests were used to assay multiple
comparisons. Tukey’s post-hoc test was exploited to evaluate
individual means. P<0.05 was considered signicant.
Results
Wet/weight of the pancreas, serum amylase and lipase
activity, cell viability, and survival rates
Compared with the SAP group, the wet/weight, activity
of amylase and lipase, and mortality in the Sham, DCQD,
anti-IL-17A, and anti-IL-23-p19 groups were obliviously
decreased (P<0.05). However, with the DCQD group, the
wet/weight, activity of amylase and lipase, and the mortality
in the markedly increased (P<0.05) (Figure 1A-1C).
AR42J cells were treated with Cerulean to simulate SAP
in rats. Figure 1C,1D shows that cell viability in the Sham,
anti-IL-17A, DCQD, and anti-IL-23-p19 groups was
signicantly up-regulated (P<0.05). Nevertheless, with the
DCQD group, cell viability in the EX527 and r-HMGB1
groups was markedly up-regulated (P<0.05) (Figure 1D,1E)
(P<0.05).
Pancreatic histopathological morphology and the expression
of CD 68 and MPO in the pancreas
HE and immunohistochemistry were used to assess
pancreatic histological morphology and CD 68 and MPO
expression, respectively. The Sham group showed that the
pancreas of some rats had slight edema, a small amount of
3034 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
inflammatory infiltration, but no necrosis or hemorrhage.
Pancreatic tissues of the SAP group had severely damaged
pancreatic tissue, widespread edema and hemorrhage, a
large number of inammatory cell inltration, and necrosis
of acinar cells with pathological injury scores higher than
those of the Sham group (P<0.05). Rat pancreatic tissues in
the anti-IL-23-p19, DCQD, and anti-IL-17A groups had
edema slightly, a small amount of inammatory inltration,
hemorrhage, and necrosis with lower pathological injury
scores than in the SAP group (P<0.05). Yet with the
DCQD group, the r-HMGB1 and EX527 groups had more
pancreatic tissue necrosis, inflammatory cell infiltration,
edema, and hemorrhage with higher pathological injury
scores (P<0.05) (Figure 2).
Compared to the sham group, the SAP group has a
higher expression of CD68 and MPO (P<0.05). Compared
to the SAP group, the DCQD group, the anti-IL-17A, and
anti-IL-23-p19 groups have lower expressions of CD68 and
MPO (P<0.05). Compared to the DCQD group, EX527
and r-HMGB1 have higher expressions of CD68 and MPO
(P<0.05) (Figure 2).
Microcirculatory function of the pancreas
Impaired pancreas microcirculation is a typical clinical
manifestation of severe pancreatitis (2,3). Compared to the
sham group, the SAP group has a lower blood ow velocity,
the number of functional blood vessels, RBC ow, and the
number of blood vessels (P<0.05). Compared to the SAP
group, the DCQD, anti-IL-17A, and anti-IL-23-p19 groups
had blood flow velocity, number of the functional blood
vessels, RBC flow, and number of blood vessels (P<0.05).
Furthermore, the r-HMGB1 and EX527 groups had a
higher blood ow velocity, the number of functional blood
vessels, the ow of RBC, and the number of blood vessels
than the DCQD group (P<0.05) (Figure 3).
Expression of IL-23, HMGB1, IL-1β, IL-17A, TNF-α
and IL-6
The immune-inammatory response plays a key role in the
progression of SAP (2,3). Therefore, the study examined
the level of IL-23, HMGB1, IL-1β, IL-17A, TNF-α, and
IL-6 in serum and pancreas at both gene and protein levels.
The SAP group has a higher level of HMGB1, IL-17A,
IL-23, IL-6, TNF-α, and IL-1β in serum and pancreases
than the Sham, DCQD, anti-IL-17A, and anti-IL-23-p19
groups (P<0.05). Furthermore, the EX527 and r-HMGB1
groups have higher levels of IL-23, HMGB1, IL-1β, IL-
17A, TNF-α, and IL-6 in serum and pancreas at gene and
protein levels compared to the DCQD group (P<0.05)
(Figure 4).
Neutrophil activation
MPO is a surface marker of neutrophils, and MIP-2, LIX,
and KC are neutrophil chemokines that reect neutrophil
activation (14). Therefore, we measured the pancreatic
MPO activity and the protein and mRNA levels of MIP-
2, LIX, and KC in serum and pancreas. The SAP group
has higher pancreatic MPO activity and more MIP-2, LIX,
and KC expression than the sham group, DCQD, anti-IL-
Table 1 The primer for IL-23, IL-17A, KC, LIX, MIP-2, IL-6, IL-1β, and TNF-α
Gene Sense group (5'-3') Antisense group (5'-3')
HMGB1 ATGGGCAAAGGAGATC ATTCATCATCATCATCTTCT
IL-17A GGAAAGCTGGAC-CACCACA CACACCCACCAGCATCTTCTC
IL-23 AGGACTTGTGCTGTTCTTGTTTTGT CTCTGGGGTTTGTTTCTTTTCTCTT
KC TCACGCTTCTGGGCCTGTTG CAGCCGACTCATTGGGATCATC
LIX TCACGCTTCTGGGCCTGTTG CAGCCGACTCATTGGGATCATC
MIP-2 GGCAAGGCTAACAGACCTGGAAAG CACATCATCAGGTACGATCCAGGCTTC
IL-6 TGCGCTGGGCTTAGATCATT TGGATGCCTTTTATGTCGTCT
IL-1βAGGGAAATCGTGCGTGACAT GAACCGCTCATTGCCGATAG
TNF-αACCAAGGATGAGGGCGACTA CAGGCTTATGCCACCACACTT
β-actin CATCCGTAAAGACCTCTATGCCAAC ATGGAGCCACCGATCCACA
3035
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
17A, and anti-IL-23-p19 groups (P<0.05). Furthermore,
the r-HMGB1 and EX527 groups have higher pancreatic
MPO activity and more expression of MIP-2, LIX, and KC
compared to the DCQD group (P<0.05) (Figure 5).
The SIRT1, acety-HMGB1 and TLR-4 expression,
HMGB1 translocation, and SIRT1-HMGB1combination
in the pancreas
The HMGB1-TLR4 signal pathway signicantly inuences
SAP, and SIRT1 regulates HMGB1 can activation through
deacetylation (7,8). Therefore, we examined the activity of
SIRT1, the combination of SIRT1 and HMGB1, and the
level of SIRT1, acety-HMGB1, TLR4, and the mitigation
of HMGB1 from nuclear to the cytoplasm in vitro and vivo.
The SAP group had a higher expression of acety-HMGB1,
TLR-4, and cytoplasmic HMGB1 than the sham group
with lower SIRT1 activity, lower level of nuclear HMGB1,
SIRT1, and lower combination of SIRT1-HMGB1
(P<0.05), while the DCQD group showed an increase in
Figure 1 The effect of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on wet/weight, amylase and lipase activity, surviving
rats, and AR42J cell viability in SAP. (A) The inuence of DCQD, EX527 and r-HMGB1 on wet/weight, amylase and lipase activity. (B)
The inuence of anti-IL-17A and anti-IL-23p19 on wet/weight, amylase and lipase activity. (C) The inuence of DCQD, EX527, anti-IL-
17A, r-HMGB1 and anti-IL-23p19 on the survive rats in SAP. (D) The effect of DCQD, EX527 and anti-IL-17A, r-HMGB1and anti-IL-
23p19 on the AR42J cell viability in SAP. (E) The effect of anti-IL-17A and anti-IL-23p19 on the AR42J cell viability in SAP; ***, P<0.001
(SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs.
SAP); #, P<0.05 (anti-IL-17A vs. SAP); ***, P<0.001 (SAP vs. Control). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
2000
1500
1000
500
0
150
100
50
0
150
100
50
0
1.0
0.8
0.6
0.4
0.2
0
2000
1500
1000
500
0
15
10
5
0
15
10
5
0
Wet/weight ratio (×10–3)
Lipase (U/mL)
Amylase (U/mL)
Wet/weight ratio (×10–3)
Lipase (U/mL)
Amylase (U/mL)
Cumulative survival
Cell Viability (% control)
Cell Viability (% control)
0 2.5 5.0 7.5 10.0 12.5
Time
Cerulein
Cerulein
Group
Sham
SAP
DCQD
EX527
r-HMGB1
anti-IL-23p19
anti-lL-17A
Sham-censor
SAP-censor
DCQD-censor
EX527-censor
r-HMGB1-censor
anti-IL-23p19-censor
arnti-IL-17A-censor
Control SAP DCQD EX527 r-HMGB1
Control
SAP
anti-IL-17A
anti-IL-23p19
A B
C
E
D
Sham
Sham
Sham
Sham
Sham
Sham
DCQD
DCQD
DCQD
EX527
EX527
EX527
r-HMGB1
r-HMGB1
r-HMGB1
SPA
SPA
SPA
SPA
SPA
SPA
anti-IL-17A
anti-IL-17A
anti-IL-17A
anti-IL-23p19
anti-IL-23p19
anti-IL-23p19
***
***
*** ***
*** ***
***
***
^
^
^
^
^
^^
^
#
#
#
#
#
#
#
#
&
&
&
&
3036 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
SIRT1 activity, a combination of SIRT1 and HMGB1, and
a level of SIRT1 and nuclear HMGB1 with a decrease in the
level of acety-HMGB1, TLR-4, and cytoplasmic HMGB1
than the SAP group (P<0.05). Furthermore, the EX527
group showed a higher level of acety-HMGB1, TLR-4, and
HMGB1 in the cytoplasm than the DCQD group with a
lower combination of SIRT1-HMGB1, lower expression of
SIRT1 and nuclear HMGB1, and lower activity of SIRT1
(P<0.05) (Figure 6).
The release of IL-23, though relying on the HMGB1/
TLR-4 way in SAP
As shown in Figure 7A,7B, r-HMGB1 up-regulated IL-
23 secretion and increased the IL-23 and TLR-4 levels
(P<0.5). Compared to the SAP group, the DCQD and
TLR-4 siRNA groups have lower IL-23 and TLR-4.
However, overexpression of TLR-4 can up-regulate IL-23
and TLR-4 expression using a TLR-4 expression plasmid,
Figure 2 The inuence of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on pancreatic histopathological morphology, and
CD68 and MPO expression in SAP. (A) Representative results from HE stains (magnication ×200) on the histopathological morphology of
the pancreas and immunohistochemistry for CD68 and MPO; (B) the damage score was evaluated by semiquantitative analysis. (C) CD68
and MPO expression were evaluated by semiquantitative analysis in SAP. ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05
(EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); @, P<0.05 (anti-IL-23p19 vs. SAP); $, P<0.05 (anti-IL-23p19 vs. SAP). The blue
arrows indicate the positive place. DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
anti-IL-17A
anti-IL-17A
anti-IL-17A
anti-IL-17A
anti-IL-23p19
anti-IL-23p19
anti-IL-23p19
anti-IL-23p19
Sham
MPO
CD68
SAP
SAP
SAP
Sham
Sham
Sham
EX527
EX527
EX527
EX527
r-HMGB1
r-HMGB1
r-HMGB1
r-HMGB1
SPA DCQD
DCQD
DCQD
DCQD
HE (200×) MPO (200×) CD68 (200×)
5
4
3
2
1
0
20
15
10
5
0
Damage score
Hrp-positive cell 200×
*** ***
***
#
#@
@
@$
$
$
&
&
&
#
^^
^
A
BC
50 μM
50 μM50 μM 50 μM
50 μM 50 μM50 μM 50 μM 50 μM50 μM50 μM
50 μM
50 μM 50 μM 50 μM50 μM50 μM 50 μM
50 μM50 μM50 μM
3037
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Figure 3 The effect of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on pancreatic microcirculatory function in SAP.
(A) The effect of DCQD, EX527, and r-HMGB1 on blood ow velocity, number of functional blood vessel, RBC ow and number of
blood vessel. (B) The inuence of anti-IL-17A and anti-IL-23p19 on the blood ow velocity, number of functional blood vessel, RBC ow
and number of blood vessel. ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1
vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP). DCQD, Dachengqi decoction; SAP, severe acute
pancreatitis.
demonstrating higher expression of IL-23 and TLR-4 in
the TLR-4 plasmid group than the SAP group (P<0.05).
The DCQD signal pathway in the pancreatic
microcirculatory system in SAP (Figure 8)
DCQD treatment improves SAP-induced pancreatic
microcirculatory dysfunction by inhibiting neutrophil-
mediated inflammation via inactivating HMGB1-TLR-4-
IL-23-IL-17A signaling by targeting SIRT1.
Discussion
One common clinical critical illness, SAP, easily caused
multiple organ failure and frequent death (1). This study
used sodium taurodeoxycholate or cerulean to induce
SAP in vivo and in vitro, regardless. In vivo, SAP caused
a disturbance in the microcirculation of the pancreas, an
increase in serum amylase and lipase activity, and an increase
in the wet weight ratio of the pancreas. In vitro, cerulean
can significantly decrease AR42J cell viability, which is
consistent with earlier studies (9). These indicated that SAP
SAP
SAP
SAP
SAP
Sham
Sham
Sham
Sham
EX527
EX527
EX527
EX527
r-HMGB1
r-HMGB1
r-HMGB1
r-HMGB1
DCQD
DCQD
DCQD
DCQD
250
200
150
100
50
0
250
200
150
100
50
0
15
10
5
0
15
10
5
0
Functional vessels number
Red blood cell flow number/min
Blood flow velocity (μm/s)
Blood vessels number
Functional vessels number
Red blood cell flow number/min
Blood flow velocity (μm/s)
Blood vessels number
anti-IL-17A
anti-IL-17A
anti-IL-17A
anti-IL-17A
anti-IL-23p19
anti-IL-23p19
anti-IL-23p19
anti-IL-23p19
SAP
SAP
SAP
SAP
Sham
Sham
Sham
Sham
^
^
^
^
^
^
^
^
&
&
&
&
***
***
***
***
***
***
***
***
#
#
#
#
#
#
#
#
#
A
B
3038 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Figure 4 The influence of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on the level of IL-23, HMGB1, IL-1β, IL-
17A, TNF-α, and IL-6 in SAP. (A) ELISA was used to assess the inuence of DCQD, EX527 and r-HMGB1 on the serum level of IL-
23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (B) RT-PCR was used to assess the effect of DCQD, EX527 and r-HMGB1 on
the pancreatic mRNA of IL-23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (C) ELISA was used to examine the effect of DCQD,
EX527, and r-HMGB1 on the pancreatic level of IL-23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (D) the ELISA was used to test
the inuence of anti-IL-17A and anti-IL-23p19 on the serum level of TNF-α, IL-6 and IL-1β in SAP; (E) ELISA was used to measure the
inuence of anti-IL-17A and anti-IL-23p19 on the pancreatic level of TNF-α, IL-6 and IL-1β in SAP; (F) RT-PCR was used to measure the
inuence of anti-IL-17A and anti-IL-23p19 on pancreatic mRNA of TNF-α, IL-6 and IL-1β in SAP; (G) ELISA and RT-PCR were used to
evaluate the inuence of anti-IL-23p19 on the level of IL-17A in SAP. *, P<0.05 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527
vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP). DCQD, Dachengqi
decoction; SAP, severe acute pancreatitis.
in vivo and in vitro models was successfully established. We
also showed that DCQD, anti-IL-17A, or anti-IL-23-p19
treatment remarkably decreased lipase and amylase activity,
reduced microcirculatory pancreatic pathological injury and
dysfunction, downregulated the weight index, and improved
cerulean -induced the decline in the cell viability of AR42J.
Furthermore, both r-HMGB1 (an HMGB1 agonist) and
EX527 (SIRT1 inhibitor) effectively abated the protective
effect of DCQD in SAP. The findings were supported, as
they showed a higher serum activity of lipase and amylase,
more serious pancreatic pathological injury, weight index
and cell viability, and worse microcirculatory function in
the EX527 and r-HMGB1 groups than in the DCQD
group. Furthermore, previous studies have confirmed
500
400
300
200
100
0
300
200
100
0
200
150
100
50
0
100
80
60
40
20
0
40
30
20
10
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
15
10
5
0
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
pg/mL
pg/mL
pg/mL
pg/mL
pg/mL
pg/mL
pg/mL
pg/mL
mRNA (fold)
mRNA (fold)
mRNA (fold)
mRNA (fold)
SAP
Serum
Serum
Tissue
Tissue
Tissue
Tissue
A
F
B
G
CDE
1
3039
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Figure 5 The inuence of DCQD, EX527, anti-IL-17A, r-HMGB1 and anti-IL-23p19 on MPO activity and expression of MIP-2, KC,
and LIX in SAP. (A) ELISA was exploited to evaluate the inuence of DCQD, r-HMGB1, and EX527 on MPO activity in SAP; (B) ELISA
was used to assess the inuence of anti-IL-17A and anti-IL-23p19 on MPO activity in SAP; (C) ELISA was used to analyze the inuence of
DCQD, r-HMGB1, and EX527 on serum MIP-2, KC, and LIX level in SAP; (D) ELISA was exploited to evaluate the inuence of DCQD,
r-HMGB1, and EX527 on MIP-2, KC and LIX expression in SAP; (E) RT-PCR was exploited to measure the inuence of DCQD, r-HMGB1
and EX527 on the MIP-2, KC and LIX mRNA expression in SAP; (F) ELISA was designated to measure the inuence of anti-IL-17A and
anti-IL-23p19 on the MIP-2, KC and LIX serum expression in SAP; (G) ELISA was designated to evaluate the inuence of anti-IL-17A and
anti-IL-23p19 on the MIP-2, KC and LIX pancreatic expression in SAP; (H) RT-PCR was used to evaluate the inuence of anti-IL-17A and
anti-IL-23p19 on the MIP-2, KC, and LIX pancreatic mRNA in SAP; ***, P<0.001 (SAP vs. Sham); ***, P<0.001 (SAP vs. Control); #, P<0.05
(SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A
vs. SAP). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis; MPO, myeloperoxidase.
that activating the HMGB1 and IL-23/IL-17A pathways
significantly aggravated pancreatic microcirculatory
impairment. Therefore, our study further confirms
that the protective effect DCQD on SAP was achieved
through down-regulation of HMGB1-mediated pancreatic
microcirculatory dysfunction, and the IL-23/IL-17A
signal pathway plays an axis in regulating microcirculatory
dysfunction in SAP.
Increasing evidence showed that the inflammatory
response significantly influences TNF-α, IL-6, and IL-1β
are the most important symbol of inammatory cytokines
in SAP (14). TNF-α is an inflammation-initiating factor
that can mediate the release of IL-6 and other inammatory
cytokines, stimulate oxygen free radicals and nitric oxide
production, promote leukocyte chemotaxis and adhesion,
and damage pancreatic tissue (15). IL-6 has a critical
Sham SAP DCQD EX527 r-HMGB1
150
100
50
0
150
100
50
0
1.0
0.8
0.6
0.4
0.2
0.0
80
60
40
20
0
80
60
40
20
0
1.0
0.8
0.6
0.4
0.2
0.0
ng/L
ng/L
ng/L
ng/L
MPO activity
(units/100 mg pancreatic tissue)
MPO activity
(units/100 mg pancreatic tissue)
Sham
SAP
anti-IL-17A
anti-IL-23p19
KC
LIX
MIP-2
KC
LIX
MIP-2
KC
LIX
MIP-2
KC
LIX
MIP-2
Serum
Serum
Tissue
Tissue Tissue
Tissue
20
15
10
5
0
20
15
10
5
0
mRNA (fold) mRNA (fold)
A
F
B
G
C
H
D E
3040 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
Figure 6 The inuence of DCQD on the SIRT1, acety-HMGB1 and TLR-4 expression, and HMGB1 translocation, SIRT1 activity, and SIRT1-HMGB1combination in the
pancreas in the SAP. Western blotting, immunoprecipitation, and ELISA were designated to evaluate SIRT1, acety-HMGB1 and TLR-4 expression, HMGB1 translocation,
SIRT1 activity, and SIRT1-HMGB1combination in vitro and in vivo. (A) Immunoprecipitation was exploited to assay the combination of SIRT1 and HMGB1 in the pancreas; (B)
immunoprecipitation was exploited to assay the combination of SIRT1 and HMGB1 in the AR42J cell; (C) the SIRT1 activity in the pancreas; (D) SIRT1 activity in AR42Jcell;
(E) typical result for Western blotting analysis SIRT1 in the pancreases; (F) rat SIRT1, Acety-HMGB1, HMGB1 in nuclear and cytoplasm, and TLR-4 expression were evaluated
by semiquantitative analysis; (G) typical result for the Western blot analysis SIRT1, Acety-HMGB1, HMGB1 in nuclear and cytoplasm, and TLR-4 in the cell; (H) the expression
of AR42J cell SIRT1 was evaluated by semiquantitative analysis: (I) typical results for Western blotting on TLR-4 in SAP in rats; (J) rat TLR-4 expression was assessed by
semiquantitative analysis; (K) typical results for Western blot on TLR-4 in the AR42J cell; (L) cell TLR-4 expression was assessed by semi-quantitative analysis. ***, P<0.001 (SAP
vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Sham SAP DCQD EX527
Sham SAP DCQD EX527
Sham SAP DCQD r-HMGB1
Sham SAP DCQD r-HMGB1
Sham
r-HMGB1
Control
r-HMGB1
SAP
SAP
DCQD
DCQD
Sham SAP DCQD EX527
Control SAP DCQD EX527
Tissue
Tissue
Tissue
Tissue
Tissue
Cell
Cell
Cell
Cell
Cell
Cell
SIRT1
SIRT1
Acetyl-
HMGB1
Acetyl-
HMGB1
Cytoplasm
HMGB1
Cytoplasm
HMGB1
TLR-4
TLR-4
TLR-4
TLR-4/β-actin
TLR-4/β-actin
TLR-4
GAPDH β-actin
β-actin
β-actin
Nucleus-
HMGB1
Nucleus-
HMGB1
Histone Histone
0.4
0.3
0.2
0.1
0.0
0.4
0.3
0.2
0.1
0.0
IP: SIRT1/HMGB1
WB: SIRT1
WB: HMGB1
IP: SIRT1/HMGB1
WB: SIRT1
WB: HMGB1
SIRTL Activity
(% Sham)
SIRTL Activity
(% control)
150
100
50
0
150
100
50
0
Sham
EX527
EX527
SAP
SAP
DCQD
DCQD
Control
2.5
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
SIRT1/GAPDH
Acety-HMGE1/GAPDH
CytoplasmHMGE1/GAPDH
TLR-4/GAPDH
NeucleusHMGE1/GAPDH
SIRT1/GAPDH
Acety-HMGE1/GAPDH
CytoplasmHMGE1/GAPDH
TLR-4/GAPDH
NeucleusHMGE1/GAPDH
Relative protein density
Relative protein density
Tissue
A
F
K
B
G
L
C
H
D
I
E
J
3041
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
influence in initiating an immune response in SAP, and
is released primarily by macrophages, T lymphocytes,
dendritic cells, and T cells (14,15). IL-1β is deeply involved
in the destruction of pancreatic tissue and the formation of
edema in SAP (15). In this study, the intervention with anti-
IL-17A, DCQD, or anti-IL-23p19 significantly decreased
TNF-α, IL-6, and IL-1β expression. However, r-HMGB1
and EX527 can eliminate the influence of DCQD on
the inflammatory response in SAP, as confirmed higher
expression of TNF-α, IL-6, and IL-1β in the r-HMGB1
and EX527 groups than in the DCQD group. The above
results implied that DCQD could reduce SAP-induced
microcirculatory dysfunction by inhibiting the HMGB1-
mediated inflammatory response, and the IL-23/IL-17A
signal pathway can improve microcirculatory dysfunction
by attenuating inammation in SAP.
Interestingly, blocking neutrophil activation has
previously been demonstrated to suppress the inammatory
response in SAP (7). Therefore, the intensity of the
inflammatory response depends on the activation of
neutrophils in SAP. In addition, MPO is a surface marker
of neutrophils, and KC, LIX, and MIP-2 are neutrophil
chemokines that reect neutrophil activation by recruitment
and migration of neutrophils. Treatment with anti-IL-
23-p19, DCQD, or anti-IL-17A decreased pancreatic MPO
expression, activity, and MIP-2., KC, and LIX. However,
the r-HMGB1 intervention can reduce the negative
inuence of DCQD on neutrophil inactivation and lessen
neutrophil chemokines, as it showed higher MPO activity
and higher expression of MIP-2, KC, and LIX in the
r-HMGB1 group than in the DCQD group. These ndings
further demonstrated that anti-IL-23-p19, DCQD, or anti-
IL-17A reduce the inflammatory response by activating
neutrophils in SAP.
IL-17A is a common molecule that critically
modulates host defense against detrimental inflammatory
stimulation (6). Controlling neutrophil activation and
recruitment to the pancreas is the main mechanism of IL-
17A activity (7). IL-17A is mainly released from NKT cells,
γδ T cells, Th17 cells, etc. (6,15). In particular, the release
of IL-17A is controlled by the heterodimeric cytokine
IL-23 through the following mechanism (16,17): IL-23
triggers naïve CD4+ T cells differentiation into Th17 and
subsequently stimulates NKT cells to release IL-17A, along
with anti-CD3. Furthermore, IL-23 promotes the release
of IL-17A from γδ T cells by working with IL-1 (17). In our
study, SAP can stimulate the activation of IL-23/IL-17A
signaling, and DCQD significantly promoted inactivation
of the IL-23/IL-17A axis, as it showed a lower level of IL-
17A and IL-23 in the DCQD group than in the SAP group.
However, r-HMGB1 can boost activating IL-23/IL-17A
signaling in SAP, demonstrating higher IL-17A and IL-
23 levels in an r-HMGB1 group than the DCQD group.
Furthermore, we also demonstrated that neutralizing IL-
23 reduced the expression of IL-17A in SAP, which further
confirmed that IL-23 could promote IL-17A secretion.
These results were in accordance with previous research
15
10
5
0
6
4
2
0
mRNA (fold)
IL-23 level in medium
(fold changs of control)
r-HMGB1
r-HMGB1
RAW264.7
RAW264.7
r-HMGB1
TLR-4
ILR-23
Control
Control
Control
SAP
SAP
SAP
DCQD
DCQD
DCQD
TLR-4 SiRNA
TLR-4 SiRNA
TLR-4 SiRNA
Plasmid-TLR-4
Plasmid-TLR-4
Plasmid-TLR-4
A
B
***
***
***
#
#
#
&
&
&
^
^
^
Figure 7 The inuence of DCQD on the expression of TLR-4 and
IL-23 in SAP. (A) The inuence of DCQD on the level of TLR-
4 and IL-23 mRNA in RAW264.7 was detected through RT-PCR;
(B) ELISA was designated to evaluate the inuence of r-HMGB1
on IL-23 expression in the supernatant in SAP. ***, P<0.001 (SAP
vs. Control); #, P<0.001, (DCQD vs. SAP); ^, P<0.05 (DCQD vs.
siRNA-TLR-4); &, P<0.05 (DCQD vs. plasmid-TLR-4). DCQD,
Dachengqi decoction; SAP, severe acute pancreatitis.
3042 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
that implied that the inhibitory effect of DCQD in
neutrophil inactivation is through the inactivation of the
IL-23/IL-17A axis through HMGB1.
HMGB1 is a non-DNA binding protein in the nucleus
of eukaryotic cells and is widely distributed in the pancreas,
lung, liver, kidney, lymph, and other tissues (2). It is known
to function as a DAMP, interacting with members of RAGE
and TLR (2). In our study, SAP only influences TLR-4.
This is inconsistent with previous research attributed to
drug dosage, purity, technology, and individual differences
in rats. TLR-4 is a pattern-recognition receptor (PRR),
and it has been extensively validated that TLR-4 mediates
HMGB1-induced pancreatic injury in SAP (2). Previous
research showed that HMGB1 stimulates the expression
of TLR-4 in the pancreas, which is positively related to
promoting macrophages and dendritic cells to secrete IL-
23 in SAP (8). Blocking TLR-4 can trigger the inactivation
of IL-23/IL-17A signaling (18). In our in vivo study,
DCQD treatment markedly advanced the inactivation of
the HMGB1/TLR-4 signal pathway, as shown by a lower
expression of TLR-4 and CD68, and lower acetylation,
migration, and release of HMGB1. Furthermore, r-HMGB1
can abolish the impact of DCQD on the signal pathway of
HMGB1/TLR-4 in SAP, as confirmed higher expression
of TLR-4 and CD68 and greater acetylation, migration,
and release of HMGB1 in the r-HMGB1 group than in the
DCQD group.
In vitro studies further confirmed that DCQD could
Figure 8 The signal pathway of DCQD on pancreatic microcirculatory in SAP. DCQD, Dachengqi decoction; SAP, severe acute
pancreatitis.
Acetylatied HMGB1 Acetylatied HMGB1
Acetylatied
HMGB1
SIRT1-HMGB1
DCQD
HMGB1 HMGB1
HMGB1
SIRT1
TLR4
Neutrophlis
Macrophages
Pancreatic cellsSAP model
Dachengqi
decoction (DCQD)
Th17, γ δ T, NK
and NKT cells
IL-23 IL-23
IL-23
IL-17A IL-17A
IL-17A
Chemokines
Inflammation
Cell apoptosis
Chemokines
Inflammation
Cell apoptosis
3043
Gland Surgery, Vol 10, No 10 October 2021
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
decrease HMGB1 secretion, and HMGB1 can promote
macrophages to secrete IL-23 and increase IL-23 and
TLR-4 expression. In addition, SiRNA TLR-4 can advance
macrophage inactivation and alleviate IL-23 and TLR-
4 expression. However, overexpression of TLR-4 has the
opposite inuence, as conrmed higher expression of LR-4
and IL-23 in the plasmid group than in the SAP group. The
above results indicated that the negative effect of DCQD
on the activation of IL-23/IL-17A signaling is through the
promotion of the inactivation of the HMGB1-TLR-4 signal
pathway through deacetylation of HMGB1.
Noteworthy, the degree of deacetylation of HMGB1 is
dependent on SIRT1, one of NAD + dependent histone
deacetylase, which can deacetylate the activities of HMGB1
through interacting with HMGB1 at promoter region (19).
In the study, DCQD can suppress the sodium taurocholate
or cerulean-induced decrease in SIRT1 activity, expression,
and interaction of HMGB1. However, EX527 can eliminate
the influence of DCQD on the SIRT-HMGB1 axis in
SAP. This indicated that DCQD could alleviate HMGB1
acetylation by regulating the SIRT-HMGB1 signaling
pathway in SAP.
In conclusion, our study provides compelling evidence
that the protective effect of DCQD on the pancreatic
microcirculatory system in SAP is through inhibiting
neutrophil-mediated inflammatory reactions. The above
action mechanism of DCQD is likely associated with the
inactivation of the HMGB1-TLR-4-IL-23-IL-17A axis by
targeting SIRT1. However, this study was only performed
on non-knockout mice with small sample size. More studies
are required with larger sample sizes, knockout mice and
clinical observations, and more cellular intervention to
conrm the efcacy and mechanism of DCQD in SAP.
Acknowledgments
Funding: The study was funded by the Foundation of
Sichuan Provincial People’s Hospital (No. 2020LY07,
No. 2017LY11), and scientic research project of Sichuan
Provincial Department of Science and Technology (No.
2020YJ0179).
Footnote
Reporting Checklist: The authors have completed the
ARRIVE reporting checklist. Available at https://dx.doi.
org/10.21037/gs-21-655
Data Sharing Statement: Available at https://dx.doi.
org/10.21037/gs-21-655
Conicts of Interest: All authors have completed the ICMJE
uniform disclosure form (available at https://dx.doi.
org/10.21037/gs-21-655). Dr. JW reported that the study
was funded by the Foundation of Sichuan Provincial
People’s Hospital (No. 2017LY11). Dr. JZ reported that the
study was funded by the Foundation of Sichuan Provincial
People’s Hospital (No. 2020LY07) and scientific research
project of Sichuan Provincial Department of Science and
Technology (No. 2020YJ0179). The other authors have no
conicts of interest to declare.
Ethical Statement: The authors are accountable for all
aspects of the work in ensuring that questions related
to the accuracy or integrity of any part of the work
are appropriately investigated and resolved. All animal
procedures were approved by the animal care committee
of the Sichuan Provincial People’s Hospital (No. 2011220),
and the experimental protocols were strictly carried out
based on NIH Guidelines for the care and use of animals.
Open Access Statement: This is an Open Access article
distributed in accordance with the Creative Commons
Attribution-NonCommercial-NoDerivs 4.0 International
License (CC BY-NC-ND 4.0), which permits the non-
commercial replication and distribution of the article with
the strict proviso that no changes or edits are made and the
original work is properly cited (including links to both the
formal publication through the relevant DOI and the license).
See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
References
1. Wu BU, Johannes RS, Sun X, et al. The early prediction
of mortality in acute pancreatitis: a large population-based
study. Gut 2008;57:1698-703.
2. Broggi A, Granucci F. Microbe- and danger-induced
inammation. Mol Immunol 2015;63:127-33.
3. Leppkes M, Maueröder C, Hirth S, et al. Externalized
decondensed neutrophil chromatin occludes pancreatic
ducts and drives pancreatitis. Nat Commun 2016;7:10973.
4. Shen X, Li WQ. High-mobility group box 1 protein and
its role in severe acute pancreatitis. World J Gastroenterol
2015;21:1424-35.
5. Yan B, Chen F, Xu L, et al. HMGB1-TLR4-IL23-
3044 Wang et al. DCQD improved pancreatic microcirculatory system in SAP
© Gland Surgery. All rights reserved. Gland Surg 2021;10(10):3030-3044 | https://dx.doi.org/10.21037/gs-21-655
IL17A axis promotes paraquat-induced acute lung injury
by mediating neutrophil inltration in mice. Sci Rep
2017;7:597.
6. Ni J, Hu G, Xiong J, et al. Involvement of interleukin-17A
in pancreatic damage in rat experimental acute necrotizing
pancreatitis. Inammation 2013;36:53-65.
7. Zhang MJ, Zhang GL, Yuan WB, et al. Treatment
of abdominal compartment syndrome in severe acute
pancreatitis patients with traditional Chinese medicine.
World J Gastroenterol 2008;14:3574-8.
8. Chen Z, Chen Y, Pan L, et al. Dachengqi Decoction
Attenuates Inammatory Response via Inhibiting HMGB1
Mediated NF-κB and P38 MAPK Signaling Pathways
in Severe Acute Pancreatitis. Cell Physiol Biochem
2015;37:1379-89.
9. Wang J, Chen G, Gong H, et al. Amelioration of
experimental acute pancreatitis with Dachengqi Decoction
via regulation of necrosis-apoptosis switch in the
pancreatic acinar cell. PLoS One 2012;7:e40160.
10. Huang J, Tian R, Yang Y, et al. The SIRT1 inhibitor EX-
527 suppresses mTOR activation and alleviates acute
lung injury in mice with endotoxiemia. Innate Immun
2017;23:678-86.
11. Li G, Wu X, Yang L, et al. TLR4-mediated NF-κB
signaling pathway mediates HMGB1-induced pancreatic
injury in mice with severe acute pancreatitis. Int J Mol
Med 2016;37:99-107.
12. Hu X, Ma R, Lu J, et al. IL-23 Promotes Myocardial I/
R Injury by Increasing the Inammatory Responses
and Oxidative Stress Reactions. Cell Physiol Biochem
2016;38:2163-72.
13. Sun J, Zhang S, Zhang X, et al. IL-17A is implicated
in lipopolysaccharide-induced neuroinammation and
cognitive impairment in aged rats via microglial activation.
J Neuroinammation 2015;12:165.
14. Kolaczkowska E, Kubes P. Neutrophil recruitment and
function in health and inammation. Nat Rev Immunol
2013;13:159-75.
15. Xu P, Wang J, Yang ZW, et al. Regulatory roles of the
PI3K/Akt signaling pathway in rats with severe acute
pancreatitis. PLoS One 2013;8:e81767.
16. Cua DJ, Tato CM. Innate IL-17-producing cells: the
sentinels of the immune system. Nat Rev Immunol
2010;10:479-89.
17. Zheng Y, Danilenko DM, Valdez P, et al. Interleukin-22,
a T(H)17 cytokine, mediates IL-23-induced dermal
inammation and acanthosis. Nature 2007;445:648-51.
18. Wang X, Sun R, Wei H, et al. High-mobility group box
1 (HMGB1)-Toll-like receptor (TLR)4-interleukin (IL)-
23-IL-17A axis in drug-induced damage-associated lethal
hepatitis: Interaction of γδ T cells with macrophages.
Hepatology 2013;57:373-84.
19. Wei S, Gao Y, Dai X, et al. SIRT1-mediated HMGB1
deacetylation suppresses sepsis-associated acute kidney
injury. Am J Physiol Renal Physiol 2019;316:F20-31.
Cite this article as: Wang J, Zou Y, Chang D, Hong DQ,
Zhang J. Protective effect of Dachengqi decoction on the
pancreatic microcirculatory system in severe acute pancreatitis
by down-regulating HMGB-TLR-4-IL-23-IL-17A mediated
neutrophil activation by targeting SIRT1. Gland Surg
2021;10(10):3030-3044. doi: 10.21037/gs-21-655
