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Phytomedicine
journal homepage: www.elsevier.com/locate/phymed
Chaiqin chengqi decoction alleviates severity of acute pancreatitis via
inhibition of TLR4 and NLRP3 inflammasome: Identification of bioactive
ingredients via pharmacological sub-network analysis and experimental
validation
Yongjian Wen
a,b,c,1
, Chenxia Han
a,1
, Tingting Liu
a
, Rui Wang
b
, Wenhao Cai
a,d
, Jingyu Yang
a
,
Ge Liang
b
, Linbo Yao
a
, Na Shi
a
, Xianghui Fu
e
, Lihui Deng
a
, Robert Sutton
d
, John A. Windsor
f
,
Jiwon Hong
c,f
, Anthony R. Phillips
c,f
, Dan Du
b,g,⁎
, Wei Huang
a,d,g,⁎
, Qing Xia
a,⁎
a
Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West
China Hospital, Sichuan University, Chengdu 610041, China
b
West China-Washington Mitochondria and Metabolism Center, West China Hospital, Sichuan University, Chengdu 610041, China
c
Applied Surgery and Metabolism Laboratory, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
d
Liverpool Pancreatitis Study Group, Royal Liverpool University Hospital and Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GE, United
Kingdom
e
Division of Endocrinology and Metabolism, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center of
Biotherapy, Chengdu 610041, China
f
Surgical and Translational Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand
g
Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu 610000, China
ARTICLE INFO
Keywords:
Acute pancreatitis
Inflammatory signaling pathways
Chaiqin chengqi decoction
Active ingredients
Pharmacology network analysis
ABSTRACT
Background: Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoc-
tion. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more
than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive
ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory
effects.
Purpose: Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family
pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a
central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we
screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3
signaling pathways followed by experimental validation.
Methods: The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets
in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice
received 7 intraperitoneal injections of cerulein (50 μg/kg; hourly) to induce AP (CER-AP), while oral gavage of
CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein.
Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction,
Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD
compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7
https://doi.org/10.1016/j.phymed.2020.153328
Received 4 March 2020; Received in revised form 8 June 2020; Accepted 22 June 2020
Abbreviations: AP, acute pancreatitis; BA, baicalin; CCK, cholecystokinin; CER, cerulein; CH, chrysin; CI, combination index; CQCQD, chaiqin chengqi decoction;
DAMPs, damage-associated molecular pattern molecules; DCQD, dachengqi decoction; DMEM, Dulbecco's modified Eagle medium; EM, emodin; ETCM, Encyclopedia
of Traditional Chinese Medicine; IL-6, interleukin-6; IL-1β, interleukin-1β; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; MPO,
myeloperoxidase; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor- kappaB; NLRP3, NOD-like receptor family pyrin domain containing 3;
PI, propidium iodide; PPI, Protein-Protein Interaction; RH, rhein; RT-qPCR, real-time quantitative polymerase chain reaction; SITICH, Search Tool for Interactions of
Chemicals; TCM, traditional Chinse medicine; TLCS, taurolithocholic acid 3-sulfate disodium salt; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-alpha
⁎
Corresponding authors: Sichuan Provincial Pancreatitis center and West China-Liverpool Biomedical Research center, West China Hospital of Sichuan University,
5B Floor 2nd Building, Tianfu Life Science Park of Hi-Tech Industrial Development Zone, No. 88 Keyuan South Road, Chengdu 610041, China.
E-mail addresses: dudan@wchscu.cn (D. Du), dr_wei_huang@scu.edu.cn (W. Huang), xiaqing@medmail.com.cn (Q. Xia).
1
These authors contributed equally as co-first authors.
Phytomedicine 79 (2020) 153328
Available online 01 September 2020
0944-7113/ © 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
T
macrophages.
Results: The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones
and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were
the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and
TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was asso-
ciated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and
chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression
of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macro-
phages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/
NLRP3-related proteins.
Conclusions: CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-in-
flammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial
effects.
Introduction
Acute pancreatitis (AP) is an inflammatory digestive system disease
with growing prevalence. It currently has an estimated global incidence
rate of 33 cases per 100,000 person-years (Xiao et al., 2016). Although
gallstones and excess alcohol consumption have been recognized as two
leading causes of AP globally (Forsmark Ch et al., 2017), recent studies
revealed that hypertriglyceridemia-associated AP is becoming the pre-
dominant etiology of AP cases in China (Ding et al., 2019;
Mukherjee et al., 2019; Shi et al., 2020; Zhang et al., 2019c).
AP cases with local complications (i.e. acute necrotic and peripan-
creatic fluid collections) and persistent organ failure are defined as
“moderately severe” or “severe” (Banks et al., 2013; Crockett et al.,
2018). Due to an increased risk of developing sepsis, and needing in-
vasive interventions, patients in these groups typically require in-
tensive-care management (Schepers et al., 2019; Shi et al., 2020). The
high morbidity and mortality rate associated with these patients results
in significant socioeconomical burdens (Peery et al., 2019). Despite an
intensive search of potential interventions, there remains no effective
pharmacological treatment for managing these AP cases (Moggia et al.,
2017).
Recent progress in translational research has deepened our under-
standing of the fundamental processes contributing to the initiation and
aggravation of AP (Lee and Papachristou, 2019). Upon exposure to
classic pancreatic toxins (i.e. bile acid), human pancreatic acinar cells
release pro-inflammatory cytokines (interleukin [IL]-1β, IL-6 and tumor
necrosis factor-alpha [TNF-α]), chemokines and chemokine receptors
(Lugea et al., 2017) as well as damage-associated molecular pattern
molecules (DAMPs; i.e. cell-free DNA, high mobility groupbox 1 and
histones) (Hoque et al., 2011; Kang et al., 2014b; Liu et al., 2017;
Merza et al., 2015) into extracellular milieu to initiate so-called sterile
inflammation. Toll-like receptors (TLRs; TLR4/9) (Chen et al., 2014;
Kang et al., 2014a; Vaz et al., 2013) and inflammasomes (Hoque et al.,
2012; Hoque and Mehal, 2015) are proteins that are essential to the
sensing of DAMPs and signal transduction, respectively, and are both
needed to completely activate the systemic inflammation cascade after
AP onset. The “TLR4” and “nucleotide-binding oligomerization domain-
like receptor (NLR) family pyrin domain containing 3 (NLRP3)” in-
flammasome are the most well studied inflammatorysignaling cassettes
in experimental AP. TLR4 is expressed in pancreatic acinar cells
(Gu et al., 2013), immune cells, epithelium of pancreatic duct and
pancreatic microcirculation (Vaz et al., 2013). Genetic knockout of
TLR4 (or its co-receptor CD14) (Sharif et al., 2009), TLR4 downstream
signaling cytosolic protein myeloid differentiation primary response 88
(MyD88) (Koike et al., 2012) and nuclear factor-kappaB (NF-κB)
(Huang et al., 2013) or pharmacologic inhibition of TLR4 by lactate
(Hoque et al., 2014) and TLR4 specific antagonist TAK242 (Awla et al.,
2011) can all significantly reduce the severity of experimental AP in
mouse models. The NLRP3 inflammasome is expressed by innate im-
mune cells, stimulated pancreatic acinar cells and especially in
macrophages (Hoque et al., 2012; Hoque and Mehal, 2015; 2011;
Sendler et al., 2020). Genetic deletion or pharmacological antagonism
of NLRP3 inflammasome elements (caspase-1, ASC, or NLRP3) also
greatly diminished pancreatic injury and systemic inflammation in AP
models (Hoque et al., 2011; Sendler et al., 2020). Since novel inhibitors
are being developed for targeting TLR4 and NLRP3 inflammasome
(Bhattacharyya et al., 2018; Coll et al., 2015), these strategies may hold
substantial promise for treating human AP.
In China, it is a common and well-established practice to use rhu-
barb-based Traditional Chinese Medicine (TCM) herbal formula such as
dachengqi decoction (DCQD) for AP care. At our hospital, we have
refined and successfully employed a DCQD-derived formula known as
chaiqin chengqi decoction (CQCQD) to manage AP patients for over 30
years (Liu et al., 2004). Systematic review (Wang et al., 2005) and
meta-analyses (Lu et al., 2014) of observation studies and trials with
small sample size have demonstrated the effectiveness of TCM in im-
proving AP's clinical outcomes, with higher quality randomized clinical
trials planned for the near future. Existing work has shown that
bioactive ingredients such as emodin, rhein, baicalin and honokiol in
DCQD and CQCQD, can significantly reduce the severity of experi-
mental AP by suppressing pro-inflammatory mediators (Lu et al., 2017;
Zhou et al., 2016). However, the exact action of individual ingredients
responsible for the various pharmacological responses and the me-
chanism underlying the synergistic protective effects remain to be
clarified.
Since TLR4/NLRP3 pro-inflammatory pathways play a central role
in determining the extent of pancreatic injury in AP, in this study we
sought to: (1) screen CQCQD bioactive ingredients that had the main
inhibitory effects on TLR4/NLRP3 pathways using a sub-network
pharmacology analysis; (2) evaluate the efficacy of CQCQD on the se-
verity of experimental AP and its pro-inflammatory signaling pathways
in vivo; (3) validate the effects of these bioactive compounds on pan-
creatic acinar cells and macrophages.
Materials and methods
Ethics and animals
All animal experiments were approved by the Animal Ethics
Committee of West China Hospital, Sichuan University (2017065A and
2019170A). Male C57BL/6J mice (22–25 g) were purchased from
Beijing Huafukang Bioscience Co., Ltd. (Beijing, China). Animals were
maintained at 22 ± 2 °C with a 12 h light-dark cycle and ad libitum
feeding of standard laboratory chow and water throughout the ex-
periment.
Materials and reagents
The detailed information of reagents, natural compound standards
and quantitative reverse transcription polymerase chain reaction (RT-
Y. Wen, et al. Phytomedicine 79 (2020) 153328
2
qPCR) primer pairs are provided in the Supplementary materials and
methods and Tables S1–2.
Preparation of CQCQD
The raw materia medica of CQCQD were purchased from Sichuan
Hospital of Traditional Chinese Medicine (Chengdu, Sichuan, China).
CQCQD formula consisted of Bupleurum marginatum Wall. ex DC,
Scutellaria baicalensis Georgi, Rheum palmatum L., Sodium Sulfate,
Magnolia officinalis Rehd. et Wils, Citrus aurantium L.,
Gardenia jasminoides Ellisand Artemisia capillaris Thunb. The detailed
information of materia medica from CQCQD is provided in Table 1. All
the dried herbs except Rheum palmatum L. and Sodium Sulfate were
first soaked in 800 ml sterile filtered water for 30 min then boiled with
1000 ml sterile filtered water for 30 min and reduced to a concentrated
solution (~200 ml). The concentrated liquid was then removed from
the dregs and stored separately. Another 1000 ml water was added to
the dregs then boiled and reduced again as described above. In the last
5 min of the boiling procedure, Rheum palmatum L. was supplemented,
while Sodium Sulfate was mixed at the end of the procedure. The twice
extracted solution was combined (~400 ml) and lyophilized into
powder using an EYELA FDU-2110 lyophilizer (Tokyo Rikakikai Co.,
Ltd.; Tokyo, Japan) and stored at −20 °C.
UPLC-QTOF/MS analysis of CQCQD and standards
Lyophilized CQCQD extractions were dissolved in hot water and
obtained as 0.5 mg/ml solution. Under optimized chromatography
conditions, all ingredients were separated within 20 min. Solution for
CQCQD and standards was analyzed on a UPLC HeClass coupled with a
Waters SYNAPT G2-Si HDMS Q-TOF (Waters Corporation; Milford, MA,
USA). Separation was performed on a BEH C18 column (2.1 × 100 mm,
1.7 μm) at 40 °C. Gradient elution of 0.1% formic acid solution (A) and
methanol (B) at a flow rate of 0.4 ml/min was employed as follows:
0–1.0 min, 5%B; 1.0–5 min, 5–50%B; 5–12 min, 50–90% B; 12–15 min,
90% B; 15–16 min, 90–5%B; 16–20 min, 5% B. The injection volume
was 1 μl. The Q-TOF system was operated using an electrospray ion
source in negative ion modes. The mass range was set at m/z 50–1200.
The capillary voltage was 1.0 KV. The source temperature was 120 °C,
and the desolvation temperature was 400 °C. Cone gas flow and deso-
lvation gas flow were 50 and 800 l/h, respectively. Data were acquired
on MS
E
mode and analyzed using a UNIFI 1.9.2 software (Waters
Corporation).
Pharmacology sub-network construction and analysis
Main pharmacology network (compounds-targets-pathways) con-
struction:
(1) Major identified compounds from CQCQD and their corresponding
149 targets were predicted using Search Tool for Interactions of
Chemicals (SITICH; http://stitch.embl.de/) and Encyclopedia of
Traditional Chinese Medicine (ETCM; http://www.nrc.ac.cn:9090/
ETCM/) databases, based on structural and functional similarities
(Tanimoto > 0.8);
(2) AP-associated 530 targets were identified from the following da-
tabase: OMIM (https://www.omim.org/), DisGeNET (http://www.
disgenet.org), NCBI, etc.;
(3) The Protein-Protein Interaction (PPI) network of con-targets (the
overlapping targets from CQCQD major identified compounds and
AP) was constructed based on STRING database (https://string-db.
org/); then pathways enrichment analysis was conducted via Kyoto
Encyclopedia of Genes and Genomes (KEGG) signaling based on
con-targets.
Sub-network establishment:
(1) TLR4/NLRP3-related pathways (TLR, NF-κB and NLR) were se-
lected based on KEGG signaling pathways enrichment analysis;
(2) Subsequently, targets related to TLR4/NLRP3 pathways were ob-
tained from PPI network of con-targets;
(3) A sub-network of TLR4/NLRP3 pathways-targets-compounds was
extracted from the main network.
Induction of experimental AP and severity assessment
Mice received seven intraperitoneal injections of a cholecystokinin
(CCK) analog cerulein (50 μg/kg) at hourly intervals, while controls
received injections of normal saline. In the treatment groups, oral ga-
vage of different doses of CQCQD (5, 10, 15 and 20 g/kg) begun at the
3rd injection of cerulein, and were given 3 times at 2 hourly intervals.
Animals were humanely culled at 12 h from the first injection of cer-
ulein/saline. Relevant organs were harvested via an abdominal midline
incision for severity assessment. Pancreatic and lung histopathology
assessment and detailed protocols for serum amylase, pancreatic trypsin
and myeloperoxidase (MPO), lung MPO and serum IL-6 were as pre-
viously described (Huang et al., 2017).
RT-qPCR, Western blot and immunohistochemistry
Detailed protocols for these assays are demonstrated in
Supplementary materials and methods.
Acinar cell isolation, RAW264.7 cell culture and cell injury assessment
Pancreatic acinar cells were freshly isolated using a collagenase IV
digestion procedure as we previously described (Huang et al., 2014).
Cells were maintained in HEPES buffer (pH 7.3; in mM: HEPES 10, D-
glucose 10, NaCl 140, KCl 4.7, MgCl
2
1.13 and CaCl
2
1.2). Cells were
pre-treated with compounds (12.5, 25 and 50 μM) for 30 min at de-
signed concentrations, followed by co-incubation with CCK (500 nM)
for 2 h, taurolithocholic acid 3-sulfate disodium salt (TLCS; 500 μM) for
30 min, or lipopolysaccharide (LPS; 1 μg/ml) for 30 min with gentle
shake at 50 rpm under room temperature; control groups received
HEPES buffer incubation. Then cells were viewed by epi‑fluorescence
microscopy ZEISS AX10 imager A2/AX10 cam HRC (Jena GmbH,
Table 1
The Ingredients list of CQCQD.
Ingredients English name Latin name Plant name Weight (g)
Zhuyechaihu Chinese thorowax root Radix Bupleuri Bupleurum marginatum Wall. ex DC. 15
Huangqin Baikal skullcap root Scutellaria baicalensis Georgi Scutellaria baicalensis Georgi 15
Dahuang Rhubarb Radix et Rhizoma Rhei Rheum palmatum L. 20
Mangxiao Sodium sulfate Sodium Sulfate Na
2
SO
4
.10H
2
O 20
Houpu Officinal magnolia bark Cortex Magnoliae Officinalis Magnolia officinalis Rehd. et Wils 15
Zhishi Unripe bitter orange Fructus Aurantii Immaturus Citrus aurantium L. 15
Zhizi Gardenia fruit Fructus Gardeniae Gardenia jasminoides Ellis 20
Yinchen Capillary wormwood herb Herba Artemisiae Scopariae Artemisia capillaris Thunb 15
Y. Wen, et al. Phytomedicine 79 (2020) 153328
3
Germany) for determination of necrotic cell death pathway activation
after staining with Hoechst 33342 (50 μg/ml) and propidium iodide (PI;
1 μM). Necrotic cells were calculated as PI positive cells divided by
Hoechst 33342 stained cells and multiplied by 100%.
RAW264.7 mouse macrophages were cultured in Dulbecco's mod-
ified Eagle medium (DMEM) medium mixed with 10% fetal bovine
serum in an incubator at 37 °C with saturated humidity and 5% CO2.
Cell viability of RAW264.7 cells was assessed by Cell Counting Kit 8.
Cells (approx. 8 × 10
3
) were cultured overnight in a 96-well plate (100
μl medium per well) and were incubated with compounds at different
concentrations (12.5, 25, 50, 100, 200 μM, final concentration) for
24 h. After changing the medium, Cell Counting Kit 8 solution (10 μl)
was added into each well and incubated for 3 h away from light.
Subsequently, the absorbance values of all the wells were measured by
the BMG Labtech CLARIOstar Plate Reader (Ortenberg, Germany) at
450 nm. Nitric oxide production was monitored by measuring the ni-
trite levels in culture medium as previously described (Joo et al., 2014).
Briefly, RAW264.7 cells were pre-treated with active compounds at
various concentrations (12.5, 25, 50 μM) for 24 h and subsequently
stimulated with LPS (1 μg/ml) for another 24 h. The supernatant was
collected to detect the nitrite levels which were calculated with re-
ference to a standard curve of sodium nitrite generated from known
concentrations. Dose-effect curves of compounds on macrophage cell
injury were analyzed using the median-effect method described
previously (Chou, 2010). The combination index (CI) was calculated
using ‘CompuSyn’ software (Cambridge, UK). Synergism (CI < 1), ad-
ditive effect (CI = 1), and antagonism (CI > 1) represent combination
effects, respectively. The suppression rate of nitric oxide production is
expressed as fraction affected (Fa).
Statistical analysis
Data were analyzed using analysis of variance (Ordinary one-way
ANOVA) and Tukey's post-hoc test for multiple comparisons with Prism
8.0 software (GraphPad Software Inc., La Jolla, CA, USA). Data are
presented as mean ± S.E.M. A two-sided p < 0.05 was considered
statistically significant.
Results
Analysis of CQCQD ingredients
The phytochemical analysis based on UPLC-QTOF/MS made a pre-
liminary identification of 70 compounds from CQCQD by matching the
molecular ions and fragment information using UNIFI 1.9.2 software.
They belonged to flavonoids, iridoids, phenols, lignans, anthraquinones
and corresponding glycosides, and those compounds with a high (>
10,000) intensity were listed in Table S3. Of 70 compounds initially
Fig. 1. Chemical ingredients analysis of CQCQD. (A) The three-dimensional UPLC-QTOF/MS of CQCQD. (B) Representative base peak intensity (BPI) chromatograms
of CQCQD and standards.
Y. Wen, et al. Phytomedicine 79 (2020) 153328
4
identified in the CQCQD, 8 compounds (shanziside, geniposide, bai-
calin, chrysin, rhein, honokiol, magnolol and emodin) were listed as the
representative active ingredients of monarch and minister drugs in
CQCQD. They were confirmed by comparing retention time, molecular
ions and fragmentions with corresponding standards in the mass spec-
trum (Fig. S1 and Table S5). The representative 3D UPLC-MS picture of
CQCQD is displayed in Fig. 1A. The representative chromatograms of
CQCQD and 8 standards are shown in Fig. 1B.
Construction of TLR4/NLRP3 related sub-network and identification of
targets-ingredients
The flowchart of pharmacology network construction was shown in
Fig. S2. A total of 27 con-targets were obtained, among them, 24 con-
targets were further filtered out by PPI network with the minimum
required interaction score of 0.7 (high confidence) (Fig. 2A). Further
KEGG pathway enrichment analysis based on con-targets indicated 62%
of all signaling pathways were associated with pro-inflammatory pro-
cess with AP, and a main “compounds-targets-pathways” network was
constructed (Fig. S3). Of them, TLR, NF-κB and NLR are related to
TLR4/NLRP3 pathways. In the TLR4/NLRP3 related sub-network, four
cross-talk targets (TLR4, NF-κB1, RelA and TNF-α) are involved
(Fig. 2B). PTGS2, TLR2 and IL-6 are targets related to NF-κB, TLR and
NLR signaling pathway, respectively. Four active compounds (emodin,
rhein, baicalin and chrysin) from CQCQD were predicted to have direct
interactive function against cross-talking targets, while isosakuranetin-
7-rutinosiede, hesperidin, geniposide, ( ± )-catechin had indirect effect
(Fig. 2C). The chemical structure of four compounds are depicted in
(Fig. 2D).
Fig. 2. Pharmacological sub-network analysis-
assisted screening of individual anti-in-
flammatory active ingredients from CQCQD.
(A) The PPI network of 24 con-targets related
to CQCQD major identified compounds and
AP. (B) Four cross-talk targets constructed by
Protein-Protein Interactions Network. (C) Sub-
network of four active compounds (emodin,
rhein, baicalin and chrysin) predicted to target
NF-κB1, TNF-α, RelA and TLR4 that are asso-
ciated with NF-κB, TLR and NOD-like receptor
(NLR) signaling pathways. Blue polygons de-
note ingredients from CQCQD, which could
modulate targets in the 3 inflammatory path-
ways. Purple frames denote three signaling
pathways involved. Red ovals denote four
cross-talk targets. (D) Chemical structures of
four compounds. (For interpretation of the re-
ferences to color in this figure legend, the
reader is referred to the web version of this
article.)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
5
Fig. 3. Effects of CQCQD on severity indices of cerulein-induced AP in mice. Mice received 7 intraperitoneal injections of cerulein (CER; 50 μg/kg) at hourly interval
to induce AP, while controls received normal saline at the same regimen. In the treatment groups, oral gavage of CQCQD (5, 10 and 15 g/kg) was implemented at 3, 5
and 7 h from the first injection of cerulein. Animals were sacrificed at 12 h after the first CER/saline injection. (A) Representative H&E images of pancreatic sections
(magnification 200 ×). (B) Pancreatic histopathology scores were exhibited as y axis. (C) Serum amylase. (D) Pancreatic trypsin activity. (E) Pancreatic myelo-
peroxidase (MPO) activity. (F) Representative H&E images of lung sections (magnification 200 ×). (G) Lung histopathology score. (H) Lung MPO activity. (I) Serum
interlukin-6 (IL-6). Data are expressed as mean ± SEM of 6–8 animals per group. * p < 0.05 vs. control group,
#
p < 0.05 vs. CER group. In the histogram: gray =
control, Red = CER, and Blue = CER with CQCQD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
6
CQCQD attenuates pancreatic injury and systemic inflammation of CER-AP
The representative hematoxylin and eosin (H&E)-stained pancreatic
images are shown in Fig. 3A. Cerulein induced typical histopathological
changes of AP manifesting as diffused edema, marked periductal and
parenchymal neutrophil filtration (indicating inflammation) as well as
scattered acinar cell necrosis that were reflected by increased histo-
pathological scores (Fig. 3B). These changes were associated with sig-
nificantly increased serum amylase (Fig. 3C), pancreatic trypsin activity
(Fig. 3D), pancreatic MPO activity (Fig. 3E). The representative H&E
lung images are shown in Fig. 3F. Cerulein injections caused sig-
nificantly increased alveolar septate thickening score (Fig. 3G), lung
MPO activity (Fig. 3H) and serum IL-6 levels (Fig. 3I) compared with
normal saline controls. Among 3 doses (5, 10, and 15 g/kg) tested,
CQCQD at 10 g/kg significantly and consistently reduced all histo-
pathological and biochemical severity indices, while 5 and 15 g/kg
were ineffective or only had partial protective effects (Fig. 3A-I).
CQCQD at 20 g/kg had no significant impact on pancreatic histo-
pathology but increased pancreatic trypsin activity, lung histo-
pathology score, lung MPO activity and serum IL-6 levels (Fig. S4),
indicating toxic effects caused by this dose.
CQCQD down-regulates expression of TLR4/NLRP3-related mRNAs and
proteins
The results from semi-quantitative qRT-PCR are summarized in
Fig. 4. Effects of CQCQD on pro-in-
flammatory signaling pathways of cer-
ulein-induced AP in mice. Mice received 7
intraperitoneal injections of cerulein (CER;
50 μg/kg) at hourly interval to induce AP,
while controls received normal saline at
the same regimen. In the treatment group,
oral gavage of CQCQD (10 g/kg) was im-
plemented at 3, 5 and 7 h from the first
injection of cerulein. Animals were sacri-
ficed at 12 h after the first CER/saline in-
jection. (A) RT-qPCR analysis for pro-in-
flammatory pathway mRNA expression in
pancreatic tissue. (B) Representative
Western blotting analysis results for pro-
tein expression in pancreatic tissue. (C)
Representative immunohistochemistry
images for pancreatic sections. Data are
expressed as mean ± SEM of 3–6 samples
per group. * p < 0.05 vs. control group,
#
p
< 0.05 vs. CER group. In the histogram:
gray = control, Red = CER, and Blue =
CER with CQCQD. (For interpretation of
the references to color in this figure le-
gend, the reader is referred to the web
version of this article.)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
7
Fig. 5. Effects of screened individual anti-inflammatory compound on necrotic cell death and TLR4/NLRP3 pro-inflammatory signaling pathway in acinar cells.
Freshly isolated mouse pancreatic acinar cells received prior incubation with emodin (EM; 50 μM), rhein (RH; 25 μM), baicalin (BA; 25 μM) and chrysin (CH; 50 μM),
or HEPES only for 30 min. Then cells were incubated with cholecystokinin (CCK; 500 nM) for 2 h or taurolithocholic acid 3-sulfate disodium salt (TLCS; 500 μM) for
30 min, respectively. Cells were further stained with Hoechst 33342 and propidium iodide (PI; 1 μM) for determination of cell death by epifluorescence microscopy.
Representative images and summary necrotic scores (%) for CQCQD active ingredients on (A) CCK- and (B) TLCS-induced necrotic cell death. (C) RT-qPCR analysis
for pro-inflammatory pathway mRNA expression in acinar cells. Data are expressed as mean ± SEM from 3 or more independent experiments. * p < 0.05, ** p <
0.01, and *** p < 0.001 vs. control group,
#
p < 0.05,
##
p < 0.01 and
###
p < 0.001 vs. CCK or TLCS group. In the histogram: gray = control, Red = CCK or TLCS,
and Blue = CCK or TLCS with different compounds. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
8
Fig. 4A. The mRNA expression related to TLR4/NLRP3 pro-in-
flammatory signaling pathway (TLR4, MyD88, IKK, RelA, p50, NLRP3,
caspase-1, IL-β and TNF-α) was significantly up-regulated in the CER-
AP compared with normal saline controls. CQCQD at 10 g/kg sig-
nificantly reduced the mRNA expression of TLR4, MyD88, IKK, NLRP3
and caspase-1 and with a tendency to reduce expression of other
mRNAs. CQCQD also suppressed cerulein-induced up-regulation of
protein expressions for p-p65, p-STAT3, caspase-1 (Western blot;
Fig. 4B) and NLRP3 (immunohistochemistry; Fig. 4C).
Validation of screened ingredients for necrotic cell death and TLR4/NLRP3-
related pathways in acinar cells
Next, we sought to validate the anti-inflammatory effects of the four
(caption on next page)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
9
compounds in both CCK- and TLCS-induced necrotic acinar cell death.
The optimal concentration of corresponding isolated authentic com-
pounds was selected from various concentrations (12.5, 25 and 50 μM)
tested with TLCS (500 μM) incubation (Fig. S5). All compounds at their
respective optimal concentration significantly decreased CCK-induced
acinar cell death with baicalin had the best protective effects followed
by rhein, chrysin and emodin (Fig. 5A). All compounds significantly
and equivalently reduced TLCS-induced acinar cell death (Fig. 5B).
Individual compound had different inhibitory effects on the expression
of TLR4, RelA, NLRP3 and TNF-α mRNAs that were up-regulated after
TLCS stimulation: baicalin significantly reduced expression of TLR4
mRNA; baicalin and chrysin significantly reduced expression of RelA
mRNA; emodin and baicalin significantly reduced expression of NLRP3
mRNA; emodin and chrysin significantly reduced expression of TNF-α
mRNA (Fig. 5C). While in the presence of LPS, only emodin sig-
nificantly decreased TLCS-induced over-expression of TLR4 and NLRP3
mRNAs (Fig. S6).
Validation of screened ingredients for cell viability and TLR4/NLRP3-
related pathways in RAW 264.7 macrophages
The cell viability showed that the four compounds had no cyto-
toxicity to RAW264.7 cells up to 50 μM (Fig. 6A). Appropriate timing
for LPS stimulation and compound pre-incubation was assessed first
(Fig. S7). Then a strategy of 24 h pre-incubation of individual com-
pounds followed by another 24 h LPS co-incubation was employed. Cell
injury of RAW264.7 cells was assessed by measuring nitric oxide pro-
duction shown in histogram (Fig. 6B). Compared with culture medium
controls, LPS caused dramatic nitric oxide release that was suppressed
by emodin in a dose-dependent manner and with the maximal effect
obtained at 50 μM. While both rhein and chrysin significantly reduced
nitric oxide production at all concentrations tested, the same dose-de-
pendent effect was not observed. Baicalin had no significant effect on
LPS-induced nitric oxide production.
The effects of emodin, rhein and chrysin on the mRNA expression
profile of the TLR4/NLRP3 pathways are shown in (Fig. 6C). LPS sti-
mulation significantly up-regulated expression of TLR4, NLRP3, RelA
and TNF-α mRNAs. Both emodin and chrysin significantly reduced
expression of TLR4, NLRP3 and TNF-α mRNAs. Rhein significantly re-
duced expression of NLRP3 mRNA but was not effective for other
mRNAs. None of the three compounds significantly altered expression
of RelA mRNA. Dose-effect curves of individual compound and com-
bination of them were plotted for nitric oxide suppression activity in
RAW264.7 cells (Fig. 6D). A synergistic suppression effect was observed
on LPS-induced nitric oxide production with Fa values (Fig. 6D).
Combination showed synergistic effects both at low dose (Fa < 0.3)
and high dose (Fa > 0.8). However, an antagonistic interaction was
observed with CI values ranging from 1.16 to1.55. Emodin, rhein and
chrysin respectively inhibited the expression of MyD88 and NLRP3
proteins (Fig. 6E). Further, their combination had even stronger in-
hibitory effects compared with individual compounds alone, high-
lighting a synergistic effect.
Discussion
For the first time, the anti-inflammatory effects of CQCQD against
AP were studied using the “multiple ingredients-targets-pathways”
approach through a network pharmacology analysis. The main findings
from this study were: (1) A pharmacological sub-network of “3 in-
flammatory pathways-4 cross talk targets-4 compounds” was built. Four
active compounds: emodin, rhein, baicalin and chrysin from CQCQD
were identified. These molecules act on the TLR4/NF-κB/NLRP3-re-
lated proteins, TLR4, RelA, NF-κB and TNF-α; (2) CQCQD significantly
reduced the severity of CER-AP and this was associated with suppres-
sion of TLR4/NLRP3 inflammasome-mediated pro-inflammatory sig-
naling pathway-related mRNAs and proteins; (3) In vitro experiments
validated the protective effects of these four compounds on acinar cells
via inhibition of TLR4/NLRP3 pathways. While baicalin was the most
effective in preventing pancreatic acinar cell injury, the other three
compounds (emodin, rhein and chrysin) reduced LPS-induced cell
death and TLR4/NLRP3 pathway activation in macrophages. The
combination of these compounds representing CQCQD bioactive in-
gredients produced a synergistic protective effect when compared to
individual compound acting on their own. These data support the po-
tential for developing a range of CQCQD-based pharmacological treat-
ments for AP.
Severe AP is characterized by the early development of persistent
the systemic inflammatory response syndrome, followed by irreversible
organ failure. It is often complicated by pancreatic necrosis, sepsis, and
death (Guo et al., 2014; Schepers et al., 2019; Shi et al., 2020). Cor-
respondingly, according to TCM theory, the early stage of severe AP is
defined as “Yangming viscera syndrome”, manifested as abdominal
distention, fullness and pain. DCQD is a classical prescription for the
treatment of this syndrome and has been shown to improve gut dys-
function and reduce severity of AP in animal models (Lu et al., 2017)
and human patients (Wan et al., 2012). This is proposed to be largely
due to its anti-inflammatory and anti-oxidative stress effects. CQCQD is
derived from DCQD with the original intention to enhance its ther-
apeutic efficacy against biliary AP. The addition of Bupleurum margin-
atum Wall. ex DC., Scutellaria baicalensis Georgi, Gardenia jasminoides
Ellis and Artemisia capillaris Thunb in CQCQD was designed to increase
the anti-inflammatory effects of DCQD (Jang et al., 2015; Jung et al.,
2008; Zhao et al., 2015). These anti-inflammatory effects of CQCQD
observed in this current study were consistent with previous reports
showing that in rats CQCQD significantly reduced lung and intestinal
injuries in sodium taurocholate-induced experimental AP (NaT-AP)
(Wu et al., 2016) and L-arginine-induced AP (Zhang et al., 2017) re-
spectively. In recent years, the incidence of hypertriglyceridemia-as-
sociated AP has rapidly increased in China (Ding et al., 2019;
Mukherjee et al., 2019; Shi et al., 2020; Zhang et al., 2019c), attributing
to a high prevalence of central obesity (Zhang et al., 2019a) or meta-
bolic syndrome (Ding et al., 2016). In addition, hypertriglyceridemia-
associated AP is often more severe when compared with other etiolo-
gies due to its more pronounced pro-inflammatory effects and worse
clinical outcomes (Ding et al., 2019; Zhang et al., 2019c). Recent stu-
dies have demonstrated that components in CQCQD such as
Fig. 6. Effects of screened compounds for cell viability, cell injury and TLR4/NLRP3 pro-inflammatory signaling pathway in RAW 264.7 cells. RAW 264.7 mac-
rophage cells received prior incubation with emodin (EM; 50 μM), rhein (RH; 25 μM), baicalin (BA; 25 μM) and chrysin (CH; 50 μM), or culture medium only for 24 h
followed by co-incubation with lipopolysaccharide (LPS; 1 μg/ml) for another 24 h. (A) Effects of four compounds representing screened ingredients on cell viability
(Cell Counting Kit 8). Solid hexagon (EM), solid rhombus (RH), solid round (BA), square (CH) (B) Dose effects of each compound on nitric oxide production after LPS
stimulation. Three concentrations (12.5, 25 and 50 μM) were tested for each compound. (C) Effects of each compound at their respective optimal concentration on
expression of TLR4, NLRP3, RelA and TNF-α mRNAs after LPS stimulation. (D) Does-effect curve of EM, RH and CH at respective optimal concentration and their
combination index (CI) values were plotted according to suppression rate of nitric oxide production (Fa). In dose-effect curve, round (EM), square (RH), regular
triangle (CH), inverted triangle (COM). In CI curve, dotted line is the reference line, where CI value equals to 1; solidline (Blue) represents CI values at different Fa
values. (E) Effects of optimal concentration of EM, RH, CH and their combination ‘COM’ (EM:RH:CH = 4:1:1) on expression of NLRP3 and MyD88 proteins (Western
blot). Data are expressed as mean ± SEM from 3 or more independent experiments. * p < 0.05, ** p < 0.01, and *** p <0.001 vs. control group,
#
p < 0.05,
##
p <
0.01 and
###
p < 0.001 vs. LPS group. In the histogram: gray = control, Red = LPS, and Blue = LPS with different compounds. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of thisarticle.)
Y. Wen, et al. Phytomedicine 79 (2020) 153328
10
Gardenia jasminoides Ellis (Ahmed et al., 2018) can antagonize pan-
creatic triglyceride lipase, a key factor for lipolysis and lipotoxicity
(de Oliveira et al., 2020) .
TLR4/NLRP3 pro-inflammatory signaling pathways play a critical
role for primary pancreatic toxin- and DAMP-induced pancreatic injury
and systemic inflammation (Chen et al., 2014; Kang et al., 2014a;
Szatmary et al., 2018). However, the specified and precise active in-
gredients of CQCQD and their synergistic protective effect on inhibiting
key pro-inflammatory pathways (i.e. TLR4/NLRP3) have remained
elusive. Through RT-qPCR, Western blot and immunohistochemistry,
we confirmed that CQCQD and its active ingredients alleviated severity
of AP at least in part through suppressing TLR4/NLRP3 related path-
ways. Natural TLR4 antagonists were mainly obtained from gram-ne-
gative bacteria, cyanobacteria, or from plants. Curcumin, sulforaphane,
iberin, xanthohumol, celastrol, berberine, atractylenolide I and zhan-
kuic acid A have been described as natural molecules for TLR4 antag-
onism (Molteni et al., 2018). A study by Li et al., (2009) showed that
combination of emodin and baicalin suppressed severity and reduced
TLR4 expression in pancreas and lung in NaT-AP in rats. Recently, a
mechanistic study revealed that about 34 phytochemicals could alter
the NLRP3 inflammasome activity in acute and chronic inflammatory
diseases (Jahan et al., 2017). Among them, emodin and curcumin were
found to be involved in modulation of NLRP3 activation in septic shock
models. One recent study indicated that emodin could attenuate the
severity of AP via the pancreatic P2X7/NLRP3 signaling pathway in
NaTC-AP in rats (Zhang et al., 2019b). Furthermore, some natural
herbal medicines can neutralize the toxicity of extracellular histones
(Isobe et al., 2016). In according with this, our team has recently found
some active ingredients (e.g. baicalin) from CQCQD demonstrated high
binding affinity to histone H3, implying a more potent detoxifying
capability of CQCQD. This research topic, therefore, warrants further
in-depth investigation to establish a potential pathway for the discovery
of novel drugs.
Pharmacological network analysis and mapping are essential tools
for exploring the dynamic ingredient-target-pathway relationship be-
tween TCM herbal formulae and diseases (Sun et al., 2020). Using these
tools, the team identified correlations between the bioactive ingredients
of CQCQD and AP targets using established disease and target data-
bases. In addition, KEGG was used to search for 21 known AP-related
pathways, including MAPK, PI3K-Akt, IL-17, TNF, TLR, NF-κB, NLR,
sphingolipid, adipocytokine, RIG-I-like receptor, T cell receptor, P53
and etc.Through analysis, we found that inflammatory pathways ac-
counted for 62% of all mechanisms involved in AP, and TLR4/NF-κB/
NLRP3 cassette plays a central role in all inflammatory pathways.
Therefore, we subsequently selected four common targets related to
TLR4/NLRP3 inflammasome-mediated pro-inflammatory pathways and
constructed a pharmacological sub-network for CQCQD. The sub-net-
work revealed that RelA, TLR4, NF-κB1 and TNF-α were crucial cross-
talk targets for TLR, NF-κB and NLR signaling pathways. Further sub-
network analysis enabled the team to filter out four active compounds
of CQCQD (emodin, rhein, baicalin and chrysin) that directly act on
these targets by using relevant TCM databases.
Among these four compounds, emodin and rhein were from Rheum
palmatum L., the principle drug in both DCQD and CQCQD. Baicalin
and chrysin were from Scutellaria baicalensis Georgi. In this study, no
protective effect of baicalin on reducing cell injury was observed in
cultured macrophages. Baicalin was the main metabolite of baicalein
after its administration in animals and human (Tian et al., 2012).
However, there is no clear evidence showing this conversion occurred
in cell culture systems (Chen et al., 2001), thus it is difficult to rule out
the possibility that baicalein may actually be responsible for the pro-
tective effects. While various studies have investigated the role of
emodin, rhein and baicalin or its aglycone baicalein in experimental
AP, the role of chrysin has not been appraised in AP settings yet.
Chrysin (5,7-dihydroxy-2-phenyl-4H-chromen-4-one) is a natural fla-
vone found in several plants, mushroom, and honeycomb. It has been
reported to be protective in inflammatory diseases such as sclerosis and
diabetes (Ramirez-Espinosa et al., 2017). Here we have now shown that
chrysin had consistent effects in reducing cell death in pancreatic acinar
cells and macrophages by down-regulating the expression of TLR4/
NLRP3 pathways related mRNAs and proteins.
Importantly in this study, in order to evaluate the synergistic effects
of these compounds that representing active ingredients in CQCQD, all
three compounds were combined at their relative ratios according to
the HPLC results. It was observed that their synergistic protective effect
was positively correlated with an increase in dosage until the maximum
safety dose was reached. This finding highlighted the existence of a
potent dose-effect relationship, but also the need for robust quality
control of raw TCM herbal ingredients and the extraction process.
Unlike their synthetic counterparts, most Chinese herbal medicines
have not been pharmaceutically evaluated to define their pharmaco-
dynamic, pharmacokinetic properties as well as key parameters such as
optimal, effective, and toxic dosing. Consequently, unduly increased
doses or prolong administrations of TCM herbal formula can cause
adverse reactions (Cheng and Leung, 2012). In this study, the safety
parameters and dose-efficacy relationship between CQCQD and severity
scale in a mouse CER-AP model were also assessed for the first time.
Using the human-mice dose equivalent coefficient, we determined that
the clinically-proven optimal human dose in mice is 5.75 g/kg. Using
this as a reference point, a four-dose regimen ranging from low to high
was derived (5, 10, 15, and 20 g/kg). The results suggest CQCQD was
most effective in reducing systematic inflammatory parameters in
pancreas and lung at 10 g/kg (approximately twice of the reference
dosage). The other CQCQD lower dosages were not as effective as 10 g/
kg, and the 20 g/kg regimen was associated with increased lung injury
and elevated serum IL-6 levels, indicating potential toxic effects.
There are several limitations to this study. As we focused on TLR4/
NLRP3 related pathways, we may have underestimated the impact of
other pro-inflammatory or signaling pathways such as the anti-oxidant
property also possess by CQCQD. Indeed, the CQCQD has a wide range
of active ingredients including flavonoids, anthraquinones, iridoids,
phenols, lignans, and corresponding glycosides, most of which have
been reported to be capable of reducing AP severity via antagonizing
reactive oxygen species by activating nuclear factor erythroid-2-related
factor 2/antioxidant response element, an intrinsic antioxidative stress
pathway (Shapiro et al., 2007). Therefore, much more work is needed
to understand the complex mechanisms and full range of presumed
therapeutic targets between AP and CQCQD.
In conclusion, the present study demonstrated that CQCQD could
alleviate the severity of experimental AP at least in part via inhibition of
TLR4 and the NLRP3 inflammasome. Pharmacological network analysis
and experimental validation identified that four CQCQD bioactive
compounds (emodin, rhein, baicalin and chrysin) are responsible for
these effects. Emodin, rhein and chrysin were also found to work sy-
nergistically to mitigate cell injury and suppress TLR4/NLRP3 related
pathways in cultured macrophages.
Author contributions
QX, WH and DD obtained funding, conceptualized this study and
supervised students; YW, CH, TL, RW, WC, JY, GL, LY and NS per-
formed experiments, acquired data and analyzed data; YW, CH and WH
drafted the manuscript; XF and LD had important intelligence input; RS,
JAW, JH and ARP critically revised the manuscript; all authors read and
approved the final version of the manuscript before submission.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Y. Wen, et al. Phytomedicine 79 (2020) 153328
11
Acknowledgments
These authors thank all the staff from Standardized Training
Technician program (Jiawang Li), Experimental Animal Center (Xijing
Yang), Laboratory of Pathology (Li Li), Core Research Facilities (Yu
Ding, Hongying Chen and Sisi Wu) at West China Hospital of Sichuan
University for their assistance. The authors give special thanks to Dr
Hsiang-Wei Wang from University of Auckland for his continuous
support in English proof-reading. The study was supported by National
Science Foundation of China (No. 81800575, TL; No. 81774120, QX;
No. 81973632, WH); NZ-China Strategic Research Alliance Award
(China: No. 2016YFE0101800, QX, TJ, WH and LD; New Zealand: JAW
and AP); China-New Zealand International Cooperation Program from
Department of Science and Technology of Sichuan Province (No.
2019YFH0157, DD); Program of Science and Technology Department of
Sichuan Province (2019YJ0047, CH).
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phymed.2020.153328.
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