Figure Legend
Figure 1: Hepato-protective effects of MDP on mice with
CCl4-induced liver fibrosis (A): The chemical structure of MDP. (B):
Representative micrographs of haematoxylin and eosin (HE) stained,
Masson stained and picrosirius red stained histological sections of
liver fibrosis tissues. (C) and (D): α-SMA, Col. I, p21 and p16
expression in liver fibrosis tissues were detected by
immunohistochemistry. (E): The serum level of ALT, AST and TBIL was
measured as described in Materials and methods. Each group consists of 6
rats. Data are shown as the mean ± SD (n=3) of one representative
experiment. ∗p < 0.05, ∗∗p < 0.01
versus normal groups/CCl4 groups.
Figure 2: MDP promoted the senescence in vivo and in vitro (A)
and (B): The expression of α-SMA, Col. I, p16 and p21 in liver tissues
with fibrosis was detected by Western Blot. (C): MTT assay was performed
to assess the effects of different concentrations of MDP on LX-2 cells
proliferation. (D): α-SMA, Col. I, p21 and p16 expression in liver
fibrosis tissues were detected by Western Blot. (E): MDP‐induced
senescence as indicated by SA‐β‐Gal activities. Representative images of
SA‐β‐Gal staining are presented. Data are shown as the mean ± SD (n=3)
of one representative experiment. ∗p < 0.05, ∗∗p< 0.01 versus normal groups/CCl4 groups/TGF-β1
groups.
Figure 3: MDP promoted the senescence in TGF-β1-induced LX-2
cells (A) and (B): The expression
of α-SMA, Col. I, p16 and p21 in liver tissues with fibrosis was
detected by immunofluorescence. (C) and (D): The expression of α-SMA,
Col. I, p16 and p21 in liver tissues with fibrosis was detected by
immunofluorescence by qRT-PCR. (E) and (F): The expression of α-SMA,
Col. I, p16 and p21 in liver tissues with fibrosis was detected by
Western Blot. Data are shown as the mean ± SD (n=3) of one
representative experiment. ∗p < 0.05, ∗∗p< 0.01 versus normal groups/ TGF-β1 groups.
Figure 4: Effect of MDP on the expression of miR-708 and Ago2
in liver tissues with fibrosis and TGF-β1-induced LX-2 cells. (A) and
(B): The expression of miR-708 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells were detected by qRT-PCR. (C): The expression
of Ago2 in liver tissues with fibrosis was detected by
immunohistochemistry. (D): The expression of Ago2 in TGF-β1-induced LX-2
cells were detected by immunofluorescence. (E) and (F): The expression
of α-SMA and Col. I in liver tissues with fibrosis was detected by
Western Blot. Data are shown as the mean ± SD (n=3) of one
representative experiment. ∗p < 0.05, ∗∗p< 0.01, ∗∗*p < 0.001 versus normal groups/
TGF-β1 groups.
Figure 5: MDP could bind to Ago2 and inhibit Ago2 activity. (A)
and (B): The expression of miR-708 in TGF-β1-induced LX-2 cells were
detected by qRT-PCR. (C): Molecular docking modeling of compound MDP and
Ago2, the small molecule and the critical interaction of 3KRW are
represented by sticks. Panel is a view into the active site cavity. (D)
and (E): The expression of Ago2 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells were detected by Western Blot. Data are shown
as the mean ± SD (n=3) of one representative experiment. ∗p< 0.05, ∗∗p < 0.01, ∗∗*p <
0.001 versus normal groups/ TGF-β1 groups.
Figure 6: miR-708 promoted senescence of TGF‐β1‐induced LX‐2
cells. (A): Cell cycle distribution was measured by flow cytometry
analysis. (B): miR‐708‐induced senescence as indicated by SA‐β‐Gal
activities. Representative images of SA‐β‐Gal staining are presented.
(C): The expression of α-SMA, Col. I, p16 and p21 in liver tissues with
fibrosis was detected by immunofluorescence. (D) and (E): The expression
of p16 and p21 in TGF-β1-induced LX-2 cells were detected by Western
Blot. (F) and (G): The expression of α-SMA and Col. I in TGF-β1-induced
LX-2 cells were detected by Western Blot. Data are shown as the mean ±
SD (n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01 versus normal groups/ TGF-β1 groups.
Figure 7: ZEB1 is identified as a directly target of miR-708.
(A): The target genes of miR‐145 were predicted by TargetScan. (B):
Luciferase reporter gene assay was performed following co-transfection
of wild-type (WT) or mutant (MUT) ZEB1 3′-UTR with miR-708 mimics in
LX-2 cells. (C): The expression of ZEB1 was detected by qRT-PCR. (D):
The expression of ZEB1 was detected by were detected by
immunofluorescence. (E): The expression of ZEB1in TGF-β1-induced LX-2
cells were detected by Western Blot. Data are shown as the mean ± SD
(n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01, ∗∗*p < 0.001,****p < 0.0001 versus normal groups/ TGF-β1 groups.
s
Figure 8: ZEB1 was upregulated in TGF-β1-induced LX-2 cells.
(A) and (B): The expression of ZEB1 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells was detected by immunofluorescence. (C) and
(D): The expression of ZEB1 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells was detected by qRT-PCR. (E) and (F): The
expression of ZEB1 in liver tissues with fibrosis and TGF-β1-induced
LX-2 cells was detected by Western Blot. Data are shown as the mean ± SD
(n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01, ****p < 0.0001 versus normal
groups/ TGF-β1 groups.
Figure 9: ZEB1 inhibited senescence of TGF‐β1‐induced LX‐2
cells. (A): Cell cycle distribution was measured by flow cytometry
analysis. (B): ZEB1‐mediated senescence as indicated by SA‐β‐Gal
activities. Representative images of SA‐β‐Gal staining are presented.
(C): The expression of α-SMA, Col. I, p16 and p21 in liver tissues with
fibrosis was detected by immunofluorescence. (D) and (E): The expression
of p16 and p21 in TGF-β1-induced LX-2 cells were detected by Western
Blot. (F) and (G): The expression of α-SMA and Col. I in TGF-β1-induced
LX-2 cells were detected by Western Blot. Data are shown as the mean ±
SD (n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01, ∗∗*p < 0.001 versus normal
groups/ TGF-β1 groups.
Figure 10: ZEB1 regulated p53 promoter activities. (A) and (B):
Effect of ZEB1 on p53 promoters. Luciferase reporter vectors were
co-transfected with p-CMV-ZEB1 in LX‐2 cells. Plasmid HSV‐TK was
co-transfected to normalize transfection efficiency. (C): The expression
of p53 in TGF-β1-induced LX-2 cells was detected by immunofluorescence.
(D): The expression of p53 in TGF-β1-induced LX-2 cells was detected by
qRT-PCR. (E): The expression of ZEB1 in TGF-β1-induced LX-2 cells was
detected by Western Blot. Data are shown as the mean ± SD (n=3) of one
representative experiment. ∗p < 0.05,∗∗p < 0.01, ***p < 0.001 versus normal
groups/ TGF-β1 groups.
Figure 11: P53 was downregulated in TGF-β1-induced LX-2 cells.
(A) and (B): The expression of p53 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells was detected by immunofluorescence. (C) and
(D): The expression of p53 in liver tissues with fibrosis and
TGF-β1-induced LX-2 cells was detected by qRT-PCR. (E) and (E): The
expression of p53 in liver tissues with fibrosis and TGF-β1-induced LX-2
cells was detected by Western Blot. Data are shown as the mean ± SD
(n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01, ***p < 0.001, ∗∗**p< 0.0001 versus normal groups/ TGF-β1 groups.
Figure 12: P53 promoted senescence of TGF‐β1‐induced LX‐2
cells. (A): Cell cycle distribution was measured by flow cytometry
analysis. (B): p53‐induced senescence as indicated by SA‐β‐Gal
activities. Representative images of SA‐β‐Gal staining are presented.
(C): The expression of α-SMA, Col. I, p16 and p21 in liver tissues with
fibrosis was detected by immunofluorescence. (D) and (E): The expression
of p16 and p21 in TGF-β1-induced LX-2 cells were detected by Western
Blot. (F) and (G): The expression of α-SMA and Col. I in TGF-β1-induced
LX-2 cells were detected by Western Blot. Data are shown as the mean ±
SD (n=3) of one representative experiment. ∗p < 0.05,∗∗p < 0.01, ∗∗*p < 0.001 versus normal
groups/ TGF-β1 groups.
Figure 13: MDP exerts its functions through regulating
Ago2/miR-708/ZEB1/p53 pathway. Schematic representation of the proposed
mechanism. MDP cold bind to the Ago2. Transcription of Ago2 gene
increases the expression of mature miR‐708 which targets the 3′‐UTR of
ZEB1 to suppress its translation. Reduced expression of ZEB1 can no
longer effectively inhibit p53 transcription factors, thereby
accelerating the expression of p53. Ultimately, the increased level of
p53 inhibits cell cycle progression and promotes the senescence of
activated HSCs, thereby inhibiting liver fibrosis.