Fuzhenghuayu Decoction ameliorates hepatic fibrosis by attenuating experimental sinusoidal capillarization and liver angiogenesis - PubMed (original) (raw)
Fuzhenghuayu Decoction ameliorates hepatic fibrosis by attenuating experimental sinusoidal capillarization and liver angiogenesis
Hong-Liang Liu et al. Sci Rep. 2019.
Abstract
Fuzhenghuayu (FZHY) is a compound extracted from natural plants. Its anti-fibrotic effect has been confirmed in experimental and clinical studies. However, precise effects and underlying mechanisms of FZHY in liver angiogenesis largely remain understood. In this study, we investigated the effects of FZHY on sinusoidal capillarization and angiogenesis with mice challenged for Carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN), in vitro human hepatic sinusoidal endothelial cells (HHSEC) and Human Umbilical Vein Endothelial Cell (HUVEC) 3D fibrin gel model. Besides its anti-fibrotic effect, FZHY ameliorated CCl4 and DMN-induced sinusoidal capillarization, angiogenesis and expression of angiogenesis-associated factors, i.e. CD31, VEGF, VEGF receptor II, phosphor-ERK and HIF-1α. Consistent with the findings based on animal models, inhibitory effects of FZHY on capillarization and angiogenesis were further confirmed in HHSEC and the HUVEC 3D fibrin gel model, respectively. These data suggest that FZHY ameliorates not only liver fibrosis but also vessel remodeling in experimental models. Therefore, FZHY might be a potentially useful drug to treat liver cirrhosis in clinical practice.
Conflict of interest statement
The authors declare no competing interests.
Figures
Figure 1
FZHY recipe ameliorated CCl4-induced liver injury in mice. (A) ALT and AST levels were measured in mice with different treatment. (B) Inflammatory and fibrotic degree were examined by hematoxylin and eosin (H&E) and Sirius red staining (original magnification ×100). (C) Sirius red positivecollagen areas were quantified. (D) Liver Hydroxyproline content was measured byJamall’s method. (E,F) Protein expression of α-SMA and collagen type I in mice livers was measured by Western blot. #And *P < 0.05, ##and **P < 0.01.
Figure 2
FZHY recipe ameliorated CCl4-induced angiogenesis and sinusoidal capillarization. (A,C) Remodeling of liver vessel was measured by ILPCI-CT in mice with different treatments as indicated. (B,D,E) CD31 expression was examined and quantified by immunofluorescence and western blot. (F,G) Liver sinusoidal structural changes were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). #And *P < 0.05, ##and **P < 0.01.
Figure 3
FZHY recipe ameliorated DMN-induced liver fibrosis, angiogenesis and sinusoidal capillarization. (A,C) Collagen deposition visualized in Sirius red and Masson staining (original magnification ×100). (B,D) Remodeling of liver vessel was measured by ILPCI-CT in mice with different administrations. (E,F) Liver sinusoidal structural changes were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM).
Figure 4
FZHY recipe reduced angiogenesis-related factors. (A) qRT-PCR was performed to analyze mRNA expression of HIF-1α, VEGF and VEGF-R2in mouse liver tissues. (B) Western Blot was used to examine protein expression of VEGF, VEGFR2, ERK1/2 and phosphorylated ERK1/2. GAPDH was used as loading control. The experiments were repeated at least three times. Data are presented as means ± SEM (B,D). #And *P < 0.05, ##and **P < 0.01.
Figure 5
FZHY recipe inhibited VEGF-induced proliferation and tube formation of HHSEC cells. (A,B) Growth and vitality of HHSEC cells was assayed by CCK8 assay. (C) Cell proliferation was analyzed by EDU assay. (D) Matrigel tube formation was measured for three times. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01.
Figure 6
FZHY maintained the number of LSEC fenestra induced by VEGF, but decreased VEGF-induced LSEC protrusion. Sinusoidal structural changes of HHSECs were observed by scanning electron microscope (SEM).
Figure 7
FZHY recipe suppressed angiogenesis via inhibiting VEGF signaling in vitro. Western blot for VEGF and VEGFR2 (A) phosphorylated and total ERK1/2 expression (C) are shown. GAPDH was used as loading control. The experiments were repeated at least three times. Data are presented as means ± SEM (B,D). #And *P < 0.05, ##and **P < 0.01.
Figure 8
FZHY recipe inhibited angiogenesis in 3D Fibrin Gel. Sprouting, branching/lumen formation, and anastomosis were visualized in HUVECs with or without FZHY treatment.
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