Effects of glycine on plasma and liver tissue changes of TNF-α, ET-1 and nitric oxide contents in rats with obstructive jaundice

Abstract

AIM: To evaluate the effect of glycine on plasma and liver tissue changes of tumor necrosis factor-α (TNF-α), endothelin-1 (ET-1) and nitric oxide (NO) contents in rats with obstructive jaundice.

METHODS: Ninety healthy Wistar rats of both sexes weighing 275 ± 25 g were randomly divided into sham-operated, bile duct-ligated, and bile duct-ligated plus glycine-treated groups, the latter was performed with 5% glycine solution substituting for tap water drunk ad libium for 5 d before and 6 d after operation. Blood and liver tissue were sampled at the time of sacrifice on the 8^th day post operation. Plasma total bilirubin, endotoxin, levels, as well as TNF-α, ET-1 and NO contents in liver tissue were determined.

RESULTS: Plasma endotoxin and total bilirubin levels were significantly higher in both bile duct-ligated and bile duct-ligated plus glycine-treated rats than in sham-operated animals (P = 0.000613, 0.00921 and 0.00737, 0.00841 respectively), whereas they did not display any statistically significant difference between the former groups (P = 0.417 and 0.374 respectively). Likewise, TNF-α, ET-1 and NO contents in both plasma and liver tissue were significantly increased in both bile duct-ligated and bile duct-ligated plus glycine-treated rats compared with sham-operated animals (P = 0.00813, 0.00793, 0.00671, 0.00804, 0.00872, and 0.00947 in plasma and 0.00531, 0.00785, 0.00912, 0.00981 and 0.00635 in liver tissue respectively). However, these inflammatory mediators in both plasma and liver tissue were significantly reduced in bile duct-ligated rats fed on 5% glycine solution compared with that without (P = 0.00953, 0.00891, 0.0795, 0.00867, 0.0697 and 0.00907 in plasma and liver tissue respectively).

CONCLUSION: Reduction of TNF-α, ET-1 and NO contents in plasma and liver tissue of rats fed on glycine may be helpful to alleviate pathological lesions in obstructive jaundice.

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INTRODUCTION

Endotoxemia is one of the major causes that can lead to complicated pathophysiologic alterations in the process of obstructive jaundice. Current research has demonstrated that severe endotoxemia in obstructive jaundice could activate immunocompetent cells such as monocytes, macrophages and endothelial cells to produce a variety of cytokines that could contribute to an uncontrollable inflammatory cascade causing multiple organ dysfunction (MODS) or even death[1-3]. Among these cytokines, TNF-α, ET-1 and NO have been considered to be the main effectors in endotoxemia[4-10]. It is also worthy to note that glycine could provide an effective protection against endotoxemia that has been recently published in several repots[11-15]. The present study, therefore, was conducted with the aim to evaluate the effect of glycine on the plasma and liver tissue changes of TNF-α, ET-1 and nitric oxide contents in rats with obstructive jaundice.

MATERIALS AND METHODS

Animal model and experimental protocol

Ninety healthy Wistar rats of both sexes weighing 275 ± 25 g were employed in the study. According to the experimental protocol, the animals were randomly divided into Group A in which the rats were performed a sham operation, Group B in which the rats were operated merely to ligate the common bile duct and Group C in which the rats were treated with both the ligation of common bile duct and a glycine regimen. Before operation, all animals were allowed access to standard rat chow and water ad libium for 5 d except that the rats in Group C drank 5% glycine solution (provided by Shanghai Institute of Biological Products Research) instead of water[2]. After a 12-h fasting period, the animals were weighed and anesthetized with 1% pentobarbitale sodium (30 mg/kg, ip) and their common bile duct was exposed and ligated to form a complete obstruction of the extrahepatic bile duct except that the common bile duct in rats of Group A was only exposed following laparotomy. When recovered for a period of 24 h, the rats were fed on their corresponding regimens for another 6 d. Rat venous blood was sampled and liver was removed surgically at the time of sacrifice on the 8^th day post operation under anesthesia. Blood samples were prepared respectively according to the procedures for determinating different variables and the resulting plasma was stored at -70 °C until use. The liver was perfused via a cannula inserted into the portal vein with sterile normal saline to remove residual blood, and then prepared into 10% tissue homogenate with 1.0 mol·L^-1 acetic acid and centrifuged at 3000 × g for 40 min. The supernatant was stored at -70 °C until analysis.

Measurements

For determination of plasma endotoxin level, 1 mL of blood sample was collected in a pyrogen-free heparinized tube and centrifuged at 500 × g for 5 min. The resulting plasma was analyzed with the limulus-amoebocyte-lysate test (LAL) kit according to the protocol of its manufacturer (Shanghai Yihua Medical Science & Technology Ltd.). The endotoxin concentration was finally expressed as Eu/mL.

Measurement of ET-1 level was performed using 1 mL of blood samples that was collected in a test tube containing 15 μL of 10% disodium edentate and 20 μL of aprotinin, and centrifuged at 3000 × g for 10 min at 4 °C. The resulting plasma and liver homogenate were determined with ET-1 radioimmunoassay kit following the protocol of Research Institute of Radioimmunotechnology, General Hospital of PLA. Heparinised blood samples were collected in a separator tube and spun at 4000 g for 10 min for detection of plasma bilirubin and TNF-α. To measure TNF-α, the resulting plasma and liver homogenate were analysed with a radioimmunoassay kit according to the instructions supplied by Research Institute of Radioimmunotechnology, General Hospital of PLA. Plasma bilirubin was determined with an automatic multifunction-biochemical analyzer.

Measurement of NO₂^-/NO₃^- content two mL of heparinised blood samples was incubated at 37 °C for 1 h and then centrifuged at 2000 × g for 5 min. The resulting plasma and liver homogenate were determined with a NO₂^-/NO₃^- assay kit following the procedures of Research Institute of Radioimmunotechnology, General Hospital of PLA.

Statistical analysis

Experimental data were processed by analysis of variance and t-tests for comparison between groups. Results were expressed as mean ± SE. P < 0.05 was selected as the level of significance.

RESULTS

Poor appetite and jaundice were observed in rats of Groups B and C on the 2^nd day after operation, the jaundice was more apparent on the ear and tail end skin. The dilation of proximal bile duct at the site of ligation, deep-brown-coloured liver and significantly elevated plasma total bilirubin (P = 0.00921 and 0.00841 in Groups B and C respectively as compared to Group A) were also noted in these animals at the time of sacrifice on the 8^nd day post operation. Animals in Group A did not exhibit obvious abnormalities. But there was no significant difference of plasma total bilirubin between rats in Groups B and C (P = 0.374).

Plasma endotoxin levels in rats of Groups B and C were significantly increased compared with that in rats of Group A (P = 0.000613 and 0.00737 respectively). However, It did not exhibit significant difference between rats in Groups B and C (P = 0.417).

Plasma levels of TNF-α, ET-1 and NO in Group B rats were significantly elevated compared with that in Group A rats (P = 0.00813, 0.00793 and 0.00671 respectively), which were significantly improved when the rats were fed on 5% glycine solution as shown in Table 1 (P = 0.00953, 0.00891 and 0.0795). The data for TNF-α, ET-1 and NO levels in liver tissue are shown in Table 2. In accordance with their plasma counterparts, significant reduction of these variables was found in bile duct-ligated rats fed on 5% glycine solution (0.00867, 0.0697 and 0.00907).

Table 1 Plasma levels of bilirubin, endotoxin, TNF-α, ET-1 and NO₂^-/NO₃^- in different groups of rats (x^-±s, n = 30).

Group	Bilirubin (μmol/L)	Endotoxin (Eu/mL)	TNF-α (pg/mL)	ET-1 (pg/mL)	NO2-/NO3- (μmol/L)
A	5.80 ± 1.65	5.98 ± 1.00	61.37 ± 3.08	88.79 ± 7.56	5.51 ± 0.44
B	45.45 ± 6.69a	11.65 ± 1.57a	352.52 ± 20.65a	183.24 ± 34.01a	12.06 ± 0.62a
C	43.18 ± 6.53a	11.27 ± 1.30a	158.63 ± 7.07ab	120.68 ± 10.99ab	8.55 ± 0.40ab

^aP < 0.01 vs group A,

^bP < 0.01 vs group B.

Table 2 Levels of TNF-α, ET-1 and NO₂^-/NO₃^- in liver tissues of different groups of rats (x^-±s).

Group	Samples	TNF-α (pg/mL)	ET-1 (pg/mL)	NO2-/NO3- (μmol/L)
A	10	43.51 ± 2.58	43.18 ± 4.47	2.15 ± 0.18
B	10	298.46 ± 18.74a	124.56 ± 11.67a	10.53 ± 0.87a
C	10	113.45 ± 6.67ab	81.49 ± 7.39ab	7.23 ± 0.34ab

^aP < 0.01 vs group A,

^bP < 0.01 vs group B.

DISCUSSION

Obstructive jaundice is associated with an increased incidence of postoperative complications such as infection, systemic inflammatory response, and even multiple organ failure due to metabolic and hemodynamic disorders, as well as depressed immune function. Current studies have revealed that endotoxemia was one of the major causes leading to high morbidity and mortality in patients with obstructive jaundice[1-3]. Therefore, the therapeutic strategy aimed at reducing plasma endotoxin level and interrupting its biological activities has become a focus of great concern[4].

It has been found that endotoxin could stimulate monocytes and macrophages to produce a variety of cytokines, because the elevated intracellular Ca²⁺ concentration in these cells was resulted from the activation of Ca²⁺ channel by endotoxin[11,12]. It has also been noted that glycine, a nonessential amino acid, could exert protective effects on animals in multiple morbid conditions by minimizing oxidative stress, as well as toxic eicosanoid cytokine production[11,13] Ding et al[14,15] demonstrated that the biological effects of endotoxin could be significantly inhibited by glycine via a mechanism of blocking TNF-α production in immunocompetent cells, the latter could play a critical role in the pathogenesis of endotoxin lesions[4-7]. ET and NO, although as the intense vasoconstrictor and vasodilator respectively, were also pleiotropic factors involveded in endotoxin-mediated pathological processes with endotoxin and TNF-α as their potent releasing irritants[3,8-10]. The present study demonstrated that severe endotoxemia could be observed in obstructive jaundice, and the TNF-α, ET and NO contents in plasma and liver tissue were all significantly increased in bile duct-ligated rats.

Persistent severe endotoxemia in obstructive jaundice could stimulate not only Kupffer cells to release TNF-α to insult directly parenchymal cells of the kidney and liver[3,16-18], but also other immunocompetent cells to produce excessive amount of ET-1 and NO[8] to aggravate the disturbance of splanchnic circulation leading to decreased oxygen delivery, and ultimately multiple organ dysfunction syndrome (MODS). Besides, NO itself is also a highly active free radical and can convert further into NO₂^- and peroxynitrite. The more vigorous oxidants could result in cell injury[9]. Although glycine could reduce the plasma and liver tissue endotoxin level in our study, it did prevent TNF-α, ET-1 and NO from excessive production, which might be beneficial to alleviate the organ injury in obstructive jaundice. However, TNF-α, ET-1 and NO levels in both plasma and liver tissue remained elevated to some extent in rats fed on glycine as rats compared with sham-operated rats. To elucidate the mechanism underlying this phenomenon is thus the purpose of our further studies.