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\begin{document}

\title{Colitis can be Improved by (-)-Epigallocatechin Gallate through
Targeting Notch in DSS-induced UC mice}



\author[1]{xiaoying yang}%
\author[1]{Jian-Fei Li}%
\author[1]{Yuan-Zhu Wang}%
\author[1]{Ming-Ming li}%
\author[1]{ke chen}%
\author[1]{Xuan-jun Wang}%
\author[1]{Jun Sheng}%
\author[1]{Qiang-Qiang Zhu}%
\affil[1]{Yunnan Agricultural University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Abstract Background: Ulcerative colitis (UC) is a chronic and
non-specific inflammatory bowel disease. Previous research shows that
Notch plays a role in the pathogenesis of UC and (-)-Epigallocatechin
Gallate(EGCG) could attenuate colitis. However, the mechanism of EGCG to
improve colitis remains unclear. Methods:The human epithelial colorectal
adenocarcinoma Caco-2 cells were intervened with EGCG (10ug/ml or
30ug/ml) with or without Lipopolysaccharide. A mouse model of UC was
induced by 3\% dextran sulfate sodium and EGCG treatment was
administered to mice at a dose of 10 and 20 mg/kg. The stool
consistency, rectal bleeding and weight were recorded daily. The disease
activity index (DAI) of mice was calculated, and the pathological injury
scores were assessed through hematoxylin and eosin staining.
Immunohistochemical analyses were performed for iNOS, F4/80, Notch1 and
hes1. Inflammatory cytokines were detected using ELISA kits .Western
blot assays were performed for TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, , \selectlanguage{english}Notch1, Cleaved-Notch1,
Notch2,, Hes-1, COX2, iNOS from colon tissues and Caco-2 cells. Results:
In this study, we found that the cytokine secretion and inflammation
protein expression were reduced with EGCG treatment in LPS induced
Caco-2 cells. And the levels of Notch1, Cleaved-Notch1, and Hes-1
expression were decreased by EGCG administration in the cell. Moreover,
we found the pro-inflammation cytokine secretion and the macrophages
accumulation were reduced by oral EGCG in DSS-induced mice colon which
indicates EGCG ameliorates colitis in vivo. And we also found the
phenotype of macrophages could alter to M1 was inhibited by oral EGCG in
vivo. In addition, we demonstrate that EGCG could attenuate the levels
of Notch1, Cleaved-Notch1, and Hes-1 expression in the colon.
Conclusion: This study demonstrates that colitis can be improved by EGCG
through targeting Notch in DSS-induced UC mice. Key words: EGCG; Notch;
colitis; inflammation%
\end{abstract}%



\sloppy


\textbf{Introduction}

Ulcerative colitis (UC) is a chronic and non-specific inflammatory bowel
disease (IBD), which is characterized by complicated and relapsing
inflammation caused enormous multidimensional burdens on patients and
health care systems\textsuperscript{1}. Over 1 million residents in the
USA and 2.5 million in Europe are estimated to have IBD and it also has
emerged in newly industrialized countries in Asia, South America, and
the Middle East and has evolved into a global disease with rising
prevalence in every continent\textsuperscript{2}. In China, the
age-standardized rate of prevalence and incidence of IBD was increased
between 1990 and 2017\textsuperscript{1}. It can impede career
aspirations, instill social stigma and impair quality of life in
patients\textsuperscript{1,3}. Some drug-like anti-inflammatory,
immunosuppressive, 5-aminosalicylic acid or corticosteroids, have been
used in clinical UC patients. But these clinical medicines are no cure
for relapsing and there are certain side effects\textsuperscript{3,4}.
Hence, it is necessary to find some new and safer treatment drugs, such
as natural products.

The pathogenesis of UC includes environmental factors, genetic
predisposition, dysregulated immune responses, mucous barrier and so
on\textsuperscript{5}. However, the pathogenesis of UC is still unclear.
The Notch signaling pathway is a major regulator of cell-fate
determination during development and cellular
differentiation\textsuperscript{6}. Some studies have been reported that
the Notch signaling pathway plays a role in the pathogenesis of
UC\textsuperscript{7,8}. And Notch could ensure integrity and
homeostasis of the intestinal epithelium\textsuperscript{7}. It also
plays a role in regulating inflammatory respond\textsuperscript{6}.
Thus, Notch maybe as a potential target to treatment UC.

The (-)-Epigallocatechin-3-gallate (EGCG) is a kind of polyphenol that
is abundant in tea\textsuperscript{9}. Previous study has been reported
that EGCG could attenuate colitis which induced by dextran sulfate
sodium (DSS)\textsuperscript{10}. However, the mechanism of EGCG to
improve colitis remains unclear. In our previous studies, we have
demonstration that Notch is a receptor of EGCG\textsuperscript{11,12}.
Thus, we hypothesized that EGCG may attenuate colitis in UC mice by
targeting Notch1 to inhibit inflammation.

To prove this hypothesis, the human epithelial colorectal adenocarcinoma
Caco-2 cell line\textsuperscript{13} and DSS-induced UC model of C57
mice\textsuperscript{4} were used to study the effects of EGCG on Notch1
to attenuate colitis. The results showed that the activation of Notch1
was attenuated by treatment with EGCG in vitro and in vivo. Furthermore,
the inflammation response and clinical symptoms of colitis in UC mice
can be also improved by EGCG. These findings suggest that EGCG can
attenuate colitis by targeting Notch1. Therefore, the identification
effect of EGCG on Notch1 provides a new idea for the treatment of UC and
EGCG as a potential drug for the treatment of IBD.

\textbf{Materials and Methods}

\textbf{Animal experimental design}

Twenty-four 7-to 8-week-old male C57BL/6 mice were purchased from CAVENS
LAB ANIMAL Co., (Changzhou, China). After two weeks of adaptive feeding,
they were randomly divided into four groups: Normal group (6 mice), DSS
group (6 mice), DSS+LE group (6 mice), DSS+HE group (6 mice). Except for
the normal group treatment with drinking water, the remaining groups
received 3\% DSS in drinking water for 8 days. From the first day of
adding DSS to the drinking water, EGCG treatment was administered to
mice at a dose of 10 and 20 mg/kg per day by gavage for 1 week. During
modelling and drug treatment, the stool consistency, rectal bleeding and
weight were recorded daily. The disease activity index (DAI), a score
used to assess the severity of colitis, was calculated as previously
described\textsuperscript{14}. All animal experiments were undertaken
according to the institutional guidelines and were approved by the
Institutional Animal Care and Use Committee of the Yunnan Agricultural
University.

At the end of the experiment, all mice were killed with sodium
pentobarbital and received colon tissue,whose length was measured, and
then portions of colon were stored at -80 for the western blot and
enzyme-linked immunosorbent assay (ELISA).Portions of colons were fixed
in formalin for histopathological and immunohistochemical analysis.

\textbf{Culture and treatment of Caco-2 cells}

The human epithelial colorectal adenocarcinoma Caco-2 cells were
purchased from KCB (Kunming, China). and were cultured in DMEM comlete
medium comprising 10\% fetal bovine serum, and 1\%
penicillin--streptomycin at 37 in a humidified 5\% CO2 atmosphere. The
Caco-2 cells were starved for 12h and intervened with EGCG (10ug/ml or
30ug/ml) with or without Lipopolysaccharide (LPS, 25ug/ml) for 24 h. The
supernatant from each treatment was used to determine the levels of
TNF-\selectlanguage{greek}α \selectlanguage{english}protein released by the cells using a ELISA kit. Cells were
collected for Western blot analysis.

\textbf{Western blot analysis}

Total proteins were extracted from colon tissues and Caco-2 cells using
RIPA buffer supplemented with 1\% PMSF (protease inhibitors), and
protein concentrations were determined by the bicinchoninic acid (BCA)
method. Equal amounts of protein (45\selectlanguage{greek}μ\selectlanguage{english}g) samples and 5\selectlanguage{ngerman}× reduction loading
buffer were mixed in the tubules and boiled at 95\selectlanguage{english} for 10min. Total
protein samples were separated by 10\% SDS-PAGE and were transferred
onto the 0.2um PVDF membrane. The membranes were blocked using 5\%
skimmed milk for 1h and were incubated for 16h at 4degC with
TNF-\selectlanguage{greek}α\selectlanguage{english}(sc-133192),IL-1\selectlanguage{greek}β\selectlanguage{english}(sc-32294),F4/80(sc-52664,Santa Cruz, USA),
Notch1(SJ205, Hangzhou HuaAn Biotechnology, China),
Cleaved-Notch1(Val1744), Notch2(D76A6, Cell Signaling Technology , MA,
USA), Hes-1 (ab-71559), COX2 (ab179800), iNOS (ab15323,abcam, MA, USA).
And then were incubated with anti-rabbit IgG (\# HAF008), anti-mouse IgG
(\# HAF007) and anti-Rat IgG (\# HAF005) secondary antibodies for 1h at
room temperature. The membrane target proteins was detected by the
enhanced chemiluminescence western blot detection system (Millipore,
Billerica, MA, USA).

\textbf{Histopathological and Immunohistochemistry analysis}

Formalin-fixed mouse colon tissues were embedded in paraffin wax and cut
into 3\selectlanguage{greek}μ\selectlanguage{english}M thick sections for histopathological and immunohistochemical
examination. For histopathology, sections were dewaxed and hydrated,
stained with hematoxylin (Sigma, USA) and eosin (Solarbio), and
photographed with a Leica microscope to evaluate the morphology.
Immunohistochemical analyses were performed for iNOS, F4/80, Notch1 and
hes1. Colon sections were dewaxed and hydrated, repaired with microwave
in PH 6, 0.1M sodium citrate buffer, washed with PBS for three times,
immunohistochemical pen drew circles on the sections, and endogenous
catalase inhibitors were dropped in the circles for 20min, and the
sections were sealed with the serum working liquid in the ABC kit for
20min. Sections were incubated overnight with anti-iNOS, anti-F4/80,
anti-Notch1 and anti-Hes1 antibodies at 4. Biotin-labeled secondary
antibody was incubated at 37 for 30 minutes, peroxidase (DAB) was
stained for 5 minutes, the positive area was stained brown, and finally
stained with hematoxylin, dehydrated, transparent and sealed. Images
were captured using a Leica microscope.

\textbf{Enzyme-linked immunosorbent assay (ELISA) analysis}

Part of the frozen colon tissue was obtained and placed in the lysate
with a tissue crusher to break the homogenate. The lysates were
centrifuged at 8000g for 10 min at 4 , and the supernatants were
transferred for ELISA. Human TNF-\selectlanguage{greek}α, \selectlanguage{english}Mouse TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}MCP-1 and IL-6
concentrations were determined using the ELISA MAX Deluxe set
(BioLegend, USA) according to the manufacturer's instructions.

\textbf{Statistical analysis}

One-way ANOVA followed by Tukey's test was used to analyse the data
expressed as means \selectlanguage{ngerman}± SEM for comparing differences among each group.
Statistical evaluations was analyzed via SPSS 22.0 (IBM, Armonk, NY,
USA) and Prism 5 (GraphPad Software, La Jolla, CA, USA). Level of
statistical significance was set to p \textless{} 0.05.

\textbf{Results}

\textbf{EGCG attenuates LPS induced inflammation by target Notch in
Caco-2 cell}

Previous studies have been reported that the inflammation could be
induced by LPS in Caco-2 cell\textsuperscript{13}. But the effect of
EGCG in LPS induced Caco-2 cells was still unclear. To evaluate the
anti-inflammatory effects of EGCG in LPS induced Caco-2 cells, we
perform the experiment as described in methods. The level of TNF-\selectlanguage{greek}α
\selectlanguage{english}secretion was increased by LPS treated compared with the normal group
(figure 1A). The expression of COX2, which is a kind of inflammation
protein, was also added by LPS treatment (figure1B and 1C). The effect
of EGCG on inflammation has been reported in some studies, and it also
plays an anti-inflammation role in LPS induced Caco-2 cells. The level
of TNF-\selectlanguage{greek}α \selectlanguage{english}secretion and COX2 expression were significantly decreased by
EGCG add compared with LPS group (figure1A, 1B and 1C). Notch was a
novel drug target of EGCG\textsuperscript{11}. To evaluate the mechanism
of EGCG anti-inflammation in the intestinal cell, we detected the
protein of Notch signaling pathway. The results showed that EGCG could
significance decrease the Notch1 expression compared with LPS treatment
(figure1D, 1E). Similarly, the expression of Notch2 also decreased by
EGCG treatment (figure1D, 1F). The LPS treatment could induce Notch1
activation (figure1D, 1G) and promote the Notch signaling transduction
through increased expression of HES-1(figure1D, 1H). But, the expression
of Notch1 and Notch2 were decreased by EGCG intervention. The activation
of Notch1 (figure1D, 1G) and the downstream signal protein
HES-1(figure1D, 1H) were reduced through EGCG treatment. These results
reveal a potential mechanism of EGCG anti-inflammation to attenuate
colitis by target to Notch pathway.

\textbf{EGCG ameliorates colitis in DSS-induced UC mice}

The typical features in the DSS-induced mice model of UC have sustained
body weight loss, diarrhea, and rectal bleeding\textsuperscript{16}. The
colitis was induced in mice by administration of 3\% DSS for 8days as
described in methods. We found that the bodyweight of mice in DSS group
continued to decrease significantly from day6 (figure2A) and EGCG
administration could dramatically improve body weight loss in mice by
treat with high dose (figure1A, p\textless{}0.05). The colon shortening
was restored by EGCG therapy compared with DSS treatment group (figure2B
and 2C). The spleen weight was generally measured as an indirect marker
of inflammation in all experiment groups. Different concentrations EGCG
could significantly reversed the DSS-mediated increase in the spleen
index (P\textless{}0.05, Figure 2D). Furthermore, the DAI score, which
is used as indicator to assessing the severity of colitis, increased
distinctly after DSS treatment and it was markedly lower in the HE group
(figure2E and 2F). In addition, we also observed epithelial crypt
damage, mucosa edema, disappearance of intestinal crypts and goblet
cells, and inflammatory cell infiltration by histopathological analysis
using H\&E staining to assessing severity of colonic ulceration and
inflammation during experiment groups. The DSS group was sharp contrast
to the normal group (figure 2G) and EGCG could exhibited obvious
protection of mucosa damage and less histological inflammation (figure
2G), which showed a lower histopathological score (figure 2H). Thus,
these data confirm that EGCG exerts therapeutic effects on DSS-induced
colitis.

\textbf{EGCG reduced the level of inflammatory cytokines in colon
tissues of DSS-treated mice}

The increased inflammation cytokines, such as TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, MCP-1,
plays a critical role in DSS-induced ulcerative
colitis\textsuperscript{4}. To investigate the effect of EGCG on
inflammation-associated molecules, ELISA assay was performed to confirm
the effects of EGCG on the protein levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and
MCP-1. Compared with the Normal group, these cytokines in colonic tissue
were upregulated markedly in colonic tissues of mice with oral
administration of DSS (figure3A, 3B, 3C and 3D). While secretion levels
of these inflammatory cytokines were significantly suppressed in mice
treated with EGCG (figure3A, 3B, 3C and 3D). Furthermore, we evaluated
the expression levels of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}using western blot assays. The
levels of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}was significantly reduced by EGCG treat
compare with those of the DSS treatment group (figure 3E, 3F and 3G).
Taken together, these data indicated that EGCG could inhibit the
overwhelming inflammatory response in colonic tissue of mice with
DSS-induced colitis.

\textbf{Macrophage phenotype could change by administration of EGCG in
colitis mice}

In our previous study, the macrophage phenotype could be changed with
EGCG treat\textsuperscript{17}. To further understand the potential
mechanism by which EGCG therapy mitigate intestinal inflammation in this
study, we analyzed the macrophages accumulation and the polarization of
M1 macrophage by IHC and western blot analyses. The results showed that
the levels of F4/80, which is marker of macrophages, were increased by
DSS treat compare with normal group. And EGCG treatment could attenuate
the macrophages accumulated (figure 4A). More importantly, the nitric
oxide synthase (iNOS, M1 specific mark) were down-regulated with EGCG
treat (figure 4A). The protein of iNOS expression was also decreased by
EGCG treatment compared with mice in the DSS group (figure 4B and 4C).
These results show that EGCG resolve inflammation of the colons partly
because of reduced macrophages, especially the type of M1 macrophages.

\textbf{EGCG attenuate the expression levels of Notch in colitis mice}

It have been confirmed that Notch is a new target of
EGCG\textsuperscript{17}. In order to further understand the mechanism
of EGCG improved the colitis, the expression levels of protein which is
belong to Notch signaling pathway were detect by WB in colon from
colitis mice. The expression levels of Notch1 and Notch2 were increase
in colitis mice colon compared with normal group (figure5A, 5B and 5C).
And the phenomenon is alleviated by EGCG treatment (figure5A, 5B and
5C). The Notch1 was activated in DSS group and deactivated with EGCG
(figure5A and 5D). The expression levels of Notch downstream target
protein HES-1 were also decreased in EGCG group compared with DSS group.
The same results were observed in the colon stained by IHC of Notch1 and
HES-1 (figure 5F). These results indicated that EGCG attenuates colitis
in mice through the Notch pathway.

\textbf{Discussion}

UC is one of the main types of IBD which is affect the
bowel\textsuperscript{5} and it could develop into colorectal
cancer(CRC)\textsuperscript{7}. EGCG is a major compound of
tea\textsuperscript{9} and it could attenuate colitis in UC
mice\textsuperscript{10}. However, the mechanism of EGCG in the
treatment of colitis should be further investigated. A previous study
reported that regulation Notch signaling pathway could enhance
intestinal barrier function in colitis mice\textsuperscript{4} and it
could be a target of chronic inflammatory diseases, such as
IBD\textsuperscript{6}. In our previous studies, we reported that Notch
is a receptor of EGCG\textsuperscript{11,17}. In this study, we focused
on the Notch signaling pathway which plays a role in the development of
UC and aberrant activation of Notch1 was attributed to the severity of
CRC\textsuperscript{18}. Our results found that the inflammation state
and Notch expression were altered by EGCG treatment in Caco-2 cell which
induced by LPS. Furthermore, we also identified the mechanism is present
in UC mice, which is induced by DSS in C57/BL6 mice. These results
suggested that EGCG could play a role to against colitis by regulation
Notch signaling pathway in UC mice.

Previous studies have been reported that Notch could regulation
inflammation\textsuperscript{19,20} and modulates macrophages
polarization\textsuperscript{21}. Some studies also propose that
targeting the Notch signaling pathway could be a potential therapeutic
strategy to improve chronic inflammatory
diseases\textsuperscript{6,22,23}. A new research reported that the
transcription levels of Notch1 and Hes-1 genes were significantly
elevated in UC patients\textsuperscript{22} which reminder us to reflect
on important of Notch in UC. And recent studies have revealed the
unexpected importance of epithelial cells in the pathophysiology of
IBD\textsuperscript{24}.~So, in this study, we take human epithelial
colorectal adenocarcinoma Caco-2 cell line as inflammation
model\textsuperscript{25} to assess the inflammatory responds with LPS
treatment. Our research found that the inflammation responds was
increased by LPS treatment in Caco-2 cell (figure 1A and 1B) or enhance
intestinal immune response in DSS induced UC mice colon (figure3 and
figure 4). And the levels of Notch1 and cleaved-Notch1 expression were
also increase by LPS treatment in Caco-2 cell (figure 1D) or DSS induced
UC mice colon. These results was similar to the previous study
report\textsuperscript{22,26,27}. And our finding also showed that Notch
could be a target to therapy hyper-inflammatory respond and UC.
Moreover, this research also proved that Notch activate could contribute
to M1 macrophages accumulation. These founds suggest that activate Notch
play a role to induced UC as other studies
report\textsuperscript{7,8,22}.

The effect of EGCG on perfect IBD has been reported in previous
studies\textsuperscript{28,29}. And our previous studies have been
demonstration that Notch was a target of EGCG\textsuperscript{11,30} to
attenuate inflammation responds or other chronic
disease\textsuperscript{17}. So, we think the Notch maybe was a target
of EGCG to attenuate UC. In this study, we found that EGCG could
decreased the cytokine level which induced by LPS in Caco-2 cell (figure
1A and 1B). The levels of Notch expression were increased and Notch was
activation by LPS stimulation in cell, but, the phenomenon was
disappeared by administration EGCG in vitro (figure 1D). Moreover, the
inflammation state of colon and macrophages accumulation were also
decreased by oral EGCG (figure 3 and figure 4). To further understand
the mechanism of EGCG on attenuate colitis, we detected Notch signaling
protein and found that the level of Notch expression, Notch activation,
and downstream protein were also significance decreased by
administration EGCG (figure 5). Our results revealed the effect of EGCG
on UC and expound the mechanism.

In conclusion, our study provides direct evidence that EGCG reduces
inflammatory response by targeting Notch and reveals the mechanism by
which EGCG alleviates colitis in UC mice (Figure 6). EGCG suppressing
inflammation may be a promising treatment option for chronic
inflammatory diseases in patients with IBD.

\textbf{Author contributions}

Q.Q.Z., J. S. and X. J. W. designed the study. X.Y.Y., J.F.L., Y.Z.W,
M.M.L. and K.C. performed the experiments. X.Y.Y. and Q.Q.Z.wrote the
manuscript. X. J. W. supervised the overall study. J. S. is the
guarantor of this work; as such, Q.Q.Z.had full access to all the data
in the study and takes responsibility for the integrity of the data and
the accuracy of the analysis.

\textbf{Ethics guidelines}

Reporting of animal data in this study followed the recommendations set
out in the ARRIVE guidelines.

\textbf{Data availability}

The datasets used and/or analysed during the current study available
from the corresponding author on reasonable request.

\textbf{Conflicts of interest}

The authors have no conflict of interest to disclose.

\textbf{Acknowledgments}

This research was funded by the Basic Research Program-young project of
Yunnan Province, grant number 202101AU070084; Basic Research project of
Yunnan Province, grant number 202301AT070505; National Natural Science
Foundation of China, grant number 31860322; and Scientific research
foundation of Yunnan Agricultural University (KY2019-24). No potential
conflicts of interest relevant to this article were reported.

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\textbf{Figure Legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1 EGCG could attenuate inflammatory responses by
targeting the Notch pathway in Caco-2 cells.} The supernatant and
samples were obtained from the Caco-2 cell line after LPS stimulation
with or without EGCG treatment for 24 h. To measure the pro-inflammatory
marker TNF-\selectlanguage{greek}α, \selectlanguage{english}the supernatant was treated as described earlier using
ELISA. 60 \selectlanguage{greek}μ\selectlanguage{english}g of each sample was applied to each well for detection of
inflammation protein and Notch signaling pathway protein. (\textbf{A} )
TNF-\selectlanguage{greek}α \selectlanguage{english}was detected to demonstrate whether EGCG attenuates inflammatory
response in Caco-2 cells. (\textbf{B, C} ) The protein expression of
COX2 was measured by western blotting, and the ODs were determined.
(\textbf{D} ) The protein levels of Notch1, Notch2, Cleaved-Notch1, and
Hes-1 were detected by western blotting, and the ODs were calculated to
demonstrate the effect of EGCG on the Notch signaling pathway in the
Caco-2 cell. Data are pooled from two experiments. Data are presented as
the means \selectlanguage{ngerman}± SEM. *P\textless{}0.05, **P\textless{}0.01,
***P\textless{}0.001 (one-way ANOVA). EGCG, (-)-epigallocatechin
gallate; LPS, Lipopolysaccharide.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2 Colitis can be improved by the administration of EGCG
in mice.} Except for the normal group, mice in other groups were given
DSS (3\% w/v) for 8 days and the mice were treated with or without EGCG,
respectively. EGCG promotes recovery of DSS-induced colitis in mice. (A)
Body weight change rate (the change rate of body weight in each group of
mice was compared with the body weight in the same group of mice on the
1st day). (B, C) Length of the colon (from the appendix to the anus)
were detected on day8. (D)The index of the spleen; (E) Disease activity
index; (F) The DAI score on day8 between groups; (G) Histopathological
images showing paraffin-embedded sections stained with H\&E in the
colon; (H)Histopathologic scoring of the inflammation, depth of
inflammation and crypt damage. Scale bars, 200 \selectlanguage{greek}μ\selectlanguage{english}m. Data are presented as
the means \selectlanguage{ngerman}± SEM. *P\textless{}0.05, **P\textless{}0.01,
***P\textless{}0.001 (one-way ANOVA). LE, Low dose of
(-)-epigallocatechin gallate; HE, High dose of (-)-epigallocatechin
gallate; DSS, Dextran sulfate sodium.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3 The level of cytokine secretion and protein expression
can be inhibited by EGCG in colon of the UC mice.} Colon in UC mice was
treated with EGCG or placebo and harvested at day 8 post DSS induced. To
measure the pro-inflammatory marker TNF-\selectlanguage{greek}α \selectlanguage{english}(A), IL-1\selectlanguage{greek}β \selectlanguage{english}(B), MCP-1 (C), and
IL-6 (D), tissues were treated as described earlier using ELISA. The
tissue was obtained from the colon of mice was homogenized in protein
extraction solution and 60 \selectlanguage{greek}μ\selectlanguage{english}g for each well for detected. The protein
expressions of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}(E) were measured by western blotting.
The value of OD (F, G) was detected by Image J (NIH). For all graphs,
bars = mean \selectlanguage{ngerman}± SEM, n = 5. Statistical analysis was performed using
one-way ANOVA. ***Mean value was significantly different for the EGCG
and normal group at the same time point compared with the model group.
LE, Low dose of (-)-epigallocatechin gallate; HE, High dose of
(-)-epigallocatechin gallate; DSS, Dextran sulfate sodium.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4 Macrophages were decreased by treat with EGCG in colon
of the UC mice.} Immunohistochemisty was performed on paraffin sections
stained for F4/80 and

iNOS(A). The Brown area (red arrows) indicates positive staining. The
tissue was obtained from the colon of mice was homogenized in protein
extraction solution and 60 \selectlanguage{greek}μ\selectlanguage{english}g for each well for detected. The protein
expressions of iNOS (B) were measured by western blotting. The value of
OD (C) was detected by Image J (NIH). Data are pooled from two
experiments (n=6 colon from six mice per group, repeated twice). Data
are presented as the means \selectlanguage{ngerman}± SEM. *P\textless{}0.05, **P\textless{}0.01,
***P\textless{}0.001 (one-way ANOVA, EGCG, and normal group vs. DSS
group). Scale bars, 100 \selectlanguage{greek}μ\selectlanguage{english}m. LE, Low dose of (-)-epigallocatechin
gallate; HE, High dose of (-)-epigallocatechin gallate; DSS, Dextran
sulfate sodium.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5 The Notch signaling pathway was weakened by treatment
with EGCG in colon of the UC mice.} The tissue obtained from the colon
of mice was homogenized in a protein extraction solution; 60 \selectlanguage{greek}μ\selectlanguage{english}g of each
sample was used for detection. (\textbf{A} ) The expression levels of
Notch1, Notch2, Cleaved-Notch1, and Hes-1 were measured by western
blotting, and the ODs (B, C, D, E) were detected. (\textbf{F} ) IHC was
performed on paraffin-embedded sections stained for Notch1 and Hes-1:
brown area (red arrows) indicates positive staining. Data are pooled
from two experiments (n=6 colon from six mice per group, repeated
twice). Data are presented as the means \selectlanguage{ngerman}± SEM. *P\textless{}0.05,
**P\textless{}0.01, ***P\textless{}0.001 (one-way ANOVA, EGCG, and
normal group vs. DSS group). Scale bars, 100 \selectlanguage{greek}μ\selectlanguage{english}m. LE, Low dose of
(-)-epigallocatechin gallate; HE, High dose of (-)-epigallocatechin
gallate; DSS, Dextran sulfate sodium.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6 Colitis can be Improved by EGCG through Targeting Notch
in mice}

\selectlanguage{english}
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\end{document}

