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\begin{document}

\title{Forsythia suspensa (Thunb.) Vahl extract ameliorates ulcerative colitis
via inhibiting NLRP3 inflammasome activation through the
TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway\selectlanguage{english}}



\author[1]{Xiao Tong}%
\author[1]{Li Chen}%
\author[2]{Shijun He}%
\author[1]{Shuangshuang Liu}%
\author[3]{Jiaying Yao}%
\author[1]{Zhenguang Shao}%
\author[1]{Yang Ye}%
\author[1]{Sheng Yao}%
\author[1]{Zemin Lin}%
\author[1]{Jianping Zuo}%
\affil[1]{Shanghai Institute of Materia Medica CAS}%
\affil[2]{Shanghai University of Traditional Chinese Medicine}%
\affil[3]{Jiangxi University of Traditional Chinese Medicine}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Ulcerative colitis (UC) is a chronic inflammatory disease
caused by abnormal immune system reactions resulting in inflammation and
ulcers in the large intestine. Phillygenin (PHI) is a natural compound
found in Forsythia suspensa (Thunb.) Vahl, known for its various
bioactivities, including anti-inflammatory, anti-obesity, and
antipyretic activities. However, the potential anti-inflammatory effects
of PHI on UC and its underlying mechanisms are still poorly understood.
Methods: In this study, we investigated the therapeutic effects of PHI
on acute UC induced by DSS and TNBS. We evaluated the effects of PHI on
disease activity index, body weight, mortality, intestinal mucosal
barrier, cytokine secretion, and macrophage infiltration into colon
tissue using various techniques such as flow cytometry,
immunofluorescence, ELISA, RT-qPCR, and Western blotting. Results: Our
findings revealed that PHI has therapeutic properties in UC treatment.
PHI was able to maintain body weight, reduce disease activity index and
mortality, restore the intestinal mucosal barrier, and inhibit cytokine
secretion. Flow cytometry assay and immunofluorescence indicated that
PHI reduces macrophage infiltration into colon tissue. Additionally,
both in vivo and in vitro results suggested that PHI may exert
anti-inflammatory effects by downregulating the TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B
pathway, inhibiting NLRP3 inflammasome activation. Conclusion: In
conclusion, PHI possesses anti-inflammatory properties and has the
potential as a therapeutic agent for the treatment of UC. Our study
provides insights into the underlying mechanisms of PHI's therapeutic
effects and highlights the potential for further research in developing
PHI-based treatments for UC.%
\end{abstract}%



\sloppy


\textbf{Title page}

\textbf{Title:}

Forsythia suspensa (Thunb.) Vahl extract ameliorates ulcerative colitis
via inhibiting NLRP3 inflammasome activation through the
TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway

\textbf{Authors:} Xiao Tong \textsuperscript{1,2}, Li
Chen\textsuperscript{2}, Shijun He \textsuperscript{3, *}, Shuangshuang
Liu\textsuperscript{1}, Jiaying Yao \textsuperscript{4}, Zhenguang
Shao\textsuperscript{1,5}, Yang Ye\textsuperscript{1,4,6}, Sheng
Yao\textsuperscript{1,4,7 *}, Zemin Lin \textsuperscript{2, *}, Jianping
Zuo \textsuperscript{1,2,8, *}

\textbf{Affiliations:}

\textsuperscript{1} University of Chinese Academy of Sciences, Beijing
100049, P.R. China

\textsuperscript{2} State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, P.R. China

\textsuperscript{3} Innovation Research Institute of Traditional Chinese
Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai
201203, P.R. China

\textsuperscript{4} College of Pharmacy, Jiangxi University of
Traditional Chinese Medicine, 818 Meiling Avenue, Nanchang 330004, P.R.
China

\textsuperscript{5} State Key Laboratory of Drug Research \& Natural
Products Research Center, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, P.R. China

\textsuperscript{6} School of Life Science and Technology, ShanghaiTech
University, Shanghai 201210, P.R. China

\textsuperscript{7} Zhongshan Institute for Drug Discovery, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan
528400, P.R. China

\textsuperscript{8} Laboratory of Immunology and Virology, Shanghai
University of Traditional Chinese Medicine, Shanghai 201203, China

\textsuperscript{*} \textbf{Corresponding Author} : Jian-ping Zuo
(jpzuo@simm.ac.cn), Ze-min Lin (linzemin@simm.ac.cn), Sheng Yao
(yaosheng@simm.ac.cn), Shi-jun He (heshijun@shutcm.edu.cn).

These authors contributed equally: Xiao Tong, Li Chen

\textbf{Abstract:}

Background:

Ulcerative colitis (UC) is a chronic inflammatory disease caused by
abnormal immune system reactions resulting in inflammation and ulcers in
the large intestine. Phillygenin (PHI) is a natural compound found in
Forsythia suspensa (Thunb.) Vahl, known for its various bioactivities,
including anti-inflammatory, anti-obesity, and antipyretic activities.
However, the potential anti-inflammatory effects of PHI on UC and its
underlying mechanisms are still poorly understood.

Methods:

In this study, we investigated the therapeutic effects of PHI on acute
UC induced by DSS and TNBS. We evaluated the effects of PHI on disease
activity index, body weight, mortality, intestinal mucosal barrier,
cytokine secretion, and macrophage infiltration into colon tissue using
various techniques such as flow cytometry, immunofluorescence, ELISA,
RT-qPCR, and Western blotting.

Results:

Our findings revealed that PHI has therapeutic properties in UC
treatment. PHI was able to maintain body weight, reduce disease activity
index and mortality, restore the intestinal mucosal barrier, and inhibit
cytokine secretion. Flow cytometry assay and immunofluorescence
indicated that PHI reduces macrophage infiltration into colon tissue.
Additionally, both in vivo and in vitro results suggested that PHI may
exert anti-inflammatory effects by downregulating the TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B
pathway, inhibiting NLRP3 inflammasome activation.

Conclusion:

In conclusion, PHI possesses anti-inflammatory properties and has the
potential as a therapeutic agent for the treatment of UC. Our study
provides insights into the underlying mechanisms of PHI's therapeutic
effects and highlights the potential for further research in developing
PHI-based treatments for UC.

\textbf{Keywords} : ulcerative colitis, Phillygenin, macrophages, NLRP3
inflammasome

\textbf{* Background}

Ulcerative colitis (UC) is an autoimmune disease that affects the colon
and rectum. It is characterized by diffuse, superficial, and localized
inflammation of the mucosa{[}1, 2{]}. Although the causative factors of
ulcerative colitis are not fully understood, multiple causes have been
identified, including changes in the intestinal flora, genetic
susceptibility, immune response disorders, and environmental factors.
Clinical treatment usually involves 5-aminosalicylic acid,
corticosteroids, thiopurines, and biological agents such as anti-tumor
necrosis factor (TNF)-\selectlanguage{greek}α \selectlanguage{english}antibodies. However, these treatments can cause
specific side effects, including diarrhea, complicated myocarditis, and
hemolytic anemia {[}3{]}.

Macrophages, which is considered as central mediators of intestinal
immune homeostasis and inflammation, exert pathological influences in
both UC and Crohn's {[}4{]}. Toll-like receptors (TLRs) are activated by
pathogen-associated molecular patterns (PAMPs), which in turn activate
macrophages leading to excessive inflammation and tissue damage, causing
colitis {[}5{]}. Moreover, innate immune signaling via cytokine
receptors and TLRs mediated macrophages activation act as a primary
response to promote NOD-like receptor family pyrin domain containing 3
(NLRP3) transcription through NF-kB activation{[}6{]}.

The NLRP3 inflammasome is a cytosolic protein complex consisting of
NLRP3, apoptosis-associated speck-like protein containing CARD (ASC),
and pro-Caspase1. This complex is present in various immune cells such
as granulocytes, macrophages, and lymphocytes and is crucial for
maintaining gut homeostasis{[}7{]}. Toll-like receptors (TLRs) activate
the NLRP3 inflammasome through the adaptor protein MyD88, which leads to
the phosphorylation of the nuclear factor kappa-B (NF-\selectlanguage{greek}κ\selectlanguage{english}B), ultimately
triggering downstream inflammation. The NLRP3 inflammasome identifies a
range of signals such as stress, foreign microorganisms, and endogenous
danger signals, producing interleukin-1\selectlanguage{greek}β \selectlanguage{english}(IL-1\selectlanguage{greek}β\selectlanguage{english}) and IL-18, thereby
promoting inflammation {[}8, 9{]}. IL-1\selectlanguage{greek}β \selectlanguage{english}plays an important role in the
colon and is mainly derived from macrophages in the lamina propria. It
has various functions such as guiding neutrophils to infected or damaged
sites, enhancing T cell proliferation, promoting phagocytosis to destroy
bacteria, and activating additional pathways to upregulate cytokines,
amongst others {[}8, 10{]}.

\emph{Forsythia suspensa} (Thunb.) Vahl is an ornamental shrub and its
fruits were used as a well-known Chinese herbal medicine with
detoxifying and heat-clearing properties. Over the past few decades,
several monomeric compounds with antioxidant, anti-inflammatory,
anti-viral, neuroprotective, and antibacterial effects have been
identified from \emph{Forsythia suspensa} {[}11{]}. One of these
compounds, phillygenin (PHI), is a lignan known to inhibit inflammation,
ameliorate liver fibrosis, inhibit epithelial-mesenchymal transition,
reduce nonalcoholic fatty liver disease, and regulate proliferation and
apoptosis {[}12-15{]}. PHI is involved in several pathways including the
SHP-1/JAK2/STAT3, AMPK/ERK/NF-\selectlanguage{greek}κ\selectlanguage{english}B, and TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways
{[}16-18{]}. However, the impact of PHI on inflammatory bowel disease
remains understudied. In the current study, we performed a detailed
study on the pharmacological impact of PHI on colitis and its mechanism
of action.

\textbf{* Methods}

\textbf{Drugs and reagents}

Preparation of PHI via phillyrin hydrolysis: Naturally occurring
phillygenin (from an in-house natural product library, \textgreater{}
97\% purity), Synthetic phillygenin (\textgreater{} 98\% purity)
for\emph{in vivo} study from phillyrin hydrolysis (FigS1 to S5). The
extraction of PHI was extracted, identified, and characterized. Briefly,
Phillyrin (3.0 g, 5.62 mmol) was dissolved in 120 ml of methanol in a
250 ml of flask, to this solution was added 2 M HCl (24 ml, 12 mmol, 8.6
eq.). The reaction was refluxed at 65\textsuperscript{o}C overnight. The
reaction mixture was cooled to room temperature and adjust pH to
5\textasciitilde{}6 with sat. NaHCO\textsubscript{3}, the resulting
mixture was evaporated under reduced pressure and then was extracted
with EtOAc (100 ml) three times. The organic phase was washed with brine
(100 ml) two times, dried with anhydrous MgSO4, and then evaporated. The
residue was subjected to a silica gel column eluting with
dichloromethane/methanol (20/1) to yield PHI (1.58 g, 4.25 mmol, 76\%).
White solid, ESI-MS \emph{m/z} (m): 373.3 {[}M +
H{]}\textsuperscript{+}, 371.2 {[}M --
H{]}\textsuperscript{-};\textsuperscript{1}H NMR (500 MHz,
CDCl\textsubscript{3}): \selectlanguage{greek}\emph{δ}\selectlanguage{english}6.94-6.84 (6H, m, Ar-H), 5.59 (1H, s,
4'-OH), 4.87 (1H, d, \emph{J} = 6.6 Hz, H-7), 4.43 (1H, d, \emph{J} =
7.2 Hz, H-7'), 4.13 (1H, dd,\emph{J} = 1.3, 9.5 Hz, H-9'ax), 3.91 (3H,
s, 3-OMe), 3.90 (3H, s, 3'-OMe), 3.89 (3H, s, 4-OMe), 3.86 (2H, m, H-9eq
and H-9'eq), 3.33 (2H, m, H-8, H-9ax), 2.91 (1H, m, H-8');
\textsuperscript{13}C NMR (125 MHz, CDCl\textsubscript{3}) \selectlanguage{greek}\emph{δ}
\selectlanguage{english}148.8 (C-3), 148.0 (C-4), 146.7 (C-3'), 145.3 (C-4'), 133.0 (C-1'),
131.0 (C-1), 119.2 (C-6'), 117.7 (C-6), 114.2 (C-5'), 111.0 (C-5), 109.0
(C-2), 108.5 (C-2'), 87.7 (C-7'), 82.0 (C-7), 71.0 (C-9'), 69.7 (C-9),
55.9 (3 \selectlanguage{ngerman}× OMe), 54.5 (C-8'), 50.2 (C-8).

Dextran Sulfate Sodium (DSS, MP Biomedicals); Urine fecal occult blood
test kit (Nanjing Jiancheng Bioengineering Institute); Goat anti-rabbit
Alexa Fluor 488 conjugate (Abcam); DAPI (Sigma-Aldrich); RNA simple
total RNA kit (Tiangen Biotech); Hifair \selectlanguage{english}II 1st Strand cDNA Synthesis
SuperMix for qPCR (gDNA digester plus, Yeasen), Hieff qPCR SYBR Green
Master Mix (High Rox, Yeasen); Protease inhibitor cocktail (Roche Life
Science); BCA protein assay kit (Thermo Fisher Scientific);
2,4,6-trinitro-Benzenesulfonic acid (TNBS, Sigma-Aldrich); Antibodies
against Occludin, TLR4 (Abcam); Antibodies against E-cadherin, NF-\selectlanguage{greek}κ\selectlanguage{english}B,
p-NF-\selectlanguage{greek}κ\selectlanguage{english}B, Cleaved Caspase-1, MyD88, NLRP3, ASC (Cell Signaling
Technology); HRP-conjugated monoclonal mouse anti-GAPDH (KangChen);
Anti-mouse CD16/32 mAb, BV510-conjugated anti-CD45 mAb,
BUV395-conjugated anti-CD11b mAb, APC-conjugated anti-CD11c mAb,
PE-conjugated anti-Ly6G mAb, PerCP-Cy5.5-conjugated anti-CD3 mAb (BD
Bioscience); FITC-conjugated anti-F4/80 mAb (Thermo Fisher Scientific);
FITC-dextran 4KD (Sigma-Aldrich); FBS (Gibco); Colony-stimulating factor
(M-CSF, Peprotech); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazoliumbromide (MTT, Sigma-Aldrich); Lipopolysaccharide (LPS,
Sigma-Aldrich); Tissue protein extraction reagent (Thermo Fisher
Scientific); Mouse TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6 (BD Biosciences); IL-1\selectlanguage{greek}β \selectlanguage{english}ELISA kit (Thermo
Fisher Scientific); Phorbol myristate acetate (PMA, Sigama-Aldrich);
Adenosine triphosphate (ATP, Sigma-Aldrich).

\textbf{Animals}

Female BALB/c mice (6-8 weeks), male and female C57BL/6 mice (8 weeks)
were purchased from Beijing Huafukang Biotechnology Co., Ltd. The
animals were kept in a specific pathogen-free environment with
controlled conditions, including a 12-hour light/dark cycle, 22 \selectlanguage{ngerman}± 1\selectlanguage{english}
temperature, and 55 +- 5\% relative humidity. All experiments were
performed according to the National Institutes of Health Guide for Care
and Use of Laboratory Animals and were approved by the Bioethics
Committee of the Shanghai Institute of Materia Medica (IACUC Protocol
\#2022-06-ZJP-169; \#2022-06-ZJP-177).

\textbf{Drug treatment and assessment of DSS-induced colitis}

For the induction of DSS-induced experimental colitis, C57BL/6 mice were
randomly divided into four groups, including a normal control group, a
vehicle control group, and two drug treatment groups (60 mg/kg PHI and
20 mg/kg PHI). The normal group received sterile water throughout the
experiment, while the other groups were given 2\% DSS for 7 days and
followed by a 3-day recovery period on regular drinking water. During
the experiment, the body-weight loss ratio, stool consistency and rectal
bleeding were assessed daily according to the described criterion (see
Table 1). The total score of these parameters represents the disease
activity index (DAI). On day 10, mice were anesthetized using
intraperitoneal injection of 4\% chloral hydrate. The peripheral blood,
colon, and mesenteric lymph nodes were collected for subsequent
analysis.

Table 1. Criteria for DAI: weight loss, stool consistency and fecal
blood\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Score & Weight loss (\%) & Stool consistency & Blood\tabularnewline
\midrule
\endhead
0 & 0 & Normal & Negative hemoccult\tabularnewline
1 & 1-5 & Soft but still formed & Weakly positive
hemoccult\tabularnewline
2 & 6-10 & Soft & Positive hemoccult\tabularnewline
3 & 11-20 & Very soft; wet & Blood traces in stool
visible\tabularnewline
4 & \textgreater{}20 & Watery diarrhea & Gross rectal
bleeding\tabularnewline
\bottomrule
\end{longtable}

\textbf{Drug treatment and assessment of TNBS-induced colitis}

For the induction of TNBS-induced experimental colitis, C57BL/6 mice
were randomly divided into three groups, including a normal group, a
vehicle control group, and a drug treatment group that received 2.5\%
2,4,6-trinitro-Benzenesulfonic acid (TNBS) by clysma on the first day of
the experiment. The drug treatment group received 40mg/kg PHI orally.
The body-weight loss ratio and survival rate were monitored. At the end
of the experiment, mice were anesthetized using intraperitoneal
injection of 4 \% chloral hydrate. The peripheral blood, colon, and
mesenteric lymph nodes were collected for subsequent analysis.

\textbf{Histological analysis}

The colons tissue specimens were fixed in 10\% formalin for 24 h,
dehydrated, and paraffin-embedded. Hematoxylin and eosin (H\&E) staining
was performed to observe the colon tissue pathology under an optical
microscope. The scoring criteria consisted of the following: 0, no
evidence of inflammation; 1, low-level inflammation with scattered
mononuclear cells (1-2 foci); 2, moderate inflammation with multiple
foci of mononuclear cells; 3, high-level inflammation with increased
vascular density and significant wall thickening; and 4, maximal
inflammation with transmural leukocyte infiltration and loss of goblet
cells. Additionally, periodic acid-Schiff stain (PAS) and Alcian blue
staining techniques were used to examine the epithelial goblet cells,
characterizing neutral mucin and acidic mucins, respectively. The Leica
DM6B laser microdissection system was utilized to obtain images.

\textbf{FITC-dextran intestinal permeability assay}

Mice were fasted overnight and orally administered FITC-dextran (600
mg/kg). After 4 h, their serum were collected and the fluorescence
intensity of FITC-dextran was measured at 480 excitation wavelength and
520 emission wavelength.

\textbf{Flow cytometry assay}

Single suspensions of mouse mesenteric lymph nodes or peripheral blood
mononuclear cells (PBMCs) were prepared. Then the single cells were
blocked with purified anti-mouse CD16/CD32 mAb for blocking and flow
cytometry antibodies: BUV395-conjugated CD11b, FITC-conjugated CD11c,
PE-conjugated Ly6G, BV510-conjugated F4/80, APC-conjugated Ly6C,
BUV395-conjugated CD4, Percy-cy5.5-conjugated CD3 and PE-conjugated
CD69. After incubation at 4 for 20 min, the cells were washed once with
phosphate buffer saline and analyzed using a BD LSR Fortessa Flow
Cytometer. The data were analyzed with FlowJo software.

\textbf{Cell culture and treatment}

Splenocytes were cultured in triplicate for 48 h in the presence or
absence of the compounds and were stimulated with 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL concanavalin A
(Con A) to induce T cell proliferation and 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL lipopolysaccharide
(LPS) to induce B cell proliferation. The cells were pulsed with 0.5
\selectlanguage{greek}μ\selectlanguage{english}Ci/well of {[}3H{]} thymidine for 8 h and harvested on glass fiber
filters. The incorporated radioactivity was counted using a Beta
Scintillation Counter (MicroBeta TriLux, PerkinElmer Life Sciences,
Boston, MA, USA).

Bone marrow-derived macrophages (BMDMs) were differentiated as described
previously. Briefly, BMDMs were separated from the femur and tibia bones
of male C57BL/6J mice and then cultured for 7 days in RPMI-1640 medium
containing 10\% FBS and 10 ng/mL of M-CSF. Differentiation of THP-1
cells was induced by 100 ng/mL PMA for 48 h. The differentiated cells
were treated with 1 \selectlanguage{greek}μ\selectlanguage{english}g/mL LPS in the absence or presence of PHI.

\textbf{Immunofluorescence}

For tissue samples, paraffin-embedded intestinal sections were dewaxed
in xylene and rehydrated through gradient alcohols. After being blocked
with 5\% BSA, the tissue sections were stained with anti-F4/80. The
signals were detected by goat anti-rabbit Alexa Fluor 647 conjugate and
then counterstained with DAPI. For BMDMs, cells are fixed by 4\%
paraformaldehyde, permeated by 1\% Triton X-100, and blocked by
immunostaining blocking solution. Anti-NLRP3 is used to incubate cells
at 4 overnight, followed by detection with goat anti-rabbit IgG Alexa
Fluro 488 conjugate staining. The cell nucleus is then stained by DAPI.
Captured images are analyzed using the Leica TCS SPS microscope and the
Leica Application Suite X.

\textbf{RNA extraction and real-time quantitative polymerase chain
reaction}

RNA lysate RZ is used to homogenize mice colons, followed by RNA
extraction using the RNAsimple total RNA kit. Reverse transcription is
carried out with HifairTM II 1st strand cDNA synthesis supermix for qPCR,
and real-time quantitative PCR is performed with SYBR Green Realtime PCR
Master Mix on an Applied Biosystems 7500 Fast Real-Time PCR system. PCR
amplification is performed with the primers listed in Table 2.

Table 2. Sequences of primers.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Genes & Forward (5'-3') & Reveres (5'-3')\tabularnewline
\midrule
\endhead
Mouse \selectlanguage{greek}β-\selectlanguage{english}actin & GGCTGTATTCCCCTCCATCG &
CCAGTTGGTAACAATGCCATGT\tabularnewline
Mouse IL-1\selectlanguage{greek}β & GCAACTGTTCCTGAACTCAACT &
ATCTTTTGGGGTCCGTCAACT\tabularnewline
Mouse IL-6 & CCAAGAGGTGAGTGCTTCCC &
CTGTTGTTCAGACTCTCTCCCT\tabularnewline
Mouse TNF-\selectlanguage{greek}α & ATGTCTCAGCCTCTTCTCATTC &
GCTTGTCACTCGAATTTTGAGA\tabularnewline
Mouse TLR4 & ATGGCATGGCTTACACCACC & GAGGCCAATTTTGTCTCCACA\tabularnewline
Mouse NLRP3 & ATTACCCGCCCGAGAAAGG & TCGCAGCAAAGATCCACACAG\tabularnewline
Mouse ASC & AGACATGGGCTTACAGGAGCTG &
CCACAAAGTGTCCTGTTCTGGC\tabularnewline
Mouse Caspase 1 & ACAAGGCACGGGACCTATG &
TCCCAGTCAGTCCTGGAAATG\tabularnewline
Human \selectlanguage{greek}β-\selectlanguage{english}actin & TTGTTACAGGAAGTCCCTTGCC &
ATGCTATCACCTCCCCTGTGTG\tabularnewline
Human IL-1\selectlanguage{greek}β & AGCTACGAATCTCCGACCAC &
CGTTATCCCATGTGTCGAAGAA\tabularnewline
Human IL-6 & ACTCACCTCTTCAGAACGAATTG &
CCATCTTTGGAAGGTTCAGGTTG\tabularnewline
Human TNF-\selectlanguage{greek}α & CCTCTCTCTAATCAGCCCTCTG &
GAGGACCTGGGAGTAGATGAG\tabularnewline
\bottomrule
\end{longtable}

\textbf{Statistical analysis}

RNA lysate RZ is used to homogenize mice colons, followed by RNA
extraction using the RNAsimple total RNA kit. Statistical differences
were detected by GraphPad Prism 8.3 using a two-tailed Student's t-test
or one-way ANOVA with Dunnet's multiple comparisons tests.
\emph{P}\textless{} 0.05 were considered significant.

\textbf{Results}

\textbf{PHI exhibited immunosuppressive activity \emph{in vitro}}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

The extraction of PHI was extracted, identified, and characterized (Fig.
1A, FigS1 to S5). In the present study, we found that PHI exhibits
immunosuppressive activity \emph{in vitro} . PHI was analyzed to
establish appropriate levels for subsequent use that showed minimal
signs of cytotoxicity on both murine splenocytes (Fig. 1B) and
THP-1cells over a 24h time course growing in cultures (Fig. 1C).

\textbf{Fig. 1. Phillygenin exhibited favorable immunosuppressive
activity \emph{in vitro}.} (A) Chemical structure of PHI. (B) The cell
viability of splenic lymphocytes with PHI treatment and
IC\textsubscript{50} on ConA or LPS-induced lymphocyte proliferation.
(C) The cell viability of THP-1 with PHI treatment. (D) Cytokine
secretion levels in LPS-induced THP-1 culture. (E) The gene expression
level of cytokine in LPS-induced THP-1.

Specifically, the CC50 of PHI on murine splenocytes was found to be
35.43 \selectlanguage{greek}μ\selectlanguage{english}g/mL, and it also exerted ideal immunosuppressive effects on
ConA- and LPS-induced lymphocyte proliferation, with IC50 values of
4.357 and 1.539 \selectlanguage{greek}μ\selectlanguage{english}g/mL, respectively (Fig. 1B). Furthermore, the
anti-inflammatory activity of PHI on PMA-induced human macrophages THP-1
was also confirmed. The ELISA assay indicated that PHI could effectively
inhibit the secretion of IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}induced by LPS (Fig. 1D). On the
genetic level, PHI was found to effectively inhibit expressions of
IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α. \selectlanguage{english}Taken together, these results indicate that PHI
is a promising candidate for immunosuppressive therapy \emph{in
vitro}(Fig. 1E).

\textbf{PHI ameliorated DSS-induced murine acute ulcerative colitis}

We first investigated the therapeutic effects of PHI in DSS-induced
colitis. 2\% Dextran Sulfate Sodium (DSS) in drinking water ad libitum
for 7 consecutive days, followed by treatment with different doses of
PHI (60 mg/kg and 20 mg/kg) throughout the whole experiment (Fig. 2A)

Using disease activity index (DAI) as a measure of UC severity, the mice
in both DSS- and 2,4,6-trinitro-Benzenesulfonic acid (TNBS)-induced
colitis experienced significant weight loss, severe diarrhea, and bloody
stool after chemical induction. Treatment with PHI resulted in a
reversal of these symptoms, as well as a restoration of immune
hyperfunction-induced splenomegaly and colon shortening (Fig. 2B and
2C). PHI also exerted positive effects on maintaining the intestinal
mucosal barrier by reducing goblet cell loss, mucous thinning, and
intestinal permeability (Fig. 2D, E).

In addition, the histologic analysis indicated that PHI treatment
restored the colon structure, prevented crypt destruction, and inhibited
inflammatory cell infiltration. The expression of tight junction
proteins in the colon was also restored, further demonstrating PHI's
beneficial effect on intestinal barrier function (Fig. 2F).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2. PHI ameliorated the experimental symptoms in DSS-induced
murine acute UC.} (A) DSS-induced murine model. (B) Body weight change
(\% initial body weight), diarrhea, stool bleeding, and disease activity
index (DAI). (C) Representative images of colon morphology, quantitative
colon length, and the spleen coefficient. (D) Representative images of
H\&E, PAS-Alcian blue staining of colon sections, and histological
scores, bar = 100 \selectlanguage{greek}μ\selectlanguage{english}m. (E) Fluorescence intensity of FITC-dextran in
serum. (F) The mRNA and protein levels of tight junction proteins in the
intestine. Compared with the model control group *\emph{p} <0.05,
**\emph{p} <0.01, ***\emph{p} <0.001, n = 6-8.

\textbf{PHI ameliorated TNBS-induced murine ulcerative colitis}

To better explore the effect of PHI on inflammatory bowel disease, we
also used TNBS-induced mouse model. Used 2.5\% TNBS to cause gut
inflammation. Mice were treated with 40 mg/kg PHI from day 0 to the
endpoint (Fig. 3A). Similarly, PHI significantly improved the survival
rate, colon length, and spleen coefficient of TNBS-exposed C57BL/6 mice.
(Fig. 3B and 3C). Histopathological examination of the colon section
revealed evident improvement in inflammatory cell infiltration in PHI
(40 mg/kg) treated colitis mice, compared with the TNBS-treated mice
(Fig. 3D).

Overall, the study provides evidence that PHI has a favorable
pharmaceutical effect in different chemically induced UC models and may
have potential as a therapeutic agent for the treatment of UC.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig 3. PHI ameliorated the experimental symptoms in TNBS-induced
murine acute UC.} (A) TNBS-induced murine model. (B) Survival rate. (C)
Representative images of colon morphology, quantitative colon length,
and the spleen coefficient. (D) Representative images of H\&E and
histological scores. Compared with the model control group *\emph{p}
<0.05, **\emph{p} <0.01, ***\emph{p} <0.001, n = 8-10.

\textbf{PHI reduced the infiltration of inflammatory cells}

Neutrophils and macrophages infiltrations are recognized as evaluative
parameters for the severity degree of colitis, and flow cytometry was
used to assess changes in the immune cell populations in MLN and
peripheral blood. The population of monocytes
(CD11b\textsuperscript{+}), macrophages
(CD11b\textsuperscript{+}F4/80\textsuperscript{+}), neutrophils
(CD11b\textsuperscript{+}Ly6G\textsuperscript{+}), and dendritic cells
(CD11b\textsuperscript{+}CD11c\textsuperscript{+}) were significantly
increased in both MLN and peripheral blood of DSS-induced UC mice.
However, both high and low doses of PHI were found to reduce the
population of macrophages in MLN and peripheral blood monocyte cells,
with high doses of PHI having an inhibitory effect on neutrophils of
PBMC (Fig. 4A-B). In TNBS-induced mice, the population of macrophages
decreased, and PHI reduced the level, which was consistent with the
findings in DSS-induced mice(Fig. 4C-D). Immunofluorescent staining with
F4/80 showed severe infiltrations of macrophages in DSS-induced mice
colon, which were reduced by PHI (Fig. 4E), suggesting that future
research may focus on macrophages' role in the mechanism of action of
PHI.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4. PHI inhibited inflammatory cells in MLN and PBMC of DSS
and TNBS-induced ulcerative colitis.} (A and B) Flow cytometry and
quantitative analysis of mesenteric lymph nodes (MLNs) and peripheral
blood mononuclear cells in the DSS-induced murine model, including
macrophages (CD11b\textsuperscript{+}F4/80\textsuperscript{+}),
neutrophils (CD11b\textsuperscript{+}Ly6G\textsuperscript{+}), and
dendritic cells (CD11b\textsuperscript{+}CD11c\textsuperscript{+}). (C
and D) Flow cytometry and quantitative analysis of mesenteric lymph
nodes (MLNs) and peripheral blood mononuclear cells in TNBS-induced
murine model. (E) Representative images of immunofluorescence staining
of F4/80, bar = 100\selectlanguage{greek}μ\selectlanguage{english}m. Compared with the model control group *\emph{p}
<0.05, **\emph{p} <0.01, ***\emph{p} <0.001, n = 3-4.

\selectlanguage{greek}\textbf{PHI improved ulcerative colitis by inhibiting the activation of
NLRP3 inflammasome through TLR4/MyD88/NF-κB pathway}\selectlanguage{english}

Proinflammatory mediators such as cytokines IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α \selectlanguage{english}play
key roles in UC, and the secretion level of these cytokines in serum or
colon was found to rise sharply in the presence of UC. After PHI
treatment, these inflammatory factors were significantly inhibited at
both the gene and protein levels in the colon and serum (Fig. 5A-C).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5. PHI ameliorated inflammation via the NLRP3 inflammasome
pathway.} (A) The gene expression level of cytokines in colonic
homogenates. (B and C) The protein levels of cytokines in colonic
homogenates and serum. (D) The gene expression level of Nlrp3 and
Caspase 1. (E) The protein expression of NLRP3 inflammasome and its
upstream pathway. Compared with the model control group *\emph{p}
<0.05, **\emph{p} <0.01, ***\emph{p} <0.001, n = 3-4.

Given the remarkable effect of PHI on IL-1\selectlanguage{greek}β, \selectlanguage{english}and NLRP3 inflammasome was
reported to be an important protein complex for IL-1\selectlanguage{greek}β \selectlanguage{english}production, we
detected the expression level of NLRP3 inflammasome relative proteins.
As shown in Fig. 5D-E, the expression of both NLRP3 and Caspase1 was
increased dramatically with DSS induction, which was restrained in
PHI-treated group.

In summary, the present study demonstrated that PHI improved UC by
inhibiting the activation of NLRP3 inflammasome through the
TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway. These findings have important implications for
the development of future UC treatments

\selectlanguage{greek}\textbf{PHI improved LPS and ATP-induced BMDM inflammation by inhibiting
the activation of NLRP3 inflammasome by TLR4/MyD88/NF-κB pathway}\selectlanguage{english}

Macrophages are known to play a critical role in both the progression of
inflammation and the eventual remission of disease during intestinal
mucosal repair. To investigate the efficacy of PHI \emph{in vitro} ,
bone marrow-derived macrophages (BMDM) were utilized in an \emph{in
vitro}experiment due to their significant impact on macrophages compared
to other myeloid cells. In the initial phase of the experiment, the
impact of PHI on cell viability was examined, and three safe
concentrations were selected (100, 50, and 25 \selectlanguage{greek}μ\selectlanguage{english}g/mL) for subsequent
research (Fig. 6A). BMDM was then stimulated with lipopolysaccharide
(LPS), a typical TLR4 agonist, to induce inflammatory cytokine
production. PHI displayed potent anti-inflammatory properties on BMDM,
which was reflected in a reduction in genetic and protein expression
levels of IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-6 under LPS induction (Fig. 6B-C).

Based on these findings, we tested the effect of PHI on NLRP3
inflammasome \emph{in vitro} . The NLRP3 inflammasome was activated by
LPS and NLRP3 agonist adenosine triphosphate (ATP). As with the results
in UC mice colon, PHI inhibited the expression of NLRP3 and Caspase1,
especially NLRP3, which was also demonstrated by using cellular
immunofluorescence (Fig. 6D-F). The influence mechanism was also proven,
as it inhibited the upstream pathway as a depressant. By combining the
results in both \emph{in vivo} and \emph{in vitro} experiments, the
conclusion was that PHI had a positive effect on ulcerative colitis, and
it inhibited inflammation by preventing NLRP3 inflammasome activation
through the TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway (Fig. 7).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 6. PHI inhibited the inflammation of BMDMs by inhibiting
the NLRP3 inflammasome pathway.} (A) The cell viability of BMDM with PHI
treatment. (B and C) The protein and gene expression levels in
LPS-induced BMDM. (D) The gene expression level of Nlrp3 and Caspase 1.
(E) The protein expression of inflammasome and its upstream pathway. (F)
Representative images of immunofluorescence staining of NLRP3, bar = 10
\selectlanguage{greek}μ\selectlanguage{english}m. Compared with the LPS or LPS\&ATP-stimulated group *\emph{p} <0.05,
**\emph{p} <0.01, ***\emph{p} <0.001, n = 3-4.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Fig. 7. Graphical summary of amelioration of ulcerative colitis
by PHI via inhibiting NLRP3 inflammasome activation through the
TLR4/MyD88/NF-κB pathway.}\selectlanguage{english}

\textbf{* Discussion}

UC is an inflammatory disease characterized by inflammation of the colon
mucosa, which affects the rectum and extends proximally{[}19{]}. The
incidence of UC has been increasing every year due to the
industrialization of various countries and the westernization of
people's eating habits {[}20{]}. Although there have been therapeutic
advancements in UC, there is still a treatment gap from people's ideal
state{[}21{]}. Consequently, an increasing number of studies have
explored the potential use of active monomers in functional food or
traditional Chinese medicine to treat colitis{[}22{]}. \emph{Forsythia
suspen} sa, a deciduous shrub with bitter and cold characteristics that
align with anti-inflammatory Chinese medicine, has been used
historically for fever, inflammation, gonorrhea, and erysipelas. Through
exploring the immunosuppressive activity of \emph{Forsythia
suspensa's}components in abnormal splenic lymphocyte proliferation
induced by ConA and LPS, we identified a beneficial component known as
PHI with an IC50 of 4.359 \selectlanguage{greek}μ\selectlanguage{english}g/mL and 1.539 \selectlanguage{greek}μ\selectlanguage{english}g/mL, respectively.
Additionally, PHI inhibited cytokine secretion in LPS-stimulated THP-1
cells, which motivated us to explore its therapeutic effect on clinical
inflammatory diseases using mouse models.

Next, significant therapeutic effects of PHI were found in mouse models
of UC, as reflected by its ability to maintain body weight, reduce
disease activity index and mortality, restore the intestinal mucosal
barrier, and inhibit cytokine secretion (Fig. 2 and Fig. 3). To
investigate the protective effects of PHI on the gastrointestinal tract,
H\&E staining, PAS-Alcian blue staining, and FITC-dextran were used to
prove the therapeutic effects of PHI on maintain intestinal morphology,
reduce goblet cell loss, inflammatory cell infiltration, and decrease
intestinal permeability. In inflammatory bowel diseases, barrier
function is maintained by the mucus layer and epithelial cells connected
by tight junction proteins. In DSS-induced colitis, the expression of
tight junction-associated protein E-cadherin and Occludin was decreased,
and PHI was able to restore their expression as shown in Fig.2E, which
reduced intestinal damage and restored mucosal barrier function.
Furthermore, Macrophages play a critical role in the progression of
inflammation and the disease's remission during intestinal repair. Flow
cytometry analysis of MLN and PBMC showed that PHI could reduce
macrophage infiltration in both colitis models. Immunofluorescence
performed on colon tissues also demonstrated that PHI significantly
inhibited macrophage infiltration in DSS-induced colitis (Fig.4E).
Cytokines are also important mediators in UC's enhancement and
continuation, and their presence directly causes mucosal and tissue
damage, inducing disease-specific immune responses in UC. The levels of
pro-inflammatory cytokines including IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and TNF-\selectlanguage{greek}α \selectlanguage{english}in serum and
colon were determined, and we found that PHI could effectively inhibit
their secretion, especially the secretion of IL-1\selectlanguage{greek}β.

\selectlanguage{english}The activation of IL-1\selectlanguage{greek}β \selectlanguage{english}in response to infection, mucosal injury, and
stress triggers a local mucosal immune response, recruiting neutrophils
to the affected site and activating the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway, which leads to
the upregulation of pro-inflammatory cytokines and chemokines {[}5{]}.
Furthermore, IL-1\selectlanguage{greek}β \selectlanguage{english}is the most likely effector molecule downstream of
the NLRP3 inflammasome. Given the significant impact of PHI on
macrophages and IL-1\selectlanguage{greek}β, \selectlanguage{english}the current study focused on the mechanism of
action of the NLRP3 inflammasome. We found that PHI inhibited
inflammasome protein levels at both the genetic and protein levels.
Starting with its upstream pathway, macrophages exposed to stimuli, such
as ligands for TLR4, activate the transcription factor NF-\selectlanguage{greek}κ\selectlanguage{english}B, which then
upregulates NLRP3 expression {[}23{]}. Specifically, TLR4 and MyD88
expression increased, and the phosphorylation of NF-\selectlanguage{greek}κ\selectlanguage{english}B increased
markedly, but PHI inhibited this pathway and restrained the activation
of the NLRP3 inflammasome. Finally, we conducted \emph{in
vitro}explorations with BMDM and found that PHI suppressed inflammation
by inhibiting the activation of the NLRP3 inflammasome through the
TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.

\textbf{* Conclusion}

In conclusion, PHI treatment exhibited significant improvements in UC by
inhibiting the activation of the NLRP3 inflammasome via the
TLR4/MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway. These findings provide potential mechanisms
underlying the therapeutic effects of PHI on UC.

\textbf{* Ethics approval and consent to participate}

All procedures followed the National Institutes of Health Guide for Care
and Use of Laboratory Animals and were approved by the Bioethics
Committee of the Shanghai Institute of Materia Medica (IACUC Protocol
\#2021-04-ZJP-145; \#2021-07-ZJP-151; \#2022-01-ZJP-161).\textbf{*
Consent for publication}

We declare that the publisher has the author's permission to publish the
relevant contribution.

\textbf{* Competing interests}

The authors declare no competing interests.\textbf{* Funding}

We are thankful to financial support from the National Natural Science
Foundation of China (81903882, 81871240, 81903485, 21920102003). Part of
the research work was also supported by the Key-Area Research and
Development Program of Guangdong Province (2020B0303070002), and
High-level new R\&D institute (2019B090904008) and High-level Innovative
Research Institute (2021B0909050003) from Department of Science and
Technology of Guangdong Province.

\textbf{* Authors' contributions}

XT, LC performed most of the experiments; SSL, JYY, SHF, XYX, CMW, DL,
FHZ, and XQY performed some experiments; SJH, SY, ZML, AND JPZ designed
the experiments; SJH, and JPZ interpreted the results; XT and ZML wrote
the paper.

\textbf{* Acknowledgements}

Not applicable.

\textbf{* Availability of data and material}

The datasets analyzed during the current study are available from the
corresponding author on reasonable request.

\textbf{Appendix A. Supplementary material}

\textbf{Abbreviations:}

UC, ulcerative colitis; IBD, inflammatory bowel disease; TNF, anti-tumor
necrosis factor; PAMPs, pathogen-associated molecular pattern; TLRs,
Toll-like receptors; NLRP3, NOD-like receptor family pyrin domain
containing 3; ASC, apoptosis-associated speck-like protein containing
CARD; NF-\selectlanguage{greek}κ\selectlanguage{english}B, nuclear factor kappa-B; PHI, phillygenin; DSS, dextran
sulfate sodium; DAI, disease activity index; TNBS,
2,4,6-trinitro-Benzenesulfonic acid; PBS, phosphate-buffered saline;
H\&E, hematoxylin, and eosin; PAS, periodic acid-Schiff stain; FITC-d,
fluorescein isothiocyanate- dextran; PBMC, peripheral blood mononuclear
cells; MLN, mesenteric lymph nodes; M-CSF, mouse colony-stimulating
factor; BMDM, bone marrow-derived macrophage; ELISA, enzyme-linked
immunosorbent assay; LPS, lipopolysaccharide; ATP, adenosine
triphosphate; DAPI, 4',6-diamidino-2-phenylindole; ConA, concanavalin A;
PMA, phorbol 12-myristate 13-acetate.

\textbf{Reference}

{[}1{]} N. Krugliak Cleveland, J. Torres, D.T. Rubin, What Does Disease
Progression Look Like in Ulcerative Colitis, and How Might It Be
Prevented?, Gastroenterology 162(5) (2022) 1396-1408.

{[}2{]} S. Jiang, D. Chen, C. Ma, H. Liu, S. Huang, J. Zhang,
Establishing a novel inflammatory bowel disease prediction model based
on gene markers identified from single nucleotide variants of the
intestinal microbiota, iMeta 1(3) (2022) e40.

{[}3{]} H. Nakase, N. Sato, N. Mizuno, Y. Ikawa, The influence of
cytokines on the complex pathology of ulcerative colitis, Autoimmun Rev
21(3) (2022) 103017.

{[}4{]} X. Han, S. Ding, H. Jiang, G. Liu, Roles of Macrophages in the
Development and Treatment of Gut Inflammation, Frontiers in Cell and
Developmental Biology 9 (2021).

{[}5{]} N. Tatiya-Aphiradee, W. Chatuphonprasert, K. Jarukamjorn, Immune
response and inflammatory pathway of ulcerative colitis, J Basic Clin
Physiol Pharmacol 30(1) (2018) 1-10.

{[}6{]} F.E.M. Ali, I.M. Ibrahim, O.M. Ghogar, E.K. Abd-Alhameed, H.S.
Althagafy, E.H.M. Hassanein, Therapeutic interventions target the NLRP3
inflammasome in ulcerative colitis: Comprehensive study, World J
Gastroenterol 29(6) (2023) 1026-1053.

{[}7{]} S.E. Corcoran, R. Halai, M.A. Cooper, Pharmacological Inhibition
of the Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome
with MCC950, Pharmacological Reviews 73(3) (2021) 968-1000.

{[}8{]} Y. Zhen, H. Zhang, NLRP3 Inflammasome and Inflammatory Bowel
Disease, Front Immunol 10 (2019) 276.

{[}9{]} X. Yao, C. Zhang, Y. Xing, G. Xue, Q. Zhang, F. Pan, G. Wu, Y.
Hu, Q. Guo, A. Lu, X. Zhang, R. Zhou, Z. Tian, B. Zeng, H. Wei, W.
Strober, L. Zhao, G. Meng, Remodelling of the gut microbiota by
hyperactive NLRP3 induces regulatory T cells to maintain homeostasis,
Nat Commun 8(1) (2017) 1896.

{[}10{]} S. Zhu, S. Ding, P. Wang, Z. Wei, W. Pan, N.W. Palm, Y. Yang,
H. Yu, H.B. Li, G. Wang, X. Lei, M.R. de Zoete, J. Zhao, Y. Zheng, H.
Chen, Y. Zhao, K.A. Jurado, N. Feng, L. Shan, Y. Kluger, J. Lu, C.
Abraham, E. Fikrig, H.B. Greenberg, R.A. Flavell, Nlrp9b inflammasome
restricts rotavirus infection in intestinal epithelial cells, Nature
546(7660) (2017) 667-670.

{[}11{]} M.H. Pan, Y.F. Su, X.J. Liu, S.Q. Xiang, Y.X. Ding, Q. Li,
Identification of Forsythia suspensa (Thunb.) Vahl in different harvest
periods using intelligent sensory technologies, HPLC characteristic
fingerprint coupled with chemometrics, Phytochem Anal 33(3) (2022)
490-501.

{[}12{]} C. Wang, C. Ma, K. Fu, Y. Liu, L. Gong, C. Peng, Y. Li,
Hepatoprotective effect of phillygenin on carbon tetrachloride-induced
liver fibrosis and its effects on short chain fatty acid and bile acid
metabolism, J Ethnopharmacol 296 (2022) 115478.

{[}13{]} Y. Lin, P. Yang, Phillygenin inhibits the inflammation and
apoptosis of pulmonary epithelial cells by activating PPARgamma
signaling via downregulation of MMP8, Mol Med Rep 24(5) (2021).

{[}14{]} H. Li, M. Chen, Z. Yang, Q. Wang, J. Wang, D. Jin, X. Yang, F.
Chen, X. Zhou, K. Luo, Phillygenin, a MELK Inhibitor, Inhibits Cell
Survival and Epithelial-Mesenchymal Transition in Pancreatic Cancer
Cells, Onco Targets Ther 13 (2020) 2833-2842.

{[}15{]} W. Zhou, X. Yan, Y. Zhai, H. Liu, L. Guan, Y. Qiao, J. Jiang,
L. Peng, Phillygenin ameliorates nonalcoholic fatty liver disease via
TFEB-mediated lysosome biogenesis and lipophagy, Phytomedicine 103
(2022) 154235.

{[}16{]} X. Ding, D. Lu, J. Fan, A natural product phillygenin
suppresses osteosarcoma growth and metastasis by regulating the
SHP-1/JAK2/STAT3 signaling, Biosci Biotechnol Biochem 85(2) (2021)
307-314.

{[}17{]} N. Hu, C. Wang, X. Dai, M. Zhou, L. Gong, L. Yu, C. Peng, Y.
Li, Phillygenin inhibits LPS-induced activation and inflammation of LX2
cells by TLR4/MyD88/NF-kappaB signaling pathway, J Ethnopharmacol 248
(2020) 112361.

{[}18{]} S. Wu, Y. Zhang, Y. Zhang, L. Chen, X. Xu, Y. Dang, X. Ti,
Phillygenin regulates proliferation and apoptosis of non-small cell lung
cancer through by AMPK/ERK/NF-kappaB axis, Pharmazie 75(10) (2020)
512-515.

{[}19{]} R. Ungaro, S. Mehandru, P.B. Allen, L. Peyrin-Biroulet, J.F.
Colombel, Ulcerative colitis, Lancet 389(10080) (2017) 1756-1770.

{[}20{]} T. Kobayashi, B. Siegmund, C. Le Berre, S.C. Wei, M. Ferrante,
B. Shen, C.N. Bernstein, S. Danese, L. Peyrin-Biroulet, T. Hibi,
Ulcerative colitis, Nat Rev Dis Primers 6(1) (2020) 74.

{[}21{]} R.P. Hirten, B.E. Sands, New Therapeutics for Ulcerative
Colitis, Annu Rev Med 72 (2021) 199-213.

{[}22{]} R.P.D. Nascimento, A. Machado, J. Galvez, C.B.B. Cazarin, M.R.
Marostica Junior, Ulcerative colitis: Gut microbiota, immunopathogenesis
and application of natural products in animal models, Life Sci 258
(2020) 118129.

{[}23{]} N. Kelley, D. Jeltema, Y. Duan, Y. He, The NLRP3 Inflammasome:
An Overview of Mechanisms of Activation and Regulation, Int J Mol Sci
20(13) (2019).

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