The Beneficial Effects of P2X7 Antagonism in Rats with Bile Duct Ligation-induced Cirrhosis

Splanchnic angiogenesis in liver cirrhosis often leads to complications as gastroesophageal variceal hemorrhage and the treatment efficacy is adversely affected by poor portal-systemic collateral vasoresponsiveness related to nitric oxide (NO). Purinergic receptor subtype P2X7 participates in the modulation of inflammation, angiogenesis, fibrogenesis and vasoresponsiveness, but the relevant influence in cirrhosis is unknown. Common bile duct-ligated (CBDL) or sham-operated Spraque-Dawley rats received brilliant blue G (BBG, a P2X7 antagonist and food additive) or vehicle from the 15th to 28th day after operations, then hemodynamics, mesenteric angiogenesis, portal-systemic shunting, liver fibrosis, and protein expressions of angiogenic and fibrogenic factors were evaluated. The influence of oxidized ATP (oATP, another P2X7 receptor antagonist) on the collateral vasoresponsiveness to arginine vasopressin (AVP) was also surveyed. BBG decreased superior mesenteric artery (SMA) flow, portal-systemic shunting, mesenteric vascular density, and mesenteric protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho (p)-VEGFR2, platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRβ), cyclooxygenase (COX)-1, COX-2, and endothelial NO synthase (eNOS) in CBDL rats. BBG also ameliorated liver fibrosis and down-regulated hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), PDGF, IL-1β, transforming growth factor-beta (TGF-β), p-extracellular-signal-regulated kinases (ERK), and alpha-smooth muscle actin (α-SMA) expressions in CBDL rats. The collateral vasocontractility to AVP was enhanced by oATP. oATP down-regulated eNOS, inducible NOS (iNOS), VEGF, Akt, p-Akt, and nuclear factor-kappa B (NF-κB) expressions in splenorenal shunt, the most prominent intra-abdominal collateral vessel in rodents. P2X7 antagonism alleviates splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and enhances portal-systemic collateral vasoresponsiveness in cirrhotic rats. P2X7 blockade may be a feasible strategy to control cirrhosis and complications.


Introduction
Liver inflammation is usually followed by fibrogenesis, cirrhosis and ultimately, complications from increased intrahepatic resistance and portal hypertension. Portal-systemic collateral vessels develop during the course and bleeding from the collaterals, especially the gastroesophageal varices, and is one of the most dreadful complications among cirrhotic patients. Angiogenesis plays a role in the process [1] and even worse, the poor vasoresponsiveness to vasoconstrictors during acute hemorrhage adversely influences the treatment efficacy [2].
Liver cirrhosis with portal hypertension is characterized by systemic and splanchnic vasodilatory substances release, especially nitric oxide (NO) and prostacyclin [3], which lead to hyperdynamic circulatory status, and further increased mesenteric blood flow and portal inflow. NO and prostacyclin are synthesized by NO synthases (NOS, inducible (iNOS) and endothelial (eNOS) forms) and cyclooxygenases (COX, COX-1 and COX-2), respectively. We have demonstrated that NO and/or prostacyclin inhibition enhanced the collateral vasoresponsiveness to arginine vasopressin (AVP) in portal hypertensive rats [4], suggesting their role in this situation. A previous study, on the other hand, indicated that NO participates in the portal hypertensive angiogenesis [5]. Increased vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), and CD31 (endothelial cell marker) expressions in mesentery of portal hypertensive mice have also been found, which provided the evidence of mesenteric angiogenesis in portal hypertension [1]. Inhibition of VEGFR2 attenuated hyperdynamic splanchnic circulation and collaterals in portal hypertensive rats, suggesting the beneficial effects of antiangiogenesis in ameliorating this pathological condition [6].
Lipopolysaccharide (LPS) and bacterial breakdown products in the gastrointestinal tract reach the liver via portal vein. The hepatic microenvironment is thus unique by the presence of bacterial endotoxin and the elicited mediators. ATP released from damaged cells as a result of inflammation serves as a cell-to-cell mediator through cell surface P2 purinergic receptors [7]. Among the purinergic receptors, P2X 7 receptor has been noticed for its role in the release of pro-inflammatory mediators: ATP induces cytokine release through P2X 7 receptor in hemopoietic cells. In mice lacking P2X 7 receptors, peritoneal macrophages failed to release interleukin-1β (IL-1β) in response to ATP [8]. It is noteworthy that a P2X 7 agonist 2',3'-(4-benzoyl) benzoyl ATP (BzATP) induced tumor necrosis factor (TNF) release much more effectively than ATP [9]. In addition, P2X 7 receptors activation in the presence of LPS stimulated iNOS expression and NO production, which elicited vasodilation [10].
Collectively, P2X 7 antagonism ameliorates inflammation, down-regulates NOS, COX and proangiogenic factors expressions and inhibits angiogenesis as well as vasodilation, but the relevant investigation on liver cirrhosis has not been performed. In this study, we aimed to survey the effects and mechanisms of selective P2X 7 antagonism on liver fibrosis, hemodynamics, mesenteric angiogenesis, severity and vasoconstrictor responsiveness of portal-systemic collaterals in rats with common bile duct ligation (CBDL)-induced cirrhosis.

Animal model
Male Sprague-Dawley rats (260-280 g) were caged at 24°C with a 12 h light/dark circle and allowed free access to food and water. Secondary biliary cirrhosis was induced by CBDL [19]. In brief, under ketamine anesthesia (100 mg/kg, intramuscularly), the common bile duct was exposed through a midline abdominal incision, and doubly ligated with 3-0 silk. The section between the ligatures was cut. The incision was then closed and the animal was allowed to recover. A high yield of secondary biliary cirrhosis was noted four weeks later [19,20]. Weekly vitamin K injection (50 μg/kg, intramuscularly) was applied to avoid coagulation defect. This study had been approved by Taipei Veterans General Hospital Animal Committee (IACUC98-170). The Guides for the Care and Use of Laboratory Animals (NIH publication no. 85-23, rev. 985, U.S.A.) were followed.

Systemic and portal hemodynamics
The right carotid artery was cannulated with PE-50 catheters, and the mesenteric vein with 18-gauge Teflon cannula. They were connected to a Spectramed DTX transducer (Spectramed Inc., Oxnard, CA, U.S.A.). The external zero reference was set at the level of the mid-portion of the animal. Recordings of mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP) were performed [21]. Superior mesenteric artery (SMA) flow was measured by a nonconstrictive perivascular ultrasonic transit-time flow probe (lRB, 1-mm diameter; Transonic Systems, Ithaca, NY, U.S.A.), expressed as mL/min per 100 g body weight [22].

Portal-systemic shunting determination
Portal-systemic shunting was evaluated as previously described [23] with a slight modification. The color microspheres substituted for radioactive microspheres. Briefly, 30,000 of 15-μm yellow microspheres (Dye Track; Triton Technology, San Diego, CA, U.S.A.) were slowly injected into the spleen. The rats were euthanized, and then the liver and lung were dissected. The number of microspheres was determined following the protocol provided by the manufacturer: 3,000 blue microspheres served as internal control. Spillover between wavelengths was corrected with the matrix inversion technique. Portal-systemic shunting was calculated as (microspheres): lung/(liver plus lung). Assuming a worst-case scenario in which two-thirds of the microspheres remain trapped in the spleen, this technique can detect a minimum shunt of 3.5%.
In situ portal-systemic collateral perfusion As previously described [4], both jugular veins were cannulated with 16-gauge Teflon cannulas as outlets of perfusate. The inlet is an 18-gauge Teflon cannula inserted in the superior mesenteric vein. To exclude the liver from perfusion, the portal vein was tied. The animal was transferred into a chamber (37±0.5°C). Perfusion was started via the mesenteric cannula by a roller pump (model 505S; Watson-Marlow Limited, Falmouth, Cornwall, UK) with Krebs solution equilibrated with 95% (v/v) O 2 and 5% (v/v) CO 2 by a silastic membrane lung [24]. Pneumothorax was created by opening slits through the diaphragm to increase the pulmonary arterial resistance and to prevent the perfusate from entering the left heart. Experiments were performed 25 min after starting perfusion at a constant rate of 12 ml/min. As the flow rate was kept constant, the measured perfusion pressure reflected collateral vascular resistance. Only one concentration-response curve was performed in each preparation and the contracting capability was challenged with a 125 mM potassium chloride solution at the end.

Liver biochemistry
Blood was centrifuged at 3,000 g for 10 min then the supernatant was collected to measure the concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin using the analyzer (Cobas C501, Roche, U.S.A.).

Histology
Five micrometer-thick sections obtained from paraffin-embedded livers were stained with Hematoxylin and Eosin (H&E) for the evaluation of structural changes, with Sirius red for fibrosis (collagen deposition), and with alpha-smooth muscle actin (α-SMA) for hepatic stellate cell (HSC) activation. The immunohistochemical staining was modified from the previous study [25]. In brief, liver sections were de-waxed with xylene and rehydrated through a series of ethanol solutions with different concentrations. Sections were subjected to microwave irradiation in citrate buffer to enhance antigen retrieval and pre-incubated with 5% normal rabbit serum in Tris-buffered saline. Primary antibody (anti-α-SMA rabbit polyclonal antibody [Abcam, Cambridge, MA, U.S.A.]) was incubated for 1 h in a humidity chamber. After rinsing twice in phosphate buffered saline, sections were incubated with fluorochrome conjugated secondary antibody (Alexa Fluor 488 fluorescent, Jackson ImmunoResearch Laboratories, Inc. Baltimore, USA) for 1 h at room temperature. Finally, slides were coverslipped with carbonate-buffered glycerol, and evaluated in an Olympus AX 80 microscope (Olympus; Hamburg, Germany) equipped with epifluorescence illumination and digital cameras. Image J software (downloaded from the National Institutes of Health (http://rsb.info.nih.gov/ij/)) was used to measure the percentage of Sirius red-stained area. Briefly, grayscale image was used, then the red area was identified using thresholding function. The thresholded area was measured and shown as the percentage of thresholded area per image [26].

Immunofluorescent study for mesenteric angiogenesis
Mesenteric angiogenesis was quantified by CD31-labelled microvascular networks in rat mesenteric windows according to the previous study with a slight modification [26]. From each rat, at least four mesenteric windows were dissected free, washed in PBS, dried on gelatin slides, and fixed in 100% MeOH (-20°C for 30 min). Slides were then incubated overnight at 4°C with the primary antibody mouse anti-rat CD31-biotin (AbD Serotec, Oxford, UK). Then secondary antibody (CY2-conjugated streptavidin; Jackson ImmunoResearch, West Grove, PA, U.S.A.) was applied for 1 h at room temperature. (×100)-magnification immunofluorescent images were assessed using an upright fluorescent microscope (AX80, Olympus, Japan) and thresholded by Image J. The vascular length was manually measured with the pencil tool and the vascular area automatically with histogram function, respectively. According to the information provided by QICAM, with the eyepiece 10X, the diameter of one pixel on an image taken with QICAM equals to 4.65 μm. Under 100X-magnification (objective 10X and eyepiece 10X), the diameter of one pixel would be 4.65 μm/10 = 0.465 μm. The vascular length could thus be determined accordingly. The unit of vascular length per unit area of mesenteric window would be μm•(μm 2 ) -1 = μm -1 and the vascular area per unit area of mesenteric window, could be pixel•pixel -1 without being converted to μm 2 •(μm 2 ) -1 .

Study protocol
Chronic treatment on CBDL or sham rats. CBDL or sham rats received either BBG (100 mg/kg, p.o., CBDL-BBG, sham-BBG) or vehicle (saline, CBDL-vehicle, sham-vehicle) since the 15 th day to 28 th day after operations. On the 29 th day, two series of experiments were performed: First, portal-systemic shunting determination; Second, after body weight (BW) and hemodynamic (MAP, PP, HR) measurements, liver, mesentery, mesenteric window were collected to survey the severity of liver fibrosis, mesenteric angiogenesis and expressions of fibrotic and angiogenic factors. Blood was collected for biochemistry survey.
Acute treatment on CBDL rats. On the 29 th day after CBDL, with an in situ collateral perfusion model, rats were randomly allocated to receive pre-incubation with Krebs solution (vehicle control) or oATP (specific P2X 7 antagonist, 50 μM) since 1 h prior to and throughout the perfusion experiments. The pre-incubation time of oATP was decided according to the previous study [10], showing that pretreatment of aortas with oATP for 1 hour inhibited the augmented LPS plus BzATP-induced iNOS protein expression. One h after oATP pre-incubation, AVP was added to the perfusate at a 3-minute interval for each escalating concentration (10 -10 , 10 -9 , 3 × 10 -9 , 10 -8 , 3 × 10 -8 , 10 -7 M) and cumulative concentration-response curves of portalsystemic collateral vascular bed to AVP were obtained. Splenorenal shunts were collected at the end of perfusion experiments.

Drugs
AVP, BBG, oATP and reagents for Krebs solution were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All solutions were freshly prepared on the days of experiments.

Statistical Analyses
All results are reported as mean ± standard error of mean (S.E.M.). An unpaired Student t test or one-way analysis of variance (ANOVA) with LSD's post-hoc test was used for the determination of statistical significance between or among experimental groups. A P value < 0.05 denotes the level of significance.

Chronic BBG treatment
Body weight and hemodynamics. Table 1 depicts the BW and hemodynamics in sham or CBDL rats after vehicle or BBG treatment. BBG did not affect sham rats but significantly decreased SMA flow in CBDL rats (CBDL-vehicle vs. CBDL-BBG: p = 0.008).
Portal-systemic shunting ratio and mesenteric vascular density. The severity of portalsystemic shunting in rats with vehicle or BBG treatment is shown in Fig 1(A). Liver histology. The H&E staining of the liver from vehicle-treated CBDL rats showed inflammatory cells infiltration with bridging necrosis and fibrotic band interposition, which was ameliorated by BBG. Nevertheless, the bile ductules proliferation was not significantly influenced by BBG. The immunohistochemical staining demonstrated that BBG reduced α-SMA expression. Sirius red staining depicted that the degree of collagen fiber deposition was significantly less in CBDL rats with BBG treatment as compared with those treated with vehicle (CBDL-vehicle vs. CBDL-BBG: fibrosis area ratio [%]: 0.1179±0.0108 vs. 0.0581±0.0084, p = 0.001). The representative images of H&E, immunohistochemistry, and Sirius red staining are shown in Fig 3. Hepatic pro-inflammatory and fibrotic mediator protein expressions.

Acute vascular response to P2X 7 inhibition
Baseline body weight and hemodynamics. Table 3 reveals that the BW, MAP, HR, PP, and baseline perfusion pressure were not significantly different in CBDL rats before vehicle or selective P2X 7 inhibitor oATP pre-incubation (all p>0.05).
The vascular response of portal-systemic collateral vascular bed.

Discussion
A salient feature of portal hypertension is the formation of an extensive network of portalsystemic collateral vessels including the gastroesophageal varices, which may rupture with a high mortality rate [27]. Angiogenesis has been ascribed for the progression of collaterals [6]. In the current study, BBG, a P2X 7 receptor antagonist as an "edible" blue dye significantly ameliorated the severity of portal-systemic shunting and mesenteric angiogenesis in CBDL rats, implying that P2X 7 inhibition can be a candidate to control the complications associated with angiogenesis in cirrhosis. P2X 7 has been noticed for its role in angiogenesis: P2X 7 expression is increased in tumor-related angiogenesis [28]. Selective P2X 7 antagonist, oATP, inhibited tumor angiogenesis via suppression of MMP-2, MMP-9, and VEGF [18]. Multiple factors are involved in angiogenesis: COX-1 up-regulation with neovascularization has been found in human ovarian cancer. Actually, selective COX-1 inhibition effectively suppressed angiogenesis [29]. COX-2 is also associated with angiogenesis in gastric cancer [30]. COX-2 down- regulation with siRNA inhibited angiogenic factor expressions in gastric cancer cell line [31]. An increased vascular area and surface in mice carrying activated PDGFRβ has also been demonstrated [32]. Intraocular antagonism of PDGF-BB significantly suppressed retinal and subretinal neovascularization [33]. A previous study also indicated that eNOS deficiency impaired angiogenesis in hind limb-ischemic mice compared with wild-type controls. Furthermore, impaired angiogenesis in eNOS -/mice was not improved by VEGF administration, implying that eNOS acts downstream of VEGF [34]. In line with the previous studies, BBG down-regulated TGF-β is the major fibrogenic cytokine in liver fibrogenesis [35], which acts through Smad transcription factors family and results in extracellular matrix production. Smad2 and Smad3 proteins are phosphorylated after TGF-β induction, and then translocate into the nucleus where they regulate the transcription of target genes. Smad7, an inhibitor of Smad signaling, prevents phosphorylation of Smad2 and Smad3 proteins [36]. TGF-β signaling also takes actions via Smad-independent pathways, such as ERK/mitogen-activated protein kinase (MAPK) pathways. Increasing evidences indicate the involvement of MAPK signaling pathway in liver fibrogenesis. It has been found that ERK activation leads to HSC activation and proliferation, resulting in tissue remodeling and hepatic fibrosis [37]. In this study with rats affected by CBDL-induced liver cirrhosis, BBG significantly alleviated the severity of hepatic collagen deposition as shown by Sirius red staining. This is supported by the previous study that P2X 7 antagonism reduced hepatic collagen formation and accumulation induced by chronic CCl 4 treatment [38]. Furthermore, it is noteworthy that BBG obviously reduced hepatic TGF-β and ERK expressions without affecting Smad family protein expressions in CBDL rats, indicating that BBG might improve hepatic fibrosis by down-regulating the TGF-β-MAPK pathway and α-SMA. Likewise, the anti-fibrotic effect of P2X 7 blockade in liver through suppression of TGF-β and α-SMA expression had been reported previously [38], suggesting the potential application of P2X 7 inhibition in controlling liver fibrogenesis.
Leukocytes amplify the inflammatory response by generating pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, along with the myofibroblasts activation elicited by PDGF and TGF-β [39]. In this study, we found that BBG significantly ameliorated liver fibrosis and down-regulated fibrosis-related pro-inflammatory cytokines IL-6, TNF-α, IL-1β, and PDGF. This is in line with a previous study that pharmacological antagonism or genetic deletion of P2X 7 receptor reduced IL-1β and TNF-α in mice bladder tissue [40]. IL-1β release suppressed by selective P2X 7 blockade [41] or P2X 7 deletion [42] have been reported as well. Taken together, BBG reduces hepatic pro-inflammatory cytokines IL-6, TNF-α, PDGF, and IL-1β expressions, down-regulates TGF-β signaling pathway, and ameliorates liver fibrosis.
There is one issue to be addressed: are the aforementioned findings linked to the anti-inflammatory effect of P2X 7 antagonism? We treated BDL rats since the 15 th day after operation, at which time point liver fibrosis rather than inflammation was the predominant histological finding [43]. The result showed that BBG was still able to ameliorate liver fibrosis. Taken the previous evidences into consideration, P2X 7 activation enhanced TGF-β1 mRNA expression in  type-2 rat brain astrocytes [44]. Furthermore, as compared with the wild type mice, there were less myofibroblasts, diminished collagen deposition, and decreased TGF-β expression in the renal interstitium of P2X 7 knockout mice subjected to unilateral ureteral obstruction [45]. Solini et al. [46] have also demonstrated the importance of P2X 7 activation on TGF-β secretion and mesangial matrix expansion induced by hyperglycemia. Another animal study also indicated the reduced collagen deposition and fibrotic pathology in lungs of bleomycin-administered P2X 7 knockout mice [42]. To sum up, P2X 7 antagonism may ameliorate fibrosis directly in rats with BDL-induced liver fibrosis. Nevertheless, it is well known that excessive inflammation initiates the cascade of fibrogenesis, so the amelioration of liver fibrosis via P2X 7 antagonism-induced anti-inflammation is not to be neglected. For instance, Vesey et al. [47] demonstrated that the pro-inflammatory cytokine IL-1β promoted fibroblast proliferation and collagen production. Regarding the potential influence on necrosis, both liver-resident cells such as Kupffer cells, HSCs, and cells that are recruited in response to injury elicit pro-inflammatory signals that contribute to hepatocyte necrosis [48]. Therefore, the reduction of hepatocyte necrosis by P2X 7 antagonism is considered to be mediated via anti-inflammation.
In the current study, pre-incubation of oATP, another selective P2X 7 antagonist [49], markedly enhanced portal-systemic collateral responsiveness to AVP in CBDL rats and downregulated splenorenal shunt iNOS and eNOS expressions. Chiao et al found that P2X 7 receptors activation in the presence of LPS stimulated iNOS expression and NO production, which elicited vasodilation and was reversed by oATP [10]. It has also been noted that iNOS was induced by NF-κB activation [50]. Moreover, absence of P2X 7 receptor reduced iNOS expression as well as NF-κB activation in lung parenchyma [41]. Compatible with the previous findings, acute oATP administration significantly reduced NF-κB and iNOS protein expressions in splenorenal shunts of CBDL rats. Regarding eNOS, P2X 7 receptor activation involves an initial upstream mechanism of LPS-induced vascular dysfunction, which is associated with IL-1βmediated eNOS activation [51]. Furthermore, P2X 7 deficiency reduced eNOS protein expression [52]. VEGF also increases eNOS activity and NO generation through Akt activation [53]. Consistently, the current study showed that oATP decreased VEGF, Akt, p-Akt, and eNOS protein expressions in splenorenal shunts of CBDL rats. The acute down-regulation of iNOS, eNOS, and their related signaling molecules by P2X 7 antagonism might be responsible, at least partly, for the improved collateral AVP vasoresponsiveness and contribute to a better therapeutic effect of vasoconstrictors in cirrhotic patients suffering from acute gastroesophageal variceal hemorrhage, though further investigation is required.
The reason why we used oATP instead of BBG for perfusion experiments is that BBG is a blue dye which stains the tubes and catheters of the in situ perfusion system. Nevertheless, the nontoxic and "edible" nature of BBG makes clinical application possible. Therefore, we used oATP to test the impacts of P2X 7 antagonism in perfusion experiments but BBG in chronic in vivo treatment. The differential effects of chronic and acute P2X 7 antagonism on mesenteric and splenorenal shunt eNOS expressions are also worth noting: Previous studies have indicated that an enhanced mesenteric eNOS expression contributed to mesenteric angiogenesis in CBDL rats [49]. Moreover, eNOS is a potent vasodilator [53]. Therefore, eNOS affects both angiogenesis and vasodilation. Actually, promising results are noted in the current study: mesenteric eNOS down-regulation by chronic P2X 7 antagonism alleviated mesenteric angiogenesis and splenorenal shunt eNOS down-regulation by acute P2X 7 antagonism enhanced collateral vasoconstrictive response to AVP.
The SMA flow was reduced by BBG in CBDL rats, which may be related to the ameliorated mesenteric angiogenesis. However, BBG did not significantly affect portal pressure. Portal pressure is determined by the net effects of portal inflow, intrahepatic resistance, and portalsystemic collateral vascular resistance. SMA, the major branch of the abdominal aorta, is responsible for about a half of portal venous blood inflow and regarded as the most dominant flow index in splanchnic circulation of portal hypertension [54]. Intrahepatic resistance is determined by dynamic and mechanical components, including enhanced vascular tone and cirrhotic change [55]. On the other hand, the portal-systemic collaterals develop with the aim to divert the stagnant portal blood flow, so the increased collateral vascular resistance may increase portal pressure. Our data show that BBG significantly lowered SMA flow, alleviated liver fibrosis, and reduced the severity of portal-systemic collaterals. Moreover, oATP enhanced the portal-systemic collateral vasoresponsiveness to vasoconstrictor, suggesting the potential of P2X 7 inhibition to increase collateral vascular resistance. Taken together, portal pressure is not influenced by P2X 7 inhibition in the current experimental setting. Nevertheless, the amelioration of portal-systemic shunting, mesenteric angiogenesis and improved collateral AVP responsiveness may be of clinical significance in alleviating gastroesophageal varices and in improving the treatment response to vasoconstrictors during acute hemorrhage. Furthermore, mean arterial pressure was not significantly influenced, suggesting that P2X 7 inhibition is hemodynamically feasible for cirrhotic patients with pre-existing hyperdynamic circulation and systemic hypotension. BBG also significantly reduced ALT in CBDL rats, which is compatible with the anti-inflammatory nature of P2X 7 antagonism [12][13][14][15][16][17].
To sum up, purinergic receptor inhibition, especially P2X 7 receptor blockade, ameliorates liver fibrosis, mesenteric angiogenesis, severity of portal-systemic shunting, and improves the portal-systemic collateral vascular responsiveness in cirrhotic rats, suggesting the potential of purinergic receptor antagonism in controlling liver cirrhosis-related complications.