Prostacyclin inhibition by indomethacin aggravates hepatic damage and encephalopathy in rats with thioacetamide-induced fulminant hepatic failure

Abstract

AIM: Vasodilatation and increased capillary permeability have been proposed to be involved in the pathogenesis of acute and chronic form of hepatic encephalopathy. Prostacyclin (PGI₂) and nitric oxide (NO) are important contributors to hyperdynamic circulation in portal hypertensive states. Our previous study showed that chronic inhibition of NO had detrimental effects on the severity of encephalopathy in thioacetamide (TAA)-treated rats due to aggravation of liver damage. To date, there are no detailed data concerning the effects of PGI₂ inhibition on the severity of hepatic encephalopathy during fulminant hepatic failure.

METHODS: Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of TAA (350 mg/(kg.d) for 3 d. Rats were divided into two groups to receive intraperitoneal injection of indomethacin (5 mg/(kg.d), n = 20) or normal saline (N/S, n = 20) for 5 d, starting 2 d before TAA administration. Severity of encephalopathy was assessed by the counts of motor activity measured with Opto-Varimex animal activity meter. Plasma tumor necrosis factor-α (TNF-α, an index of liver injury) and 6-keto-PGF_1α (a metabolite of PGI₂) levels were measured by enzyme-linked immunosorbent assay.

RESULTS: As compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving indomethacin (20% vs 5%, P<0.01). Inhibition of PGI₂ created detrimental effects on total movement counts (indomethacin vs N/S: 438±102 vs 841±145 counts/30 min, P<0.05). Rats treated with indomethacin had significant higher plasma levels of TNF-α (indomethacin vs N/S: 22±5 vs 10±1 pg/mL, P<0.05) and lower plasma levels of 6-keto-PGF_1α (P<0.001), but not total bilirubin or creatinine (P>0.05), as compared with rats treated with N/S.

CONCLUSION: Chronic indomethacin administration has detrimental effects on the severity of encephalopathy in TAA-treated rats and this phenomenon may be attributed to the aggravation of liver injury. This study suggests that PGI₂ may provide a protective role in the development of fulminant hepatic failure.

INTRODUCTION

Hepatic encephalopathy is a neuropsychiatric syndrome associated with acute liver failure, chronic parenchymal liver disease, or portal-systemic anastomosis[1-3]. The spectrum of symptoms may vary from subtle mental changes or a disrupted circadian rhythm to hepatic coma. The pathogenesis of hepatic encephalopathy has not been fully established and a delineation of pathogenetic factors for hepatic encephalopathy, as well as developing therapeutic modalities, are highly warranted today.

Hyperdynamic circulation observed in portal hypertension is characterized by pronounced vasodilatation, increased systemic and regional blood flows, and augmented cardiac index[4]. Vasodilatation and increased capillary permeability have been proposed to be involved in the pathogenesis of acute and chronic form of hepatic encephalopathy[5-8]. Recent studies demonstrated that excessive formation of nitric oxide (NO) and prostacyclin (PGI₂), the endogenous vasodilators synthesized by the vascular endothelium[9], are important contributors to the hyperdynamic circulation[10-13]. Our recent study[14] demonstrated that chronic inhibition of NO had detrimental effects on the severity of encephalopathy in thioacetamide (TAA)-treated rats due to aggravation of liver damage, suggesting a protective role for NO in the development of hepatic encephalopathy. Concerning the relationship between prostaglandins and the liver, some studies suggested that prostaglandins could protect the liver against the damage by a wide variety of toxins (carbon tetrachloride, acetaminophen, galactosamine, alcohol), hypoxia, ischemia, and immune injury[15-23] although the mechanisms remain unclear. To date, there are no detailed data concerning the effects of PGI₂ inhibition on the severity of hepatic encephalopathy during the condition of fulminant hepatic failure.

The aim of this study was to investigate the effects of indomethacin, a PGI₂ inhibitor[24], on the severity of encephalopathy in rats with TAA-induced fulminant hepatic failure[25-27]. For more accurate quantification of the degree of hepatic encephalopathy, motor activities of rats were measured in automated open field boxes equipped with infrared cells[28-30]. Plasma levels of tumor necrosis factor-α (TNF-α, an index of liver injury), 6-keto-prostaglandin F_1α (6-keto- PGF_1α a metabolite of PGI₂), aminotransferase (ALT), total bilirubin, and creatinine were also determined.

MATERIALS AND METHODS

Animal model

Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of TAA (350 mg/(kg.d) in normal saline, Sigma Chemical Co, St. Louis, MO, USA) for 3 consecutive days. Rats were divided into two groups to receive intraperitoneal injection of indomethacin (5 mg/(kg.d), n = 20)[24] or placebo (normal saline, N/S, n = 20) from two days prior to the starting of TAA administration and lasted for 5 d. To avoid hypoglycemia and electrolyte imbalance, subcutaneous injections of a solution containing 10% glucose water mixed with lactate ringer (25 mL/kg) was performed every 12 h after the first injection of TAA[31]. Motor activity measurements and blood sample collection were performed 3 d after the first administration of TAA. All rats were caged at 24 °C with a 12-h light-dark cycle and allowed free access to water and food. The experiments reported here were conducted according to the American Physiological Society guiding principles for the care and use of laboratory animals.

Measurement of motor activities

Motor activities in an open field were determined by using the Opto-Varimex animal activity meter (Columbus Instruments Inc., Columbus, OH, USA)[28-30]. The Opto-Varimex activity sensors utilize high-intensity, modulated infrared light beams to detect animal motion. Animals were housed in transparent cages (17 inches×17 inches×8 inches) through which 30 infrared beams pass in the horizontal plane, 15 on each axis. This device differentiates non-ambulatory 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 the vertical movements. During the activity measurements, animals had no access to food or chow. All studies were performed under strictly standardized conditions in the dark room for 30 min. The number of total movements, ambulatory movements, and vertical movements was separately recorded to reflect the motor activities of rats with fulminant hepatic failure.

Determination of plasma TNF-α, 6-keto-PGF_1α, ALT, total bilirubin, and creatinine

After the abdominal skin was cleaned twice with iodine tincture and 70% alcohol, the abdomen was opened using a sterile technique. A 3 mL of blood sample was collected from the inferior vena cava into a pyrogen-free syringe containing 75 units of heparin sodium, then placed in an ice bath and transported immediately to the laboratory. Plasma was centrifuged at 4 °C and 2935 g for 10 min, then stored at -70 °C in pyrogen-free polyethylene tubes for subsequent analysis within 6 wk.

We measured plasma TNF-α levels[32] with a commercially available solid phase sandwich enzyme-linked immunosorbent assay (rat TNF-α kits; R&D systems, Minneapolis, MN, USA) according to the protocol supplied by the manufacturer. The standards and samples were incubated with a TNF-α antibody coating a 96-well microtiter plate. The wells were washed with buffer and then incubated with anti-TNF-α antibody conjugated to horseradish peroxidase for 2 h. This was washed away and a yellow-brownish color was developed in the presence of tetramethyl benzidine chromogen substrate. The intensity of the color was measured in a Bio-kinetics reader (Bio-Tek Instruments Inc., Winooski, VT, USA) by reading the absorbance at 450 nm with a correction wavelength of 570 nm. We compared the samples against the standard curve to determine the amount of TNF-α present. All samples were run in duplicate. The lower limit of sensitivity for TNF-α by this assay was 5 pg/mL. The intra-assay and inter-assay coefficients of variation were 5.1% and 9.7%, respectively.

The measurement of 6-keto-PGF_1α was based on a competitive binding technique in which 6-keto-PGF_1α present in a sample competes with a fixed amount of alkaline phosphatase-labeled 6-keto-PGF_1α for sites on a sheep polyclonal antibody (6-keto-PGF_1α Kits; R&D systems, Minneapolis, MN, USA). During the incubation, the polyclonal antibody became bound to the donkey anti-sheep antibody coated onto the microplate. Following a wash to remove excess conjugates and unbound samples, a substrate solution was added to the wells to determine the bound enzyme activity. The color development was stopped and the absorbance was read at 405 mm in a Bio-kinetics reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The intensity of the color was inversely proportional to the concentration of 6-keto-PGF_1α in the sample. All samples were run in duplicate. The lower limit of the sensitivity for 6-keto-PGF_1α by this assay was 1.4 pg/mL.

We determined plasma levels of ALT, total bilirubin and creatinine by colorimetric assay with a Clinical Systems 700 analyzer (Beckman Instruments, Fullerton, CA, USA).

Statistical analysis

Results were expressed as mean±SE. Statistical analyses for categorical variables were performed by Chi-square and Fisher exact tests. Student’s t-test was used for continuous variables. Results were considered statistically significant at P<0.05.

RESULTS

Effects of chronic indomethacin administration on mortality rate in rats with fulminant hepatic failure

Four rats in the indomethacin group and one rat in the N/S group died during chronic administration of TAA before the measurement of motor activities. As compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving chronic indomethacin administration (20% vs 5%, P<0.01).

Effects of chronic indomethacin administration on degree of hepatic encephalopathy in rats with fulminant hepatic failure (Figure 1)

Figure 1.

Comparison of the degree of hepatic encephalopathy by total, ambulatory and vertical movement counts between rats with fulminant hepatic failure treated with indomethacin (n = 19) or N/S (n = 16).

In rats receiving indomethacin, the total movements were significantly decreased (indomethacin vs N/S: 438±102 vs 841±145 counts/30 min, P<0.05). The counts of ambulatory movements (256±62 vs 385±95 counts/30 min, P = 0.29) and vertical movements (28±12 vs 68±27 counts/30 min, P = 0.20) in the rats treated with indomethacin tended to be lower; however, the differences did not reach a significant level.

Effects of chronic indomethacin administration on plasma levels of 6-keto-PGF_1α, TNF-α, ALT, total bilirubin, and creatinine in rats with fulminant hepatic failure (Figure 2)

Figure 2.

Comparison of the plasma levels of 6-keto-PGF_1α, TNF-α, total bilirubin, and creatinine between rats with fulminant hepatic failure treated with indomethacin (n = 19) or N/S (n = 16).

Rats treated with indomethacin had significantly lower plasma levels of 6-keto-PGF_1α (indomethacin vs N/S: 32±11 vs 213±47 pg/mL, P<0.001) and higher plasma levels of TNF-α (indomethacin vs N/S: 22±5 vs 10±1 pg/mL, P<0.05) as compared to N/S-treated rats. The indomethacin group of rats had a higher serum ALT (indomethacin vs N/S: 172±35 vs 141±31 IU/L, P>0.05); however, it did not reach a significant level. Serum levels of total bilirubin (indomethacin vs N/S: 0.3±0.1 vs 0.4±0.1 mg/dL, P>0.05) and creatinine (indomethacin vs N/S: 0.2±0.1 vs 0.3±0.1 mg/dL, P>0.05) were comparable between these two groups.

DISCUSSION

Regarding the relationships between the liver and arachidonic acid derivatives, in the last decade much has been learned between the interactions of liver and prostaglandins (PGs) under normal and pathological conditions. However, the puzzle is far from complete. Physiologically, the liver synthesizes all the major metabolites of the arachidonate cascade, including PGs. It has been found that PGs are involved in various physiological processes in the liver, including vasoregulation, platelet aggregation and mediation of inflammation[33]. As we stated previously, some studies suggested that PGs could protect the liver against damage by a wide variety of toxins (carbon tetrachloride, acetaminophen, galactosamine, alcohol), hypoxia, ischemia, and immune injury[15-23]. Although the precise mechanism underlying the cytoprotective effects of PGs in acute liver injury remains to be precisely defined, it may be related to the alleviation of vasoconstriction with tissue hypoxia, lipid peroxidation, and release of inflammatory cytokines or cytotoxic factors[34-36]. In agreement with previous studies[15-23], our current study showed that inhibition of PGI₂ by indomethacin during acute liver injury could aggravate hepatic damage. In addition, this is the first study which demonstrated that indomethacin administration could result in detrimental effects on encephalopathy in rats with TAA-induced fulminant hepatic failure. This finding was similar to NO inhibition in our previous research[14] suggesting that maintaining tissue oxygenation by vasodilators is important in fulminant hepatic failure. Besides, evidence from animal studies suggested indomethacin administration might affect neurotransmission and concentrations of neuropeptides in the brain[37-39]. Since our current study did not have data on the above issues, it is hard to tell whether the deleterious effects of indomethacin injection on encephalopathy may be partially attributed to this and further studies are warranted.

Physiologically, TNF-α has been found to be a peptide mediator released by mononuclear cells on activation by endotoxin, tissue injury and tumor cells[40]. The metabolism of TNF-α was located within the liver after it was bound to a specific receptor[41]. Therefore, plasma levels of TNF-α, together with serum bilirubin, could be regarded as an index for evaluating the degree of hepatocellular damage and tissue necrosis.

Grading of hepatic encephalopathy is crucial in the research field for causally related pathogenetic factors. Various tests such as equilibrium test, corneal, auditory startle, and righting reflexes have been used but the interpretation of these tests may be subjective and cannot be quantified. Thus, a mechanical scoring system was applied in the present study. These tests are of great benefit in monitoring the minor changes in motivated behavior. On the basis of consecutive interruption of the infrared monitoring beams, we could differentiate non-ambulatory (scratching, gnawing) from ambulatory movements. The additional row of infrared cells above the plane could provide us more information concerning the movements toward the vertical direction. In the current study, the time interval from the first dose of TAA administration to the measurement of motor counts was fixed (72 h). The advantage of this model is that it can provide a therapeutic window for treatment modalities.

Indomethacin acts by inhibiting cyclooxygenase (COX), the rate-limiting enzyme in metabolic conversion of arachidonic acids into PGs. Two isoforms of COX have been identified[42] and both can be inhibited by indomethacin. COX-1 is expressed constitutively in most tissues and plays an important function in the protection of gastric mucosa and maintaining physiological homeostasis. COX-2 is thought to be the enzyme induced in inflammatory states and tumorigenesis. Recently, the intervention of selective COX-2 inhibitors was shown to preserve the anti-inflammatory efficacy of non-steroid antiinflammatory drugs (NSAIDs) and to achieve greater safety than traditional NSAIDs[43-45]. It is unknown whether using selective COX-2 inhibitors may have different effects on hepatic encephalopathy other than detrimental effect and this issue deserves further evaluations.

Results of preliminary clinical trials using PGs as therapeutic agents in patients with severe acute liver injury have been encouraging[46]. However, most of these trials were lack of control groups. To accept that PGs as a standardized treatment in some forms of liver injury, there is still much to learn about the intimate mechanisms responsible for the hepatoprotective effect. We acknowledge that this study may not be 100% extrapolated to patients with acute or chronic forms of hepatic encephalopathy in conditions of liver insufficiency. However, we want to point out that using NSAIDs in patients who potentially suffer from hepatic encephalopathy should be closely monitored.

In summary, chronic indomethacin administration had detrimental effects on the severity of encephalopathy in TAA-treated rats and this phenomenon may be partly attributed to the aggravation of liver injury. It is possible that prostacyclin inhibition may affect neurotransmission, brain microcirculation, or changes of blood-brain-barrier permeability. However, the detailed responsible mechanisms remained to be elucidated. Our current study suggests that PGI₂ may provide a protective role in the development of fulminant hepatic failure.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Chun-Ching Tai, Yu-Yao Kau and Yun-Ni Hsieh for their excellent technical assistance.