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\begin{document}

\title{Tectorigenin alleviates intrahepatic cholestasis by inhibiting hepatic
inflammation and bile accumulation via activation of PPARg}



\author[1]{Jiaqing Xiang}%
\author[1]{Guangyan Yang}%
\author[2]{Chuanrui Ma}%
\author[3]{Linlin Wei}%
\author[1]{Han Wu}%
\author[3]{Xiuhua Tao}%
\author[1]{Lingyun Jiang}%
\author[1]{Zhen Liang}%
\author[1]{Lin Kang}%
\author[1]{shu yang}%
\affil[1]{Shenzhen People's Hospital}%
\affil[2]{First Teaching Hospital of Tianjin University of Traditional Chinese Medicine}%
\affil[3]{Jiangxi Academy of Agricultural Sciences}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and purpose: Increasing evidence has shown that human
cholestasis is closely related to hepatic macrophage accumulation and
activation. Research has indicated that peroxisome
proliferator-activated receptor-g (PPARg) activation exerts liver
protection in cholestatic liver disease (CLD), particularly by
ameliorating inflammation and fibrosis, thus limiting disease
progression. However, existing PPARg agonists, such as troglitazone and
rosiglitazone, have significant side effects that impede their clinical
application in the treatment of CLD. In this study, we found that
tectorigenin (TEC) can alleviate intrahepatic cholestasis in mice by
activating PPARg. Experimental approach: Wild-type mice received
intragastric administration of a-naphthylisothiocyanate (ANIT) or were
fed a diet containing 0.1\% 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC) to establish an experimental intrahepatic cholestasis model and
TEC intervention simultaneously, followed by determination of
intrahepatic cholestasis and the involved mechanisms. In addition, PPARg
deficient mice were administered ANIT and/or TEC to determine whether
TEC exerts its liver protection effect by activating PPARg. Key results:
Our results demonstrated that TEC intervention alleviated intrahepatic
cholestasis by inhibiting hepatic macrophage recruitment and activation
as well as promoting the expression of bile transporters through
activating PPARg. Furthermore, our results show that TEC increased bile
salt export pump (Bsep) expression through enhanced PPARg binding to the
Bsep promoter. We also demonstrated that PPARg deficiency blocked the
hepatocyte protective effect of TEC during cholestasis. Conclusions and
implications: In conclusion, TEC reduced hepatic macrophage recruitment
and activation, and enhanced bile acid export by activating PPARg. Taken
together, our results suggest that TEC is a potential drug for the
prevention of CLD.%
\end{abstract}%



\sloppy


\textbf{Lay summary}

Intrahepatic cholestasis includes primary biliary cholangitis and
primary sclerosing cholangitis. It can cause fibrosis, cirrhosis and
eventually liver failure. Effective drugs for PBC and PSC are still
limited and new treatment strategies are urgently needed. This study
defines tectorigenin, an isoflavone, have the potential to alleviate
cholestatic liver disease, which is achieved mainly by relieving liver
inflammation and promoting the secretion of bile in hepatocyte.

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

\end{center}
\end{figure}

TEC alleviate CLD by activating PPAR\selectlanguage{greek}γ, \selectlanguage{english}and its molecular mechanisms are
mainly as follows: 1) restrain macrophage activation and its derived
inflammatory factor, such as IL-1\selectlanguage{greek}β; \selectlanguage{english}2) upregulated the expression of FXR
and LXR as well as FXR-regulated and LXR-regulated genes through
activating PPAR\selectlanguage{greek}γ \selectlanguage{english}to block NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 mediated inhibition of FXR and LXR in
hepatocyte; 3) enhanced the PPAR\selectlanguage{greek}γ \selectlanguage{english}binding to Bsep promoter and increased
its expression.

\textbf{Introduction}

Cholestatic liver disease (CLD) manifests as a multitude of etiological
heterogeneous hepatobiliary disorders, mainly including primary biliary
cholangitis (PBC) and primary sclerosing cholangitis (PSC), in adults.
CLD, which is also called cholestasis, describes a range of conditions
caused by the accumulation of bile acids in the liver, resulting in
hepatocellular necrosis, apoptosis, progressive fibrosis, and even
end-stage liver disease\textsuperscript{{[}1{]}}.

Accumulated evidence has proved that ursodeoxycholic acid (UDCA) can
decrease the progression of PBC\textsuperscript{{[}2{]}}; however,
approximately one-third of patients do not respond to UDCA
treatment\textsuperscript{{[}3{]}}. In 2016, obeticholic acid (OCA), a
farnesoid X receptor (FXR) agonist, was found could decrease serum
alkaline phosphatase (AP) level in CLD patient, was approved by the FDA
for UDCA nonresponders\textsuperscript{{[}4, 5{]}}. In spite of this,
long-term follow-up is needed to confirm the safety and effectiveness of
this novel treatment. At present, there is no clinical evidence that
there is any medical therapy can alter the course of PSC. Current
treatment regimens focus on symptom management and treatment of
cholangitis\textsuperscript{{[}6{]}}. Effective drugs for PBC and PSC
are still limited and new treatment strategies are urgently needed.

Increasing evidence shows that human cholestasis is closely related to
the disorder of microbiome composition\textsuperscript{{[}7, 8{]}},
increased intestinal permeability, enhanced translocation of pathogenic
bacteria and bacterial toxins, such as lipopolysaccharide (LPS) into the
liver\textsuperscript{{[}9, 10{]}}\emph{.} Inflammasomes and
proinflammatory cytokines are then activated due to these
microbe-derived products, which are recognized by the innate immune
system via pathogen recognition receptors (TLRs and
NLRs)\textsuperscript{{[}11{]}}\emph{.}When persistent liver
inflammation is unresolved, the proinflammatory milieu can play a
detrimental role in parenchymal and nonparenchymal liver cells resulting
in fibrosis and ultimately loss of function\textsuperscript{{[}12{]}}.

Macrophages, which are composed of resident tissue macrophages and
monocyte-derived recruited cells, can differentiate into either
classically activated macrophages (a pro-inflammatory phenotype, also
called M1 polarity) or alternatively activated macrophages (M2 polarity)
which express anti-inflammatory cytokines\textsuperscript{{[}13{]}}.
Recent studies have demonstrated that the intestinal microbiome leads to
the process of cholestasis-mediated cell death and inflammation by
activating the mechanisms of the inflammasome in
macrophages\textsuperscript{{[}14{]}}. Additionally, clinical studies
have suggested that in cholestasis patients, the recruitment of
monocytes and macrophages in diseased liver is significantly
increased\textsuperscript{{[}15{]}}. The expression of various monocyte
chemotactic proteins, such as monocyte chemoattractant protein (MCP)-1
is significantly increased in the livers of patients with
cholestasis\textsuperscript{{[}16{]}}. In parallel to these findings,
C--C chemokine receptor type-2 (CCR-2) expressed by liver macrophages is
accompanied by increasing macrophage numbers in the livers of
cholestasis patients\textsuperscript{{[}17{]}}.

Importantly, macrophages in CLD patients are more susceptible to
stimulation signals such as endotoxin (for example, LPS), which have
also been shown to be increased in cholestasis
patients\textsuperscript{{[}18{]}}. In line with the above findings, the
expression and activity of TLR4 (the primary receptor of LPS) are
upregulated on the monocytes of CLD patients, leading to LPS
hyperreactivity and increased production of proinflammatory cytokines
{[}i.e., interleukin (IL)-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and
IL-8{]}\textsuperscript{{[}19{]}}. It is reported that
macrophage-derived IL-1\selectlanguage{greek}β \selectlanguage{english}is a key cytokine activating hepatocyte nuclear
factor \selectlanguage{greek}κ\selectlanguage{english}B (NF\selectlanguage{greek}κ\selectlanguage{english}b), which is the master inflammation regulator. Activation
of NF\selectlanguage{greek}κ\selectlanguage{english}b can also interfere with FXR and liver X receptor (LXR)
signaling, which results in transcriptional suppression of bile and
sterol transporters, finally culminating in
cholestasis\textsuperscript{{[}20{]}}. The above evidence indicates that
hepatic macrophages, namely Kupffer cells (KCs), exert a pivotal role
during the development of CLD.

Traditional Chinese medicines (TCMs) have been demonstrated to be an
important source for potential drug discovery\textsuperscript{{[}21{]}}.
Tectorigenin (TEC), a plant isoflavone, has attracted much attention due
to its multiple activities such as antiproliferation, antiinflammatory
and antioxidant effects. We and others have demonstrated that TEC can
inhibit macrophage activation (M1 polarity) in vivo and in
vitro\textsuperscript{{[}22, 23{]}}. However, the underlying molecular
mechanisms still require further investigation. It is noteworthy that
TEC has been widely reported to provide protective functions in the
liver\textsuperscript{{[}24-26{]}}. This evidence prompted us to
hypothesize that TEC could alleviate CLD by suppressing hepatic
macrophage recruitment and activation. Thus, in the present study, we
investigated whether TEC intervention could improve the development of
CLD in the ANIT-induced and DDC-induced mouse models. We also
investigated the molecular mechanism by which TEC regulates the
polarization of bone marrow-derived macrophages (BMDMs) and primary
mouse KCs.

\textbf{Results}

TEC alleviated ANIT-induced and DDC-induced experimental intrahepatic
cholestasis

A previous study reported that ANIT and DDC treatment could cause
intestinal permeability, leading to the translocation of bacteria or
their products (LPS) from the leaky intestine into the
liver\textsuperscript{{[}14{]}}. Additionally, whilst murine
experimental models may not fully represent human disease, ANIT and
feeding with 0.1\% DDC are well-accepted models of sclerosing
cholangitis relevant to PSC\textsuperscript{{[}27, 28{]}}. Thus, the
hepatoprotective effect of TEC was further confirmed in an experimental
model of ANIT-induced and DDC-induced CLD.

The ANIT-induced mouse model exhibits increased cholestasis-serum
markers and multiple large areas of necrosis, which can be significantly
relieved by TEC treatment (Figure 1A and 1B). In line with these
results, TEC inhibited ANIT-induced apoptotic cell death, as seen by
fewer positive areas on TUNEL staining and reduced caspase-3 activity
after TEC intervention (Figure 1C and 1D). Additionally, TEC alleviated
ANIT-induced inflammation as shown by a significant decrease in the
recruitment of macrophages in the liver (Figure 1E and 1F). Consistent
with this, our results showed that ANIT treatment significantly
increased the content of IL-1\selectlanguage{greek}β \selectlanguage{english}in mouse serum (Figure 1G) and the mRNA
level of inflammatory factors in the liver (Figure 1H). As expected, TEC
intervention reduced the serum level of IL-1\selectlanguage{greek}β \selectlanguage{english}and the expression of
hepatic inflammatory factors.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1. TEC alleviates ANIT-induced intrahepatic
cholestasis.}C57BL/6J mice were pre-treated with TEC for 3 days followed
by ANIT treatment for 48 h. (A) Serum level of AST, ALT, \selectlanguage{greek}γ-\selectlanguage{english}GT, and AP
were measured by ELISA kits, n=6. (B) Representative images of hepatic
HE staining were shown. (C) Representative images of hepatic TUNEL
staining (left) and quantitative statistics (right) are shown in the
figure. (D) The hepatic activity of caspase-3 was determined by
following the kit instructions, n=6. (E-F) E: liver-isolated immune
cells were stained for CD11b and F4/80 and analyzed by flow cytometry,
representative dot plots are shown. F: further quantification of flow
cytometry. (G) ELISA kits were used to detect inflammatory factors
(IL-1\selectlanguage{greek}β\selectlanguage{english}) in serum, n=6. (H) The mRNA levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}CCR2 and MCP-1 were
measured by qRT-PCR, n=6. *p\textless{}0.05. The data represent the mean
\selectlanguage{ngerman}± SD.

Further experiments in mice fed with TEC in a diet containing 0.1\% DDC
for 2 weeks confirmed hepatocyte protection by TEC in cholestatic liver.
DDC-fed mice showed marked liver injury compared to wild-type mice fed
normal chow, as shown by increased AP, aspartate transaminase (AST),
alanine transaminase (ALT) and \selectlanguage{greek}γ-\selectlanguage{english}glutamyltransferase (\selectlanguage{greek}γ-\selectlanguage{english}GT) (Figure 2A).
The results of HE staining and Sirius red staining also supported these
findings, as seen by decreased areas of tissue damage and collagen
deposition in mouse liver after TEC intervention (Figure 2B and 2C).
Flow cytometry analysis showed that the 0.1\% DDC diet increased the
recruitment of macrophages in the liver of mice (Figure 2D and 2E). In
accordance with this result, the mRNA level of inflammatory and
profibrogenesis factors were also dramatically increased in 0.1\%
DDC-fed mice (Figure 2F and 2G). As expected, TEC significantly reduced
hepatic macrophage recruitment, and the expression of inflammatory and
profibrogenesis factors in the liver.

Overall, our results show that TEC treatment attenuated liver injury
induced by ANIT or DDC.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2. TEC protects mice from 0.1\% DDC diet-induced liver
injury and inflammation.} C57BL/6J mice were fed with 0.1\% DDC and/or
TEC for 1 week, and then sacrificed at the end of the experiment. (A)
Serum level of AST, ALT, \selectlanguage{greek}γ-\selectlanguage{english}GT, and AP were measured by ELISA kits, n=6.
(B) Representative images of hepatic HE staining are shown. (C)
Representative images of hepatic Sirius red staining are shown. (D-E) D:
liver-isolated immune cells were stained for CD11b and F4/80 and
analyzed by flow cytometry, representative dot plots are shown. E:
further quantification of flow cytometry. (F-G) Hepatic mRNA levels of
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}Col1\selectlanguage{greek}α\selectlanguage{english}1, TIMP-1 and MMP9 were measured by qRT-PCR, n=5.
*p\textless{}0.05. The data represent the mean \selectlanguage{ngerman}± SD.

TEC rescued bile metabolic dysfunction in mice with intrahepatic
cholestasis induced by ANIT or DDC

We then undertook experiments to verify bile metabolism after TEC
intervention in the CLD experimental model. Consistent with previous
studies\textsuperscript{{[}29{]}}, ANIT treatment alone resulted in the
accumulation of bile acids in the liver and serum of mice, and reduced
the content of bile acids in feces, which suggested that bile flow was
mainly obstructed from the liver to the intestine (Figure 3A). CLD in
mice was associated with reduced hepatic gene expression of canalicular
transporters of bile salts (Bsep encoded by Abcb11), bilirubin (Mrp2,
encoded by Abcc2), and phytosterols (the heterodimer sterolin1 and
sterolin2, encoded by ABCG5 and ABCG8)\textsuperscript{{[}20, 29,
30{]}}. Concomitant with biochemical cholestasis, mRNA expression of
export transporters (Mrp2 and Bsep), phytosterol transporters (ABCG5 and
ABCG8) and uptake transporters (Ntcp, Oatp) were significantly
suppressed in mice with ANIT-induced CLD (Figure 3B). TEC significantly
decreased the bile acid pool size in the liver and serum, and markedly
increased the bile acid content in feces. Similarly, ANIT-induced
inhibition of transporters was reversed after TEC intervention. FXR is
the master regulator of bile acids transporters (Bsep, Mrp2 and Ntcp),
while LXR is an important regulator of ABCG5 and
ABCG8\textsuperscript{{[}20{]}}. Therefore, western blotting analysis
was performed, and the results showed that TEC intervention restored
ANIT-induced decrease in FXR and LXR (Figure 3C).

Additionally, in 0.1\% DDC-treated mice bile acids accumulated in the
liver and serum, but decreased in feces (Figure 3D). Analysis of the
expression of hepatic bile acid and phytosterol export transporters
confirmed the expected reduction of the bile acid uptake transporters
(Ntcp and Oatp), bile acid export transporters (Mrp2 and Bsep) and
phytosterol export transporters (ABCG5 and ABCG8) after 0.1\% DDC
treatment (Figure 3E). Notably, TEC significantly rescued the bile acid
pool size in the liver, serum and feces. In line with this finding, TEC
improved the expression of bile acid uptake transporters (Oatp and
Ntcp), bile acid export transporters (Mrp2 and Bsep) and phytosterol
export transporters (ABCG5 and ABCG8) in 0.1\% DDC fed mice. The
expression of FXR and LXR was determined by western blot analysis, and
the results showed that TEC restored DDC-induced downregulation of FXR
and LXR in mouse liver (Figure 3F).

Taken together, these results showed that TEC attenuated ANIT-induced
and DDC-induced bile metabolic dysfunction, and exerted a protective
effect on CLD.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3. TEC restored ANIT and DDC-induced hepatic bile acid
metabolic dysfunction.} (A-C) Liver, serum and feces samples were
collected from ANIT and/or TEC treatment mice in Figure 1. A: bile acid
pool size in the liver, serum and feces, n=6, was determined by ELISA
kits. B: bile acid uptake (Oatp, Ntcp), bile acid export (Mrp2, Bsep)
and sterol transporters (ABCG5 and ABCG8) genes were determined by
qRT-PCR, n=5. C: western blot analysis was performed to determine the
protein level of FXR and LXR in mouse liver, and quantitative statistics
are shown in the right panel, n=5. (D-F) Liver, serum and feces samples
were collected from DDC and/or TEC treated mice in Figure 2. D: bile
acid pool size in the liver, serum and feces, n=5, was determined by
ELISA kits. E: bile acid uptake (Oatp, Ntcp), bile acid export (Mrp2,
Bsep) and sterol transporters (ABCG5 and ABCG8) genes were determined by
qRT-PCR, n=5. F: western blot analysis was performed to determine the
protein level of FXR and LXR in mouse liver, and quantitative statistics
are shown in the right panel, n=5. *p\textless{}0.05. The data represent
the mean \selectlanguage{ngerman}± SD.

TEC regulates macrophage activation dependent on PPAR\selectlanguage{greek}γ

\selectlanguage{english}Previous studies have shown that TEC is a partial peroxisome
proliferator-activated receptor-\selectlanguage{greek}γ \selectlanguage{english}(PPAR\selectlanguage{greek}γ\selectlanguage{english}) agonist with an
IC\textsubscript{50} value of 13.3 \selectlanguage{greek}µ\selectlanguage{english}M\textsuperscript{{[}31{]}}. PPAR\selectlanguage{greek}γ
\selectlanguage{english}activation inhibits the production of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}in monocytes and
macrophages, and promotes alternative macrophage
activation\textsuperscript{{[}32{]}}.

To confirm the regulation of TEC in KCs, we used primary KCs isolated
from C57BL/6J mice. In vitro, TEC treatment inhibited LPS-induced
activation of KCs, which proved that TEC markedly increased the M2
markers (IL-10, Arg-1, Retnla, CD163 and CD80) and decreased the M1
markers (TNF-\selectlanguage{greek}α, \selectlanguage{english}INF-\selectlanguage{greek}γ, \selectlanguage{english}CCL2, CXCL1 and iNOS) (Figure 4A). Then, GW9662
(PPAR\selectlanguage{greek}γ \selectlanguage{english}inhibitor) was used to confirm that TEC regulation of KCs
polarity was dependent on PPAR\selectlanguage{greek}γ. \selectlanguage{english}Our results showed that TEC treatment
did not further influence macrophage activation (TNF-\selectlanguage{greek}α, \selectlanguage{english}CXCL1, iNOS and
CCL2) and macrophage alternative activation (IL-10, Arg-1, Retnla,
CD206) when treated with GW9662 in the presence of LPS (Figure 4B).
Analogical results were obtained by cotreatment with GW1929 (PPAR\selectlanguage{greek}γ
\selectlanguage{english}agonist) and TEC (Figure S1A and S1B). Importantly, PPAR\selectlanguage{greek}γ \selectlanguage{english}expression
deficiency dramatically suppressed TEC-mediated inhibition of macrophage
activation in the presence of LPS (Figure 4C and 4D).

It is worth noting that BMDMs infiltrated the liver under CLD
conditions. Thus, BMDMs were isolated from mice, and treated with TEC
and/or ILPS. As expected, TEC increased the ratio of CD206/CD80,
decreased M1 marker genes (TNF-\selectlanguage{greek}α, \selectlanguage{english}iNOS, IL-1\selectlanguage{greek}β \selectlanguage{english}and CCL2) and upregulated
the expression of M2 marker genes (IL-10, Arg-1, Retnla and CD163)
(Figure S2A). BMDMs treated with shPPAR\selectlanguage{greek}γ \selectlanguage{english}and TEC supported our findings
that TEC did not inhibit LPS-induced macrophage activation when PPAR\selectlanguage{greek}γ
\selectlanguage{english}was knocked out (Figure S2B).

Accumulated evidence suggests that PPAR\selectlanguage{greek}γ \selectlanguage{english}attenuates inflammation by
suppressing NF\selectlanguage{greek}κ\selectlanguage{english}b activity. Mechanically, PPAR\selectlanguage{greek}γ \selectlanguage{english}is an E3 ligase that
induces the degradation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 to terminate NF\selectlanguage{greek}κ\selectlanguage{english}B signaling
pathway-elicited inflammation\textsuperscript{{[}33{]}}. In light of
this, we further determined whether TEC-regulated macrophage activation
was dependent on the PPAR\selectlanguage{greek}γ/\selectlanguage{english}NF\selectlanguage{greek}κ\selectlanguage{english}b pathway. BAY 11-7085 (NF\selectlanguage{greek}κ\selectlanguage{english}b inhibitor)
and/or TEC were used to treat KCs in the presence of LPS (Figure S1B).
Our results showed that inhibition of NF\selectlanguage{greek}κ\selectlanguage{english}b activity abolished the
TEC-mediated regulation of KCs activation.

Taken together, these results indicate that TEC notably inhibited
LPS-induced activation of macrophages. Moreover, TEC regulated
macrophage activation which was dependent on activating PPAR\selectlanguage{greek}γ.

\selectlanguage{english}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 4. TEC inhibits macrophage activation by activating
PPARγ.} \selectlanguage{english}(A) The primary culture of KCs was treated with LPS (0.1 \selectlanguage{greek}µ\selectlanguage{english}g/mL)
with or without TEC (10 \selectlanguage{greek}µ\selectlanguage{english}M) for 24 h. The mRNA levels of macrophage
activation markers (TNF-\selectlanguage{greek}α, \selectlanguage{english}iNOS, IL-1\selectlanguage{greek}β \selectlanguage{english}and CCL2) and macrophage
alternative activation markers (IL-10, Arg-1, Retnla, CD206 and CD163)
were measured by qRT-PCR, n=5. (B) The primary culture of KCs was
treated with TEC and/or GW9662 (PPAR\selectlanguage{greek}γ \selectlanguage{english}antagonist, 10 \selectlanguage{greek}µ\selectlanguage{english}m) for 24 h in the
presence of LPS. The mRNA level of TNF-\selectlanguage{greek}α, \selectlanguage{english}iNOS, IL-1\selectlanguage{greek}β, \selectlanguage{english}CCL2, IL-10,
Arg-1, Retnla and CD163 was determined by qRT-PCR, n=5. (C-D) KCs
treated with shPPAR\selectlanguage{greek}γ, \selectlanguage{english}TEC and LPS as shown in the figure. C:
representative images of western blot assay. D: the mRNA levels of
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}CCL2, IL-10, Arg-1 and Retnla were detected by qRT-PCR,
n=5. *p\textless{}0.05. The data represent the mean \selectlanguage{ngerman}± SD.

\emph{TEC directly improved the LPS-induced insults on FXR and LXR in
hepatocytes}

The molecular basis for LPS-induced cholestasis has mainly been examined
in hepatocytes and has been attributed to decreased expression of
several transporters important for canalicular bile
formation\textsuperscript{{[}34, 35{]}}. Mechanically, it was previously
reported that NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 can bind to FXR and LXR promoters, inhibit the
expression of FXR and LXR, and lead to the decreased expression of bile
transporters, resulting in the retention of bile acids and phytosterol.
Phytosterol would then further inhibit the activity of
FXR\textsuperscript{{[}36{]}}. Therefore, we investigated whether TEC
could directly suppress LPS-induced activation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 in
hepatocytes. Primary hepatocytes were treated with LPS and/or TEC, and
it was found that TEC reversed LPS-induced NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 activation, as shown
by decreased phosphorylation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 and reduced NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 binding to
the FXR and LXR promoter (Figure 5A-C). In addition, TEC reversed the
LPS-inhibited expression of FXR and LXR, as well as the expression of
Bsep and ABCG5 (Figure 5D). To determine whether TEC regulates the
expression and activity of FXR and LXR via PPAR\selectlanguage{greek}γ, \selectlanguage{english}hepatocytes were
treated with LPS, shPPAR\selectlanguage{greek}γ \selectlanguage{english}and TEC as indicated in Figure 5E. The results
showed that PPAR\selectlanguage{greek}γ \selectlanguage{english}expression deficiency in hepatocytes blocked the
regulatory effects of TEC on NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 activity, the expression of FXR,
Bsep, LXR and ABCG5 (Figure 5E and 5F). Furthermore, when NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 was
inhibited, TEC treatment could not further enhance the expression of FXR
and LXR as well as FXR-regulated and LXR-regulated genes in the presence
of LPS (Figure S3A-C). Finally, FXR or LXR deficiency abolished the
influence of TEC on the expression of Mrp2, ABCG5 and ABCG8, but not
Bsep (Figure S4A-B). Combined with the results of Bsep in Figure 5D and
5F, this suggested that TEC regulated the expression of Bsep, which was
partly dependent on FXR and completely relied on PPAR\selectlanguage{greek}γ.

\selectlanguage{english}TEC directly restored LPS-induced dysfunction of hepatocytes by
activating PPAR\selectlanguage{greek}γ.

\selectlanguage{english}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 5. TEC restored LPS-induced insults on bile transporters
in hepatocytes through PPARγ.} \selectlanguage{english}(A-D) Hepatocytes cultured with LPS
and/or TEC for 24 h. A, B: western blot was used to detect the
phosphorylation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65, n=5. B, C: Chromatin immunoprecipitation
(ChIP) of hepatocytes with specific antibodies for NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 subunits
binding to the promotor of mouse FXR (B) or the promotor of mouse LXR
(C) represented as semiquantitative data (left) and quantitative PCR
data with specific primers (right). D: the mRNA levels of FXR, LXR, Bsep
and ABCG5 were determined by qRT-PCR, n=5. (E and F) Hepatocytes were
transfected with shPPAR\selectlanguage{greek}γ \selectlanguage{english}and/or TEC in the presence of LPS. E:
phosphorylation and protein level of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 was measured by western
blot and quantitative data are shown on the right, n=5. F: qRT-PCR was
used to determine the mRNA expression of FXR, LXR, Bsep and ABCG5, n=5.
*p\textless{}0.05. The data represent the mean \selectlanguage{ngerman}± SD.

TEC promoted PPAR\selectlanguage{greek}γ \selectlanguage{english}binding to the Bsep promoter region and up-regulated
its expression

In vitro studies have found that thiazolidinedione compounds (TZDs) can
increase the expression of Bsep, but the exact molecular mechanism is
still not fully understood\textsuperscript{{[}37{]}}. Consistent with
our current research results, TEC regulated the expression of Bsep
independent of NF\selectlanguage{greek}κ\selectlanguage{english}b activity in hepatocytes (Figure 5D). Western
blotting analysis showed that TEC increased the expression of Bsep, but
not PPAR\selectlanguage{greek}γ \selectlanguage{english}in HepG2 cells (Figure 6A). When HepG2 cells were treated with
TEC and GW1929 or TEC and GW9662, TEC did not further regulate the
expression of Bsep compared with GW1929 or GW9662 treatment alone
(Figure 6B). Further analysis revealed that TEC regulation of Bsep was
completely dependent on PPAR\selectlanguage{greek}γ, \selectlanguage{english}as PPAR\selectlanguage{greek}γ \selectlanguage{english}deficiency blocked the
regulation of Bsep by TEC (Figure 6C). Importantly, the luciferase assay
showed that TEC enhanced the transcriptional activity of PPAR\selectlanguage{greek}γ \selectlanguage{english}in a
dose-dependent manner (Figure 6D).

Thus, we hypothesized that TEC increased the binding of PPAR\selectlanguage{greek}γ \selectlanguage{english}to the
Bsep promoter and promoted its transcription. To gain further insight
into the molecular mechanism of the regulation of Bsep by TEC, a series
of Bsep promoter deletion mutants were cloned into the luciferase
reporter system, and it was found that overexpression of PPAR\selectlanguage{greek}γ \selectlanguage{english}enhanced
Bsep activity only in the Luc1 constructs, suggesting that the PPAR\selectlanguage{greek}γ
\selectlanguage{english}binding site within the Bsep promoter was between the --2948 and --2235
base pairs (bp) (Figure 6E). Chromatin immunoprecipitation (ChIP) assay
was used to confirm that PPAR\selectlanguage{greek}γ \selectlanguage{english}was recruited to the --2948 and --2235 bp
(Figure 6F). The promoter analysis tool JASPAR predicted putative PPAR\selectlanguage{greek}γ
\selectlanguage{english}binding motif sequences (-2560 bp to -2541 bp: ttggtccacagtgacctcca)
based on the PPAR\selectlanguage{greek}γ \selectlanguage{english}consensus sequence outlined (Figure 6G). These
results collectively indicated that TEC treatment increased the binding
of PPAR\selectlanguage{greek}γ \selectlanguage{english}to the Bsep promoter region, thereby upregulating its
expression.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 6. PPARγ positively regulates Bsep transcription through
direct binding to the Bsep promoter.}\selectlanguage{english} (A) HepG2 cells were incubated
with TEC for 24 h. The protein level of Bsep and PPAR\selectlanguage{greek}γ \selectlanguage{english}were detected by
western blotting, n=5. (B) HepG2 cells were incubated with TEC and/or
GW1912 (GW9662) for 24 h, and the mRNA level of Bsep was determined by
qRT-PCR, n=5. (C) HepG2 cells were transfected with shPPAR\selectlanguage{greek}γ \selectlanguage{english}for 24 h,
then treated with TEC for another 24 h, and the mRNA level of Bsep was
determined by qRT-PCR, n=5. (D) HepG2 cells were incubated with various
concentrations of TEC for 24 h, and PPAR\selectlanguage{greek}γ \selectlanguage{english}transcriptional activity was
measured by luciferase assay, n=5. (E) Luciferase reporter assays of a
series of Bsep promoter fragments cloned into luciferase reporter gene
(Luc1 to Luc3) with or without PPAR\selectlanguage{greek}γ-\selectlanguage{english}transfection of HepG2 cells, n=5.
(F) Chromatin immunoprecipitation (ChIP) assay--qPCR analysis in the
region -3218 bp/-2178 bp of the Bsep promoter in PPAR\selectlanguage{greek}γ-\selectlanguage{english}transfected HepG2
cells with or without TEC treatment. IgG, immunoglobulin G, n=5. (G)
Depiction of the predicted PPAR\selectlanguage{greek}γ \selectlanguage{english}binding sites at -2560 to -2541 base
pairs (bp) in the human Bsep gene. *p\textless{}0.05. The data represent
the mean \selectlanguage{ngerman}± SD.

PPAR\selectlanguage{greek}γ \selectlanguage{english}is essential for TEC to alleviate ANIT-induced experimental
intrahepatic cholestasis

To further confirm the physiological roles of PPAR\selectlanguage{greek}γ \selectlanguage{english}in TEC-mediated
hepatic protection in vivo, Ad-shPPAR\selectlanguage{greek}γ \selectlanguage{english}was injected into C57BL/6J mice
via the tail vein. Next, the mice were treated with ANIT and/or TEC as
described in the experimental method. As expected, TEC did not exert
hepatic protection in ANIT-induced CLD of PPAR\selectlanguage{greek}γ \selectlanguage{english}deficient mice, which
was supported by the fact that there was no obvious difference in AST,
ALT, AP and necrosis area after TEC treatment in PPAR\selectlanguage{greek}γ-\selectlanguage{english}deficient mice
(Figure 7A and 7B). Quantification of the bile acid pool size showed
that there was no significant influence on the content of bile acid in
the liver, serum and feces of PPAR\selectlanguage{greek}γ-\selectlanguage{english}deficient mice after TEC treatment
(Figure 7C). ANIT treatment alone markedly promoted macrophage
recruitment compared with NC, as evidenced by a significant increase in
F4/80 mRNA level in the liver (Figure 7D). The mRNA levels of
proinflammatory factors (Figure 7E) and profibrogenesis factors (Figure
7F) also supported these findings. PPAR\selectlanguage{greek}γ \selectlanguage{english}deficiency aggravated
inflammation in mouse liver, as shown by increased F4/80,
proinflammatory factors and profibrogenesis factors compared with ANIT
treatment alone, while TEC did not significantly decrease these genes in
PPAR\selectlanguage{greek}γ \selectlanguage{english}deficient mice. Furthermore, the expression of FXR and LXR in
mouse liver was analyzed, and the results showed that TEC did not
enhance the expression of FXR, LXR and bile transporters in
PPAR\selectlanguage{greek}γ-\selectlanguage{english}deficient mice (Figure 7G and 7H). Taken together, the lack of
PPAR\selectlanguage{greek}γ \selectlanguage{english}expression abolished hepatic protection by TEC.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\selectlanguage{greek}\textbf{Figure 7. TEC treatment protects against ANIT-induced
intrahepatic cholestasis dependent on activating PPARγ.} \selectlanguage{english}C57BL/6J mice
were treated as previously mentioned (Figure 1). (A) The serum levels of
aspartate transaminase (AST), alanine transaminase (ALT) and AP were
determined by an ELISA kit, n=6. (B) Representative sections stained
with HE were used to estimate the area of necrosis. (C) Bile acid pool
size was determined by ELISA kits in the liver, serum and feces, n=6.
(D-F) The mRNA level of macrophage recruitment (F4/80), inflammatory
factors (TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}MCP-1 and CCR2) and fibrosis-related genes
(TGF\selectlanguage{greek}β\selectlanguage{english}1, MMP9, TIMP-1 and col1\selectlanguage{greek}α\selectlanguage{english}1) were measured by qRT-PCR, n=6. (G)
qRT-PCR analysis was used to determine the mRNA level of Oatp, Ntcp,
Mrp2, Bsep, ABCG5 and ABCG8, n=6. (H) Western blot analysis was used to
confirm the expression of PPAR\selectlanguage{greek}γ, \selectlanguage{english}FXR and LXR, n=5. *p\textless{}0.05.
The data represent the mean \selectlanguage{ngerman}± SD.

\textbf{Discussion}

Various studies have demonstrated that PPAR\selectlanguage{greek}γ \selectlanguage{english}agonists have potential in
the treatment of CLD. Immune modulation by PPAR\selectlanguage{greek}γ \selectlanguage{english}ligands may be of
therapeutic benefit in reducing biliary inflammation in
PBC\textsuperscript{{[}38{]}}. In addition, PPAR\selectlanguage{greek}γ \selectlanguage{english}inhibits the
transcriptional activation of inflammatory response genes and represses
cellular toll-like receptor signaling in inflammatory cells as well as
in cholangiocytes\textsuperscript{{[}39{]}}. Importantly, recent studies
have shown that rosiglitazone improve intrahepatic cholestasis and
cholestasis-associated dyslipidemia induced by
ANIT\textsuperscript{{[}29{]}}. These data indicated that PPAR\selectlanguage{greek}γ
\selectlanguage{english}activation may be an effective strategy for the treatment of CLD,
especially for the improvement of liver fibrosis and inflammation,
thereby limiting disease progression. Despite this, troglitazone, a
PPAR\selectlanguage{greek}γ \selectlanguage{english}ligand, was withdrawn from the market due to hepatotoxicity and no
experimental or clinical data on other glitazones are
available\textsuperscript{{[}40{]}}. Although this evidence suggests the
feasibility of PPAR\selectlanguage{greek}γ \selectlanguage{english}agonists as a therapeutic strategy for CLD, new
agonists still need to be developed.

In this study, we demonstrated that TEC, a partial PPAR\selectlanguage{greek}γ
\selectlanguage{english}agonist\textsuperscript{{[}31{]}}, can alleviate cholestasis in an
experimental mouse model (Figure 1 and 2) without obvious side effects.
Importantly, we confirmed that TEC alleviation of ANIT-induced liver
injury was dependent on PPAR\selectlanguage{greek}γ \selectlanguage{english}to reduce the recruitment and activation
of macrophages and enhance bile transporter expression in hepatocytes
(Figure 7). Taken together, we found that TEC may be a potential
therapeutic strategy for the treatment of CLD as a PPAR\selectlanguage{greek}γ \selectlanguage{english}agonist.

It was shown in vitro that TEC inhibited LPS-induced macrophage
activation as well as LPS-induced hepatocyte dysfunction via the
PPAR\selectlanguage{greek}γ/\selectlanguage{english}NF\selectlanguage{greek}κ\selectlanguage{english}b pathway (Figure 4 and 5). In accordance with our findings,
multiple studies have suggested that activation of NF\selectlanguage{greek}κ\selectlanguage{english}b plays an
important role in the development of CLD\textsuperscript{{[}20, 41{]}}.
Genetic or pharmacological inhibition of NF\selectlanguage{greek}κ\selectlanguage{english}b prevented
cholestasis-induced liver damage in various experimental
mice\textsuperscript{{[}42, 43{]}}. However, inhibition of NF\selectlanguage{greek}κ\selectlanguage{english}b was
found to lead to an increase in hepatocyte apoptosis after bile duct
ligation (BDL)\textsuperscript{{[}44{]}}. Additionally, IKK1 and IKK2
are I\selectlanguage{greek}κ\selectlanguage{english}B kinases which are important for NF\selectlanguage{greek}κ\selectlanguage{english}b activation and genetic
ablation of I\selectlanguage{greek}κ\selectlanguage{english}B kinases could lead to inflammatory damage to portal bile
ducts\textsuperscript{{[}45{]}}. Therefore, appropriate targets or the
identification of drugs that either exert only a moderate effect on NF\selectlanguage{greek}κ\selectlanguage{english}b
activity or that can be specifically delivered to nonparenchymal cells
are essential to avoid the increase in liver injury associated with
complete NF\selectlanguage{greek}κ\selectlanguage{english}b blockade in hepatocytes\textsuperscript{{[}46{]}}.

Interestingly, PPAR\selectlanguage{greek}γ \selectlanguage{english}is highly expressed in macrophages where it has an
important role in immune modulation, while PPAR\selectlanguage{greek}γ \selectlanguage{english}expression is
relatively low in hepatocytes under normal physiological
conditions\textsuperscript{{[}47, 48{]}}. Our results showed that TEC
treatment almost blocked the phosphorylation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 induced by LPS
in KCs (Figure S1C) and BMDMs (Figure S2D). The phosphorylation of
NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 was still 1.78-fold higher than that in control hepatocytes
after TEC treatment in the presence of LPS (Figure 5A). An in vivo study
also supported these findings, where TEC decreased rather than increased
the apoptosis of hepatocytes in ANIT-induced model mice, as shown by the
down-regulation of the positive area of TUNEL staining and reduced
caspase-3 activity (Figure 1C and D). Our results suggest that TEC had a
much stronger inhibitory effect on NF\selectlanguage{greek}κ\selectlanguage{english}b in macrophages compared with
hepatocytes which could be attributed to the different levels of PPAR\selectlanguage{greek}γ
\selectlanguage{english}expression in KCs and hepatocytes. Additionally, NF\selectlanguage{greek}κ\selectlanguage{english}b inhibitor (BAY
11-7085) treatment at the same dose as TEC markedly decreased the
phosphorylation of NF\selectlanguage{greek}κ\selectlanguage{english}b-p65 induced by LPS in hepatocytes, to an even
lower level than that in the control group (Figure S3A). Collectively,
this dose of TEC had a stronger inhibitory effect on NF\selectlanguage{greek}κ\selectlanguage{english}b activation in
macrophages compared with hepatocytes, and avoided liver injury induced
by complete NF\selectlanguage{greek}κ\selectlanguage{english}b blockade in hepatocytes.

Bsep, encoded by the gene ABCB11, is a member of the adenosine
triphosphate (ATP)-binding cassette (ABC) transporters. It is mainly
expressed on hepatocyte canalicular membranes and is basically
responsible for the secretion of bile acids, and it deficiency may
result in progressive familial intrahepatic cholestasis type
2\textsuperscript{{[}49{]}}. De novo or retargeted canalicular
expression of Bsep has been confirmed to play an important role in bile
acid canalicular export in the treatment of
cholestasis\textsuperscript{{[}50, 51{]}}. Previous studies have shown
that troglitazone can induce intrahepatic cholestasis by increasing
serum bile salt concentrations and inhibiting Bsep expression in rat
liver\textsuperscript{{[}40, 52{]}}. In contrast, another study showed
that troglitazone, but not rosiglitazone or pioglitazone, regulated the
expression of the FXR target gene Bsep\textsuperscript{{[}37{]}}. In
summary, this evidence could support further investigation of the
relationship between Bsep and TEC. According to our findings, TEC
promoted the binding of PPAR\selectlanguage{greek}γ \selectlanguage{english}and Bsep promoter regions and promoted
their expression (Figure 5). As TEC directly increased the expression of
Bsep, this may be another molecular mechanism of TEC in the treatment of
CLD.

Although we found that TEC (50 mpk) significantly alleviated liver
injury in ANIT and DDC-induced CLD without significant side effects,
additional cholestatic models and different doses are still needed to
verify the efficacy and toxicity of TEC. In conclusion, we have
demonstrated that TEC exert liver protection in a PPAR\selectlanguage{greek}γ-\selectlanguage{english}dependent
manner, which in turn inhibit macrophage activation and hepatocyte
dysfunction through restrain NF\selectlanguage{greek}κ\selectlanguage{english}b activation as well as enhance Bsep
expression, thus alleviated intrahepatic cholestasis.



\textbf{Materials and methods}

\textbf{Reagents}

Antibodies for FXR (Cat\#: ab129089), LXR (Cat\#: ab176323), GAPDH
(Cat\#: ab181602), PPAR\selectlanguage{greek}γ\selectlanguage{english}(Cat\#: ab178860), p-NF\selectlanguage{greek}κ\selectlanguage{english}b/P65 (Cat\#: ab76302),
NF\selectlanguage{greek}κ\selectlanguage{english}b/P65 (Cat\#: ab16502), and Bsep (Cat\#: ab155421) were obtained from
Abcam (Cvambridge, MA). Reagents of Tectorigenin (TEC) (Cat\#:
HY-N0792), GW9662 (Cat\#: HY-16578), GW1929 (Cat\#: HY-15655),
BAY11-7085 (Cat\#: HY-10257) were obtained from MedChemExpress (Monmouth
Junction, NJ), and Lipopolysaccharides (LPS) (Cat\#: L2630) and
alpha-naphtylisocyanate (ANIT) were obtained from Sigma-Aldrich
(Shanghai, China). Mouse AST ELISA Kit (Aspartate Aminotransferase)
(Cat\#: ab263882), ALT Assay Kit (Cat\#: ab241035), Caspase-3 Activity
Assay Kit (Cat\#: ab252897), AP Assay Kit (Cat\#: ab267583), and Gamma
Glutamyl Transferase (\selectlanguage{greek}γ-\selectlanguage{english}GT) Assay Kit (Cat\#: ab241029) were obtained
from Abcam (Cambridge, MA). Mouse IL-1\selectlanguage{greek}β \selectlanguage{english}ELISA KIT (Cat\#: SEKM-0002) was
obtained from Solarbio \& Technology Co, Ltd (Beijing, China).
Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit (Cat\#: G1501),
Sirius Red dyestuff (Cat\#:G1018), and HE dyestuff (Cat\#: G1005) were
obtained from servicebio (Wuhan, China). FXR shRNA(m) (Cat\#:
sc-155894), LXR\selectlanguage{greek}α \selectlanguage{english}shRNA(m) (Cat\#: sc-38829), PPAR\selectlanguage{greek}γ \selectlanguage{english}shRNA(h) (Cat\#:
sc-29455-V), and PPAR\selectlanguage{greek}γ \selectlanguage{english}shRNA(m) (Cat\#: sc-29456) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were
obtained from Sigma-Aldrich except where indicated.

\textbf{In vivo studies with animals}

All animal care and experimental protocols for in vivo studies conformed
to the Guide for the Care and Use of Laboratory Animals published by the
NIH (NIH publication no. 85-23, revised 1996) and approved by the Ethics
Committee of the Second Clinical Medical College of Jinan University,
Shenzhen People's Hospital. C57BL/6J wild type mice (8 weeks old) were
purchased from the Animal Center of Nanjing University (Nanjing,
Jiangsu, China). These mice were maintained at the Animal Center of
Shenzhen People's Hospital with free access to food and water. Based on
the previous study, the dose of TEC used to mice was converted as 75
mg/day/kg body weight (mpk). Cholestasis was induced was induced in
C57BL/6J mice by administration of ANIT for 48 hr by oral gavage or fed
with a diet containing 1\% 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC) for 2 weeks. C57BL/6J mice were treated either with vehicle (corn
oil) or ANIT (100mg/kg) by oral gavage, while a subset of C57BL/6J mice
were treated with TEC (75 mpk) for 5 days and another subset of C57BL/6J
mice treated with TEC by oral gavage 3 days before, at the time of the
administration of ANIT, and 24h after. During the DDC feeding, TEC (75
mpk) was administered daily after DDC feeding for one week. All ANIT
treated animals were sacrificed 48 hr after gavage, while all 1\%DDC
treated mice 2 weeks after feeding. For constructed PPAR\selectlanguage{greek}γ \selectlanguage{english}knockdown
mice, mice were injected i.v. through the tail vein with control
adenovirus expressing shorthairpin (sh)RNA against luciferase
(Ad-shCtrl) or adenovirus expressing shRNA against PPAR\selectlanguage{greek}γ \selectlanguage{english}(Ad-shPPAR\selectlanguage{greek}γ\selectlanguage{english})
(0.5--1.5 \selectlanguage{ngerman}× 10\textsuperscript{9} active viral particles in 200 \selectlanguage{greek}μ\selectlanguage{english}l
saline {[}54 mmol/l NaCl{]}). At 2 weeks after infection, mice performed
experiments as mentioned before. Extraction and determination of total
bile acids (TBA) in mouse liver and intestine was performed as
previously reported\textsuperscript{{[}29{]}}.

\textbf{Cell Culture}

Primary KCs were prepared from the livers of 8- to 9-week-old male
C57BL/6 mice and cultured in 10\% FBS RPMI 1640 medium .A single-cell
suspension form livers was prepared as described
previously\textsuperscript{{[}53{]}}. BMDMs were isolated from femoral
and tibia bone marrow and then cultured in RPMI1640 medium(10\%FBS)
with 100 ng/mL M-CSF (Peprotech) for 5 days to fully differentiate
macrophages. Primary mouse hepatocytes were obtained from the livers of
male C57BL/6J mice (8 weeks of age) and cultured as previously
reported\textsuperscript{{[}54{]}}. HepG2 cells, a human hepatic cell
line, were purchased from ATCC (Manassas, VA, USA, RRID: CVCL 0027) and
cultured in DMEM medium containing 10\% fetal bovine serum (FBS), 50
mg/mL penicillin/streptomycin and 2 mmol/L glutamine. Cells
(\textasciitilde{}90\% confluence) received treatment in serum-free
medium.

\textbf{Quantitative real-time polymerase chain reaction (qRT--PCR),
Western blotting, ChIP and ELISA}

qRT--PCR was performed using the ABI
StepOnePlus\textsuperscript{TM}Real-time PCR system (Applied Biosystem)
with specific primers (Table 1). The relative mRNA level of target genes
was analyzed using the equation 2--\selectlanguage{greek}Δ\selectlanguage{english}Ct (\selectlanguage{greek}Δ\selectlanguage{english}Ct = Ct of the target gene --
Ct of \selectlanguage{greek}β-\selectlanguage{english}actin) and normalized using the level detected in the control
group as 1. Western blotting experiments were performed using antibodies
for FXR, LXR, and PPAR\selectlanguage{greek}γ \selectlanguage{english}etc., as described
previously\textsuperscript{{[}22{]}}. ChIP was performed as
described\textsuperscript{{[}21{]}}. ChIP was done using specifc
antibodies for NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 (Abcam, cat\# ab16502) or PPAR\selectlanguage{greek}γ\selectlanguage{english}(Cat\#:
ab178860), and amplification of promoter sequences from the Nr1h3
(LXR\selectlanguage{greek}α\selectlanguage{english}), Nr1h4 (FXR) and Bsep genes using specific primer sets and
subsequent PCR; After elution and purification, the chromatin DNA was
subjected to real time PCR analysis with primers, LXR\selectlanguage{greek}α: \selectlanguage{english}forward: 5'-
aaggaagctcaggcacaaaa -3' and reverse: 5'- gaggctgtgcttgtgaaaca -3', FXR:
forward: 5'- ccactggagatccaaaagga -3' and reverse: 5'-
aatctatgcaaagcgctggt -3', Bsep primer sequences: forward: 5'-
tccaaattggtccacagtga -3' and reverse: 5'- agcagcagcctcctcattac -3'. The
levels of level of AST, ALT, \selectlanguage{greek}γ-\selectlanguage{english}GT, and AP etc., were measured using a
commercial ELISA kit (Abcam) according to the manufacturer's protocol.

\textbf{Table 1. The sequences of primers for qRT-PCR analysis}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene & Forward & Backward\tabularnewline
\midrule
\endhead
\selectlanguage{greek}\emph{mTNF-α} & GACGTGGAACTGGCAGAAGAG &
TTGGTGGTTTGTGAGTGTGAG\tabularnewline
\emph{mCCR2} & ATCCACGGCATACTATCAACATC &
CAAGGCTCACCATCATCGTAG\tabularnewline
\emph{mMCP-1} & TTAAAAACCTGGATCGGAACCAA &
GCATTAGCTTCAGATTTACGGGT\tabularnewline
\emph{mCol1a1} & GCTCCTCTTAGGGGCCACT &
CCACGTCTCACCATTGGGG\tabularnewline
\emph{mTIMP-1} & GCAACTCGGACCTGGTCATAA &
CGGCCCGTGATGAGAAACT\tabularnewline
\emph{mMMP9} & CTGGACAGCCAGACACTAAAG &
CTCGCGGCAAGTCTTCAGAG\tabularnewline
\emph{mOatp} & GGGAACATGCTTCGTGGGATA &
GGAGTTATGCGGACACTTCTC\tabularnewline
\emph{mNtcp} & CAAACCTCAGAAGGACCAAACA &
GTAGGAGGATTATTCCCGTTGTG\tabularnewline
\emph{mMrp2} & GTGTGGATTCCCTTGGGCTTT &
CACAACGAACACCTGCTTGG\tabularnewline
\emph{mBsep} \emph{hBsep} & TCTGACTCAGTGATTCTTCGCA GCCGCAGCTCGTCAGATAC &
CCCATAAACATCAGCCAGTTGT GAATTGCAGTCAAACCACCCTAT\tabularnewline
\emph{mABCG5} & AGGGCCTCACATCAACAGAG &
GCTGACGCTGTAGGACACAT\tabularnewline
\emph{mABCG8} & CTGTGGAATGGGACTGTACTTC &
GTTGGACTGACCACTGTAGGT\tabularnewline
\emph{miNOS} & GTTCTCAGCCCAACAATACAAGA &
GTGGACGGGTCGATGTCAC\tabularnewline
\selectlanguage{greek}\emph{mIL-1β} & GCAACTGTTCCTGAACTCAACT &
ATCTTTTGGGGTCCGTCAACT\tabularnewline
\emph{mCCL2} & TTAAAAACCTGGATCGGAACCAA &
GCATTAGCTTCAGATTTACGGGT\tabularnewline
\emph{mIL10} & GCTCTTACTGACTGGCATGAG &
CGCAGCTCTAGGAGCATGTG\tabularnewline
\emph{mArg1} & CTCCAAGCCAAAGTCCTTAGAG &
AGGAGCTGTCATTAGGGACATC\tabularnewline
\emph{mRetnla} & CCAATCCAGCTAACTATCCCTCC &
ACCCAGTAGCAGTCATCCCA\tabularnewline
\emph{mCD206} & CTCTGTTCAGCTATTGGACGC &
CGGAATTTCTGGGATTCAGCTTC\tabularnewline
\emph{mCD163} & ATGGGTGGACACAGAATGGTT &
CAGGAGCGTTAGTGACAGCAG\tabularnewline
\emph{mFXR} & GCTTGATGTGCTACAAAAGCTG &
CGTGGTGATGGTTGAATGTCC\tabularnewline
\emph{mLXR} & ATGTCTTCCCCCACAAGTTCT &
GACCACGATGTAGGCAGAGC\tabularnewline
\emph{mF4/80} & TGACTCACCTTGTGGTCCTAA &
CTTCCCAGAATCCAGTCTTTCC\tabularnewline
\selectlanguage{greek}\emph{mTGFβ1}\selectlanguage{english} & CTCCCGTGGCTTCTAGTGC &
GCCTTAGTTTGGACAGGATCTG\tabularnewline
\bottomrule
\end{longtable}

\textbf{Flow cytometry}

KCs were incubated with anti-CD16/CD32 (BD 553141) FC receptor for 5 min
at RT. And then cells were labeled with the following antibody at
indicate dilutions. CD45-APC cy7(BD 557659),CD11b-FITC(BD
557391),F4/80-PE(BD 565410),after the 30 minute incubationat 4. KCs
were washed and fixed in stain buffer FBS (BD 554656). Finally, analyses
were performed on a Beckman DXFLEX Flow Cytometer.

\textbf{Dual luciferase reporter gene assay}

The full-length mouse Bsep gene was amplified by PCR from HepG2 cDNA.
The mouse Bsep gene promoter (-3218 to -198 bp) was amplified by PCR
using mouse genomic DNA and inserted into a pGL3-basic luciferase
reporter vector (-3218 Luc). A series of 5' truncated constructs of the
Bsep gene promoter (-3218 Luc, -2178 Luc, -1363 Luc) were prepared by
PCR using -3218 Luc as a template. All constructs were confirmed by DNA
sequencing analysis. For dual luciferase reporter gene assays, HepG2
were transfected with corresponding plasmids, as well as renilla
luciferase. Firefly and renilla luciferase activities were then measured
by a dual-luciferase reporter gene system (Promega, Madison, WI, USA).

\textbf{Histological analysis}

The mice tissues were preserved with 4\% paraformaldehyde solution,
dehydrated and embedded in paraffin.The sections (4 \selectlanguage{greek}µ\selectlanguage{english}m) were used for
histological analysis. HE, Sirus red and TUNEL staining performed as
standard instructions. Positive cells were morphometrically quantified
with image processing software (ImageJ).

\textbf{Data analysis}

All the data were generated from at least 3 independent experiments. All
the raw data were initially subjected to a normal distribution anal
analysis with SPSS software (1-sample K-S of nonparametric test). The
data in normal distribution were conducted the parametric statistic,
post hoc test of 1-way analysis of variance.

\textbf{Abbreviations}

ALT, alanine transaminase; ANIT, \selectlanguage{greek}α-\selectlanguage{english}naphthylisothiocyanate; AP, alkaline
phosphatase;AST, aspartate transaminase; ATP,adenosine triphosphate ;
BDL, bile duct ligation; BMDMs, bone marrow-derived macrophages; Bsep,
bile salt export pump; CCR-2, C--C chemokine receptor type-2; CLD,
cholestatic liver disease; DDC,
3,5-diethoxycarbonyl-1,4-dihydrocollidine; FXR, farnesoid X receptor;
\selectlanguage{greek}γ-\selectlanguage{english}GT, \selectlanguage{greek}γ-\selectlanguage{english}glutamyltransferase; IL-1\selectlanguage{greek}β, \selectlanguage{english}interleukin 1 beta; KCs, Kupffer
cells; LPS, lipopolysaccharide; LXR, liver X receptor; MCP, monocyte
chemoattractant protein; NF\selectlanguage{greek}κ\selectlanguage{english}b, nuclear factor \selectlanguage{greek}κ\selectlanguage{english}B; OCA, obeticholic acid;
PBC, primary biliary cholangitis; PPAR\selectlanguage{greek}γ, \selectlanguage{english}peroxisome
proliferator-activated receptor-\selectlanguage{greek}γ; \selectlanguage{english}PSC, primary sclerosing cholangitis;
TBA, total bile acids; TCMs, traditional chinese medicines; TEC,
tectorigenin; TZDs,thiazolidinedione compounds; UDCA, ursodeoxycholic
acid.

\textbf{Acknowledgments}

This work was supported by grants from Natural Science Foundation of
Tianjin (19JCQNJC12600); Research project of Tianjin education
commission (2019KJ044).

\textbf{Contributions}

JX, GY and CM contributed equally to this work. JX and CM performed in
vivo experiments; GY, HW and LJ completed molecular biological
experiments and biochemical assays; LK, ZL, LW and XT edited the
manuscript; SY designed experiments, interpreted results and wrote the
manuscript.

\#: These authors make equal contribution to this work.

\textbf{Competing interests}

All the authors state no conflict of interest.

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