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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.
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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.