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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
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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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.
Gene Primer sequence Size (bp) Accession number
75 BC059110.1
68 NM_019160
60 NM_020537
69 NM_053304.1
75 NM_133530.1
70 NM_053819.1
61 NM_001613.2
100 NM_021578.2
Tab.1  Primers for quantitative real-time PCR analysis
n = 6
n = 10
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)
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
n = 6
n = 10
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).
1 RJ Groszmann, JG Abraldes. Portal hypertension: from bedside to bench. J Clin Gastroenterol 2005; 39(4 Suppl 2): S125–S130 pmid: 15758647
2 M McConnell,  Y Iwakiri. Biology of portal hypertension. Hepatol Int 2018; 12(Suppl 1):11–23 PMID:29075990
3 M Vilaseca, H García-Calderó, E Lafoz, O García-Irigoyen, MA Avila, JC Reverter, J Bosch, V Hernández-Gea, J Gracia-Sancho, JC García-Pagán. The anticoagulant rivaroxaban lowers portal hypertension in cirrhotic rats mainly by deactivating hepatic stellate cells. Hepatology 2017; 65(6): 2031–2044 pmid: 28142199
4 M Vilaseca, H García-Calderó, E Lafoz, M Ruart, CI López-Sanjurjo, MP Murphy, R Deulofeu, J Bosch, V Hernández-Gea, J Gracia-Sancho, JC García-Pagán. Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats. Liver Int 2017; 37(7): 1002–1012 pmid: 28371136
5 DC Rockey, L Fouassier, JJ Chung, A Carayon, P Vallee, C Rey, C Housset. Cellular localization of endothelin-1 and increased production in liver injury in the rat: potential for autocrine and paracrine effects on stellate cells. Hepatology 1998; 27(2): 472–480 pmid: 9462646
6 Y Iwakiri. Pathophysiology of portal hypertension. Clin Liver Dis 2014; 18(2): 281–291 pmid: 24679494
7 Y Nishimura, T Ito, K Hoe, JM Saavedra. Chronic peripheral administration of the angiotensin II AT(1) receptor antagonist candesartan blocks brain AT(1) receptors. Brain Res 2000; 871(1): 29–38 pmid: 10882779
8 RS Ames, HM Sarau, JK Chambers, RN Willette, NV Aiyar, AM Romanic, CS Louden, JJ Foley, CF Sauermelch, RW Coatney, Z Ao, J Disa, SD Holmes, JM Stadel, JD Martin, WS Liu, GI Glover, S Wilson, DE McNulty, CE Ellis, NA Elshourbagy, U Shabon, JJ Trill, DW Hay, EH Ohlstein, DJ Bergsma, SA Douglas. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 401(6750): 282–286 pmid: 10499587
9 B Ross, K McKendy, A Giaid. Role of urotensin II in health and disease. Am J Physiol Regul Integr Comp Physiol 2010; 298(5): R1156–R1172 pmid: 20421634
10 G Thanassoulis, T Huyhn, A Giaid. Urotensin II and cardiovascular diseases. Peptides 2004; 25(10): 1789–1794 pmid: 15476947
11 W Kemp, S Roberts, H Krum. Increased circulating urotensin II in cirrhosis: potential implications in liver disease. Peptides 2008; 29(5): 868–872 pmid: 17913301
12 D Liu, J Chen, J Wang, Z Zhang, X Ma, J Jia, Y Wang. Increased expression of urotensin II and GPR14 in patients with cirrhosis and portal hypertension. Int J Mol Med 2010; 25(6): 845–851
pmid: 20428787
13 DG Liu, J Wang, ZT Zhang, Y Wang. The urotension II antagonist SB-710411 arrests fibrosis in CCl4 cirrhotic rats. Mol Med Rep 2009; 2(6): 953–961
pmid: 21475927
14 M Clozel, C Binkert, M Birker-Robaczewska, C Boukhadra, SS Ding, W Fischli, P Hess, B Mathys, K Morrison, C Müller, C Müller, O Nayler, C Qiu, M Rey, MW Scherz, J Velker, T Weller, JF Xi, P Ziltener. Pharmacology of the urotensin-II receptor antagonist palosuran (ACT-058362; 1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt): first demonstration of a pathophysiological role of the urotensin system. J Pharmacol Exp Ther 2004; 311(1): 204–212 pmid: 15146030
15 M Mejias, L Coch, A Berzigotti, E Garcia-Pras, J Gallego, J Bosch, M Fernandez. Antiangiogenic and antifibrogenic activity of pigment epithelium-derived factor (PEDF) in bile duct-ligated portal hypertensive rats. Gut.  2015; 64(4): 657–666 PMID:24848263
16 M Clozel, P Hess, C Qiu, SS Ding, M Rey. The urotensin-II receptor antagonist palosuran improves pancreatic and renal function in diabetic rats. J Pharmacol Exp Ther 2006; 316(3): 1115–1121 pmid: 16267137
17 SJ Hsu, FY Lee, SS Wang, IF Hsin, TY Lin, HC Huang, CC Chang, CL Chuang, HL Ho, HC Lin, SD Lee. Caffeine ameliorates hemodynamic derangements and portosystemic collaterals in cirrhotic rats. Hepatology 2015; 61(5): 1672–1684 pmid: 25557829
18 MG Delgado, J Gracia-Sancho, G Marrone, A Rodríguez-Vilarrupla, R Deulofeu, JG Abraldes, J Bosch, JC García-Pagán. Leptin receptor blockade reduces intrahepatic vascular resistance and portal pressure in an experimental model of rat liver cirrhosis. Am J Physiol Gastrointest Liver Physiol 2013; 305(7): G496–G502 pmid: 23886859
19 AS Darlington, DW Dippel, GM Ribbers, R van Balen, J Passchier, JJ Busschbach. A prospective study on coping strategies and quality of life in patients after stroke, assessing prognostic relationships and estimates of cost-effectiveness. J Rehabil Med 2009; 41(4): 237–241 pmid: 19247542
20 A Mülsch, M Oelze, S Klöss, H Mollnau, A Töpfer, A Smolenski, U Walter, JP Stasch, A Warnholtz, U Hink, T Meinertz, T Münzel. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation 2001; 103(17): 2188–2194 pmid: 11331261
21 TD Schmittgen, KJ Livak. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3(6): 1101–1108 pmid: 18546601
22 GA Ramm. Isolation and culture of rat hepatic stellate cells. J Gastroenterol Hepatol 1998; 13(8): 846–851 pmid: 9736182
23 DC Rockey, RA Weisiger. Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance. Hepatology 1996; 24(1): 233–240 pmid: 8707268
24 J Liu, H Gong, ZT Zhang, Y Wang. Effect of angiotensin II and angiotensin II type 1 receptor antagonist on the proliferation, contraction and collagen synthesis in rat hepatic stellate cells. Chin Med J (Engl) 2008; 121(2): 161–165
pmid: 18272044
25 W Kemp, H Krum, J Colman, M Bailey, T Yandle, M Richards, S Roberts. Urotensin II: a novel vasoactive mediator linked to chronic liver disease and portal hypertension. Liver Int 2007; 27(9): 1232–1239 pmid: 17919235
26 W Kemp, A Kompa, A Phrommintikul, C Herath, J Zhiyuan, P Angus, C McLean, S Roberts, H Krum. Urotensin II modulates hepatic fibrosis and portal hemodynamic alterations in rats. Am J Physiol Gastrointest Liver Physiol 2009; 297(4): G762–G767 pmid: 19797237
27 PN Sidharta, K Rave, L Heinemann, E Chiossi, S Krähenbühl, J Dingemanse. Effect of the urotensin-II receptor antagonist palosuran on secretion of and sensitivity to insulin in patients with type 2 diabetes mellitus. Br J Clin Pharmacol 2009; 68(4): 502–510 pmid: 19843053
28 J Trebicka, L Leifeld, M Hennenberg, E Biecker, A Eckhardt, N Fischer, AS Pröbsting, C Clemens, F Lammert, T Sauerbruch, J Heller. Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats. Hepatology 2008; 47(4): 1264–1276 pmid: 18318439
29 J Heller, M Schepke, M Neef, R Woitas, C Rabe, T Sauerbruch. Increased urotensin II plasma levels in patients with cirrhosis and portal hypertension. J Hepatol 2002; 37(6): 767–772 pmid: 12445417
30 L Leifeld, C Clemens, J Heller, J Trebicka, T Sauerbruch, U Spengler. Expression of urotensin II and its receptor in human liver cirrhosis and fulminant hepatic failure. Dig Dis Sci 2010; 55(5): 1458–1464 pmid: 19582578
31 L Yadav,  N Puri,  V Rastogi,  P Satpute,  R Ahmad,  G Kaur. Matrix metalloproteinases and cancer-roles in threat and therapy. Asian Pac J Cancer Prev  2014; 15(3): 1085–1091. doi10.7314/APJCP.2014.15.3.1085 PMID:24606423
32 LM Liu, DY Liang, CG Ye, WJ Tu, T Zhu. The UII/UT system mediates upregulation of proinflammatory cytokines through p38 MAPK and NF-kB pathways in LPS-stimulated Kupffer cells. PLoS One 2015; 10(3): e0121383
33 R Wiest, RJ Groszmann. The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough. Hepatology 2002; 35(2): 478–491 pmid: 11826425
34 L Verbeke, R Farre, J Trebicka, M Komuta, T Roskams, S Klein, IV Elst, P Windmolders, T Vanuytsel, F Nevens, W Laleman. Obeticholic acid, a farnesoid X receptor agonist, improves portal hypertension by two distinct pathways in cirrhotic rats. Hepatology 2014; 59(6): 2286–2298 pmid: 24259407
35 A Mallat,  S Lotersztajn. Targeting cannabinoid receptors in hepatocellular carcinoma. Gut  2016; 65(10):1582–1583 PMID:27342953
36 K Cheng, N Yang, RI Mahato. TGF-b1 gene silencing for treating liver fibrosis. Mol Pharm 2009; 6(3): 772–779 pmid: 19388665
37 RJ Chen, HH Wu, YJ Wang. Strategies to prevent and reverse liver fibrosis in humans and laboratory animals. Arch Toxicol 2015; 89(10): 1727–1750 pmid: 25963329
38 J Trebicka, M Hennenberg, W Laleman, N Shelest, E Biecker, M Schepke, F Nevens, T Sauerbruch, J Heller. Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase. Hepatology 2007; 46(1): 242–253 pmid: 17596891
[1] Xuefu Wang, Zhigang Tian. γδ T cells in liver diseases[J]. Front. Med., 2018, 12(3): 262-268.
[2] James S. Park,Calvin Pan. Current recommendations of managing HBV infection in preconception or pregnancy[J]. Front. Med., 2014, 8(2): 158-165.
[3] Jiangyi Zhu, Yongquan Shi, Xinmin Zhou, Zengshan Li, Xiaofeng Huang, Zheyi Han, Jianhong Wang, Ruian Wang, Jie Ding, Kaichun Wu, Ying Han, Daiming Fan. Observation on therapeutic efficacy of ursodeoxycholic acid in Chinese patients with primary biliary cirrhosis: a 2-year follow-up study[J]. Front Med, 2013, 7(2): 255-263.
[4] Cong Tong, Xinsen Xu, Chang Liu, Tianzheng Zhang, Kai Qu. Assessment of liver volume variation to evaluate liver function[J]. Front Med, 2012, 6(4): 421-427.
[5] Lili LIU MM , Jiyao WANG MD , Weimin SHE MM , . Correlation between viral load and liver cirrhosis in chronic hepatitis B patients[J]. Front. Med., 2009, 3(3): 271-276.
[6] Ling YANG, Rui ZHU, Qingjing ZHU, Dan DAN, Jin YE, Keshu XU, Xiaohua HOU. Influence of β-elemene on the secretion of angiotensin II and expression of AT1R in hepatic stellate cells[J]. Front Med Chin, 2009, 3(1): 36-40.
[7] CHU Deyong, LI Conglei, SHEN Jilong, WU Qiang. Paeoniflorin prevents hepatic fibrosis of by inhibiting TGF-&#946;1 production from macrophages in mice[J]. Front. Med., 2008, 2(2): 154-165.
[8] LIU Jie, WANG Jiyao, WEI Liming, LU Ye, Jin Hong. Effects of Decoction on plasma proteome in cirrhosis: preliminary experimental study with rats[J]. Front. Med., 2008, 2(1): 39-44.
[9] CHEN Jisheng, HUO Jinshan, ZHANG Hongwei, SHANG Changzhen, CHEN Rufu, ZHANG Jie, Obetien Mapudengo, CHEN Yajin, ZHANG Lei. Splenic autotransplantation and oesophageal transection anastomosis in patients with portal hypertension (26 years clinical observation)[J]. Front. Med., 2007, 1(1): 30-35.
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