Role of estrogen receptor β selective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis - PubMed (original) (raw)
Role of estrogen receptor β selective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis
Cheng-Gang Zhang et al. World J Gastroenterol. 2016.
Abstract
Aim: To investigate the role of diarylpropionitrile (DPN), a selective agonist of estrogen receptor β (ERβ), in liver cirrhosis with portal hypertension (PHT) and isolated hepatic stellate cells (HSCs).
Methods: Female Sprague-Dawley rats were ovariectomized (OVX), and liver cirrhosis with PHT was induced by CCl4 injection. DPN and PHTPP, the selective ERβ agonist and antagonist, were used as drug interventions. Liver fibrosis was assessed by hematoxylin and eosin (HE) and Masson's trichrome staining and by analyzing smooth muscle actin expression. Hemodynamic parameters were determined in vivo using colored microspheres technique. Protein expression and phosphorylation were determined by immunohistochemical staining and Western blot analysis. Messenger RNA levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Collagen gel contraction assay was performed using gel lattices containing HSCs treated with DPN, PHTPP, or Y-27632 prior to ET-1 addition.
Results: Treatment with DPN in vivo greatly lowered portal pressure and improved hemodynamic parameters without affecting mean arterial pressure, which was associated with the attenuation of liver fibrosis and intrahepatic vascular resistance (IHVR). In CCl4-treated rat livers, DPN significantly decreased the expression of RhoA and ROCK II, and even suppressed ROCK II activity. Moreover, DPN remarkedly increased the levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS, and promoted the activities of protein kinase G (PKG), which is an NO effector in the liver. Furthermore, DPN reduced the contractility of activated HSCs in the 3-dimensional stress-relaxed collagen lattices, and decreased the ROCK II activity in activated HSCs. Finally, in vivo/in vitro experiments demonstrated that MLC activity was inhibited by DPN.
Conclusion: For OVX rats with liver cirrhosis, DPN suppressed liver RhoA/ROCK signal, facilitated NO/PKG pathways, and decreased IHVR, giving rise to reduced portal pressure. Therefore, DPN represents a relevant treatment choice against PHT in cirrhotic patients, especially postmenopausal women.
Keywords: Estrogen receptor; Hepatic stellate cells; Nitric oxide; Portal hypertension; Rho-kinase signaling.
Figures
Figure 1
Therapeutic effects of diarylpropionitrile on hepatic fibrosis in CCl4-treated rats. Histological images of rat livers stained with HE (A) or Masson’s staining (B) (magnification × 100) and semi-quantitative measurement of Masson’s staining (C). a_P_ < 0.05 vs control group; c_P_ < 0.05 vs DPN group. DPN: Diarylpropionitrile.
Figure 2
Diarylpropionitrile downregulates α-SMA expression in the livers of CCl4-treated rats. A and B: Immunohistochemical staining for α-SMA (magnification × 400); C and D: Analysis of α-SMA protein expression by Western blot (each group n = 5). a_P_ < 0.05 vs control group; c_P_ < 0.05 vs DPN group. DPN: Diarylpropionitrile.
Figure 3
There were no statistically significant differences between the hepatic estrogen receptor β protein expression levels of all groups, as determined by Western blot analysis (A-B) (each group n = 5). a_P_ < 0.05 vs control group; c_P_ < 0.05 vs DPN group. DPN: Diarylpropionitrile; ERβ: Estrogen receptor β.
Figure 4
Diarylpropionitrile inhibits the protein (A, B) and mRNA (C, D) expression of RhoA and ROCKII, and even suppresses the site-specific phosphorylation of moesin (Thr558) in CCl4-treated rats (D, E). Shown are the relative densitometric quantifications of all experiments (mean ± SE), with values from the sham-operated controls set to 100 DU. a_P <_ 0.05 vs control group; c_P <_ 0.05 vs DPN group. DPN: Diarylpropionitrile.
Figure 5
Diarylpropionitrile increases the hepatic expression of NO/PKG pathway proteins and increases their activity but inhibits hepatic iNOS expression in CCl4-treated rats. A: Western blot analysis of eNOS, p-eNOS, VASP, p-VASP, and iNOS protein expression; B: Relative densitometric quantifications of all experiments (mean ± SE), with the values from the controls set to 100 DU (each group n = 5). a_P <_ 0.05 vs control group; c_P <_ 0.05 vs DPN group.
Figure 6
Diarylpropionitrile inhibits the phosphorylation of MLC in the livers of CCl4-treated rats. A: Western blot analysis of total MLC and p-MLC; B: Relative densitometric quantifications of all experiments (mean ± SE), with the values of the controls set to 100 DU (each group n = 5) a_P <_ 0.05 vs control group; c_P <_ 0.05 vs DPN group. DPN: Diarylpropionitrile.
Figure 7
Diarylpropionitrile inhibits collagen lattice contraction in hepatic stellate cells and decreases ET-1 induced moesin and MLC phosphorylation. A: Appearance of collagen lattices 4 h after drug treatment; B: The percentage of remaining lattice area 4 h after drug treatment (each group n = 12); C, D: Western blot analysis of ERβ protein expression in hepatic stellate cells (HSCs) (each group n = 5); E: Western blot analysis of the total and phosphorylated moesin and MLC in HSCs; F: Relative densitometric quantifications of moesin and MLC experiments (mean ± SE), with the values of the controls set to 100 DU (each group n = 5). a_P <_ 0.05 vs control group; c_P <_ 0.05 vs DPN group. OVX: ovariectomized; SVR: Systemic vascular resistance; DPN: Diarylpropionitrile.
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