Accepted Manuscript Title: Inhibition of Soluble Epoxide Hydrolase Lowers Portal Hypertension in cirrhotic Rats by Ameliorating Endothelial Dysfunction and Liver Fibrosis Authors: Wensheng Deng, Yiming Zhu, Jiayun lin, Lei Zheng, Chihao Zhang, Meng Luo PII: DOI: Reference:

S1098-8823(17)30080-1 http://dx.doi.org/doi:10.1016/j.prostaglandins.2017.08.004 PRO 6241

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Prostaglandins and Other Lipid Mediators

Received date: Revised date: Accepted date:

4-6-2017 25-7-2017 8-8-2017

Please cite this article as: Deng Wensheng, Zhu Yiming, lin Jiayun, Zheng Lei, Zhang Chihao, Luo Meng.Inhibition of Soluble Epoxide Hydrolase Lowers Portal Hypertension in cirrhotic Rats by Ameliorating Endothelial Dysfunction and Liver Fibrosis.Prostaglandins and Other Lipid Mediators http://dx.doi.org/10.1016/j.prostaglandins.2017.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of Soluble Epoxide Hydrolase Lowers Portal Hypertension in cirrhotic Rats by Ameliorating Endothelial Dysfunction and Liver Fibrosis Wensheng Deng1, Yiming Zhu1, Jiayun lin1, Lei Zheng1, chihao Zhang1, Meng Luo1. 1Department of General Surgery, Shanghai Ninth People’s Hospital, No. 639 Zhizaoju RD, Shanghai, China

Address for Correspondent: Prof. Dr. Meng Luo Department of General Surgery, Shanghai Ninth People’s Hospital, No. 639 Zhizaoju RD, Shanghai, China Tel: 13817882266 E-mail:[email protected]

Highlights 1. Inhibition of soluble epoxide hydrolase decreased intrahepatic vascular resistance and portal pressure in liver cirrhosis 2. Inhibition of soluble epoxide hydrolase attenuated endothelial dysfucntion and hepatic fibrosis 3. Inhibiting soluble epoxide hydrolase may be an alternative method to treat portal hypertension in patients with cirrhosis Abstract Epoxyeicostrienoic acids (EETs) are arachidonic acid derived meditators which are catalyzed by soluble epoxide hydrolase (sEH) to less active dihydroeicostrienoics acids (DHETS). The aim of our study is to investigate the effects of sEH inhibition on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. The sEH inhibitor,trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid (t-TUCB) was administered to stabilize hepatic EETs by gavage at a dose of 1 mg/kg/d. Our results showed that hepatic sEH expression was markedly increased in portal hypertension,and led to a lower ratio of EETs/DHETs which was effectively reversed by t-TUCB administration. t-TUCB significantly decreased portal pressure without significant changes in systemic hemodynamics, which was associated with the attenuation of intrahepatic vascular resistance (IHVR) and liver fibrosis. t-TUCB ameliorated endothelial dysfunction, increased hepatic endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. In addition, t-TUCB significantly reduced alpha-Smooth Muscle Actin (α-SMA) expression and liver fibrosis, which was associated with a decrease in NF-κB signaling. Taken together, inhibition of sEH reduces portal pressure, liver fibrosis and attenuates hepatic endothelial dysfunction in cirrhotic rats. Our results indicate that sEH inhbitors may be useful in the treatment of portal hypertension in patients with cirrhosis.

Abbreviations EETs, epoxyeicosatrienoic acids;sEH,soluble epoxide hydrolase,

DHETs, dihydroxyeicosatrienoic acids; t-TUCB, trans-4-{4-[3-(4-trifluorometh oxyphenyl)-ureido]cyclohexyloxy}benzoic acid; IHVR, intrahepatic vascular resistance;α-SMA, alpha-Smooth Muscle Actin;eNOS, endothelial nitric oxide synthase; NO, nitric oxide; PEG400, polyethylene glycol 400; HSCs, hepatic stellate cells Keywords: sEH; EETs; endothelial dysfunction; liver fibrosis; portal hypertension Introduction Increased intrahepatic vascular resistance is the primary factor in the pathophysiology of portal hypertension (PHT) with liver cirrhosis[1]. In addition to morphological changes, reversible functional alterations including activated hepatic stellate cells (HSCs), exaggerated production of vasoconstrictors and decreased levels of NO vasodilator, contribute to increased IHVR [2, 3]. Apart from cyclooxygenase- and lipoxygenase-dependent pathways, the third pathway for arachidonic acid metabolism is cytochrome P-450 (CYP)/epoxygenase-dependent production of EETs. The EETs are rapidly degraded by sEH, producing a low activity of DHETs [4]. Stabilization of EETs through inhibition of sEH has shown anti-inflammatory, anti-hypertensive, anti-fibrotic and analgesic effects. For example, sEH pharmacological inhibition ameliorates experimental acute pancreatitis in mice[5], exhibited antihypertensive and renoprotective actions[6], and inhibits angiotensin II induced ventricular remodeling[7]. These studies support that EETs are protective in pathological conditions,so sEH is a potentially important therapeutic target, especially in inflammatory diseases. Increasing EETs in vivo has been reported to exert protective effects in experimental models of liver injury induced by high-fat diet or CCl4. Restoring hepatic and circulating EET levels significantly attenuated hepatic inflammation and injury in a mouse model of non-alcoholic fatty liver disease[8]. Moreover, endothelial-specific CYP2J2 transgenic mice showed significantly improved liver function, reduced inflammatory responses, and a decrease in hepatic oxidative stress [9]. In addition, sEH inhibition reduced endoplasmic

reticulum stress by decreasing endogenous fatty acid epoxide chemical mediators, and resulted in a dramatic attenuation of CCl4-induced liver fibrosis in mice [10]. Although many evidence have demonstrated the protective action of sEH inhibition in liver injury, the effects of sEH inhibition in portal hypertension with chronic liver cirrhosis is still unknown. In cirrhotic livers, NO plays a key role in the pathogenesis of the increased IHVR. Insufficient NO availability resulting from decreased eNOS activity has been observed within cirrhotic livers, leading to constriction of hepatic sinusoids and increased IHVR to portal blood flow [11]. It has been reported that EETs relax vessels largely through its their ability to activate endothelial NO synthase (eNOS) and NO release [12]. Moreover, EETs enhance eNOS function via promoting eNOS phosphorylation. Overall, our hypothesis is that the inhibition of sEH decrease IHVR in cirrhosis by affecting eNOS function and NO production in liver. The aim of the present study was to evaluate the effects of long-term administration of sEH inhibitor in CCl4-cirrhotic rats with portal hypertension.

Materials and Methods Animal models and treatment

Male Sprague-Dawley rats (6 weeks; 180–220 g) were obtained from Experimental Animal Center of School of Medicine, Shanghai Jiaotong University (shanghai, China) and maintained in our specific pathogen-free conditions with a 12-h light/dark cycle and unlimited supplies of food and water. All protocols were approved by Ethical Committee of Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). A total of 48 rats were randomly divided into four groups: control group, control + t-TUCB group, model group, and model+ t-TUCB group. To induce liver cirrhosis, rats were subjected to subcutaneous administration of CCl4 (1:1 in olive oil) at a dose of 3 ml/kg body weight, twice a week for 12 weeks. Control mice were given the same volume of olive oil alone for the indicated time

intervals. In all experiments, rats from control and model groups received vehicle or sEH inhibitor, t-TUCB. t-TUCB was solubilized in in 20% PEG-400 in saline and administered at a dose volume of 10 ml/kg, body weight [13]. At the end of the 8th week, rats received a dose of 1 mg/kg of t-TUCB or vehicle by gavage once a day for 4 weeks.

Hemodynamic studies Rats were anesthetized with ketamine and xylazine. The left femoral artery and ileocolic vein were introduced with a PE-50 catheter for the measurement of mean arterial pressure (MAP; mmHg) and portal pressure (PP, mmHg). The catheters placed in the portal vein and femoral artery were connected to pressure transducers (ALC-MPA Acquisition and Analysis System for Life Science Research, Alcott Biotech, Shanghai, China) for monitoring portal and systemic blood pressure respectively. After 20 min stabilization, MAP and PP were recorded. The Dye-Trak microsphere technique was performed as previously described [14]. In brief, the 1-min withdrawal (0.65 mL/min) of a reference sample was conducted with a continuous extraction pump (ALC-IP900, shanghai, china). Suspending in the solution of 0.3 mL saline with 0.05% Tween, approximately 300000 yellow microspheres of 15.5 μm in diameter (Triton Technologies, San Diego, California, United States) were injected into the left ventricle within 10 s of starting blood withdrawal. Suspending in a solution as same as yellow ones, 150000 blue microspheres was injected into an ileocaecal vein within 30s to evaluate mesenteric portal-systemic shunt volume. The rats were sacrificed by injection of KCl intravenously. The tissues were carefully weighted and completely digested by boiling them in 5 M KOH solution containing 10% Tween 80. The microspheres were then collected and dissolved into 0.2 ml dimethylformamide and measured by absorption spectrophotometry. Hemodynamic parameters were measured and calculated according to

standard methods [15]. Afterwards, organ blood flow were calculated and expressed based on 100 g per body weight with the software of Triton Technologies. Portal blood flow (PBF) was presented by the sum of blood flow to the stomach, spleen, intestines, pancreas, colon and mesentery. Mesenteric portal-systemic shunt flow was derived from the fraction in the lung out of total injected blue microspheres. Hepatic portal-vascular resistance was estimated as PP divided by the sum of gastrointestinal and splenic perfusion minus mesenteric portal-systemic shunt flow. Evaluation of endothelial function In situ liver perfusion study was performed as previously described [16]. Briefly, Rats were anesthetized and livers were quickly isolated and perfused by a flow-controlled perfusion system. To evaluate intrahepatic vascular responsiveness to vasoconstrictors, a1-adrenergic agonist methoxamine (10-4 M) was added to the perfusate after 20 min stabilization period. After 5 min incubation period with methoxamine, concentration–response curves to increasing concentrations of acetylcholine (Ach: 10-7, 10-6, and 10-5 mol/L) were performed to evaluate endothelial function. The concentration of Ach was increased by 1 log unit every 1.5 min interval. Responses to Ach were calculated as the percentage change in portal perfusion pressure. The gross appearance of the liver, stable perfusion pressure, bile production over 0.4 μl /min/g of liver and a stable buffer pH (7.4 ± 0.3) were monitored during this period. If any viability or stability criteria were not satisfied, the experiment was discarded. LC-MS/MS analysis of EETs and DHETs The quantification of hepatic tissue EETs by LC-MS/MS was performed as previous description [17]. EETs and DHETs were extracted with ethyl acetate(Sigma, USA) following alkali hydrolysis of the phospholipids to release esterified EETs and quantified with a Q-trap 3200 linear ion trap quadruple LC/MS/MS (ABScieX; Q-trap 3200) equipped with a Turbo V ion source operated in negative electrospray mode (Applied Biosystems, Foster City, CA),

according to the instruction. Protein concentration of samples was determined by the BCA method (Beyotime, China) and was used to normalize the detected lipids. Total EETs and DHETs levels were expressed as ng/mg protein. Measurement of NO According to the instructions of NO assay kit (Beyotime), we used Griess method to measure the NO2- and NO3- levels in liver, which serve as markers of NO production. The NO2- and NO3- levels were expressed as nmol /mg protein. Protein concentration of liver samples was determined by a BCA protein assay kit (Beyotime). Histological and immunohistochemical examination The liver sections were fixed in 10% neutral formalin, embedded in paraffin, cut into 5 μm-thick sections with a microtome and mounted on glass slides. Hematoxylin and eosin (H&E) staining and Sirius red staining were performed to some liver slices. Liver sections were evaluated randomly by an accomplished liver pathologist yet unfamiliar with animal groups. For immunohistochemistry, liver sections were incubated with anti-sEH antibody (1:100, Santa Cruz, CA, USA) and anti-a-smooth muscle actin (a-SMA) antibody (1:100, Abcam, Cambridge, United Kingdom) overnight at 4°C, or, as a negative control, with phosphate-buffered saline. The slides were incubated with appropriate HRP-conjugated goat anti-rabbit secondary antibody for 60 min and with DAB as a substrate, and were then counterstained with hematoxylin. The content of collagen deposition was quantified with an image analyzer (Image-Pro Plus, Media Cybernetics), and the stained areas are reported to be its ratio to the total area. The liver sections were then averagely valued among five rats from each group. Western Blot Analysis Liver tissue was pulverized with a high throughput tissue-grinding apparatus. According to instruction of Nuclear and Cytoplasmic Extraction kit

(Thermo Fisher Scientific, USA), Nuclear and cytosolic proteins from the tissues were prepared. The total cellular protein was extracted as a radioimmunoprecipitation assay lysate containing the protease inhibitor phenylmethanesulfonyl fluoride. Total protein was quantified by BCA Protein Assay Kit (Beyotime). Samples (40 μg of protein/lane) were assayed by 10% SDS-PAGE. After electrophoresis, protein was shifted to a polyvinylidene difluoride membrane which was blocked in 5% (wt/vol) non-fat dry milk for 2 h and treated as below in primary antibodies at the temperature of 4 °C overnight: eNOS, p-eNOS (Ser1177), and caveolin-1(1:1000 dilution) from Cell Signaling Technology(CST, MA, USA) ; α-SMA(1:1000 dilution) from Abcam; sEH (1:200 dilution) from Santa Cruz; NF-κBp65 and IκBα (1:200 dilution) from Santa Cruz. The membrane was then processed for 1 h with appropriate secondary antibodies (CST) at a 1:5000 dilution. The bands were detected using an enhanced chemiluminescence system (Fusion FX7 Spectra; Vilber Lourmat, Eberhardzell, Germany). Statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed using SPSS version 19.0 software. Data were analyzed by using one way ANOVA and P < 0.05 was considered statistically significant.

Results sEH is up-regulated in cirrhotic livers The first is to investigate the sEH expression in portal hypertensive rats with liver cirrhosis. Immunohistochemical (IHC) staining showed an increase of the intracellular sEH staining in all untreated cirrhotic rats, compared to healthy controls (fig. 1A). Meanwhile, western blot date showed that the level of hepatic sEH expression was significantly increased in cirrhotic rats comparing with the normal rats (fig.1B). Relative protein levels are shown in figure 1B.

Hepatic EETs/DHETs ratio is increased in CCl4-induced cirrhosis rats

after t-TUCB treatment As shown in the table. 1, the ratio of EETs/DHETs in cirrhotic rat livers was significantly lower compared with the control livers, because increased sEH activity led to more EETs degradation and DHETs production. Moreover, total EETs levels were greatly decreased in cirrhotic rat livers. t-TUCB-treated cirrhotic rats had a significantly higher ratio of EETs/DHETs than vehicle-treated cirrhotic rats (95.123±18.312 vs 30.165±14.951), reveling that sEH inhibitor, t-TUCB could effectively reverse the ratio EETs/DHETs and cause an increase in EETs levels.

Effect of t-TUCB on hepatic and systemic hemodynamics in control and CCl4 cirrhotic rats Compared with the control rats, cirrhotic rats had higher portal pressure and lower arterial blood pressure (table. 2). Cirrhotic rats also displayed increased PBF and intrahepatic vascular resistance, indicating the presence of the hyperdynamic circulatory state. t-TUCB had no significant effects on hepatic and systemic hemodynamics in control rats. In contrast, cirrhotic rats receiving long-term treatment with t-TUCB had a significantly lower PP than cirrhotic rats treated with vehicle (16.1 ± 1.5 vs.13.0 ± 1.8 mmHg; p < 0.01; mean decrease 19.3%). This reduction was associated with a significant decrease in PBF, which indicates a reduction in intrahepatic vascular resistance. In addition, MAP was lower in cirrhotic rats with t-TUCB treatment, but the difference did not reach statistical significance (p = 0.075). Effect of t-TUCB on Endothelial Function in cirrhotic rat livers Under normal vasoactive endothelial function, a dose–dependent vasodilatation could be observed in normal intrahepatic vascular bed by increasing each concentration (fig. 2). Cirrhotic rat livers displayed endothelial dysfunction, which was an impaired vasorelaxation at any given concentration of ACh. As shown in fig. 2, cirrhotic rats receiving treatment of t-TUCB showed

a significant decrease in perfusion pressure in response to each given concentration of ACh. These results support that administration of t-TUCB ameliorates endothelial dysfunction in cirrhotic rat livers.

Effect of t-TUCB on hepatic fibrosis and hepatic stellate cells (HSCs) As shown in fig. 3A, cirrhotic rats exhibited a distorted architecture with extensive fibrosis combined with the development of micronodules. Rats receiving t-TUCB showed a significant reduction in hepatic fibrosis (fig. 4A). This could be proved by Sirius Red staining from a histological perspective. Collagen content quantification demonstrated that the collagen volume fraction decreased in t-TUCB-treated cirrhotic rats (13.5 ± 3.5%) in comparison with vehicle-treated cirrhotic rats (28.3 ± 7.7%%). α-SMA is a marker of HSCs activation and serves as the sensitive index of the degree of cirrhosis. Immunohistochemical staining and Western blot analysis against α-SMA consistently showed a significant increase in α-SMA expression in cirrhotic rat livers, while t-TUCB treatment markedly reduced the expression of α-SMA (fig. 4C), indicating that t-TUCB reduces the activation of HSCs.

t-TUCB induced-portal hypertensive reduction relates to increased eNOS activation and decreased NF-κB signaling The hepatic eNOS expression did not change between normal and cirrhotic rats, but a significant decrease in hepatic eNOS phosphorylation was observed in cirrhotic rats. This indicates that a reduction in NO release in cirrhotic livers (fig.4A ) corresponded with western blot data which showed that NO levels in cirrhotic rat livers were significantly decreased compared to the control group (fig. 4C). These results demonstrate that NO bioavaibility is insufficient in liver cirrhosis. After t-TUCB administration, p-eNOS (Ser1177) levels in cirrhotic livers were significantly increased without changing eNOS expression. Furthermore, the expression of Caveolin-1 (an intracellular eNOS inhibitor) was observed to be significantly higher in cirrhotic rat livers compared

with control rats. In contrast, treatment with t-TUCB resulted in significant downregulation of Caveolin-1 expression in comparison with vehicle treatment. As shown in fig. 4C, cirrhotic rats given t-TUCB treatment showed an increase in NO levels compared with vehicle treatment. The above results suggest that increased NO release is caused by t-TUCB treatment which indicates that eNOS function was restored. NF-κB signaling plays a pivotal role in driving the progress of hepatic inflammation and HSCs activation. The expression levels of the cytoplasmic proteins NF-κBp65 and IκBα were significantly decreased in cirrhotic rats compared with the control group, whereas nucleic NF-κBp65 expression was markedly increased in cirrhotic rats (fig. 5). As shown in fig. 5, t-TUCB administration restored cytoplasmic proteins NF-κBp65 and IκBα to normal levels. Additionally, t-TUCB significantly prevented NF-κBp65 from entering the nucleus and inhibit the activation of NF-κB signaling indicating its anti-inflammatory/fibrotic effects. Discussion The salient findings of the present study are that 1) The level of the sEH expression is high in cirrhotic rat livers, then the administration of sEH inhibitor, t-TUCB for 4 weeks reduces portal hypertension, and decreases portal blood flow which reduces hepatic vascular resistance; 2) Treatment with t-TUCB improves endothelial dysfunction via enhancement of hepatic eNOS phosphorylation and NO release; 3) t-TUCB exhibits an effective reduction in hepatic fibrosis, due to the inhibition of HSCs activation and NF-κB signalling. In cirrhosis, increased intrahepatic vascular resistance to portal blood flow is still the primary factor in the development of portal hypertension[18]. Hence, focusing on decreasing portal pressure via reducing intrahepatic vascular resistance is a very attractive therapeutic strategy. In this study, we found that utilizing t-TUCB to inhibit sEH activity has a beneficial effect on hepatic circulation, as shown by the reduction in portal pressure due to decreased intrahepatic vascular resistance. It is noteworthy that sEH inhibitor also has an

adverse effect on blood pressure due to vasodilatation, although it is not statistically significant. Under physiological condition, increasing EET bioavailability via deficiency/inhibition of sEH potentiates vasodilator responses, leading to significantly lower blood pressure [19]. Furthermore, inhibition of sEH exhibits antihypertensive and cardioprotective actions in this transgenic model of angiotensin II-dependent hypertension which is associated with the increased ratio of EETs/DHETEs [20]. There are some common features between liver cirrhosis and hypertension, such as reduced endothelial NO availability. In cirrhotic portal hypertension, decreased intrahepatic eNOS activity is considered a main factor in the pathogenesis of increased intrahepatic vascular resistance[21]. Thus, increasing NO availability leading to lower portal pressure is current advocated treatment methods. In fact, improved eNOS function from t-TUCB treatment not only attenuated endothelial dysfunction in cirrhotic livers but also resulted in a reduction in intrahepatic vascular resistance. In cirrhotic liver, increased caveolin-1 expression in perisinusoidal cells may promote caveolin-eNOS binding and reduce the activity of eNOS, leading to a reduction in NO production and an increase in intrahepatic vascular resistance[22]. A possible mechanism caveolin-1 involvement in the downregulation of eNOS activity is that caveolin-1 interacts with G-Protein– Coupled Receptor Kinase-2, thus clustering eNOS within a complex that inhibits eNOS activity[23]. In this study, inhibition of sEH could enhance eNOS phosphorylation and downregulate the expression of Caveolin-1. Therefore, the decreased caveolin-1 serves as one of reasons that t-TUCB increases the activity of eNOS in cirrhotic rats. The interaction between EETs and NO in vascular homeostasis is quite complicated. NO decreases CYP450 activity and content, and inhibits CYP450-dependent production of arachidonic acid metabolites that dilate the vasculature [24]. CYP-450-dependent production of EETs exhibit its crucial

biological function after NOS inhibition [25]. On the other hand, EETs at least in part upregulate the function of eNOS in cardiovascular diseases and diabetes [12, 26]. For example, increasing EETs restores high fat-induced decrease in vascular heme oxygenase-1, phosphorylated eNOS, and phosphorylated protein kinase B, thus attenuates endothelial dysfunction in diet-induced obese mice [27]. Inconsistent with previous research, the present study demonstrates that increasing EETs with sEH inhibitor also improves endothelial dysfunction, which is probably associated with increased up-regulation of eNOS function. In addition to improved endothelial dysfunction, t-TUCB contributes to the reduction in hepatic vascular resistance via the amelioration of liver fibrosis. Indeed, t-TUCB significantly reduced hepatic fibrosis, as shown by the reduction in fibrosis area in Sirius Red stained liver sections and the decrease in α-SMA expression. Hammock et al [10] also found that sEH inhibition prevents the development of fibrosis in a CCl4-induced mouse model, but there is currently no study about the effect of t-TUCB on HSCs activation. It is now widely accepted that the activation of HSCs is vital to liver injury and liver fibrosis [28]. Therefore, aiming at the HSC activation is crucial to prevent or reverse hepatic fibrosis. In the present study, our results show a decline in α-SMA expression in liver tissue, demonstrating that a decrease in HSCs activation is due to the anti-fibrotic function of t-TUCB. It have been reported that activation of the NF-κBs, especially the NF-κBp65 subunit, is closely associated with HSC activation in liver fibrosis[29]. These results suggest that inhibition of NF-κB/IκBα signaling might be a potential anti-fibrosis method. In this study, we firstly observed that t-TUCB suppressed liver fibrosis through the NF-κB/IκBα signaling pathway. In unstimulated conditions, NF-κB is associated with IκBα in cytoplasm. Once stimulated by stressors, IKK is activated leading to the phosphorylation of IκB, which is then degraded through the ubiquitin-proteasome pathway. Subsequently, NF-κB is transported into the nuclei, leading to the activation of gene transcription[30]. As expected, t-TUCB administration corrects the abnormal changes in the

subcellular distribution of NF-κB, indicating that t-TUCB could inhibit NF-κB/IκBα signaling in liver cirrhosis. Thus, we come to a conclusion that t-TUCB could suppress the activation of HSCs and reduce hepatic fibrosis, in part due to the inhibition of NF-κB/IκBα signaling. Although we have made many achievements, there are some limitation which should be addressed in the future work. For instance, it is recently reported that inhibition of EETs production reduces portal hypertension through reducing splanchnic vasodilation[31]. Indeed, it is not contradictory to our study. Because sEH inhibitors exhibit anti-inflammatory/ fibrotic effects, which may overwhelm these deleterious effects of EETs. Therefore, we will continue to focus on the properties of EETs in my future work, and explore the potential mechanism that EETs stimulate NO production. In addition, EETs contain four regioisomers: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. We need to confirm which one is mainly responsible for the attenuated portal hypertension. The last is we only explore the effects of sEH inhibiton on intrahepatic vascular system, but the role of sEH inhibitors on splanchnic vascular vasodilation should also be evaluated. In conclusion, our current data show that long-term t-TUCB administration to cirrhotic rats reduces portal pressure both by causing a reduction in hepatic fibrosis and the amelioration of hepatic endothelial dysfunction, without affecting systemic hemodynamics. Inhibition of sEH may be a useful supplement in the treatment of patients with cirrhosis and portal hypertension, due to the biological properties of EETs, including as anti-oxidation, anti-inflammation, activating eNOS enzyme and anti-fibrosis. Conflict of Interests The authors declare that there is no conflict of interest in relation to this article. Acknowedgements This study was supported by a grant from the Natural Science Foundation of China (No. 81370548). Authors’ Contribution

Wensheng Deng performed the majority of experiments and wrote the paper; Yiming Zhu and Jiayun Lin established portal hypertensive rats model. Meng Luo designed this study and revised the paper. Chihao Zhang and Lei Zheng provided analytical tools, and analyzed data. Meng Luo provided financial support for this work.

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Figures:

Fig.1. (A) Comparative IHC sEH staining showing an increase above detection level of the typical cytoplasmic staininig pattern in cirrhotic rats (n=5 in all groups). Data are indicated as mean ± SEM. (B) Western blot analysis showing a significant increase in sEH expression in cirrhotic rats. Data are shown as mean ± SEM.

Fig.2. In the healthy control group, a significant decrease in perfusion pressure was observed at all ACh concentrations ranging from 10-7 to 10-5 M after preincubation with methoxamine 10-5 M (P

Inhibition of soluble epoxide hydrolase lowers portal hypertension in cirrhotic rats by ameliorating endothelial dysfunction and liver fibrosis.

Epoxyeicostrienoic acids (EETs) are arachidonic acid derived meditators which are catalyzed by soluble epoxide hydrolase (sEH) to less active dihydroe...
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