Discussion
Various studies have demonstrated that PPARγ agonists have potential in
the treatment of CLD. Immune modulation by PPARγ ligands may be of
therapeutic benefit in reducing biliary inflammation in
PBC[38]. In
addition, PPARγ inhibits the transcriptional activation of inflammatory
response genes and represses cellular toll-like receptor signaling in
inflammatory cells as well as in
cholangiocytes[39].
Importantly, recent studies have shown that rosiglitazone improve
intrahepatic cholestasis and cholestasis-associated dyslipidemia induced
by ANIT[29]. These
data indicated that PPARγ 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troglitazone, a PPARγ ligand, was withdrawn from the market due to
hepatotoxicity and no experimental or clinical data on other glitazones
are available[40].
Although this evidence suggests the feasibility of PPARγ agonists as a
therapeutic strategy for CLD, new agonists still need to be developed.
In this study, we demonstrated that TEC, a partial PPARγ
agonist[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 PPARγ 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 PPARγ agonist.
It was shown in vitro that TEC inhibited LPS-induced macrophage
activation as well as LPS-induced hepatocyte dysfunction via the
PPARγ/NFκb pathway (Figure 4 and 5). In accordance with our findings,
multiple studies have suggested that activation of NFκb plays an
important role in the development of
CLD[20,
41]. Genetic or pharmacological
inhibition of NFκb prevented cholestasis-induced liver damage in various
experimental mice[42,
43]. However, inhibition of NFκb was
found to lead to an increase in hepatocyte apoptosis after bile duct
ligation (BDL)[44].
Additionally, IKK1 and IKK2 are IκB kinases which are important for NFκb
activation and genetic ablation of IκB kinases could lead to
inflammatory damage to portal bile
ducts[45].
Therefore, appropriate targets or the identification of drugs that
either exert only a moderate effect on NFκb activity or that can be
specifically delivered to nonparenchymal cells are essential to avoid
the increase in liver injury associated with complete NFκb blockade in
hepatocytes[46].
Interestingly, PPARγ is highly expressed in macrophages where it has an
important role in immune modulation, while PPARγ expression is
relatively low in hepatocytes under normal physiological
conditions[47,
48]. Our results showed that TEC
treatment almost blocked the phosphorylation of NFκb-p65 induced by LPS
in KCs (Figure S1C) and BMDMs (Figure S2D). The phosphorylation of
NFκb-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 NFκb in macrophages compared with
hepatocytes which could be attributed to the different levels of PPARγ
expression in KCs and hepatocytes. Additionally, NFκb inhibitor (BAY
11-7085) treatment at the same dose as TEC markedly decreased the
phosphorylation of NFκb-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 NFκb activation in
macrophages compared with hepatocytes, and avoided liver injury induced
by complete NFκb 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[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[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[40,
52]. In contrast, another study showed
that troglitazone, but not rosiglitazone or pioglitazone, regulated the
expression of the FXR target gene
Bsep[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 PPARγ 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 PPARγ-dependent
manner, which in turn inhibit macrophage activation and hepatocyte
dysfunction through restrain NFκb activation as well as enhance Bsep
expression, thus alleviated intrahepatic cholestasis.