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\begin{document}

\title{Changes in cytokines and signaling pathways in different stages of
hepatic fibrosis in rats}



\author[1]{Qiangzhong Pi}%
\author[2]{Xiaoping Hua}%
\author[3]{Liping Wang}%
\author[4]{Dali Fu}%
\author[2]{Li Zhang}%
\author[2]{Wanying Tan}%
\author[2]{Zhenghuai Tan}%
\affil[1]{Department of Respiratory and Critical Care Medicine, Southwest Hospital, Army Medical University }%
\affil[2]{Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of Chinese Medicine Sciences}%
\affil[3]{Department of Pharmacy, Chengdu Jinjiang Hospital for Women and Children Health}%
\affil[4]{Clinical Pharmacy Department of Western Theater General Hospital}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background \& Purpose: Liver fibrosis is a disease that seriously
threatens people's health, and its etiopathogenesis has not been
described clearly. Experimental Approach: Female rats were subjected to
common bile duct ligation (BDL) for one month, and male rats were
treated with thioacetamide for 23 weeks. The expression of cytokines,
signal pathways, histopathology in liver and biochemical indexes in
serum were detected. Key Results: The levels of transaminases in serum
and hydroxyproline and \selectlanguage{greek}α-\selectlanguage{english}smooth muscle actin in the liver were
remarkably increased in both models, although the degree of liver
fibrosis was more severe in thioacetamide rats than in BDL rats.
However, the levels of IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-4, IL-10, TNF\selectlanguage{greek}α, \selectlanguage{english}MCP-1, PDGF-BB, p-Akt
and p-STAT5 decreased, and the levels of IL-18, TGF\selectlanguage{greek}β\selectlanguage{english}1 and p-p70s6k
increased in the livers of BDL rats, while the levels of IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-4,
IL-10, IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and IL-12p70, TNF\selectlanguage{greek}α, \selectlanguage{english}MCP-1, PDGF-BB, p-CREB, p-JNK,
p-NF\selectlanguage{greek}κ\selectlanguage{english}B, p-Akt, p-p70s6k, p-STAT3 and p-STAT5 decreased, and the levels
of IL-18 and TGF-\selectlanguage{greek}β\selectlanguage{english}1 increased in the livers in thioacetamide rats.
Conclusion and Implications: These data suggest that TGF\selectlanguage{greek}β\selectlanguage{english}1 and IL-18 may
be the main fibrogenic factors at different stages of liver fibrosis and
that the levels of inflammation-associated cytokines and signaling
pathway components decrease as the severity of hepatic fibrosis
progresses. Therefore, it may be better to apply anti-inflammatory drugs
in the early stage or use these drugs which facilitating more
inflammatory cells or cytokines into liver tissue at the end stage of
hepatic fibrosis.%
\end{abstract}%



\sloppy


\textbf{Changes in cytokines and signaling pathways in different stages
of hepatic fibrosis in rats}

Qiangzhong Pi\textsuperscript{1a}, Xiaoping Hua\textsuperscript{2a},
Liping Wang\textsuperscript{3a}, Dali Fu\textsuperscript{4}, Li
Zhang\textsuperscript{2}, Wanying Tan\textsuperscript{2*}, Zhenghuai
Tan\textsuperscript{2*,}

\textsuperscript{1} Department of Respiratory and Critical Care
Medicine, Southwest Hospital, Army Medical University ,Chongqing
400038, China.

\textsuperscript{2} Institute of Traditional Chinese Medicine
Pharmacology and Toxicology, Sichuan academy of Chinese Medicine
Sciences, Chengdu 610041, China

\textsuperscript{3} Department of Pharmacy, Chengdu Jinjiang Hospital
for Women and Children Health, Chengdu 610021, China

\textsuperscript{4} Clinical Pharmacy Department of Western Theater
General Hospital

\textsuperscript{a}The same contributions

*Corresponding authors. Tel.: +86 28 85258982.

mail addresses: Tanwy58@163.com (W. Tan), tanzhh616@163.com (Z. Tan)

\textbf{Funding information:}

This work was supported by Science \& technology Department of Sichuan
Province (grant numbers 11010119) and National Natural Science
Foundation of China (21861142007).

\textbf{Data availability:}

The data is available on request from the authors.

\textbf{Competing interests:}

The authors declare no competing interests.

\textbf{Authors contributions:}

All authors contributed to the manuscript. Q.Pi, X.Hua and L.Wang
performed the experiments, analyzed data and wrote the manuscript. They
contribute the same. D.Fu and L.Zhang performed the experiments and
graphed the data. Z. Tan and W.Tan contributed to the design of the
experiments and supervised the project. All authors read and approved
the final version of the manuscript.

\textbf{Ethics approval:}

All animal experiments were performed in accordance with the guidelines
of the Ethical Committee for the Care and Welfare of Laboratory Animals
from the Sichuan province government.

\textbf{Abbreviations:} BDL, bile duct ligation; \selectlanguage{greek}α-\selectlanguage{english}SMA, \selectlanguage{greek}α-\selectlanguage{english}smooth muscle
actin; TAA, thioacetamide; HBV, hepatitis B virus; HCV, hepatitis C
virus; NASH, nonalcoholic steatohepatitis; HSCs, hepatic stellate cells;
NK, natural killer; TNF\selectlanguage{greek}α, \selectlanguage{english}tumor necrosis factor \selectlanguage{greek}α; \selectlanguage{english}ILs, interleukins;
TGF-\selectlanguage{greek}β, \selectlanguage{english}transforming growth factor \selectlanguage{greek}β; \selectlanguage{english}ALT, alanine aminotransferase; AST,
aspartate amino transferase; ALP, alkaline phosphatase; TBIL, total
bilirubin; DBIL, direct bilirubin; H\&E, hematoxylin and eosin; SABC,
streptavidin-biotin complex; DAB, 3,3'-diaminobenzidine; PDGF,
platelet-derived growth factor; IFN, interferon; TMB,
Tetramethylbenzidine; MCP-1, monocyte chemoattractant protein-1; MIP,
macrophage inflammatory protein; MFI, median fluorescent intensity;
EMCs, extracellular matrix components; FN, fibronectin; LN, laminin;
NLRP, NOD-like receptor protein;

\textbf{Bullet point summary:}

\textbf{What is already known:}

Inflammatory factors increase in early stage of liver fibrosis.

\textbf{What this study adds:}

Most inflammatory cytokines decrease in advanced hepatic cirrhosis.

\textbf{Clinical significance:}

According to the different degree of liver fibrosis, change the
treatment strategy.

\textbf{Background \& Purpose:}

Liver fibrosis is a disease that seriously threatens people's health,
and its etiopathogenesis has not been described clearly.

\textbf{Experimental Approach:}

Female rats were subjected to common bile duct ligation (BDL) for one
month, and male rats were treated with thioacetamide for 23 weeks. The
expression of cytokines, signal pathways, histopathology in liver and
biochemical indexes in serum were detected.

\textbf{Key Results:}

The levels of transaminases in serum and hydroxyproline and \selectlanguage{greek}α-\selectlanguage{english}smooth
muscle actin in the liver were remarkably increased in both models,
although the degree of liver fibrosis was more severe in thioacetamide
rats than in BDL rats. However, the levels of IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-4, IL-10, TNF\selectlanguage{greek}α,
\selectlanguage{english}MCP-1, PDGF-BB, p-Akt and p-STAT5 decreased, and the levels of IL-18,
TGF\selectlanguage{greek}β\selectlanguage{english}1 and p-p70s6k increased in the livers of BDL rats, while the levels
of IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-4, IL-10, IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and IL-12p70, TNF\selectlanguage{greek}α, \selectlanguage{english}MCP-1, PDGF-BB,
p-CREB, p-JNK, p-NF\selectlanguage{greek}κ\selectlanguage{english}B, p-Akt, p-p70s6k, p-STAT3 and p-STAT5 decreased,
and the levels of IL-18 and TGF-\selectlanguage{greek}β\selectlanguage{english}1 increased in the livers in
thioacetamide rats.

\textbf{Conclusion and Implications:}

These data suggest that TGF\selectlanguage{greek}β\selectlanguage{english}\textsubscript{1} and IL-18 may be the main
fibrogenic factors at different stages of liver fibrosis and that the
levels of inflammation-associated cytokines and signaling pathway
components decrease as the severity of hepatic fibrosis progresses.
Therefore, it may be better to apply anti-inflammatory drugs in the
early stage or use these drugs which facilitating more inflammatory
cells or cytokines into liver tissue at the end stage of hepatic
fibrosis.

\textbf{Key words} : liver fibrosis; cytokines; common bile duct
ligation (BDL); thioacetamide (TAA); signal pathway;

\textbf{Introduction}

Liver fibrosis is a wound healing response to various types of injury,
and it can progress into liver cirrhosis and even to hepatocellular
carcinoma (HCC). Alcohol abuse, hepatitis B virus (HBV) and hepatitis C
virus (HCV) infections, and nonalcoholic steatohepatitis (NASH) are the
main causes of liver fibrosis. In the European Union, 0.1\% of the
population is affected by cirrhosis, which leads to approximately
170,000 deaths each year.(1) In China, the morbidity rate of HBV was
9.75\% in 1992, and approximately 300,000 patients died of HBV-related
diseases (2). Unfortunately, there are currently no effective drugs for
liver fibrosis, especially liver cirrhosis.

The etiopathogenesis of liver fibrosis is very complex. Many cells,
including hepatic stellate cells (HSCs), fibroblasts, bone
marrow-derived myofibroblasts, hepatocytes, Kupffer cells, and natural
killer (NK) cells, are involved in this pathological processs(3-7) .
These cells interact with each other through cytokines, resulting in the
accumulation of extracellular matrix, as it is formed faster than it is
degraded, and the gradual formation of fibrotic tissue that can further
develop into cirrhosis. In particular, tumor necrosis factor \selectlanguage{greek}α \selectlanguage{english}(TNF\selectlanguage{greek}α\selectlanguage{english}),
interleukins (ILs), and transforming growth factor \selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}) are
involved in the formation and development of liver fibrosis(8-11).
However, whether these cytokines exhibit obvious changes with the
development of liver fibrosis or whether those changes differ depending
on the fibrosis stimulus is not fully understood.

Biliary cirrhosis is caused by biliary obstruction and cholestasis (12).
Cholestasis leads to the sedimentation of bilirubin, bile salts, bile
acids, cholesterol and other substances in the liver. Long-term
cholestasis causes liver fibrosis and even cirrhosis(13). Bile duct
ligation (BDL) is a common way to establish an animal model of
cholestatic liver fibrosis (12). The animal model can simulate human
liver fibrosis caused by long-term cholestasis, with good repeatability
and a high success rate.

Thioacetamide (TAA) has hepatotoxicity and leads to the destruction of
liver cells (14). Long-term and low-dose exposure to TAA may promote
gradual hepatocyte necrosis, which is similar to the pathological
process of human liver fibrosis, meaning that it is a good model for
chronic liver injury and liver fibrosis(15).

This paper focuses on comparing the early stage of liver fibrosis or
cirrhosis (caused by choledochal ligation for 1 month) with the late
stage of cirrhosis (treated with TAA for 3 months) and exploring the
similarities and differences in the main cytokines and signaling
pathways in female and male rats to identify the common factors
promoting liver fibrosis and find new targets or target groups for the
treatment of liver fibrosis and cirrhosis.

\textbf{Materials and Methods}

\textbf{Animals}

Female and male SD rats (body weight 200-250 g) were supplied by the
Center of Experimental Animals of Sichuan Academy of Chinese Medicine
Sciences (eligibility certification no. SCXK (chuan) 2013-19). The rats
were maintained under standard conditions with a 12 h:12 h light--dark
cycle in a temperature- and humidity-controlled environment. The rats
were provided food and water \emph{ad libudum.} All animal experiments
were performed in accordance with the guidelines of the Ethical
Committee for the Care and Welfare of Laboratory Animals from the
Sichuan province government.

\textbf{Induction of liver fibrosis by BDL}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

BDL operation was performed under sterile conditions. 20 female SD rats
were randomly into control and BDL groups, rats were anesthetized by
intraperitoneal injection of pentobarbital 50mg/kg, the abdomen was
opened by a median incision, and the common bile duct was double-ligated
and cut between the ligatures. Sham-operated control animals underwent
similar manipulation without bile duct ligation. On the
29\textsuperscript{th} day, the animals were sacrificed, and sampling
was performed to detect related indicators.

\textbf{Induction of liver fibrosis with TAA}

20 male SD rats were randomly into control and TAA treated groups. The
model animals were fed 0.035\% TAA solution for 23 weeks, and the
controls were fed pure water. Twenty-four hours after the last
administration, the animals were sacrificed, and sampling was performed
to detect related indicators.

\textbf{Serum biochemistry assays}

Blood samples were taken from the abdominal aorta of anesthetized
animals, and the serum levels of alanine aminotransferase (ALT),
aspartate amino transferase (AST), alkaline phosphatase (ALP), total
bilirubin (TBIL), and direct bilirubin (DBIL)(Sichuan Maker
Biotechnology Co. Ltd, Chengdu, China) were measured using a standard
automatic biochemical analyzer (HITACHI 7020).

\textbf{Measurement of hepatic hydroxyproline content}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Liver tissue (60 mg) was mixed with 1.0mL of 6 N NaOH and incubated at
100\selectlanguage{ngerman}°C for 20 min with frequent vigorous shaking. After cooling, 1.0 mL
of 3N HCl was added to the solution, the pH was regulated to
6.0\textasciitilde{}6.8, and then 10 mL of water was added and mixed.
Then, 3.0 mL of the suspension was added to 20\textasciitilde{}30 mg
active carbon, and the sample was mixed and centrifuged at 3500 rpm for
10 min. The supernatant was taken to measure the hydroxyproline contents
using a colorimetric hydroxyproline test kit according to the
manufacturer's instructions (Institute of Jiancheng Bioengineering,
Nanjing, China). The hydroxyproline content was expressed as micrograms
per gram of liver weight (\selectlanguage{greek}μ\selectlanguage{english}g/g liver).

\textbf{Hepatic histological and pathological}

\textbf{assay}

The wet weight of the liver or spleen was measured and then divided by
the body weight to obtain the liver or spleen index. Then, the liver
tissues were fixed in 4\% paraformaldehyde and embedded in paraffin.
Tissue sections (4-\selectlanguage{greek}μ\selectlanguage{english}m thick) were prepared and placed on glass slides.
They were then stained with hematoxylin and eosin (H\&E) or Masson's
trichrome stain following standard protocols. A pathologist who was
blinded to the experimental groups scored liver fibrosis under a
microscope according to the METAVIR scoring system (F0, no fibrosis; F1,
portal fibrosis without septa; F2, portal fibrosis with rare septa; F3,
numerous septa without cirrhosis; F4, liver cirrhosis) (16).

\selectlanguage{greek}\textbf{The expression of α-SMA in the liver was detected by
immunohistochemistry}\selectlanguage{english}

Tissue sections (4-\selectlanguage{greek}μ\selectlanguage{english}m thick) were prepared and placed on glass slides.
Endogenous enzymes were inactivated, and antigen retrieval was performed
using heat (in 0.01 M citrate salt buffer, pH 6.0, heating to 100\selectlanguage{ngerman}°C two
times for 10 mins). Then, the sections were incubated with 5\% BSA at
room temperature for 20 min, followed by the addition of anti-\selectlanguage{greek}α-\selectlanguage{english}SMA
(1:40) antibody (Santa Cruz Biotechnology, Inc., CA, USA) at 2--8\selectlanguage{ngerman}°C
overnight. They were then incubated with biotinylated goat anti-rabbit
IgG (1:100) (Wuhan Boster, Wuhan, Hubei, China) at 37°C for 30 min.
Next, streptavidin-biotin complex (SABC) detection reagent (1:100)
(Wuhan Boster, Wuhan, Hubei, China) was added dropwise, and the samples
were incubated for 30 min before 3,3'-diaminobenzidine (DAB) reagent
(Wuhan Boster, Wuhan, Hubei, China) was added. After washing with
distilled water, the sections were stained with hematoxylin, and a
pathologist who was blinded to the treatment groups counted the number
of positively stained cells under a microscope.

\textbf{Extraction of protein}

Segments (100 mg) of frozen livers were ground up at 0°C, and then 500
\selectlanguage{greek}μ\selectlanguage{english}l diluted cell signaling lysis buffer supplemented with 5 \selectlanguage{greek}μ\selectlanguage{english}l protease
inhibitor cocktail set III (Millipore Corporation, USA) was added. The
mixture was vortexed for 3 cycles of 20 min on/3 min off. The suspended
liver tissues were sheared with an ultrasonic cell disruptor and
centrifuged at 4\selectlanguage{ngerman}°C (14000 rpm for 20 min). The supernatant was stored at
-80°C until measurement of the levels of cytokines and phosphorylation
of the key protein in the signal pathways by ELISA or
MILLIPLEX\textsuperscript{®}MAP assay.

\textbf{Protein assay}

The BCA Protein Assay Kit (Thermo Scientific Pierce Protein Research
Products, USA) is a comprehensive and robust protein quantification
assay based on the colorimetric detection of copper ions that are
chelated with proteins. The protein content in the liver samples was
measured according to the manufacturer's instructions.

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

The contents of platelet-derived growth factor (PDGF)-BB, interferon
(IFN)-\selectlanguage{greek}γ \selectlanguage{english}and TGF-\selectlanguage{greek}β\selectlanguage{english}1 (R\&D Systems Inc., Minneapolis, USA) in the protein
extracts were measured by ELISA according to the manufacturer's
instructions\textbf{.} Briefly, the protein extracts were lysed with 100
mM Tris (pH: 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1\% Triton X-100,
and 0.5\% sodium deoxycholate. Then, 50 \selectlanguage{greek}µ\selectlanguage{english}L of standard, control, or
sample was added to each well of the microplate, which was precoated
with capture antibody; A second HRP-labeled antibody was added and bound
to the antigen-antibody complex.Unbound detection antibody was washed
away. Tetramethylbenzidine (TMB) substrate solution was added to the
wells, and a blue color developed in proportion to the amount of analyte
present in the sample. Color development was stopped, turning the color
in the wells yellow. The absorbance of the color at 450 nm was measured.
The contents of PDGF-BB, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TGF\selectlanguage{greek}β\selectlanguage{english}\textsubscript{1} were calculated
by means of standard curves.

\textbf{Multiplex bead assay for detecting cytokines and phosphorylated
proteins of the signaling pathway}

Multiplex bead assays were used to quantify the amounts of IL-10, IL-13,
IL-12p70, IL-4, IL-6, IL-12, IL-18, IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}monocyte
chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein
(MIP)-1\selectlanguage{greek}α, \selectlanguage{english}MIP-2, and TNF\selectlanguage{greek}α \selectlanguage{english}(the MILLIPLEX MAP Rat Cytokine/Chemokine
Magnetic Bead Panel; Cat \# RECYTMAG-65K-12, Billerica, MA, USA) and
P-CREB, P-JNK, P-NF-\selectlanguage{greek}κ\selectlanguage{english}B, P-P38MAPK, P-ERK1/2, P-Akt, P-p70S6K, P-STAT3
and P-STAT5 (MILLIPLEX\selectlanguage{ngerman}® MAP 9-Plex Multi-Pathway Signaling Magnetic Bead
Kit for Phosphoprotein, 96-well; Cat \# 48-680MAG, Billerica, MA, USA)
in liver protein extracts. All assays were performed and analyzed
according to the manufacturer's instructions. The plate was analyzed
using a Luminex 200\selectlanguage{english} analyzer and xPONENT (Billerica, MA, USA) software,
and the median fluorescent intensity (MFI) data were saved and analyzed
using a five-parameter logistic or spline curve-fitting method to
calculate the analytical concentrations in the samples.

\textbf{Statistical analysis}

All the data are expressed as the mean+-SD. The differences in the
biomarkers between groups were analyzed using one-way analysis of
variance test, and then the individual differences between groups were
evaluated using Dunnett's test. The differences in histopathology
between groups were analyzed using the Kruskal-Wallis test and Dunn's
multiple comparison test.

\textbf{Results}

\textbf{TAA and BDL caused significant liver dysfunction in rats and led
to obvious liver fibrosis}

Elevated serum levels of ALT and AST are indicators of liver injury(17).
ALP levels are related to chronic liver injury(18). The serum levels of
ALT, AST and ALP and the liver weights of normal female rats were
significantly lower than those of male rats. The levels of ALT, AST, and
ALP and the liver weights or liver indexes were significantly increased
by BDL for 1 month and were much higher than those in rats treated with
TAA for 23 weeks. These data suggest that BDL for 1 month induced an
obvious inflammatory reaction in the livers of rats that was more potent
than that of TAA (Figure 1).

Fibrosis is the excessive accumulation of collagen and other
extracellular matrix components (EMCs) such as fibronectin (FN) and
laminin (LN), due to chronic injury (19-21). Hydroxyproline is a
characteristic component of collagen, and the content of hydroxyproline
is related to the level of tissue fibrosis(22). As shown in Table 1,
hydroxyproline in the livers of female rats was significantly higher
than that in those of male rats. In addition, 1 month after BDL, the
increase in hydroxyproline was significantly higher in female rats than
in male rats treated with TAA for 23 weeks but BDL for 1 month showed
lower pathological scoring, suggesting that the hydroxyproline level in
liver was not completely proportional to the degree of fibrosis. The
mediastinal form and connective tissue hyperplasia in the liver were
more obvious in TAA rats than in BDL rats (Table 1, Figure 2), and
\selectlanguage{greek}α-\selectlanguage{english}SMA, the marker of HSC activation, was upregulated in the livers of
BDL and TAA rats (Figure 3).

\textbf{The effect of BDL on the levels of ILs in female rats is
different from the effect of TAA in male rats}

ILs include many cytokines that play an important role in liver injury
or fibrosis. Many cells can synthesize or secrete ILs. Many cells can
synthesis or secretion of ILs. As Figure 4 shows, the levels of
interleukins, especially IL-4, IL-10, IL-13, IL-18 and L-12p70, in liver
tissue of female rats were higher than those in the liver tissue of male
rats. After BDL or TAA treatment, IL-1\selectlanguage{greek}α, \selectlanguage{english}IL- 4 and IL-10 decreased, but
IL-18 increased; BDL for a month upregulated IL-1\selectlanguage{greek}β \selectlanguage{english}in the livers of
female rats but had no significant effect on IL-6 and IL-12p70, while
TAA decreased IL-1 \selectlanguage{greek}β, \selectlanguage{english}IL-6 and IL-12p70 levels in the livers of male
rats (Figure 4).

\par\null

\selectlanguage{greek}\textbf{BDL significantly increases MIP-1α but decreases TNFα
expression, while TAA markedly decreases TNFα and MCP-1 expression in
the livers of rats}\selectlanguage{english}

Macrophages are key regulators of fibrosis development(23). The liver
contains macrophages including Kupffer cells and macrophages filtered
from the blood. When activated, they can produce and release many
proinflammatory cytokines, such as TNF\selectlanguage{greek}α, \selectlanguage{english}MIP-2, MIP-1\selectlanguage{greek}α \selectlanguage{english}and MCP-1. These
cytokines may attract other proinflammatory cells and recruit
macrophages themselves to sites of inflammation. As Table 4 shows, the
TNF\selectlanguage{greek}α, \selectlanguage{english}MIP-2 and MCP-1 levels were significantly higher in the livers of
control female rats than in the livers of male rats. BDL or TAA
treatment significantly reduced TNF\selectlanguage{greek}α \selectlanguage{english}and MCP-1 levels and slightly
increased MIP-2 levels; BDL significantly increased the level of MIP-1 \selectlanguage{greek}α
\selectlanguage{english}in female rats, but TAA had no obvious effect on MIP-1\selectlanguage{greek}α \selectlanguage{english}in male rats
(Figure 5).

\selectlanguage{greek}\textbf{BDL significantly increases TGFβ1 expression in female rats}\selectlanguage{english}

PDGF-BB possesses mitogenic(24), differentiation-promoting (25),
chemotactic(26) and angiogenic(27) properties, and is produced by
osteoblasts, platelets and monocytes/macrophages, PDGF-BB and TGF-\selectlanguage{greek}β\selectlanguage{english}1both
activate the HSCs and promote liver fibrosis(28). TGF-\selectlanguage{greek}β\selectlanguage{english}1is ubiquitously
present in both normal cells and transformed cells and almost all cells
have receptors for it(22)\textbf{.} IFN-\selectlanguage{greek}γ \selectlanguage{english}plays critical roles in
promoting pathologic inflammatory processes(29). In Table 5, the levels
of PDGF-BB, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TGF-\selectlanguage{greek}β \selectlanguage{english}1 in the liver of female and male rats
showed no significant difference; BDL or TAA slightly decreased PDGF-BB
and increased in TGF-\selectlanguage{greek}β \selectlanguage{english}1 but had no obvious effect on IFN-\selectlanguage{greek}γ. \selectlanguage{english}The
increase in TGF-\selectlanguage{greek}β \selectlanguage{english}1 in livers of female rats with BDL was significantly
greater than that with TAA treatment (Figure 6).

\textbf{BDL and TAA have similar inhibitory effects on signaling pathway
components except p-p70S6K in the livers of rats}

Many signaling pathway components, such as CREB, c-JNK, NF-\selectlanguage{greek}κ\selectlanguage{english}B, P38MAPK,
ERK1/2, Akt, p70S6K, STAT3 and STAT5, are involved in the occurrence,
development and dissolution of liver fibrosis(30-37). Activation of the
signaling pathways occurs via phosphorylation of these key proteins, so
the levels of phosphorylated protein represent the level of signaling
pathway activation.

As shown as Table 6, p-CREB, p-JNK, p-NF-\selectlanguage{greek}κ\selectlanguage{english}B, p-P38MAPK, p-ERK1/2, p-Akt,
p-p70s6k, p-STAT3 and p-STAT5 expression was significantly higher in the
livers of female rats than those of male rats; BDL significantly
decreased p-NF\selectlanguage{greek}κ\selectlanguage{english}B p-P38MAPK, p-Akt, p-STAT5 expression and increased
p-p70s6k expression in the livers of female rats. TAA significantly
decreased the levels of p-CREB, p-JNK, p-NF\selectlanguage{greek}κ\selectlanguage{english}B, p-Akt, p-p70s6k, p-STAT3
and p-STAT5 in male rats but had no significant effect on the levels of
p-P38MAPK and p-ERK1/2 (Figure7).

\textbf{Discussion}

The TAA- and BDL-induced models of liver fibrosis in rats are good
models for exploring the pathogenesis of liver fibrosis and evaluating
the action of drugs for liver fibrosis and cirrhosis. Here, we found
that BDL for one month significantly increased the ALT, AST, ALP, and
Hyp levels, liver indexes and pathological score of liver fibrosis in
female rats. TAA treatment for 23 weeks has similar effects on the male
rats. However, the level of Hyp in the livers of BDL-treated rats was
higher than that in those of TAA-treated rats, but the pathological
score of liver fibrosis was much lower than that in TAA-treated rats,
which suggested that the composition of liver fibrosis in BDL rats was
different from that in TAA-treated rats, which may be due to the
difference in sex.

Interleukins (IL) including a lot of cytokines which play an important
role in liver injury or fibrosis.IL-1\selectlanguage{greek}α \selectlanguage{english}and IL -6 correlates with
severity of liver diseases (38, 39) while IL-4 levels can be used to
predict advanced fibrosis in chronic hepatitis C (40). IL-1\selectlanguage{greek}β, \selectlanguage{english}Il-6,
IL-9, IL-10, IL-13, IL-18 and L-12 are higher in patients with cirrhosis
(41-47), However, we found that with BDL or TAA treatment, IL-1\selectlanguage{greek}α, \selectlanguage{english}IL- 4,
IL-10, TNF\selectlanguage{greek}α, \selectlanguage{english}MCP-1 and PDGF-BB decreased, but IL-18 and TGF-\selectlanguage{greek}β \selectlanguage{english}1
increased; BDL for a month upregulated IL-1\selectlanguage{greek}β \selectlanguage{english}and MIP-1\selectlanguage{greek}α \selectlanguage{english}in the livers of
female rats, while TAA decreased IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6 and IL-12p70 in the livers
of male rats.

BDL significantly decreased p-NF\selectlanguage{greek}κ\selectlanguage{english}B, p-P38MAPK, p-Akt, and p-STAT5 and
increased p-p70s6k in the livers of female rats. TAA significantly
decreased the levels of p-CREB, p-JNK, p-NF\selectlanguage{greek}κ\selectlanguage{english}B, p-Akt, p-p70s6k, p-STAT3
and p-STAT 5 in male rats but had no significant effect on the levels of
p-P38MAPK and p-ERK1/2.

IL-1 refers to two similar cytokines (IL-1\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β\selectlanguage{english}) that bind to the
same receptor and play an important role in acute and chronic
inflammation. IL-1\selectlanguage{greek}β \selectlanguage{english}is produced by monocytes, macrophages and
neutrophils; it also recruits and activates these cells and induces
local inflammation. In addition, IL-1\selectlanguage{greek}β \selectlanguage{english}can activate HSCs, which
contributes to fibrosis. When IL-1\selectlanguage{greek}β \selectlanguage{english}levels are elevated, depletion of
IL-1R1 ameliorates this fibrotic phenotype in mouse models of liver
fibrosis, and the absence of IL-1 signaling attenuates
thioacetamide-induced liver fibrogenesis in rats. IL-1 may regulate
fibrosis and tissue remodeling by modulating the expression of matrix
metalloproteinases and tissue inhibitors of matrix metalloproteinases.
IL-18 is constitutively expressed in myeloid cells and epithelial cells;
it signals via a heteromeric receptor, increases the production of
nitric oxide (NO) and chemokines, and increases cell adhesion molecules
for leukocyte trafficking. The maturation and secretion of IL-1\selectlanguage{greek}β \selectlanguage{english}and
IL-18 are mediated by inflammatory caspases within inflammasomes
containing the NOD-like receptor (NLR) protein (NLRP) 3. Large amounts
of IL-1\selectlanguage{greek}β \selectlanguage{english}are produced by Kupffer cells, which also express most NLRs.
Inflammasome signaling often induces pyroptosis that allows the passive
release of alarmins, including IL-1\selectlanguage{greek}α. \selectlanguage{english}Inflammasomes may regulate liver
fibrosis directly by mediating inflammasome expression in HSCs or
indirectly by HSC activation via Kupffer cell-derived IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-18.
In addition, depletion of Nlrp3 significantly reduced the expression of
TGF-\selectlanguage{greek}β\selectlanguage{english}1 and collagen-1\selectlanguage{greek}α\selectlanguage{english}1 in carbon tetrachloride-induced or TAA-induced
liver fibrosis mouse models.

p-p70s6k is the active form of p70s6k, which plays an important role in
protein synthesis and cell cycle control. p70s6k is activated by growth
factors and hormones through the phosphatidylinositol 3-kinase
(PI3K)-dependent signaling pathway. IL-1\selectlanguage{greek}β \selectlanguage{english}increases the p-p70s6k protein
level in a time-dependent manner(48) .

Overall, BDL for one month increased the degree of liver fibrosis by
augmenting the production of TGF\selectlanguage{greek}β\selectlanguage{english}1, induced the activation of HSCs by
increasing the production of IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-18 to enhance inflammasome
function and accelerated the formation of the collagenous connective
tissue via the p70s6k signaling pathway. However, a key function of
IL-18 is that it cooperates with IL-12 to induce IFN-\selectlanguage{greek}γ \selectlanguage{english}production from T
helper cells and NK cells, leading to NK cell activation, but BDL
slightly decreased the levels of IL-12p70 and IFN-\selectlanguage{greek}γ.

\selectlanguage{english}However, the level of MIP-1\selectlanguage{greek}α \selectlanguage{english}markedly increased in the livers of BDL
rats. MIP-1\selectlanguage{greek}α \selectlanguage{english}is also known as CCL3 and is strongly expressed on T cells,
macrophages, neutrophils, endothelial cells and HSCs. It may recruit
several cell types, such as T cells, neutrophils and eosinophils, to the
site of inflammation by interacting with its receptors CCR1 and CCR5.
HSCs also express CCR5, which is the target of CCL3 in the liver. CCR1
and CCR5 deficiency can alleviate liver fibrosis from chronic carbon
tetrachloride treatment or BDL.

CREB, JNK, NF\selectlanguage{greek}κ\selectlanguage{english}B, P38MAPK, ERK1/2, Akt, STAT3 and STAT5 are related to
liver injury or fibrosis. Unexpectedly, most of the ILs and
phosphorylated signaling pathway proteins we assessed were decreased by
BDL for one month or by TAA treatment for 23 weeks. Under a microscope,
we found that liver tissue slices from TAA-treated male rats exhibited a
high degree of hyperplasia of connective tissue and scar tissue
formation, and this effect was more serious than that of BDL female
rats. There was no inflammation or necrotic tissue, which may be the
cause of the decrease in inflammation-related signaling pathways in the
two liver fibrosis groups, especially in TAA-treated rats.

\textbf{Conclusion}

The levels of most inflammation-associated cytokines and signaling
pathway components in the livers of normal young female rats are higher
than those in the livers of male rats, and liver fibrosis induced by BDL
in female rats or TAA in male rats is analogous; however, due to the
different degrees of fibrosis, there are some variations in the levels
of inflammation-associated cytokines and signaling pathway components.
As the severity of hepatic fibrosis increases, the scar form and
connective tissue hyperplasia become more obvious, and the levels of
inflammation-associated cytokines and signaling pathways decrease, with
some levels falling even lower than those in normal controls. These data
suggest that the therapeutic strategies for liver fibrosis must change
along with the state of liver fibrosis: anti-inflammatory drugs should
be used at the early stage to prevent liver fibrosis progress, and
improved liver microcirculation drugs should be used at the end stage of
liver fibrosis (liver cirrhosis); this strategy may facilitate the
recruitment of more inflammatory cells or cytokines into liver tissue
activating these enzymes such as matris metalloproteinases to eliminate
the scar structure and reverse liver cirrhosis.

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\textbf{Table 1. Effects of BDL or TAA on the liver fibrosis in rats}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Groups & N & Hydroxyproline (mg\selectlanguage{ngerman}·g) & Degree of liver fibrosis & Degree
of liver fibrosis & Degree of liver fibrosis & Degree of liver fibrosis
& Degree of liver fibrosis & Total scores\tabularnewline
& & & 0 & 1 & 2 & 3 & 4 &\tabularnewline
Female & 10 & 276.1\selectlanguage{ngerman}±17.4 & 10 & 0 & 0 & 0 & 0 & 0\tabularnewline
DBL & 10 & 496.3\selectlanguage{ngerman}±79.8\textsuperscript{**} & 0 & 1 & 7 & 0 & 2 &
23\emph{\textsuperscript{***}}\tabularnewline
Male & 10 & 228.84\selectlanguage{ngerman}±21.80 & 10 & 0 & 0 & 0 & 0 & 0\tabularnewline
TAA & 9 & 376.57\selectlanguage{ngerman}±160.27\textsuperscript{*} & 0 & 0 & 2 & 3 & 4 &
29\textsuperscript{***}\tabularnewline
\bottomrule
\end{longtable}

Compared with the same sex controls,
\emph{\textsuperscript{*}p\textless{}0.05} , \emph{\textsuperscript{**}
p} \textless{}\emph{0.01,\textsuperscript{***} p \textless{}0.001}

Hydroxyproline in the livers of female rats was significantly higher
than that in those of male rats. And liver hydroxyproline level in BDL
female rats was significantly higher but the degree of fibrosis is much
less than the male rats treated with TAA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 1. Effects of BDL or TAA on the liver functions in rats.}

(A) ALT, (B) AST and (C) ALP in Serum and (E) liver index significantly
increased by BDL or TAA. * p\textless{}0.05, ** p \textless{}0.01 and
*** p \textless{}0.001 compared with the same sex control group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 2. Effects of BDL or TAA on histopathology of liver in
rats.}

A, C and E, G are normal pictures of hepatic tissue stained by H\&E or
masson under microscope, while B, D, F, H are liver fibrosis induced by
BDL or TAA, however, the mediastinal form in the liver in TAA rats are
more seriousness than that in BDL rats.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 3. Effects of BDL or TAA on the expression of α-SMA in
the liver.}\selectlanguage{english}

Brown color indicates positive. (A) negligible positive immunostaining
in female normal rat livers; (B) strong \selectlanguage{greek}α-\selectlanguage{english}SMA expression in liver of BDL
rats; (C) negligible positive immunostaining in male normal rat livers;
(D) \selectlanguage{greek}α-\selectlanguage{english}SMA expression markedly increased in TAA rat livers. (\selectlanguage{greek}α-\selectlanguage{english}SMA
immunohistochemistry, haematoxylin counterstain, \selectlanguage{ngerman}×100).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 4. Effects of BDL or TAA on the levels of ILs in livers
in rats}

BDL significantly decrease the levels of (A) IL-1\selectlanguage{greek}α, \selectlanguage{english}(C) IL-4, (E) IL-10
in livers in female rats, while TAA decreased the levels of (A) IL-1\selectlanguage{greek}α,
\selectlanguage{english}(B) IL-1\selectlanguage{greek}β, \selectlanguage{english}(C) IL-4, (D) IL-6, (E) IL-10, and (H) IL-12p70 in livers in
male rats, however, BDL or TAA both significantly increased the levels
of (G) IL-18 in livers in female or male rats. * p\textless{}0.05, ** p
\textless{}0.01 and *** p \textless{}0.001 compared with the same sex
control group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 5. Effects of BDL or TAA on the levels of MIP-1α, MIP-2,
MCP-1and TNF-α in livers in rats.}\selectlanguage{english}

BDL markedly increased the levels of (A) MIP-1\selectlanguage{greek}α \selectlanguage{english}and decreased the level
of TNF\selectlanguage{greek}α \selectlanguage{english}in liver in female rats, while TAA decreased the levels of MCP-1
and TNF\selectlanguage{greek}α\selectlanguage{english}in liver in male rats. * p\textless{}0.05, ** p \textless{}0.01
and *** p \textless{}0.001 compared with the same sex control group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf0/imagf0}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 6. Effects of BDL or TAA on the levels of PDGF-BB, IFN-γ
and TGF-β1 in livers in rats.}\selectlanguage{english}

BDL markedly increased the level of (C) TGF\selectlanguage{greek}β\selectlanguage{english}1 in liver in female rats.
But has no effects on the levels of (A) PDGF-BB and (B) IFN-\selectlanguage{greek}γ,\selectlanguage{english}while TAA
has no the levels of (A) PDGF-BB, (B) IFN-\selectlanguage{greek}γ\selectlanguage{english}or TGF-\selectlanguage{greek}β\selectlanguage{english}1. *
p\textless{}0.05, ** p \textless{}0.01 and *** p \textless{}0.001
compared with the same sex control group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf1/imagf1}

\end{center}
\end{figure}

\textbf{Figure 7. Effects of BDL or TAA on the phosphorylated protein
levels of signaling pathway-related peotein in liver in rats.}

BDL markedly decreased the level of (C) p-NF\selectlanguage{greek}κ\selectlanguage{english}B, (D)p-P38MAPK, (F) p-Akt,
(I) p-STAT5and increased (G) p-70S6K in liver in female rats, and TAA
markedly decreased the level of (A) p-CREB, (B) p-JNK, (C)p-NF\selectlanguage{greek}κ\selectlanguage{english}B, (F)
p-Akt, (G) p-p70S6K, (H) p-STAT3 and (I) p-STAT5 in liver in male rats.
* p\textless{}0.05, ** p \textless{}0.01 and *** p \textless{}0.001
compared with the same sex control group.

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