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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (3) : 398-408    https://doi.org/10.1007/s11684-019-0689-5
RESEARCH ARTICLE
Urotensin II receptor antagonist reduces hepatic resistance and portal pressure through enhanced eNOS-dependent HSC vasodilatation in CCl4-induced cirrhotic rats
Ruoxi Zhang1, Jing Chen2, Diangang Liu1(), Yu Wang3
1. Department of General Surgery, Xuanwu Hospital, Capital Medical University, Beijing 100053, China
2. Department of Gastroenterology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150010, China
3. Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
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Abstract

Increased serum urotensin II (UII) levels in human cirrhotic populations have been recently shown, but the long-term effects of UII receptor antagonist on the cirrhosis have not been investigated. To investigate the therapeutic effects of urotensin II receptor (UT) antagonist palosuran on rats with carbon tetrachloride (CCl4)-induced cirrhosis, the hepatic and systemic hemodynamics, liver fibrosis, the metalloproteinase-13 (MMP-13)/ tissue inhibitor of metalloproteinase-1 (TIMP-1) ratio, hepatic Rho-kinase activity, and the endothelial nitric oxide synthase (eNOS) activity are measured in CCl4-cirrhotic rats treated with palosuran or vehicle for 4 weeks. Primary hepatic stellate cells (HSCs) are used to investigate the changes in UII/UT expression and the in vitro effect of palosuran. Compared with the vehicle-treated cirrhotic rats, treatment with palosuran can reduce the portal pressure (PP), decrease the risk of liver fibrosis and the level of α smooth muscle actin, collagen-I (COL-I), and transforming growth factor β expression. However, treatment with palosuran can increase MMP-13/TIMP-1, p-vasodilator-stimulated phosphoprotein (p-VASP), and p-eNOS expression. Moreover, in vitro UII/UT mRNA expression increases during HSC activation. MMP-13/TIMP-1, COL-I, and p-VASP are inhibited after palosuran treatment. Our data indicate that long-term administration of palosuran can decrease PP in cirrhosis, which results from decreased hepatic fibrosis and enhanced eNOS-dependent HSC vasodilatation.

Keywords portal hypertension      cirrhosis      urotensin II      palosuran      hepatic stellate cell     
Corresponding Authors: Diangang Liu   
Just Accepted Date: 28 January 2019   Online First Date: 28 February 2019    Issue Date: 05 June 2019
 Cite this article:   
Ruoxi Zhang,Jing Chen,Diangang Liu, et al. Urotensin II receptor antagonist reduces hepatic resistance and portal pressure through enhanced eNOS-dependent HSC vasodilatation in CCl4-induced cirrhotic rats[J]. Front. Med., 2019, 13(3): 398-408.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-019-0689-5
http://academic.hep.com.cn/fmd/EN/Y2019/V13/I3/398
Gene Primer sequence Size (bp) Accession number
GADPH F: 5′-CCTGCCAAGTATGATGACATCAAGA-3′
R: 5′- GTAGCCCAGGATGCCCTTTAGT-3′
75 BC059110.1
UII F: 5′-CAGAAGCAGAGGGAAGCCTA-3′
R: 5′-CAAGCTTCCCCTTGGAGTG-3′
68 NM_019160
UT F: 5′-CATTGGGCTGCTCTATGTCC-3′
R: 5′-AAAGAAGCTTGCTGAGATAGCC-3′
60 NM_020537
COL-I F: 5′- CCTTTCTCCACCCCCTCTT-3′
R: 5′- TGTGTCTTTGGGGGAGACTT-3′
69 NM_053304.1
MMP-13 F: 5′- CCCTGGAGCCCTGATGTTT-3′
R: 5′- CTCTGGTGTTTTGGGGTGCT-3′
75 NM_133530.1
TIMP-1 F: 5′- CAGCAAAAGGCCTTCGTAAA-3′
R: 5′- TGGCTGAACAGGGAAACACT-3′
70 NM_053819.1
a-SMA F: 5′- TGCCATGTATGTGGCTATTCA-3′
R: 5′- ACCAGTTGTACGTCCAGAAGC-3′
61 NM_001613.2
TGF-b1 F: 5′- CCTGGAAAGGGCTCAACAC-3′
R: 5′- CTGCCGTACACAGCAGTTCT-3′
100 NM_021578.2
Tab.1  Primers for quantitative real-time PCR analysis
Normal
n = 6
Vehicle
n = 10
Palosuran
n = 10
HR (beats/min) 438.25±14.66 447.25±11.13 442.00±14.45
MAP (mmHg) 113.38±12.86 78.25±11.30# 89.25±10.78
PP (mmHg) 8.00±1.31 17.13±2.42 11.25±1.03*
Portal flow (mL·min1·100 g1)
SMA flow (mL·min1·100 g1)
6.51±0.32
1.9±0.12
6.85±0.39
5.1±0.21
7.20±0.44
3.2±0.23*
HVR (mmHg·mL1·min·100 g) 1.3±0.14 2.5±0.23 1.4±0.10*
Body weight (g) 450±11.23 387±10.11 392±12.34
Tab.2  Effects of palosuran on hepatic and systemic hemodynamics in CCl4-cirrhotic rats
Normal
n = 6
Vehicle
n = 10
Palosuran
n = 10
AST (IU/L) 98.63±28.75 2242.86±560.67# 1288.42±350.29*
ALT (IU/L) 36.76±6.36 932.05±213.89# 538.10±53.55*
TBIL (mg/dL) 0.61±0.17 25.54±5.33# 18.618±2.85
ALB (g/L) 31.06±3.03 27.63±2.39 28.38±2.67
Tab.3  Effects of palosuran on biochemical parameters in CCl4-cirrhotic rats
Fig.1  Reduction of hepatic fibrosis with palosuran treatment. (A) Histologic findings ((H&E, Gordon–Sweet reticulum and Masson trichrome, immunohistochemistry): a comparison of liver architecture of normal control, extensive portal-portal and portal-central fibrous linkage, distortion of liver architecture and marked regeneration nodules showed much more positive expressions for α-SMA and TGF-β1 were observed. (B) Palosuran treatment markedly reduced liver fibrosis change score compared with vehicle group. (C) Hepatic Hyp concentrations in the three experimental groups were analyzed by the sample alkaline hydrolysis method. (D) COL-I mRNA in rat liver tissue and plasma COL-I concentration. (E) Number of α-SMA positive cells. (F) Number of TGF-β1 positive cells. *P<0.05, compared with the vehicle; #P<0.05, compared with the normal control group.
Group Fibrosis score
0 1 2 3 4 5 6
Normal 6 0 0 0 0 0 0
Vehiclea 0 0 0 0 2 4 4
Treatmentb 0 0 4 3 3 0 0
Tab.4  Fibrosis change score of CCl4-induced cirrhosis treated with palosuran.
Fig.2  Effects of palosuran on UII/UT system, MMP-13/TIMP-1, Rho-kinase, and eNOS pathways in CCl4-cirrhotic rats. (A) Plasma UII was upregulated in CCl4-cirrhotic rats, and UII levels were significantly decreased in palosuran-treated groups compared with vehicle group. *P<0.05, versus vehicle. (B) UII/UT mRNAs in liver were upregulated in CCl4-cirrhotic rats, and UII/UT mRNAs were markedly reduced in palosuran-treated group. (C) Serum MMP-13 and TIMP-1 concentration levels in CCl4-cirrhotic rats treated with vehicle or palosuran. MMP-13 was upregulated in CCl4-cirrhotic rats, and MMP-13 levels were significantly decreased in palosuran-treated groups compared with vehicle group. TIMP-1 levels were not significantly different among groups. (D) MMP-13 and TIMP-I mRNAs in liver were upregulated in CCl4-cirrhotic rats, and palosuran treatment tended to decrease expression of MMP-13 and TIMP-1 mRNAs. (E) Palosuran treatment tended to attenuate the increase in ratio of MMP-13/TIMP-1 compared with the vehicle group. (F) Western blot analysis revealed a decreased p-eNOS in CCl4 cirrhotic rats compared with the normal controls, and palosuran treatment significantly increased p-eNOS. (G) p-VASP levels were not significantly different between normal group and vehicle group. Palosuran treatment significantly increased p-VASP compared with vehicle-treated cirrhotic rats. *P<0.05, versus vehicle; #P<0.05, versus normal.
Fig.3  Direct effect of palosuran on primary hepatic stellate. mRNA expression was determined by real-time PCR in the HSC cells; protein expression of collagen I, MMP-13, and TIMP-1 in HSC cell supernatant was determined by ELISA. (A) Trypan blue staining (left). Primary HSC at 48 h, 72 h (middle); stimulated at 327 nm wavelength, HSCs had blue–green intrinsic auto fluorescence (right). Fluorescence staining with α-SMA. (B) UII gene expression was upregulated during HSC activation (α-SMA as indicated by the measurement of its activation). (C) Palosuran inhibited HSC COL-I gene expression of HSC and protein expression in HSC cell supernatant. (D, E) Palosuran inhibited the expression of MMP-13 and TIMP-1 mRNA as well as the secretion of MMP-13 and TIMP-1mRNA in HSC cells. (F) Protein levels of p-VASP in HSC were quantified by Western blot; palosuran increased the protein levels of p-VASP in a dose-dependent manner. *P<0.05 (10−5 mol/L).
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