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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld

Liver, Pancreas and Biliary Tract

Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats Dong Wang a , Jikai Yin a , Rui Dong a , Jian Zhao b , Qing Wang a , Nan Wang a , Shouli Wang c , Xilin Du a,∗ , Jianguo Lu a,∗ a

Department of General Surgery, TangDu Hospital, Fourth Military Medical University, Xi’an, China Department of Orthopedics, TangDu Hospital, Fourth Military Medical University, Xi’an, China c Laboratory of General Surgery Department, TangDu Hospital, Fourth Military Medical University, Xi’an, China b

a r t i c l e

i n f o

Article history: Received 19 August 2014 Accepted 31 December 2014 Available online xxx Keywords: AG490 Cirrhosis JAK2/STAT3 signalling Portal hypertension

a b s t r a c t Background and aims: JAK2/STAT3 signalling promotes fibrosis, angiogenesis and inflammation in many diseases; however, the role of this pathway in portal hypertension remains obscure. This study aimed to explore the function of JAK2/STAT3 signalling in portal hypertension and estimate the potential therapeutic effect of treatment with the specific inhibitor AG490. Methods: Rats induced by partial portal vein ligation and common bile duct ligation were treated with AG490 for two weeks. Haemodynamic parameters were assessed. The levels of phospho-STAT3 protein and related cytokines were detected by western blotting of splanchnic organs. Liver, spleen and intestine characterization was performed using histological analyses. Peripheral blood cell counts were also detected. Results: High levels of phospho-STAT3 protein were detected in portal hypertensive rats. AG490 effectively inhibited JAK2/STAT3 signalling and its downstream cytokines and provided protective effects by decreasing splanchnic neovascularization and inflammation and by attenuating portal pressure and hyperdynamic splanchnic circulation. In cirrhosis rats, AG490 inhibited intrahepatic fibrosis, angiogenesis and inflammation. AG490 improved the peripheral blood cell counts and the splenomegaly observed in these rats. Conclusions: JAK2/STAT3 signalling is essential in portal hypertension, and targeting JAK2/STAT3 may be a promising therapy to treat this condition. © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

1. Introduction Portal hypertension, a major complication of liver cirrhosis [1], is primarily characterized by elevated portal pressure, hyperdynamic splanchnic circulation, an extensive network of portosystemic collaterals, intestine inflammation and splenomegaly. Non-selective beta-blockers such as propranolol are the only drugs used in clinics to treat portal hypertension; however, only a third to half of patients have a satisfactory reduction in portal pressure after treatment [2]. Surgeries to alleviate portal hypertension are usually limited by their disadvantages or by the physical condition of

∗ Corresponding authors at: Department of General Surgery, TangDu Hospital, Fourth Military Medical University, 569 Xin Si Road, Xi’an, China. Tel.: +86 029 84777431; fax: +86 029 84777431. E-mail addresses: [email protected] (X. Du), [email protected] (J. Lu).

the patient. Until recently, no radical medical therapy was implemented to treat portal hypertension. Several cytokines are involved in pathological changes such as fibrosis, angiogenesis and inflammation in portal hypertension. Specifically, transforming growth factor-␤ (TGF-␤) is a profibrogenic cytokine involved in the activation of hepatic stellate cell (HSC) [3], a major contributor to collagen deposition in liver fibrosis, and is also a contributor to splanchnic inflammation. Vascular endothelial growth factor (VEGF), an important proangiogenic factor, plays a crucial role in both intra and extra hepatic circulations. Overexpressed VEGF also accelerates intestinal inflammation [4] and promotes liver fibrosis by regulating monocyte infiltration [5] and HSC activation [6]. Activated HSC enhances the activation of liver sinusoidal endothelial cells by releasing VEGF [7]. Splanchnic nitrogen monoxide (NO) overproduction primarily derived from endothelial nitric oxide synthase (eNOS) contributes to hyperdynamic splanchnic circulation. Therefore, the effective inhibition of

http://dx.doi.org/10.1016/j.dld.2014.12.017 1590-8658/© 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wang D, et al. Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2014.12.017

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these cytokines may promote the alleviation of portal hypertension. Janus kinase/signal transducers and activators of transcription (JAK/STAT) signalling is one of the major pathways involved in cytokine signal transduction. JAK2, the most conserved isoform of the JAK family, functions upstream of STAT3 in JAK/STAT signalling. Studies have shown that JAK2/STAT3 signalling plays an important role in fibrogenesis, angiogenesis and immune/inflammatory reactions by regulating factors such as VEGF, TGF-␤, eNOS and inducible nitric oxide synthase (iNOS) in numerous diseases [8–11]. However, the function of JAK2A/STAT3 signalling in portal hypertension remains unclear. Considering that the factors mentioned above play an important role in portal hypertension, we hypothesized that JAK2/STAT3 signalling has increased activity and participates in the development of portal hypertension. The aim of our study was to explore whether JAK2/STAT3 signalling contributes to the pathogenesis of portal hypertension and to determine whether portal hypertension is improved by JAK2/STAT3 pathway inhibition. AG490, a specific inhibitor of JAK2/STAT3 signalling that has been used in other studies [12,13], was utilized in our study. These results shed light on the mechanism underlying portal hypertension development and the design of novel strategies to prevent/ameliorate portal hypertension in clinics. 2. Materials and methods 2.1. Materials The phospho-STAT3 (p-STAT3, Tyr705) ELISA Kit and TGF-␤ antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). STAT3 and p-STAT3 (Tyr705) antibodies were purchased from Epitomics (Burlingame, CA, USA). VEGF, eNOS, phospho-eNOS (S1177), iNOS, lamin B and ␤-actin antibodies were purchased from Abcam (Boston, MA, USA). The CD43 primary antibody (GTX37449) was purchased from GeneTex (San Antonio, TX, USA). CD31 and alpha-smooth muscle actin (␣-SMA) antibodies were purchased from Boster (Wuhan, China). AG490 was purchased from InvivoGen (San Diego, CA, USA). 2.2. Animals In this study, male Sprague-Dawley (SD) rats (280–300 g) were used. All procedures were approved by the Committee for Care and Use of Laboratory Animal of the Fourth Military Medical University, and all animals received humane care in accordance with the institution’s ethical guidelines. Prehepatic portal hypertension was performed by partial portal vein ligation (PPVL) as described [14]. Briefly, under anaesthesia with phenobarbital sodium (50 mg/kg, Sigma, St. Louis, MO, USA), the portal vein was freed from the surrounding tissue. A ligature (silk 3-0) was placed around a 20-gauge blunt-tipped needle lying along the portal vein. The needle was then removed, leaving a calibrated constriction of the portal vein. In sham-operated animals, the portal vein was isolated and similarly manipulated but not ligated. Secondary biliary cirrhosis with intrahepatic portal hypertension was induced by common bile duct ligation (CBDL) as described [14]. When each animal was under anaesthesia, the common bile duct was occluded by double ligature with 5-0 silk thread. Then, between the two ligatures, the bile duct was resected. Two or three days after surgery, the urine turned to a dark brown colour, indicating the presence of bilirubin and a successful ligation operation. In the sham animals, the common bile duct was similarly manipulated but not ligated.

2.3. Treatments The PPVL/CBDL or sham rats were treated with (1) vehicle (VEH) (45% dimethyl sulfoxide (DMSO) and 55% 0.9% sodium chloride) (PPVL, n = 8; CBDL, n = 7; sham, n = 6); or (2) AG490 (5 mg/kg/day) (PPVL, n = 9; CBDL, n = 8; sham = 6). Therefore, the rats were divided into the following six groups: (1) sham-VEH, (2) sham-AG490, (3) PPVL-VEH, (4) PPVL-AG490, (5) CBDL-VEH, and (6) CBDL-AG490. All the treatments were administered intraperitoneally, once a day, over a two-week period, beginning on the day of the PPVL operation or 2 weeks after the CBDL operation. The AG490 dosage and schedule of administration were chosen according to the significant inhibition of JAK2 phosphorylation detected in our preliminary experiment. 2.4. Haemodynamic studies PE-50 catheters were introduced into the right femoral arteries to obtain the heart rate (HR; beats/min) and mean arterial pressure (MAP, mmHg) and into the portal vein through an ileocolic vein for the measurement of portal pressure (mmHg). The superior mesenteric artery (SMA) was isolated from connective tissue, and a nonconstrictive perivascular flow probe (Transonic Inc., Ithaca, NY, USA) was placed around the artery to continuously measure the SMA blood flow (SMABF, mL/min 100 g). The SMA resistance (SMAR, mmHg/mL min 100 g) was calculated as (MAP-portal pressure)/SMABF. The external zero reference was at the level of the midportion of the rat. Continuous recordings were recorded by a Data Acquisition & Analysis System (model BL420S, TME Technology Co., Ltd., Chengdu, Sichuan, China). 2.5. Sirius red staining Livers and spleens were fixed in 10% formaldehyde, embedded in paraffin, sectioned and stained with 0.1% Sirius red, photographed, and analyzed using a light microscope equipped with a Nikon digital camera (Nikon Instruments, Tokyo, Japan). The red-stained area per total area of the image was measured using NIS-Elements software (Nikon Instruments). 2.6. Immunohistochemistry (IHC) analysis Immunostaining of paraffin-embedded liver sections was performed with anti-p-STAT3 and anti-CD43 antibodies diluted 1:100 or with phosphate-buffered saline as a negative control. The final detection was performed using 3,3 -diaminobenzidine as the chromogen and 3% haematoxylin as the counterstain. 2.7. Western blot analysis Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and western blotting was performed using corresponding primary antibodies and an enhanced chemiluminescence detection system. 2.8. Enzyme linked immunosorbent assay (ELISA) analysis The ELISA was performed according to the manufacturer’s ELISA protocol. 2.9. Haematological analysis Blood samples were obtained from the rats through the femoral artery. Blood cell count analysis was performed using a haematology analyzer.

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Fig. 1. Activation and inhibition of JAK2/STAT3 signalling in partial portal vein ligation and common bile duct ligation rats. (A) Immunostaining for phospho-STAT3 protein (brown stain; magnification 200×), and western blot analysis of phospho-STAT3 and total STAT3 protein in common bile duct ligation rats. (B) Western blot analysis of phospho-STAT3 and total STAT3 protein in spleens and intestines from partial portal vein ligation and common bile duct ligation rats. (C) ELISA analysis of phospho-STAT3 protein in serum from partial portal vein ligation and common bile duct ligation rats. p-STAT3, phospho-STAT3; VEH, vehicle; CBDL, common bile duct ligation; PPVL, partial portal vein ligation.

2.10. Statistical analysis All data are expressed as mean ± standard deviation. Differences between experimental groups were analyzed using Student’s t test for unpaired data. Statistical significance was established at P < 0.05. 3. Results 3.1. Activation and Inhibition of JAK2/STAT3 signalling in portal hypertensive rats Phospho-STAT3 (Tyr705) (p-STAT3) protein was observed as a marker of JAK2/STAT3 overactivation. IHC in livers show that p-STAT3 protein was detected both in the cytoplasm and in the nucleus (brown stain) in different types of cells (hepatocytes, hepatic stellate cells and biliary epithelial cells) of the liver in the

CBDL-VEH group. After AG490 treatment, the shallower and less staining in the nucleus was observed, indicating that p-STAT3 protein expression decreased and less p-STAT3 protein was present in the nuclei where these proteins act as transcriptional factors (Fig. 1A). P-STAT3 protein expression in the liver nucleus was detected by western blot, which supported our finding from IHC (Fig. 1A). In PPVL-VEH rats and CBDL-VEH rats, the livers, spleens and small intestines exhibited elevated expression of p-STAT3 compared with sham-VEH animals. Impressively, after 2 weeks of AG490 treatment, p-STAT3 expression significantly decreased. No differences in p-STAT3 protein expression were observed between the sham-VEH and sham-AG490 group. No significant changes in the total STAT3 protein levels were detected in the livers, spleens and small intestines between the PPVL/CBDL groups and the sham group (Fig. 1A and B). ELISA analyses showed greatly increased p-STAT3 protein expression in serum from PPVL-VEH rats and CBDL-VEH rats

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4 Table 1 Haemodynamic data of the different animal groups.

Sham group

Number of animals HR, beats/min MAP, mmHg PP, mmHg SMABF (mL/min/100 g) SMAR (mmHg/mL/min/100 g)

PPVL group

CBDL group

VEH

AG490

VEH

AG490

VEH

AG490

6 292 ± 11 102 ± 4 7.5 ± 1.0 3.2 ± 0.2 29.2 ± 3.1

6 283 ± 14 103 ± 5 7.2 ± 0.9 3.3 ± 0.3 28.6 ± 2.7

8 323 ± 15* 93 ± 6* 13.2 ± 1.1* 5.4 ± 0.4* 15.1 ± 2.6*

9 312 ± 22 97 ± 4 9.6 ± 1.3** 4.4 ± 0.4** 20.2 ± 1.8**

7 325 ± 17* 92 ± 7* 16.4 ± 1.2* 6.0 ± 0.3* 13.9 ± 1.8*

8 315 ± 20 96 ± 4 12.7 ± 0.8*** 4.4 ± 0.2*** 19.6 ± 1.7***

HR, heart rate; MAP, mean arterial pressure; PP, portal pressure; VEH, vehicle; PPVL, partial portal vein ligation; SMABF, superior mesenteric artery blood flow; SMAR, superior mesenteric artery resistance. All of the values represent the means ± SD (standard deviation). * P < 0.05 vs. sham-VEH. ** P < 0.05 vs. PPVL-VEH. *** P < 0.05 vs. CBDL-VEH.

compared to sham-VEH rats, and this elevation was significantly inhibited after AG490 injection (P < 0.01, Fig. 1C).

3.2. Effect of AG490 on haemodynamics (Table 1) In PPVL/CBDL-VEH rats, higher portal pressure and splanchnic hyperdynamic circulation (increased SMABF and decreased SMAR) were observed compared with the sham-VEH rats (P < 0.05). Fortunately, 2-week AG490 treatment significantly reduced portal pressure and splanchnic hyperdynamic circulation in PPVL and CBDL rats (P < 0.05). In systemic circulation, a significantly increased HR and decreased MAP were observed in the PPVL-VEH groups and CBDL-VEH groups compared with the sham-VEH group (P < 0.05). However, AG490 had no effect on the HR or MAP in the PPVL and CBDL groups (P > 0.05).

3.3. Effect of AG490 on liver 3.3.1. Effects on intrahepatic fibrosis and liver function CBDL resulted in significant histological changes in the liver, including a distortion of the normal architecture, expansion of portal tracts with extensive bile-duct proliferation and collagen deposition (Fig. 2A). AG490 attenuated liver fibrosis compared with the CBDL-VEH rats as shown by Sirius red staining (P < 0.01, Fig. 2A). Western blotting showed that ␣-SMA, a HSC activation marker [15], was expressed more strongly in the livers of the CBDL-VEH group compared with the sham-VEH group (P < 0.05, Fig. 2B). Notably, AG490 significantly attenuated the overexpression of ␣-SMA in the livers of CBDL rats (P < 0.05, Fig. 2B). Western blot analyses showed that TGF-␤ is more highly expressed in the livers of CBDL-VEH rats compared with the sham-VEH rats, and this expression was inhibited after AG490 injection (P < 0.05, Fig. 2B). AG490 also decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in CBDL rats. However, no effect on total serum bilirubin (TBIL) was observed after AG490 treatment (Table 2), which may be due to the limitations of the CBDL model. AG490 had no effect on the serum biochemical parameters in PPVL rats (Table 2).

3.3.2. Effects on intrahepatic neovascularization Overexpression of VEGF, a pro-angiogenic factor, and CD31, a vascular endothelial cell marker, was demonstrated by western blot analyses of the livers of CBDL-VEH rats compared with sham-VEH animals (Fig. 2C). Notably, the expression of VEGF and CD31 significantly decreased compared with the CBDL-VEH animals after AG490 treatment (both P < 0.05, Fig. 2C).

3.3.3. Effects on intrahepatic eNOS expression Marked decreased phospho-eNOS expression was observed in the livers of the CBDL-VEH group compared with the sham-VEH group. No detectable changes in phospho-eNOS were observed between the CBDL-VEH and CBDL-AG490 groups (P < 0.05, Fig. 2C). 3.3.4. Effects on intrahepatic inflammation IHC to detect CD43 was used to observe the infiltration of inflammatory cells into the liver. We found increased CD43-positive cells surrounding the proliferating bile ducts in the CBDL-VEH livers compared with the sham group. AG490 attenuated CD43-positive inflammatory cell infiltration (Fig. 2D). 3.4. Effect of AG490 on splenomegaly Splenomegaly is typically found in patients with portal hypertension in the clinic. In our study, a large amount of fibrosis was observed in spleens from PPVL/CBDL-VEH animals compared with the sham-VEH rats (P < 0.01, Fig. 3B) as detected by Sirius red staining. Western blot analysis showed significantly higher expression of VEGF and ␣-SMA, the perivascular cell marker [16], in the PPVL/CBDL-VEH group compared with the sham-VEH group (P < 0.05, Fig. 3C). The greatly enlarged area of white pulps, a gathering place for immune cells in the spleen, was identified by haematoxylin and eosin (H&E) staining in the PPVL-VEH and CBDLVEH groups compared with the sham-VEH group (P < 0.01, Fig. 3D). Strongly expressed TGF-␤ was detected in the PPVL/CBDL-VEH groups compared with the sham-VEH rats (P < 0.05, Fig. 3D). Due to the high levels of fibrogenesis, angiogenesis and inflammation, the spleens in the PPVL/CBDL rats reflected an increased splenic index (the ratio of splenic weight to body weight) compared with the sham-VEH rats. After AG490 treatment, fibrogenesis, angiogenesis and inflammation were effectively inhibited, leading to decreased spleen size (P < 0.01, Fig. 3A). 3.5. Effects of AG490 on peripheral blood cell count The numbers of red blood cells (RBC), white blood cells (WBC) and blood platelets (PLT) are typically decreased in portal hypertensive patients with splenomegaly. In our study, the RBC and PLT counts in the PPVL-VEH group and CBDL-VEH group decreased compared with the sham-VEH rats. After AG490 injection, the RBC and PLT counts were significantly elevated compared with the levels in the PPVL/CBDL-VEH rats. No significant change in WBC counts was observed between the PPVL/CBDL-VEH rats and sham-VEH rats. AG490 had no effect on the peripheral blood cell count in the sham group (see Supplementary Figure S1).

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Fig. 2. Effects of AG490 on the livers of common bile duct ligation rats. (A) Histological images of livers stained with H&E or Sirius red and the quantification of liver fibrosis (Sirius red staining area/total area). (B) Western blot analysis of alpha-smooth muscle actin and transforming growth factor-␤. (C) Western blot analysis of vascular endothelial growth factor, CD31 and phospho-endothelial nitric oxide synthase. (D) Immunostaining for CD43 (brown stain; magnification 200×). VEH, vehicle; CBDL, common bile duct ligation; ␣-SMA, alpha-smooth muscle actin; TGF-␤, transforming growth factor-␤; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase.

Table 2 Serum biochemical parameters of different groups. Group

ALT (U/L)

Sham-VEH Sham-AG490 PPVL-VEH PPVL-AG490 CBDL-VEH CBDL-AG490

32.0 25.5 33.7 26.3 175.0 58.0

± ± ± ± ± ±

2.8 12.7 3.1 11.4 11.3* 14.1**

AST (U/L) 87.0 119.8 89.4 103.8 1089.0 145.0

± ± ± ± ± ±

TBIL (␮mol/L) 36.8 42.9 38.7 46.4 304.1* 28.3**

0.4 0.5 0.4 0.3 130.2 92.3

± ± ± ± ± ±

0.2 0.2 0.3 0.4 25.9* 22.8

Albumin (g/L) 30.3 26.9 29.5 28.9 27.1 28.6

± ± ± ± ± ±

2.1 2.8 2.5 3.2 3.1 2.6

ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin. All values are given as means ± SD (standard deviations). * P < 0.05 vs. Sham-VEH. ** P < 0.05 vs. CBDL-VEH.

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Fig. 3. Effects of AG490 on the spleens of partial portal vein ligation and common bile duct ligation rats. (A) Photographs of spleens in partial portal vein ligation and common bile duct ligation rats treated with the vehicle or AG490. Quantification of the spleen index. (B) Photomicrographs of Sirius red-stained spleen sections from AG490- or vehicle-treated partial portal vein ligation and common bile duct ligation rats (magnification 100×). Quantification of spleen fibrosis, (C) Western blot analysis of vascular endothelial growth factor and alpha-smooth muscle actin in partial portal vein ligation and common bile duct ligation rats. (D) Photographs of H&E-stained spleen sections (magnification 50×) showing white pulp (black arrow) and red pulp (white arrow). Quantification of white pulp area of spleens. Western blot analysis of transforming growth factor-␤ in partial portal vein ligation and common bile duct ligation rats. VEH, vehicle; CBDL, common bile duct ligation; PPVL, partial portal vein ligation; ␣-SMA, alpha-smooth muscle actin; TGF-␤, transforming growth factor-␤; VEGF, vascular endothelial growth factor.

Please cite this article in press as: Wang D, et al. Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2014.12.017

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Fig. 4. Effects of AG490 on the mesenteries and intestines of partial portal vein ligation and common bile duct ligation rats. (A) Histological images of mesenteries from partial portal vein ligation and common bile duct ligation rats (H&E stain; magnification 100×) and neovascularization quantification. (B) Photographs of H&E-stained intestines (magnification 100×). (C) Western blot analysis of vascular endothelial growth factor, CD31, alpha-smooth muscle actin, phosphorylated endothelial nitric oxide synthase, inducible nitric oxide synthase and transforming growth factor-␤ in partial portal vein ligation and common bile duct ligation rats. VEH, vehicle; CBDL, common bile duct ligation; PPVL, partial portal vein ligation; ␣-SMA, alpha-smooth muscle actin; TGF-␤, transforming growth factor-␤; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; p-eNOS, phosphorylated endothelial nitric oxide synthase.

3.6. Effect of AG490 on mesenteries and on intestines 3.6.1. Effects on mesenteric vascularization The vascularization area analyzed with H&E staining by Image-Pro Plus 6.0 software (Media Cybernetics Cooperation, Silver Spring, MD, USA) in sectional mesenteric tissues showed

that the vascular area was greatly increased in the PPVL-VEH and CBDL-VEH groups compared with the sham-VEH group (P < 0.01, Fig. 4A). The vascular area was markedly reduced after AG490 injection (P < 0.01, Fig. 4A); however, no difference was observed between the sham-VEH and sham-AG490 groups.

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3.6.2. Effects on intestines Histological studies of the intestines were performed using H&E staining. Both widened villi and a thickened muscle layer of small intestine were observed in the PPVL and CBDL group compared with the sham-VEH group. AG490 treatment produced a marked attenuation of the intestinal inflammation response (Fig. 4B). In addition, western blot analyses showed the strong expression of TGF-␤, iNOS VEGF, CD31, ␣-SMA and phospho-eNOS protein in the PPVL/CBDLVEH group compared with the sham-VEH group (P < 0.05, Fig. 4C); however, the levels of these proteins were significantly reduced after AG490 treatment (P < 0.05, Fig. 4C). No significant change in TGF-␤, iNOS VEGF, CD31, ␣-SMA and phospho-eNOS expression was found between the sham-VEH and sham-AG490 groups.

4. Discussion Portal hypertension is a syndrome involving fibrosis, angiogenesis and inflammation; therefore, the simultaneous inhibition of all these processes may be an effective way to alleviate portal hypertension. The JAK2/STAT3 signalling pathway is turned on by the activation of a cell surface receptor, leading to the recruitment and activation of JAK2 proteins, which in turn recruit and phosphorylate STAT3. Phosphorylated STAT3 homodimerizes and translocates to the nucleus where it binds to the promoter of target genes and activates their transcription to participate in fibrosis, angiogenesis and inflammation in numerous diseases. AG490 inactivates JAK2/STAT3 signalling by inhibiting JAK2 protein phosphorylation. Our research initially showed that JAK2/STAT3 signalling is overactivated and may be inhibited by AG490 in portal hypertensive rats. Although the role of JAK2/STAT3 signalling in liver fibrogenesis is controversial [17], the STAT3 pathway is crucial for the activation of HSC, the key step of liver fibrogenesis [18,19]. Deletion of the suppressor of cytokine signalling (SOCS3, a STAT3 signalling inhibitor) in the liver promotes dimethylnitrosamineinduced liver fibrosis in mice [20], and IL-17 exacerbates liver fibrosis through STAT3-mediated HSC activation in mice [21]. However, IL-22 protects against liver fibrosis through STAT3 pathway overactivation [22]. Our study showed that STAT3 signalling promotes ␣-SMA expression and fibrogenesis in the livers of CBDL rats. Similarly, recent research has shown [23] that p-STAT3 protein is overexpressed in the livers of SD rats and mice treated with CBDL or CCl4, and liver fibrogenesis was shown to improve after AG490 treatment. Therefore, the activation of JAK2/STAT3 may promote liver fibrosis and may be effectively inhibited by AG490. Our results demonstrated that the inhibition of JAK2/STAT3 signalling by AG490 contributed to the improvement of the liver with regard to fibrosis, angiogenesis and inflammation. The mechanism by which AG490 attenuates fibrosis is most likely mediated by the inhibition of HSC activation, as demonstrated by the strongly diminished ␣-SMA expression. The expression of TGF␤, which is crucial for fibrogenic myofibroblast activation, was also effectively diminished by AG490, which contributed to the inhibition of HSC activation [24]. Irregular angiogenesis promoted by VEGF in liver fibrosis may contribute to an increase in intrahepatic circulation resistance [25]. VEGF inhibition by AG490 greatly decreased intrahepatic neovascularization, as shown by diminished CD31 expression. The inhibition of HSC activation may also contribute to the reduction of VEGF expression [26]. Moreover, the inhibition of VEGF expression may also inhibit fibrosis by diminishing HSC activation [6]. A reduction in NO production by the endothelium is considered an important mechanism that results in an increase in intrahepatic resistance in

liver cirrhosis [27,28]. Although JAK2/STAT3 was reported to promote eNOS protein expression in many studies [10], decreased p-eNOS expression was found in the CBDL-VEH rats compared with the sham-VEH rats, indicating that JAK2/STAT3 signalling may not be the primary mechanism involved in eNOS regulation in liver fibrosis. Therefore, the diminished intrahepatic resistance may be due to the liver fibrosis attenuation and neovascular inhibition. Angiogenesis and increased eNOS-derived NO in splanchnic organs in portal hypertension were thought to be important factors in the maintenance of higher portal pressure [29,30]. Blockage of VEGF promoted portal pressure reduction [31,32], and the inhibition of JAK2/STAT3 signalling by AG490 greatly decreased the expression of VEGF and phospho-eNOS, which contributed to the inhibition of angiogenesis and the increase of the splanchnic vascular tone. Thus, in splanchnic circulation, the decreased formation of blood vessels and increased SMAR may have decreased SMABF. The decrease of SMABF and the number of blood vessels as well as the SMAR increase together contributed to the portal pressure decrease. In addition, reduction of intrahepatic resistance due to the improvement of liver fibrosis in the CBDL-AG490 group ameliorated the increase in haemodynamics. Therefore, we deduced that JAK2/STAT3 signalling promoted the development and maintenance of portal hypertension by stimulating vascular proliferation and decreasing vascular tone in splanchnic organs. The improvement of haemodynamic parameters in splanchnic circulation such as portal pressure, SMAR and SMABF benefited from the inhibition of JAK2/STAT3 signalling by AG490. Splanchnic inflammation is typically found in portal hypertensive patients, especially in patients with advanced portal hypertension and spontaneous bacterial peritonitis. Splanchnic inflammation aggravates both endothelial dysfunction and angiogenic processes [33]. Our data showed that increased JAK2/STAT3 signalling accelerates intestine inflammation in portal hypertensive rats by up-regulating TGF-␤ and iNOS expression. Moreover, the enhanced VEGF expression by JAK2/STAT3 signalling, though as an angiogenesis promoter, accelerated intestine inflammation [4]. In addition, high iNOS expression also contributed to the hyperdynamic splanchnic circulation in portal hypertension by regulating splanchnic vascular smooth muscle contractions [34]. Splenomegaly, a common complication in portal hypertensive patients [35], is thought to be caused by the difficulty of the splenic blood outflow to enter the portal vein due to portal hypertension. However, recent studies [36] showed that splenomegaly is related to several complicated and complementary mechanisms, including blood congestion, inflammation, angiogenesis and fibrogenesis. In our research, inflammation, angiogenesis and fibrogenesis related to splenomegaly were effectively inhibited after AG490 treatment. The improvement of splenomegaly decreases splenic vein blood flow, contributing to portal pressure reduction. Moreover, the enlarged spleen sequesters more circulating blood, leading to a decrease in the number of peripheral hemocytes. The improvement in the RBC and PLT counts may due to the inhibition of splenomegaly by AG490. In summary, our present results documented the role of the JAK2/STAT3 pathway in tissue inflammation, fibrosis and angiogenesis during portal hypertension. This study also initially demonstrated that specifically targeting JAK2/STAT3 signalling using AG490 improved portal pressure and haemodynamics and ameliorated tissue inflammation, fibrosis and angiogenesis. In conclusion, blocking the JAK2/STAT3 signalling pathway using AG490 may provide a new potential target for portal hypertensive therapeutic intervention.

Please cite this article in press as: Wang D, et al. Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2014.12.017

G Model YDLD-2801; No. of Pages 9

ARTICLE IN PRESS D. Wang et al. / Digestive and Liver Disease xxx (2015) xxx–xxx

Conflict of interest None declared. Funding The paper is supported by the Key Natural Science Foundation of Shaanxi Province, China (2012JM4002) and the National Natural Science Foundation of China (81172287). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dld.2014.12.017. References [1] Bosch J, Abraldes JG, Groszmann R. Current management of portal hypertension. Journal of Hepatology 2003;38(Suppl. 1):S54–68. [2] Tripathi D, Hayes PC. Beta-blockers in portal hypertension: new developments and controversies. Liver International 2014;34:655–67. [3] Tu K, Li J, Verma VK, et al. VASP promotes TGF-beta activation of hepatic stellate cells by regulating Rab11 dependent plasma membrane targeting of TGF-beta receptors. Hepatology 2014;61:361–74. [4] Chen X, Yang G, Song JH, et al. Probiotic yeast inhibits VEGFR signaling and angiogenesis in intestinal inflammation. PLoS ONE 2013;8:e64227. [5] Yang L, Kwon J, Popov Y, et al. Vascular endothelial growth factor promotes fibrosis resolution and repair in mice. Gastroenterology 2014;146:1339–50. [6] Nakamura I, Zakharia K, Banini BA, et al. Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling. PLOS ONE 2014;9:e92273. [7] Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as hypoxiadependent factors stimulating migration of human hepatic stellate cells. American Journal of Pathology 2007;170:1942–53. [8] Joung YH, Na YM, Yoo YB, et al. Combination of AG490, a Jak2 inhibitor, and methylsulfonylmethane synergistically suppresses bladder tumor growth via the Jak2/STAT3 pathway. International Journal of Oncology 2014;44:883–95. [9] Liu RY, Zeng Y, Lei Z, et al. JAK/STAT3 signaling is required for TGF-beta-induced epithelial-mesenchymal transition in lung cancer cells. International Journal of Oncology 2014;44:1643–51. [10] Wang SG, Xu Y, Chen JD, et al. Astragaloside IV stimulates angiogenesis and increases nitric oxide accumulation via JAK2/STAT3 and ERK1/2 pathway. Molecules 2013;18:12809–19. [11] Chong HC, Chan JS, Goh CQ, et al. Angiopoietin-like 4 stimulates STAT3mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice. Molecular Therapy 2014;22:1593–604. [12] Yang J, Xiao Z, Li T, et al. Erythropoietin promotes the growth of pituitary adenomas by enhancing angiogenesis. International Journal of Oncology 2012;40:1230–7. [13] Gyurkovska V, Stefanova T, Dimitrova P, et al. Tyrosine kinase inhibitor tyrphostin AG490 retards chronic joint inflammation in mice. Inflammation 2014;37:995–1005. [14] Abraldes JG, Pasarin M, Garcia-Pagan JC. Animal models of portal hypertension. World Journal of Gastroenterology 2006;12:6577–84. [15] Rosado E, Rodriguez-Vilarrupla A, Gracia-Sancho J, et al. Terutroban, a TPreceptor antagonist, reduces portal pressure in cirrhotic rats. Hepatology 2013;58:1424–35. [16] Hirschberg RM, Plendl J, Kaessmeyer S. Alpha smooth muscle actin in the cycling ovary – an immunohistochemical study. Clinical Hemorheology and Microcirculation 2012;50:113–29.

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Please cite this article in press as: Wang D, et al. Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats. Dig Liver Dis (2015), http://dx.doi.org/10.1016/j.dld.2014.12.017

Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats.

JAK2/STAT3 signalling promotes fibrosis, angiogenesis and inflammation in many diseases; however, the role of this pathway in portal hypertension rema...
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