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, 8 (49), 86206-86216
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Modulation of HIF-1α and STAT3 Signaling Contributes to Anti-Angiogenic Effect of YC-1 in Mice With Liver Fibrosis

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Modulation of HIF-1α and STAT3 Signaling Contributes to Anti-Angiogenic Effect of YC-1 in Mice With Liver Fibrosis

Tzung-Yan Lee et al. Oncotarget.

Abstract

Hypoxia has been shown to have a role in the pathogenesis of several forms of liver disease. The aim of the study was to evaluate the mechanisms of HIF-1α inhibitor, YC-1, during bile duct ligation (BDL)-induced liver fibrosis in mice. Liver fibrosis was induced in mice, and YC-1 was then given intraperitoneally (50 mg/kg) once daily following 5 days. Liver injuries mice that were treated with YC-1 showed improved inflammatory response and diminished angiogenesis and hepatic fibrosis. YC-1 treatment inhibited liver neutrophil infiltration, while a decreased in TNF-α signaling as well as macrophage aggregation. In addition, YC-1 downregulates iNOS and COX-2 levels by inhibiting the activation of NF-κB and STAT3 phosphorylation by negative regulation the expression of SOCS1 and SOCS3 signaling. On the other hand, YC-1 decreased angiogenesis, as shown by the downregulation of hypoxia-inducible cascade genes, i.e. VEGF. YC-1 treatment resulted in a significant decrease in hepatic fibrogenesis, α-SMA abundance, and TGF-βR1 expression as well as hypoxia were assessed using VEGFR1, vWF and HIF-1α immunostaining. These results suggest that multi-targeted therapies directed against angiogenesis, hypoxia, and fibrosis. Therefore, it may be suggested that YC-1 treatment may be a novel therapeutic agent for the treatment of liver disease.

Keywords: YC-1; angiogenesis; fibrosis; hypoxia-inducible factor-1α; inflammation.

Figures

Figure 1
Figure 1. YC-1 attenuated cholestasis-induced liver injury in mice
(A) Pronounced hepatoatrophy and necrosis is observed in livers after bile duct ligation. Mice treated with YC-1 (50 mg/kg) daily for 5 days show minor signs of hepatic necrosis in contrast to BDL mice. H&E staining (40× magnification) showing liver injury. (B) Mice were subjected to BDL, and liver injury were assessed at 5 days after surgery. ALT, AST and TNF-ɑ levels in plasma were increased to an extent in BDL mice, YC-1 attenuated BDL induced hepatic injury as analyzed with ALT, AST and TNF-ɑ levels. (C, D) Immunohistochemical identification of CK19 and protein expressions in Mice treated with YC-1 in contrast to BDL mice. (E) Immunohistochemical identification of HIF-1ɑ. (F) The gene expression induction of HIF-1ɑ in liver tissue exposed to BDL or with YC-1 for 5 days. (G) Western blotting of HIF-1ɑ. Densitometry analyses are represented as a relative ratio of HIF-1ɑ to β-actin. Bars represent mean±SEM from 5 samples per liver tissue type. (*p < 0.05 vs. sham; #p < 0.05 vs. BDL.)
Figure 2
Figure 2. Effects of YC-1 on neutrophil and macrophage accumulation in the liver after BDL
The extent of liver injury in mice given BDL were evaluated by antibody staining. Mice treated with YC-1 were also analyzed. Effective suppression is observed in the two strains (neutrophil and F4/80 staining as activated macrophage) treated with YC-1 in BDL mice (A, B). The response of NF-κB, Nrf2 (C) and pSTAT3 (E), protein activity in fibrotic mice treated with YC-1. (D) Hepatic tissue mRNA expression was determined by RT-PCR using GAPDH as the quantity control. The mRNA densitometry values were normalized to GAPDH levels. Data are presented as the mean ± SEM of at least three independent experiments. (*p < 0.05 vs. sham; #p < 0.05 vs. BDL.)
Figure 3
Figure 3. Effects of YC-1 on protein levels of plasminogen activator inhibitor-1 (PAI-1) and osteopontin (OPN) in the BDL mice
Immunohistochemical analysis of liver sections from BDL or BDL plus YC-1-treated mice. The expression of PAI-1 (A) and OPN (C) were analyzed using PAI-1 or OPN antibodies, respectively. Immunoblot analysis of total liver lysates of BDL mice. BDL leads to a significant increase in PAI-1 (B) or OPN (D) protein levels, whereas YC-1 treatment shows more prominent changes. Equal amounts of lysate from sham or BDL mice were loaded in each lane and normalized against the control β-actin. The results are presented as the mean ± SEM from three independent experiments. (*p < 0.05 vs. sham; #p < 0.05 vs. BDL.)
Figure 4
Figure 4. Effect of YC-1 on angiogenesis signaling in BDL mice
(A) Livers were sectioned and staining with VEGFR1. VEGFR1-positive cells were observed adjacent to the central vein and portal triad. In contrast, after YC-1 treatment, the cells display a more ordered pattern and a lower VEGFR1-positive signals of pathologic sections (40× magnification). (B) Western blot analysis of the hepatic proteins VEGF, VEGFR1,2 and PDGFR-ɑ in the liver injury of mice. The densitometry values were normalized to β-actin. (C) Livers were sectioned and staining with vWF. In contrast, after YC-1 treatment, the cells display a more ordered pattern and a lower vWF-positive signals of pathologic sections (40× magnification). (D) Western blot analysis of the hepatic proteins VCAM-1, iNOS and COX-2 in the liver injury of mice. The densitometry values were normalized to β-actin. The results are presented as the mean ± SEM from three independent experiments. (*p < 0.05 vs. sham; #p < 0.05 vs. BDL.)
Figure 5
Figure 5. Fibrotic mice treated with YC-1 exhibited a significant reduction in hepatic levels of angiogenesis and symptom of fibrosis
(A) Fluorescent immunostaining of liver sections of BDL mice. Nuclei were counterstained with DAPI (blue). The expression and localisation of ɑ-SMA (green), a marker of activated HSCs, were analysed using anti-ɑ-SMA antibodies, respectively. Mice overexpressing ɑ-SMA in the livers of BDL mice. Western blot analysis of hepatic ɑ-SMA (B), TGF-βR1 (C) proteins in the livers of sham, BDL, and BDL plus YC-1-treated mice. The densitometry values were normalized to that of β-actin. Hepatic tissue procollagen I, III mRNA (D) expression were determined by RT-PCR using GAPDH as the internal control. The results are presented as the mean ± SEM from three independent experiments. (*p < 0.05 vs. sham; #p < 0.05 vs. BDL.)

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