Psidium Guajava Flavonoids Prevent NLRP3

WSPC/174-AJCM 2150094 ISSN: 0192-415X
FA
The American Journal of Chinese Medicine, Vol. 49, No. 8, 2001–2015

© 2021 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine
DOI: 10.1142/S0192415X21500944
Psidium guajava Flavonoids Prevent NLRP3

by FUDAN UNIVERSITY on 03/18/22. Re-use and distribution is strictly not permitted, except for Open Access articles.
Inflammasome Activation and Alleviate the

Pancreatic Fibrosis in a Chronic Pancreatitis
Mouse Model
Am. J. Chin. Med. 2021.49:2001-2015. Downloaded from www.worldscientific.com
Guixian Zhang,*,a Liming Tang,†,a Hongbin Liu,*,a Dawei Liu,* Manxue Wang,*
Jun Cai,* Weijun Liu,‡ Wei Nie,* Yi Zhang‡ and Xiaomeng Yu*
*
Department of Cancer Pharmacology, Tianjin Institute of Medical & Pharmaceutical Sciences
Tianjin Medicine and Health Research Center
Tianjin 300020, P. R. China
†
Department of Traditional Chinese Medicine, Tianjin Santan Hospital
‡
Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair
Tianjin NanKai Hospital
Published 11 November 2021
Abstract: Chronic pancreatitis (CP) is a multifactorial, inflammatory syndrome characterized

by acinar atrophy and fibrosis. Activation of NOD-like receptors family pyrin domain-con-
taining 3 (NLRP3) inflammasome is a central mediator of multiple chronic inflammatory
responses and chronic fibrosis including pancreatic fibrosis in CP. The Psidium guajava
leaf is widely used in traditional medicine for the treatment of chronic inflammation, but
the anti-inflammatory effect of Psidium guajava leaf on CP has not yet been revealed. In
this study, we investigated whether the extract of total flavonoids from Psidium guajava
leaves (TFPGL) plays a therapeutic mechanism on CP through NLRP3 inflammasome sig-
naling pathway in a mouse CP model. The H&E and acid-Sirius red staining indicted that
TFPGL attenuated the inflammatory cell infiltration and fibrosis significantly. The results
of immunohistological staining, western blot and RT-qPCR showed that the expressions of
NLRP3 and caspase-1 were significantly increased in the CP model group, while TFPGL
significantly decreased the NLRP3 and caspase-1 expression at both the gene and protein
levels. Moreover, ELISA assay was used to examine the levels of NLRP3 inflammasome
target genes, such as caspase-1, IL-1b and IL-18. We found that TFPGL treatment decreased
Correspondence to: Dr. Hongbin Liu, Department of Cancer Pharmacology, Tianjin Institute of Medical &
Pharmaceutical Sciences, Tianjin Medicine and Health Research Center, No. 79 Duolun Road, Tianjin 300020,
P. R. China. Tel: (+86) 22-2731-3851, E-mail: jtss@sina.com
a
These authors contributed equally to this work.
2001
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FA WSPC/174-AJCM 2150094 ISSN: 0192-415X
2002 G. ZHANG et al.
the expression of caspase-1, IL-1b and IL-18, which is critical for the NLRP3 inflammasome
signaling pathway and inflammation response significantly. These results demonstrated that
TFPGL attenuated pancreatic inflammation and fibrosis via preventing NLRP3 inflam-
masome activation and TFPGL can be used as a potential therapeutic agent for CP.
Keywords: Total Flavonoids From Psidium Guajava Leaves; Chronic Pancreatitis; Fibrosis;
NLRP3.
Introduction
Chronic pancreatitis (CP) is characterized by ongoing inflammation of the pancreas that

results in progressive destruction of exocrine parenchymal tissue and replacement with
fibrotic tissue ultimately leading to endocrine and exocrine insufficiency (Habtezion,
2015; Kunnumakkara et al., 2018; Singh et al., 2019; Beyer et al., 2020). The underlying
molecular mechanism that leads to development of CP remains elusive. More recently, the
NOD-like receptors family pyrin domain-containing 3 (NLRP3) inflammasome signaling
pathway received particular attention due to its involvement as a key regulatory element
of tissue or organ fibrosis (Mridha et al., 2017; Lv et al., 2018; Wree et al., 2018). For
instance, it was found that NLRP3 inflammasome pathway activation was associated with
the inflammatory cytokine expression in hepatic stellate cells and liver fibrosis (Inzaugarat
et al., 2019; Dong et al., 2020; Li et al., 2020). Also, NLRP3 inflammasome activation
was demonstrated to be related with renal tubulointerstitial fibrosis and pulmonary fibrosis
(Lasithiotaki et al., 2016; Chi et al., 2017; Seo et al., 2019). However, the exact mecha-
nism of NLRP3 inflammasome in CP and its fibrosis process remains unclear and has not
yet been established, and the interaction between NLRP3 inflammasome and the fibrosis
process in the development of CP fibrosis is not fully understood.
Inflammasomes are multiprotein cytoplasmic complexes that serve as pattern recogni-
tion receptors and regulate the production of proinflammatory cytokines, such as IL-1b and
IL-18 (Rathinam and Fitzgerald, 2016; Man et al., 2017; Hardeland, 2019). NLRP3 inflam-
masome is the most extensively investigated inflammasome identified so far, and activation
of the NLRP3 inflammasome may occur in response to diverse stimuli (Karasawa and
Takahashi, 2017; Song and Li, 2018; Yi, 2020). The ability of NLRP3 inflammasome to
respond to a variety of endogenous danger signals via a single common pathway makes it
an attractive therapeutic target (Haneklaus and O’Neill, 2015; Swanson et al., 2019).
Psidium guajava is a well-known traditional medicinal plant widely used in folk med-
icine (Diaz-de-Cerio et al., 2017; Chao et al., 2020). Although clinic and animal studies
have shown that aqueous and organic extracts of guava leaves have various kinds of ther-
apeutic mechanisms, of which the most important one is its inhibiting effects on chronic
inflammation (Jang et al., 2014; Jayachandran et al., 2018; Luo et al., 2019). For example,
Psidium guajava leaf extracts were demonstrated to play a substantial role against cancer
and down-modulate inflammatory nuclear factor-kb (NF-kB) (Ashraf et al., 2016; Vas-
concelos et al., 2020). Psidium guajava leaf extracts also could decrease the expression of
lipopolysaccharide-inducible nitric oxide synthase and cyclooxygenase-2 proteins level,
two pro-inflammatory mediators, through the down-regulation of NF-kB transcriptional
2150094.indd 2002 21-Dec-21 2:15:27 PM

WSPC/174-AJCM 2150094 ISSN: 0192-415X FA
TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2003
activity (Han et al., 2011; Sen et al., 2015). In addition, triterpenoids from guava leaves
were suggested as a potential therapeutic approach for treating diabetic peripheral neurop-
athy, as they enhanced physical functions and offered neuronal protection toward the sup-
pression of the expression of pro-inflammatory cytokines (Jayachandran et al., 2018). As
we now know, CP is a kind of chronic and nonresolving inflammation process, and we put
forward a hypothesis that Psidium guajava leaves have therapeutic effects on CP; however,
the evidence is lacking.

In this study an extract of total flavonoids from Psidium guajava leaves (TFPGL) is used
to prevent NLRP3 inflammasome activation and alleviate the pancreatic fibrosis in a mouse
model of CP as a potential alternative drug approach for the treatment of CP.
Materials and Methods
Extraction of TFPGL
The dry Psidium guajava leaves were purchased from Hebei Anguo Tongyi Traditional
Chinese Medicine Pieces Co., Ltd. (lot number: 13220422). The air-dried powdered leaves
(5 kg) were extracted with 10-fold 70% ethanol in reflux condenser for twice, each time for
2 h. The extracts were filtrated using Whatman filter paper, and the filtrates were concen-
trated by rotary evaporator to remove the ethanol. The ethanolic extracts (1 g crude drug
per milliliter with distilled water) were eluted with 2-fold water-saturated ethyl acetate
solution for three times in a separating funnel, after which the ethyl acetate layer was col-
lected and concentrated under reduced pressure. The resulting filtrates were dried using a
freeze drier and stored at −80°C until further analysis.
Determination of TFPGL
Falconoid content was determined by Al(NO3)3 colorimetric method (Ma et al., 2007). Each
extract (3 mL) was added to a test tube containing 3 mL of methanol, 1 mL of 5% NaNO2, 1
mL of 10% Al(NO3)3, 10 mL of 1mol/L NaOH, and 7 mL of H2O to total 25 mL. After 15 min
at room temperature, the absorbance was measured at 500 nm. The content of total falconoid
was determined with rutin (lot number: 100080-201610) as the standard (5–50 mg/mL).
Cerulein-Induced Murine Model of Pancreatitis and TFPGL Treatment
Six week old male C57BL/6 mice were purchased from the Vital River Laboratory Animal
Technology Co, Ltd (Beijing, China). All animal experimental protocols were conducted
in accordance with the Regulations for the Administration of Affairs Concerning Experi-
mental Animals of Tianjin Institute of Medical & Pharmaceutical Sciences. After 1 week
of acclimatization, the mice were randomly divided into four groups: Normal control, CP
Model, and TFPGL high-dose and low-dose groups, with 10 mice per group. Except for the
control group, the cerulein (Sigma-Aldrich, St Louis, Mo) was administered through six
intraperitoneal injections (50 mg cerulein/kg body weight) at hourly intervals, three times
a week for six weeks. The TFPGL high-dose group and low-dose group received TFPGL
2150094.indd 2003 21-Dec-21 2:15:27 PM

(0.372 g/kg and 0.186 g/kg), respectively. At the same time, the normal control and CP
model group received saline injections. Eight weeks after the first intraperitoneal injection,
all the mice were sacrificed.
Histological Examinations and Quantitative Analysis of Pancreatic Fibrosis

Partial pancreatic tissues were fixed in 10% formaldehyde solution, paraffin embedding 24
h later, and 4-mm sections were stained with hematoxylin-eosin (H&E) and picric acid-Sir-
ius red (Sigma-Aldrich) according to standard methods. The quantification of histological
acinar loss, fibrosis and inflammation were analyzed as previously described (Zhang et al.,
2017). To evaluate these three parameters, pancreas sections were randomly selected from
each mouse, and 10 randomly chosen microscopic fields were examined for each tissue
section.
Immunohistochemical Staining of a-SMA, NLRP3, Caspase-1
Pancreas specimens were embedded in paraffin for histological analyses, as previously

described (Zhang et al., 2017). Immunohistochemical staining of pancreatic a-SMA
(1:200), NLRP3 (1:200), and caspase-1 (1:400) was performed using the peroxidase ABC
kit (Vector Laboratories, Burlingame, Calif). Microscopy analyses were performed, and
images were recorded using a Leica DM4000B microscope (×200). Quantification of
labeled cells was performed in at least five fields per slides of three slides for each mouse.
Western Blot Assay for the Expression of NLRP3 and Caspase-1 in Pancreas
Pancreatic tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail,
and the protein concentrations were quantified using a BCA protein assay kit. Then, the
proteins were resolved by SDS-PAGE and blotted onto PVDF membranes, and the mem-
branes were incubation with primary antibodies overnight. The dilution of NLRP3 and
caspase-1 was 1:500 and 1:1000, respectively. Bands were visualized using the chemilu-
minescence detection and quantified with Quantity One image analysis software (Bio-Rad
Laboratories).
Quantitative Real-Time PCR Reaction of NLRP3 and Caspase-1 in Pancreas
The total RNA was extracted from the frozen pancreatic tissue with Trizol reagent. cDNA
was generated from RNA extracts using a reverse transcription kit (Transgen, Beijing,
China). Quantitative real-time PCR was performed with SYBR Green Master Mix kit
(TIANGEN Biotech, Beijing, China). The amplification was carried out in the iCycler
Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). After the reaction, GAPDH was
used as the endogenous control gene, and the relative abundance of each gene mRNA was
calculated by the 2−DDCT method. Primer nucleotide sequences of transcripts evaluated by
quantitative real-time PCR are shown as Table 1.
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Table 1. Primers used in Quantitative Real-time PCR Amplification

Gene Primer Nucleotide Sequences
NLRP3 Forward 5′-GTGGATGGGTTTGCTGGGAT-3′
Reverse 5′-CCACACTCTCACCTAGACGC-3′
Caspase1 Forward 5′-CCGTGGAGAGAAACAAGGAGT-3′
Reverse 5′-AGCCCCTGACAGGATGTCTC-3′
GAPDH Forward 5′-GGGTCCCAGCTTAGGTTCATC-3′
Reverse 5′-TGAGGTCAATGAAGGGGTCG-3′
Measurement of Levels of Caspase-1, Interleukins 1β and 18 Protein Concentrations in

the Pancreas by ELISA
After the fresh pancreatic tissue was washed away with pre-cooled PBS, 50 mg pancreatic
tissue was weighed and added with 500 mL of precooled PTR buffer. The tissue homoge-
nate was incubated on ice for 20 min, then centrifuged at 4°C for 18,000 g for 20 min. The
protein concentration of supernatant was detected by BCA method. The concentrations for
each sample were calculated from the standard curve generated and were corrected by the
concentration of protein. The levels of caspase-1 (LifeSpan BioSciences, Seattle, Wash),
interleukin 1b (IL-1b) (Abcam, Cambridge, MA), and IL-18 (MyBioSource, San Diego,
CA) were measured by ELISA kit and expressed as the content per milligram of protein of
the tissue (pg/mg protein).
Statistical Analysis
Quantified results are shown as means ± S.D. SPSS 22.0 was used for statistical analysis.
In all experiments, statistical analysis was performed using one-way ANOVA followed by
two-tailed t-tests with p values < 0.05 being considered statistically significant.
Results
Histological Findings and Scoring
Compared with the control group, the pancreatic tissues of CP model mice showed abnor-
mal architecture, diffuse acinar atrophy, heavy infiltration of inflammatory cells and signif-
icant collagen deposition around the pancreatic ducts and blood vessels (red arrows). On
the other hand, the H&E staining showed that the degrees of acinar atrophy, fibrosis, and
inflammatory cell infiltrate were all decreased in TFPGL groups (Fig. 1).
Fibrotic Changes in the Pancreas
Picric acid-Sirius red staining was used for quantitative analysis of the collagen content
in the pancreas. Collagen I appeared to be yellowish red, and collagen III appeared to be
2150094.indd 2005 21-Dec-21 2:15:28 PM

(A)
(B)
Figure 1. Histological assessment of pancreatic injury and fibrosis after TFPGL treatment. H&E staining (A)
and histological score (B) of pancreas after the mice were treated with TFPGL (0.372 g/kg and 0.186 g/kg) from
weeks 7 to 8, all markers of pancreatic damage were significantly attenuated (*p < 0.01, all for the comparisons
with normal group; †p < 0.05, all for the comparisons with CP model group). (×200, scale bar = 100 mm).
2150094.indd 2006 21-Dec-21 2:15:52 PM

(A)
(C)
(B)
(D)
Figure 2. Observation of pancreatic fibrosis stained by Picric acid-Sirius red under the polarized light (A) and
light microscope (B). Data are expressed as means ± S.D. *p < 0.01, compared with the normal control group;
†
p < 0.05, compared with the CP model group. (×200, scale bar = 100 mm).
green with polarized light illumination. Under the light microscope, collagen specific stain-
ing was red in model group, and the two TFPGL groups. The quantitative analysis results
showed that collagen contents in the CP group had obviously increased and mainly were
type I collagen (Figs. 2A and 2B), whereas in the two TFPGL groups, mice pancreatic
collagen volume fractions were remarkably alleviated (Figs. 2C and 2D).
In particular, a-SMA immunohistochemistry was performed to quantify the number of
activated PSCs, the putative cells responsible for fibrosis in the pancreas. The number of
stellate cells increased during CP induction, and this increase in stellate cells was attenu-
ated by TFPGL treatment (Fig. 3).
2150094.indd 2007 21-Dec-21 2:16:09 PM

(A)
(B)
Figure 3. Immunochemical staining of pancreatic tissue sections for a-SMA. (A) Identification of PSCs by
immunohistochemical staining of a-SMA. (B) Quantitative analysis of the number of activated PSCs. Values are
expressed as means ± S.D. *p < 0.01, compared with the normal control group, and †p < 0.05 compared with the
CP model group.
NLRP3 and Caspase-1 Protein Expression in the Pancreas
Immunohistological staining of NLRP3 and caspase-1 were examined in cerulein-induced

mice (Fig. 4). Compared with the normal group, the expression of NLRP3 and caspase-1 in
pancreatic tissue of model group was significantly increased (p < 0.01), while the expres-
sion of NLRP3 and caspase-1 in pancreatic tissue of mice treated with TFPGL was lower
than the model group, p < 0.05.
The expression of NLRP3 and caspase-1 was also detected by Western blot (Fig. 5).
NLRP3, and caspase-1 protein levels were increased greatly in pancreatic tissue of the CP
model group detected by Western blot compared with the normal control group, and when
mice were treated with TFPGL after establishment of the CP model, the levels of these
proteins were obviously downregulated.
NLRP3 and Caspase-1 mRNA Expression in the Pancreas
Repeated cerulein injections of the CP model group enhanced the mRNA expression of
NLRP3 and caspase-1 significantly. In the TFPGL treatment groups, NLRP3 and caspase-1
mRNA expressions were decreased for the comparisons with the CP model group (Fig. 6).
Changes of Caspase-1, IL-1β, and IL-18 Protein Concentrations in the Pancreas
Caspase-1 has been implicated as the rate-limiting enzyme in IL-1b and IL-18 production
among the NLRP3–caspase-1 signal pathway. Induction of CP was associated with an
increase in active caspase-1, IL-1b, and IL-18 concentrations in pancreas homogenates.
Compared with the normal group, the concentrations of caspase-1, IL-1b and IL-18 protein
2150094.indd 2008 21-Dec-21 2:16:19 PM

(A)
(B) (C)
(B)
Figure 4. Immunohistological staining and quantitative analysis of NLRP3 and Caspase-1 in pancreatic tissue.
(×200, scale bar = 100 mm). Data are expressed as means ± S.D. *p < 0.01, compared with the normal control
group; †p < 0.05, compared with the CP model group.
in pancreatic tissue of the model group were significantly increased (p < 0.01). As shown in
Fig. 7, the TFPGL treatment prevented the rise in pancreatic caspase-1, IL-1b, and IL-18
levels in mice administered repeated cerulein injections (p < 0.05).
Discussion
CP is characterized by progressive, irreversible chronic inflammatory and fibrotic changes of

the pancreas resulting in exocrine and endocrine insufficiency and, often, chronic disabling pain
(Whitcomb et al., 2016; Robinson et al., 2019; Ramakrishnan et al., 2020). Its incidence and
prevalence worldwide seem to be on the rise because of advances in pancreatic imaging leading to
earlier diagnosis of CP (Anaizi et al., 2017; Lew et al., 2017; Vujasinovic et al., 2020). Currently,
there is still no definite medical treatment for pancreatic inflammation, fibrosis or pain of CP.
Pancreatic fibrosis is the hallmark of CP and is also a major concern for the successful
treatment of CP (Lew et al., 2017; Barry, 2018; Kojayan et al., 2019). Although multiple
overlapping mechanisms have been shown to be involved in pancreatic fibrogenesis, it is
believed that pancreatic stellate cells (PSCs) are the main fibroblastic cells of the pancreas
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(A)
(B)
Figure 5. Western blot examination and quantitative analysis of NLRP3 and caspase-1 protein expression in
pancreatic tissue. Data are expressed as means ± S.D.; *p < 0.01, compared with the normal control group; †p <
0.05, compared with the CP model group.
which are responsible for producing the desmoplasia in CP (Xue et al., 2015; Bynigeri
et al., 2017; Haeberle et al., 2018). PSCs are activated by proinflammatory cytokines to
induce pancreatic fibrogenesis (Li et al., 2018; Sun et al., 2018).
Formed in response to tissue injury and inflammation, inflammasomes are large cytoso-
lic protein complexes composed of intracellular pattern recognition receptors (PRRs) and
adaptor proteins that trigger caspase enzyme (for instance caspase-1) activation, leading to
maturation of the proinflammatory cytokines interleukin (IL)-1b and IL-18 (Rathinam and
Fitzgerald, 2016; Vince et al., 2018; Bo et al., 2020). Nod-like receptor pyrin domain con-
taining 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation and proin-
flammatory cytokine production (Cordaro et al., 2020; Ferrero-Andres et al., 2020; Sendler
et al., 2020). Our previous research demonstrated that blocking P2 × 7R (purinergic 2 × 7
receptor)-NLRP3 inflammasome-dependent IL-1b/IL-18 release can significantly decreased
pancreatic chronic inflammation and fibrosis in a mouse model of CP (Zhang et al., 2017).
Psidium guajava (Myrtaceae), commonly known as guava, has long been used in folk
medicines in East Asian and other countries and as a therapeutic agent for the treatment
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Figure 6. The relative expression of NLRP3 and Caspase-1 mRNA in the pancreas of mice in each group. *p <
0.01, compared with the normal control group; †p < 0.05, compared with the CP model group.
Figure 7. Evaluation of the Caspase-1, IL-1b and IL-18 protein concentrations in pancreas in all groups. *p <
0.01, compared with the normal control group; †p < 0.05 compared with the CP model group.
2150094.indd 2011 21-Dec-21 2:17:14 PM

of numerous diseases e.g., as an anti-inflammatory, oxidative damage for diabetes, cancer,

rheumatic pain, hypertension, diarrhea, wounds, ulcers, and reducing fever (Ashraf et al.,
2016; Morais-Braga et al., 2016; Jayachandran et al., 2018). The main constituents of
guava leaf extract are a variety of polyphenolics, flavonoids, and triterpenoids (Fernandes
et al., 2014; Ashraf et al., 2016; Diaz-de-Cerio et al., 2017). In this study, total flavonoids
were extracted from Psidium guajava leaves and were tested to determine whether TFPGL
can alleviate the pancreatic fibrosis via preventing NLRP3 inflammasome activation.
Our study demonstrated that (1) in the TFPGL treatment group, the degrees of acinar
atrophy, fibrosis, and inflammatory cell infiltrate were all decreased significantly; (2) pan-
creatic fibrosis stained by Picric acid-Sirius red examined under polarized light and light
microscope shows that 0.372 g/kg TFPGL and 0.186 g/kg TFPGL treatment markedly
reduced the expression of collagen I and collagen III; (3) the expression of PSCs marker-a-
SMA examined by immunohistochemical staining was decreased significantly by TFPGL
treatment; (4) the NLRP3 and caspase-1 protein expression and the relative expression of
NLRP3 and caspase-1 mRNA expression in pancreatic tissue were all decreased signifi-
cantly in TFPGL treatment group; (5) the caspase-1, IL-1b and IL-18 protein concentra-
tions in pancreas tissue were markedly reduced in the TFPGL treatment group.
Taken together, NLRP3 inflammasome activation was involved in CP and its fibrosis
process. Once activated, NLRP3 inflammasome could mediate caspase-1 activation, which
is then able to cleave pro–IL-1b and pro–IL-18 to their mature forms. These cytokines
probably by autocrine and paracrine signals up-regulate various signaling pathways, result-
ing in an increase in profibrotic transforming growth factor b1, a central mediator of the
fibrotic response, and the activation of PSC, a pivotal profibrotic cell in CP. In this study,
we found that TFPGL suppresses pancreatic NLRP3 and caspase-1 expressions at both the
gene and protein levels and decreases the pancreatic concentrations of caspase-1, IL-1b
and IL-18. Furthermore, TFPGL attenuates the pancreatic chronic inflammation and fibro-
sis degree. TFPGL has potential therapeutic value for CP and its fibrosis via preventing
NLRP3 inflammasome activation.
Acknowledgments
This work was supported by Natural Science Foundation Project of Tianjin Municipal of
China, China (No.17JCYBJC27700); the National Natural Science Foundation of China
(No.81373854) Scientific Research Project of Integrated Traditional Chinese and Western
Medicine of Tianjin Municipal Health Committee and Tianjin Administration of Tradi-
tional Chinese Medicine (No. 2019109); The Science and Technology Talent Cultivation
Project of Tianjin Municipal Health Committee (No. KJ20026).
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Psidium Guajava Flavonoids Prevent NLRP3

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WSPC/174-AJCM 2150094 ISSN: 0192-415X

The American Journal of Chinese Medicine, Vol. 49, No. 8, 2001–2015

Psidium guajava Flavonoids Prevent NLRP3

Inflammasome Activation and Alleviate the

Published 11 November 2021

Abstract: Chronic pancreatitis (CP) is a multifactorial, inflammatory syndrome characterized

2150094.indd 2001 21-Dec-21 2:15:27 PM

2002 G. ZHANG et al.

Chronic pancreatitis (CP) is characterized by ongoing inflammation of the pancreas that

2150094.indd 2002 21-Dec-21 2:15:27 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2003

the evidence is lacking.

Materials and Methods

Cerulein-Induced Murine Model of Pancreatitis and TFPGL Treatment

2150094.indd 2003 21-Dec-21 2:15:27 PM

2004 G. ZHANG et al.

Histological Examinations and Quantitative Analysis of Pancreatic Fibrosis

Immunohistochemical Staining of a-SMA, NLRP3, Caspase-1

Pancreas specimens were embedded in paraffin for histological analyses, as previously

Quantitative Real-Time PCR Reaction of NLRP3 and Caspase-1 in Pancreas

2150094.indd 2004 21-Dec-21 2:15:28 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2005

Table 1. Primers used in Quantitative Real-time PCR Amplification

Measurement of Levels of Caspase-1, Interleukins 1β and 18 Protein Concentrations in

Histological Findings and Scoring

Fibrotic Changes in the Pancreas

2150094.indd 2005 21-Dec-21 2:15:28 PM

2006 G. ZHANG et al.

2150094.indd 2006 21-Dec-21 2:15:52 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2007

2150094.indd 2007 21-Dec-21 2:16:09 PM

2008 G. ZHANG et al.

NLRP3 and Caspase-1 Protein Expression in the Pancreas

Immunohistological staining of NLRP3 and caspase-1 were examined in cerulein-induced

NLRP3 and Caspase-1 mRNA Expression in the Pancreas

Changes of Caspase-1, IL-1β, and IL-18 Protein Concentrations in the Pancreas

2150094.indd 2008 21-Dec-21 2:16:19 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2009

CP is characterized by progressive, irreversible chronic inflammatory and fibrotic changes of

2150094.indd 2009 21-Dec-21 2:16:34 PM

2010 G. ZHANG et al.

2150094.indd 2010 21-Dec-21 2:16:50 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2011

2150094.indd 2011 21-Dec-21 2:17:14 PM

2012 G. ZHANG et al.

of numerous diseases e.g., as an anti-inflammatory, oxidative damage for diabetes, cancer,

2150094.indd 2012 21-Dec-21 2:17:14 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2013

2150094.indd 2013 21-Dec-21 2:17:14 PM

2014 G. ZHANG et al.

Karasawa, T. and M. Takahashi. Role of NLRP3 inflammasomes in atherosclerosis. J. Atheroscler.

2150094.indd 2014 21-Dec-21 2:17:14 PM

TFPGL ATTENUATED PANCREATIC INFLAMMATION AND FIBROSIS 2015

Robinson, S.M., S. Rasch, S. Beer, I. Valantiene, A. Mickevicius, E. Schlaipfer, J. Mann, P. Maison-

Gemigliptin attenuates renal fibrosis through down-regulation of the NLRP3 inflammasome.

2150094.indd 2015 21-Dec-21 2:17:15 PM

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