Hepatic encephalopathy (HE) is a neuropsychiatric derangement associated with acute or chronic hepatic decompensation with or without portal-systemic shunts.1,2 The disorder consists of a diversity of symptoms, varying from trivial mental instability or memory impairment to confusion, coma, and death. The initiation and maintenance of HE have been ascribed to several substances, such as ammonia, gamma-aminobutyric acid, benzodiazepines, aromatic amino acids, false neurotransmitters, endotoxin, and tumor necrosis factor-α.1–3 However, the true nature mechanism remains not totally clarified.
Prostacyclin (PGI2), a vasodilatory prostaglandin, has been postulated to play a role in the pathogenesis of hepatic failure with encephalopathy. PGI2 participates in the mechanism of hyperdynamic circulation in portal hypertensive status. For instance, increased levels of 6-keto-prostaglandin-F1α (6-keto-PGF1α), a stable metabolite of PGI2, have been noted in animals with portal hypertension and patients with liver cirrhosis.4,5 The vasodilatory nature of PGI2 may contribute to HE6: cerebral vasculature dilatation increases capillary surface and subsequently facilitates the diffusion of noxious gut-derived compounds, such as ammonia.7 It has also been shown that PGI2 alters the permeability of the blood–brain barrier.8 Therefore, it is reasonable that PGI2 may participate, at least partly, in the pathophysiology of HE.
The role of PGI2 during hepatic injury seems to be quite controversial. Although raised blood prostaglandin levels have been noted in massive hepatic necrosis,9 prostaglandins have been shown to ameliorate hepatic insult in terms of prolonged survival and histological improvements.10 Our previous study showed a detrimental effect of indomethacin administration on HE in rats with thioacetamide (TAA)-induced fulminant hepatic failure, which is believed to be derived from nonselective prostaglandin synthesis inhibition.11 Because indomethacin exerts a universal blockade of two prostaglandin synthesis isoenzymes, cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2), the roles of COX isoforms in HE, indeed, require further clarification.
As chronic liver parenchymal disease is a more prevalent form of hepatic failure and encephalopathy in clinical practice, we used bile duct-ligated cirrhotic rats to quantitatively survey the degree of HE with motor activities.12–14 The plasma levels of 6-keto-PGF1α and liver biochemistry parameters were also measured, and the hepatic COX isoform expressions were evaluated. Furthermore, the correlations between them were assessed.
2.1. Animal model
Male Sprague–Dawley rats weighing 240–270 g were used for this study. The rats were caged at 24°C, with a 12-hour light–dark cycle and free access to food and water until the time of the experiments. Rats with secondary biliary cirrhosis were induced by common bile duct ligation (BDL).15 Under ketamine anesthesia (100 mg/kg, intramuscularly), the common bile duct was exposed through a midline abdominal incision, catheterized by a PE-10 catheter and doubly ligated with 3–0 silk. The first ligature was made below the junction of the hepatic ducts and the second above the entrance of the pancreatic duct. Ten percent formalin (˜10 μL/100 g) was slowly injected into the biliary tree to prevent the subsequent dilatation of the ligated residual bile duct. Thus, this animal model is abbreviated as FBDL. The PE-10 catheter was then removed and the ligatures were tightened, followed by sectioning of the common bile duct between the ligatures. The incision was then closed, and the animal was allowed to recover. A high yield of secondary biliary cirrhosis was noted at least 4 weeks after the ligation.15–17 To avoid coagulation defects, FBDL rats received weekly vitamin K injection (50 μg/kg, intramuscularly).18 The studies were performed in overnight-fasted rats, 6 weeks after the operation. Two series of studies were performed. In the first series, after measurements of the motor activities, heparinized blood samples were obtained from the inferior vena cava for alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALK-P), total bilirubin, and PGF1α measurements (n = 10 for each group). In the second series, livers were dissected and removed for RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR; n = 9 for each group). The animal experiments were conducted according to Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985).
2.2. Measurement of motor activities
The severity of HE was quantified with motor activities. Motor activities in an open field were determined using the Opto-Varimex animal activity meter (Columbus Instruments, Inc., Columbus, OH, USA).12,18 The Opto-Varimex activity sensors utilize high-intensity, modulated infrared light beams to detect animal motion. Animals were housed in transparent cages (17 × 17 × 8 inches), through which 30 infrared beams passed in the horizontal plane, 15 on each axis. This device differentiated nonambulatory movements (scratching, gnawing) from ambulation on the basis of consecutive interruption of the infrared monitoring beams. An additional row of infrared beams in the horizontal plane (15 on each axis) about 10 cm above the floor was used to count vertical movements. During the activity measurements, animals had no access to food or chow. All studies were performed under strictly standardized conditions in a dark room for 30 minutes. Counted numbers of total movements, ambulatory movements, and vertical movements were separately recorded to reflect the motor activities of the rats.
2.3. Determination of plasma 6-keto-PGF1α levels
Heparinized blood was centrifuged at 1789g for 10 minutes, then the plasma was separated, added with indomethacin (10 μg/ml), and stored under −80°C. At the time of measurement, samples were thawed, and 6-keto-PGF1α was determined with a commercially available enzyme immunoassay according to the protocol supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA).
2.4. RNA extraction and RT-PCR
Total RNA was extracted from the vessel with an RNeasy Mini Kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany).19,20 A one-step RT-PCR kit (Qiagen) was used with the following components: 10 μL RT-PCR buffer containing Tris–HCl, KCl, (NH4)2SO4, and 2.5mM MgCl2; 2 μL deoxynucleotide mixture containing 400μM dATP, dCTP, dGTP, and dTTP, respectively; 2 μL enzyme mixture containing Omniscript reverse transcriptase, Sensiscript reverse transcriptase, and HotStar Taq DNA polymerase; 1 μL RNase inhibitor (40 U); 3 μL of each random primer (10 pmol/μL), and 1 μg substrate RNA. RNase-free water was added in each reaction to the final volume of 50 μL. The sequences of primers for COX1 were 5′-TGCATGTGGCTGTGGATGTCATCAA-3′ (sense) and 5′-CACTAAGACAGACCCGTCATCTCCA-3′ (antisense), respectively. Those for COX2 were 5′-ACACTCTATCACTGGCATCC- 3′ (sense) and 5′-GAAGGGACACCCTTTCACAT-3′ (antisense), respectively. A constitutively expressed gene, β-actin, was analyzed as internal control, and the primers for β-actin were 5′-TTGTAACCAACTGGGACGATATGG-3′ (sense) and 5′-GATCTTGATCTTCATGGTGCTAGG-3′ (antisense), respectively. The primers for COX1, COX2, and β-actin were designed to allow amplification of 450, 584, and 764 base-pair (bp) fragments, respectively.20,21 A negative control was included in each set of experiments. The tubes were placed in a thermocycler (Biometra T Gradient thermocycler; Biometra GmbH, Göttingen, Germany) at 50°C for 30 minutes for RT, 95°C for 15 minutes for initial denaturation, followed by 35 cycles of the following sequential steps: 30 seconds at 94°C (denaturation), 45 seconds at 56°C for COX-1, 57.2°C for COX-2, 59.6°C for β-actin (annealing), respectively, and 45 seconds at 72°C (extension). The final extension was performed at 72°C for 10 minutes. Then, 10 μL of the PCR-amplified mixture was subjected to electrophoresis on a 2% agarose gel, and DNA was visualized by ethidium bromide staining. Location of the predicted PCR products (base pairs) was confirmed with a 100-bp ladder (Gibco BRL, Gaithersburg, MD, USA) as standard size marker. The gel was then photographed, and the PCR products were quantitated by a digitalized software (Kodak Digital Science ID Image Analysis Software; Eastman Kodak Company, Rochester, NY, USA). The intensities of COX1 and COX2 signals were standardized against that of β-actin from the same RNA sample and expressed as COX1/β-actin and COX2/β-actin ratio for comparison.
2.5. Data analysis
All results are expressed as mean ± standard error of the mean. Statistical analyses were performed using Student t test. Results were considered statistically significant at p < 0.05.
3.1. Motor activities
Fig. 1 shows the motor activities of the FBDL and control (sham) groups. The FBDL group showed lower motor activity counts than those of the control group in total (FBDL vs. control: 1472 ± 156 vs. 2174 ± 262 counts/30 min; p = 0.034), ambulatory (824 ± 99 vs. 1443 ± 206 counts/30 min, p = 0.014), and vertical movements (431 ± 69 vs. 849 ± 145 counts/30 min, p = 0.018).
3.2. Plasma ALT, AST, ALK-P, bilirubin, and 6-keto-PGF1α levels
Table 1 shows the plasma concentrations of ALT, AST, ALK-P, bilirubin, and 6-keto-PGF1α of the two experimental groups. They were significantly higher in the FBDL group (all p < 0.05).
3.3. Cyclooxygenases expression
Fig. 2 shows the hepatic COX1 and COX2 mRNA expressions. Hepatic COX2 expression was significantly higher in the FBDL group (COX2/β-actin: 0.41 ± 0.04 vs. 0.13 ± 0.03, p < 0.001). However, the hepatic COX1 mRNA expression (COX1/β-actin: 1.13 ± 0.11 vs. 0.90 ± 0.08, p = 0.117) was not significantly different between the two groups.
3.4. Correlations between severity of hepatic injury, HE, and hepatic COX expression
Figs. 3 and 4 depict the correlations among the aforementioned parameters. Plasma ALK-P and bilirubin levels were negatively correlated with total movements (p = 0.035, γ = −0.474; p = 0.009, γ = −0.569, respectively). In addition, hepatic COX2 mRNA expression was positively correlated with AST, ALK-P, bilirubin, and 6-keto-PGF1α (p = 0.004, γ = 0.608; p < 0.001, γ = 0.745; p = 0.001, γ = 0.700; and p < 0.001, γ = 0.722, respectively). However, there was no significant relationship between hepatic COX2 mRNA expression or 6-keto-PGF1α level and total movements.
Different from animal models with acute hepatic damage, the BDL model is an applicable model of chronic liver disease with moderate to severe degree of hepatic injury and modest or moderate degree of shunting.17,22,23 In our previous study, we demonstrated that BDL rats steadily developed HE.24 Compatible with that previous finding, we also found markedly decreased motor activities in the current FBDL group.
In the present study, the FBDL rats showed significantly elevated plasma levels of ALT, AST, ALK-P, and bilirubin, compatible with cholestatic liver injury. The 6-keto-PGF1α level was also elevated, consistent with previous studies: The vasodilatory prostanoids, especially PGI2, have been implicated as mediators of the systemic and splanchnic hyperemia characteristic of portal hypertension.5,25 The increased portal venous PGI2 levels may maintain the portal hypertensive state in cirrhotic patients.26 In addition, plasma and urinary levels of 6-keto-PGF1α were higher in rats with portal vein stenosis and cirrhosis.27 The urinary excretion of 2,3-dinor-6-keto-PGF1α in cirrhotic patients was also increased.4
PGI2 has been postulated to take part in the pathogenesis of HE.6 PGI2 altered the permeability of the blood–brain barrier.8 In addition, cerebral vasculature dilatation may increase capillary surface and subsequently facilitate the diffusion of noxious gut-derived compounds, such as ammonia.7 The absence of cerebral blood flow autoregulation in TAA-induced acute liver failure appears to arise from cerebral arteriolar dilatation28–30 and brain edema has been found in cirrhotic patients with HE.31 In addition, indomethacin, an inhibitor of cyclooxygenase, has been considered a cerebral vasoconstrictor.32 The administration of indomethacin decreased cerebral blood flow, brain water, and intracranial pressure in rats that underwent portocaval anastomosis.33 A retrospective study also revealed a strong relationship among cerebral blood flow, brain swelling, outcome, and depth of coma.34 Therefore, it is reasonable that PGI2 may participate in the pathophysiology of hepatic coma, and pharmacological manipulation of the cerebral circulation might prevent the development of brain edema in hepatic failure. However, according to our recent study,11 indomethacin, a nonselective COX inhibitor, exerted detrimental effects in terms of mortality and severity of encephalopathy in rats with TAA-induced acute liver failure. By contrast, no detrimental or therapeutic effect was identified in BDL rats receiving indomethacin.35 These findings suggest different roles of COX in acute and chronic liver failure-induced HE.
Interestingly, a previous study identified that a COX1 inhibitor, SC-560, significantly improved HE in rats with TAA-induced fulminant hepatic failure,36 implying that COX1 plays a significant role in HE related to acute liver failure. In the current study, although hepatic COX2 expression was upregulated in FBDL rats with HE, there was no significant correlation between hepatic COX2 expression and motor activities. Rather, the motor activities correlated with ALK-P and bilirubin, two parameters of cholestatic liver damage. Furthermore, hepatic COX1 expression was not significantly different between FBDL and sham rats. The results indicated that first, compatible with our previous finding,37 HE in FBDL-cirrhotic rats is correlated with the severity of cholestatic liver injury rather than portosystemic shunting; second, the pathophysiological mechanism of HE in cirrhosis is multifactorial and not limited to prostaglandins.
Prostaglandins have been shown to reverse acute experimental hepatic damage.10 Indomethacin administration following liver damage induced by carbon tetrachloride also increased liver cell death in rats.38 The previous investigations have indicated that PGI2 may exert a protective effect on liver damage attributed to its lysosomal membrane stabilization effect.39,40 Another study addressed that although PGI2 did not induce vasodilatory effects on the hepatic circulation, it exerted a beneficial action by inducing redistribution of flow to certain areas of the liver or by decreasing hepatic shunt flow.40 We found in the present study that hepatic COX2 mRNA expression was enhanced in BDL rats. Interestingly, the hepatic COX2 expression correlated with AST, ALK-P, and bilirubin levels. Whether it is activated following liver injury to modulate the hepatic circulation or the COX2 overexpression actually damages liver function remains to be clarified, and further studies are required.
In conclusion, although hepatic COX2 mRNA overexpression and an increased plasma level of PGF1α were found in FBDL rats with HE, they were not significantly correlated with the severity of HE, suggesting a multifactorial and complicated nature of HE in cirrhosis.
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