3.1 PIC Ⅱ significantly alleviates hepatic fibrosis and
inflammation in Mdr2−/− mice
To broadly analyze the potential pharmacological effects of PIC Ⅱ on
hepatic fibrosis, we attempted to use the Mdr2-/- mice
that mimics the immunopathogenesis and symptoms of patients with liver
fibrosis in the clinic (Fig. 1A , left panel ). As shown
in Fig. 1A , right panel and Fig. S1A , there
was little difference in the weight loss, ratio of liver or spleen to
body weight between different groups. We further applied H & E
and
Masson’ s trichrome staining to
evaluate the pathological change before or after PIC Ⅱ administration
and found that the obvious inflammatory infiltration, ECM deposition,
and fibrous scar were observed in the liver
of
Mdr2−/− mice but
were markedly alleviated by different doses of PIC Ⅱ to varying degrees
(Fig. 1B ). Consistently, although it had less effects
on γ-glutamyl transpeptidase (γ-GGT)
(a cell surface enzyme for indicating hepatobiliary injury, Fig.
S1B ), PIC Ⅱ at different concentrations also significantly
downregulated the serum levels of aspartate aminotransferase (AST) and
alanine aminotransferase (ALT, two typical markers for hepatic injury),
total biliary acid (TBA), total bilirubin (TBIL) and alkaline
phosphatase (AKP, three markers for reflecting bile acid accumulation
and liver fibrosis degree) induced by the deficiency of Mdr2
(Fig. 1C and Fig. S1B ). To better figure out the
protective effects and underlying mechanisms of PIC Ⅱ, we further
performed RNA-sequencing analysis of
fibrotic livers in
Mdr2−/− mice and
plotted DEGs implicated in fibrosis-related genes as a heatmap
(Fig. 1D ). A total of 8274 DEGs with P -value <
0.05 was identified between three different groups. In cluster 1, the
expression of genes was downregulated in the Mdr2−/−mice and further decreased in Mdr2−/− + PIC Ⅱ group,
such as Stat3 ,
ribosomal protein S6
(Rps6 ) and small mother against decapentaplegic family member 3
(Smad3 ), which often early increased and sustained in liver
injury. The gene expression in cluster 2 was upregulated in the
Mdr2−/− group and further increased in
Mdr2−/− + PIC Ⅱ group, such as the key genes involved
in inhibiting HSC proliferation and activation like peroxisome
proliferator activated receptor gamma (Pparg ) and Smad7 as
well as collagen degradation related gene including matrix
metallopeptidase 13 (Mmp13 ).
The expression of DEGs in cluster 3 including
cellular communication network
factor 2 (Cnn2 ) as well as collagen formation genes such as
tissue inhibitor of metalloproteinase 1 (Timp1 ) and
collagen type XXIII alpha
1 chain (Col23a1 ) in cluster 4 were remarkably downregulated in
the Mdr2-/- + PIC Ⅱ group when compared to the
Mdr2-/- group (Fig. 1D ). Consistently, the
qPCR results validated the mRNA changes of representative
fibrosis-related genes in the heatmap and showed that PIC Ⅱ mainly
inhibited the expression of the collagen-synthesis related genes
(Fn1 , Acta2 , H19 and Col1a1 ) and positively
supported the collagen-degradation related ways (increased Mmp13and Timp1 ) (Fig. 1E ). Furthermore, the
immunofluorescence staining also confirmed the anti-fibrotic effects of
PIC Ⅱ on the Mdr2-/- mouse model, as illustrated by
the lower distribution of Fibronectin, α-SMA and Collagen1 (Fig.
1F and Fig. S1C ). Of noted, the direct inhibition of PIC Ⅱ on
the protein level of a-SMA and fibronectin in aHSC had not been observed
clearly (Fig. S2A and S2B ), which suggested that PIC Ⅱ
might possess a suppressive effect on aHSCs through another indirect
way.