Original Paper Pharmacology 2014;93:155–165 DOI: 10.1159/000360181
Received: November 5, 2013 Accepted after revision: January 31, 2014 Published online: April 30, 2014
Preconditioning with Glutamine Protects against Ischemia/Reperfusion-Induced Hepatic Injury in Rats with Obstructive Jaundice Feng Xu Chao-Liu Dai Song-Lin Peng Yang Zhao Chang-Jun Jia Yong-Qing Xu Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital, China Medical University, Shenyang, China
Abstract Objective: To ascertain whether glutamine (Gln) pretreatment protects rats with obstructive jaundice from hepatic ischemia/reperfusion (I/R) injury and to determine the underlying molecular mechanisms. Methods: An obstructive jaundice rat model was developed by bile duct ligation. On the first day after the operation, all rats were randomized into two groups and received oral Gln or normal saline (NS) daily for 7 days. Then both groups underwent a 15-min liver ischemia via the Pringle maneuver. Blood samples as well as liver and intestinal tissues were harvested and measured after 1, 6 and 24 h of reperfusion. Results: The results showed that the histological morphology of the liver and intestinal tissues significantly improved in the Gln group after I/R injury compared with the NS group. Serum proteins and enzymes associated with hepatic function also significantly improved in the Gln group. The level of glutathione increased and the levels of malondialdehyde and myeloperoxidase
© 2014 S. Karger AG, Basel 0031–7012/14/0934–0155$39.50/0 E-Mail [email protected] www.karger.com/pha
decreased in the Gln group. The levels of interleukin-1β and tumor necrosis factor-α decreased in the Gln group. Moreover, bcl-2 protein expression was upregulated and intercellular adhesion molecule 1 and bax protein expression downregulated in the Gln group; the caspase 3 mRNA level significantly increased in the Gln group. Conclusions: The study demonstrates that preconditioning with Gln significantly improves hepatic structure and function after I/R injury in rats with obstructive jaundice. The protective effect of Gln was mediated by the inhibition of reactive oxygen species and inflammation as well as a reduction in hepatocyte apoptosis. © 2014 S. Karger AG, Basel
Introduction
Most of the patients with malignant tumors at the liver hilum present with obstructive jaundice due to extrahepatic biliary obstruction. Radical surgical resection, i.e. major liver resection, remains the best treatment option for these people. To prevent massive blood loss during the hepatectomy, intermittent or continuous total hepatic inflow occlusion (the Pringle maneuver) is widely used as Chao-Liu Dai, MD, PhD Department of Hepatobiliary and Splenic Surgery General Surgery, Shengjing Hospital, China Medical University 36 Sanhao Street, Heping District, Shenyang 110004 (China) E-Mail daichaoliusjh @ 126.com
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Key Words Obstructive jaundice · Reactive oxygen species · Liver · Ischemia/reperfusion injury · Glutamine · Inflammation · Apoptosis
Materials and Methods Animals Male Wistar rats (weighing 250–300 g) were housed on standard laboratory diet and tap water ad libitum, and in an environmentally controlled room under a 12-hour light-dark cycle. The protocol was conducted according to ethical standards and approved by the local animal care and use committee.
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Development of Hepatic I/R Injury Model in Rats with Obstructive Jaundice All procedures were performed under general anesthesia induced by intraperitoneal injection of 10% chloral hydrate (3 ml/ kg). Extrahepatic biliary obstruction was induced in the rats by common bile duct ligation. Briefly, their abdomen was shaved and disinfected with 1% antiseptic povidone-iodine. After an epigastric midline laparotomy of 1.5–2 cm, the common bile duct was ligated and obstructive jaundice induced within 1 week. The abdominal incisions were closed in two layers using a 5-0 silk suture. The rats were randomly allocated to two main groups: (1) a normal saline (NS)-treated group (n = 18) and (2) a Gln-treated group (n = 18). In each group, the rats were further divided into three subgroups (n = 6 in each group) depending on different reperfusion times: (a) 1 h, (b) 6 h and (c) 24 h. Gln granules (Chongqing Yaoyou Pharmaceutical Co., Ltd., China; 0.5 mg/kg body weight) dissolved in 3 ml of NS were given to the rats by oral gavage once on the first day after bile duct ligation; 3 ml of NS were administered to the control rats. The treatment lasted 7 days. After this, the animals were anesthetized again, followed by laparotomy at the original incision site, as described above. The dilated common bile duct was freed after severing the peripheral hepatic ligaments. Warm ischemia of 15 min duration was induced by total hepatic inflow occlusion (Pringle maneuver) using an atraumatic arterial clip; then the artery clamp was removed to restore the liver’s blood supply. A wet cotton swab was used to loosen the liver hilum. An epidural catheter (diameter: 0.9 mm) was inserted into the common bile duct and ligated with a 2-0 silk suture. The other side of the catheter was inserted into the duodenum about 1.5–2 cm deep and buried with a purse-string suture. After liver reperfusion for 1, 6 or 24 h, 3 ml of blood were drawn from the abdominal aorta with sterilized pyrogen-free syringes. Then the blood was placed on ice for 20 min, and centrifuged at 3,000 rpm for 10 min. The serum was collected and stored at –20 ° C for later assays of serum total protein (TP), albumin (ALB), biochemical enzyme and bilirubin levels. The right lobe of the liver was harvested and stored at –80 ° C for later ELISA and RT-PCR assays. The left lateral lobe of the liver (1 cm3) and the terminal ileal segment (2 cm) were harvested and fixed in 10% formaldehyde for later histological staining. Another 2-cm ileal segment was fixed in 2.5% glutaraldehyde solution for electron microscopic scanning.
Histology Samples from the liver and a segment of the ileum were embedded in paraffin and then sections measuring 5 μm in thickness were cut. The specimens were stained with hematoxylin-eosin and examined under a light microscope. Electron Microscopy Biofilm samples from the segment of the ileum were prepared for high-resolution transmission electron microscopy by drying small segments of the ileum on copper mesh-supported grids coated with Formvar. The samples were then coated with gold and examined with a Hitachi S-450 Field Emission Scanning Electron Microscope (3 kV). Assessment of Hepatocellular Injury Blood samples were collected from the animals after 1, 6 and 24 h of reperfusion. Serum levels of TP, ALB, total bilirubin (TBIL),
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an efficient method, but this causes hepatic warm ischemia. Liver ischemia/reperfusion (I/R) injury is inevitable upon restoring the inflow blood supply. Many factors including proinflammatory cytokines, reactive oxygen species (ROS), lipid mediators, intracellular calcium overload, activated leukocytes and platelet aggregation are involved in the pathophysiology of hepatic I/R injury. Cholestasis has been identified as a major risk factor for oxidative stress and renders these livers more susceptible to I/R than any normal liver. ROS play an important role in the pathophysiology of hepatic I/R injury [1–4]. Glutathione (GSH) is one of key enzymatic defense mechanisms of eliminating ROS in rodents and the human body. Three amino acids are responsible for GSH synthesis: glycine, cysteine and glutamate (derived from glutamine, Gln). Decreased Gln levels are closely correlated with reduction in intracellular GSH [5, 6]. Gln is a conditionally essential amino acid. It does not possess an antioxidant effect on its own. However, it has been shown to protect cells and tissues against a variety of stressful stimuli including inflammation [7– 9], ischemia [10–12], infection, fibrosis and cancer [13– 16]. In recent years, the protective effects of Gln against I/R-induced organ injury have attracted more and more attention in the scientific field [10–12]. For rodents, some reports have demonstrated that pretreatment with Gln helps to reduce I/R injury in a variety of organs by using different I/R animal models [11, 17–20]. Nevertheless, few studies have been carried out on this topic using the obstructive jaundice hepatic I/R model. Our current study is designed to ascertain whether or not Gln pretreatment protects rats with obstructive jaundice from I/R-induced hepatic injury and to determine the underlying molecular mechanisms. It shows that pretreatment of rats with Gln significantly reduces I/R-induced hepatic injury, which is evidenced by improved morphology, reduced mortality, induction of proapoptotic proteins and inhibition of inflammatory chemokines. It demonstrates that Gln protects rats with obstructive jaundice against I/R-induced hepatic injury.
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Fig. 1. Hepatic morphological changes at
direct bilirubin (DBIL), indirect bilirubin (i.e. unconjugated BIL, UDBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were determined by standard spectrophotometry using a clinical automated chemistry analyzer. Immunohistochemistry The paraffin-embedded sections were cleared and immunohistochemically stained with antibodies against intercellular adhesion molecule 1 (ICAM-1; 1: 100), bcl-2 (1: 75) and bax (1: 75; all purchased from Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA) using the standard immunohistochemical protocol. Briefly, following antigen retrieval using 0.01 mol/l EDTA, pH 9.0, and inactivation of endogenous peroxidase using 3% hydrogen peroxide, sections were blocked for 10 min in 10% goat serum. The sections were first incubated with specific primary antibody for 1 h,
Gln Protects against Hepatic Injury in Rats
then with SP-9000 (Beijing Zhongshan Biological Technology Co., Ltd., Beijing, China) for 30 min at 37 ° C, after which they were developed with DAB (Beijing Zhongshan Biological Technology) for 5 min and counterstained with hematoxylin. Images were obtained using an Olympus BX5 microscope (Olympus, Tokyo, Japan). Bcl2- and bax-positive cells were referred to the cells with brown granules in the cytoplasm. ICAM-1-positive cells were referred to the cells with brown granules in both the cytoplasm and cell membrane. The integral optical densities of ICAM-1, bcl-2 and bax expression were calculated by image analysis software (integrated optical density average).
Assays Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the rat liver were determined by ELISA according to the manufacturer’s protocols. Tissue levels of GSH, myeloperoxidase (MPO)
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different reperfusion time points after bile duct ligation in rats pretreated with NS or Gln. ×400.
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Fig. 2. Morphological changes to the ileum shown 24 h after bile duct ligation in rats pretreated with NS or Gln. a Light microscopic images. b Electron microscopic images.
and malondialdehyde (MDA) were detected using GSH, MDA or MPO detection kits, which were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The TNF-α and IL-1β ELISA kits were purchased from Shanghai Senxiong Technology Industry Co., Ltd. (Shanghai, China). Three independent experiments were performed. RNA Extraction, Reverse Transcription and Semi-Quantitative RT-PCR Amplification Approximately 500 mg of frozen liver tissue was homogenized in 1 ml TRIzol reagent and total RNA was prepared according to the procedures suggested by the manufacturer. First-strand cDNA was synthetized by using an RT-PCR kit [TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China] using 5 μg total RNA as a template in a GeneAmp PCR System 9700. Amplification of caspase 3 was performed by using the following primers: forward – 5′-CTG GAC TGC GGT ATT GAG-3′; reverse – 5′-GGG TGC GGT AGA GTA AGC3′. β-Actin was used as an internal reference. The primers for β-actin were: forward – 5′-CAC CCT GTG CTG CTC ACC GAG GCC-3′; reverse – 5′-CCA CAC AGA TGA CTT GCG CTC AGG-3′. Statistical Analysis The data in all the figures are expressed as means ± SD. Data analysis was performed with a statistical software package (SPSS version 11.5). Student’s t test was used to compare differences between groups. p < 0.05 was considered statistically significant.
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Normal ileum without pretreatment (×200)
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Results
Gln Significantly Improves the Histological Morphology of the Liver after I/R Injury Study under the light microscope revealed that at all 3 time points after reperfusion, the hepatic morphology was significantly improved in the Gln group compared with that seen in the NS group, as evidenced by the reduced swelling of liver cells in hematoxylin-eosin-stained sections. The architectures of the liver cell cord and hepatic sinusoid were well organized, accompanied by an expanded hepatic sinusoid. The structure was abnormal in the NS group. The swelling and necrosis of hepatocytes around the central veins were obvious, especially 6 h after hepatic reperfusion (fig. 1). Gln Significantly Improves the Histological Morphology of the Intestine after I/R Injury Observation by light microscopy revealed that the number of small intestinal villi had significantly decreased in the NS group. Distorted and deformed structures were visible, accompanied by hemorrhage, necrosis and shedXu/Dai/Peng/Zhao/Jia/Xu
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ding, damage to intestinal crypt epithelial cells and mucosa, and lamina propria fibrosis. Compared with the NS group, the number of intestinal villi significantly increased in the Gln group, and they had a more organized structure and small gaps between the villi. The villi were thick and long. The mucosal hyperemia and edema significantly receded (fig. 2a). Study by electron microscopy showed that microvilli in the normal ileum are well organized with tight junctions. The intestinal microvilli in the rats treated with NS lost their normal structure and were sparsely ar-
Gln Significantly Improves Hepatic Function after I/R Injury To test whether Gln could improve hepatic function after I/R-induced injury, the serum levels of proteins and enzymes associated with liver function were assayed by us-
Gln Protects against Hepatic Injury in Rats
Pharmacology 2014;93:155–165 DOI: 10.1159/000360181
ranged with big gaps in between. In the Gln group, the intestinal microvilli were tied closely together, with small gaps and an enlarged surface. These changes are similar to what can be seen in the normal ileum (fig. 2b).
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Fig. 4. Hepatic expression of the inflammatory cytokine ICAM-1 stained by immunohistochemistry in rats pretreated with NS or Gln after 1, 6 and 24 h of reperfusion. a Light microscopic images.
× 400 b The average integral optical density of ICAM-1 proteins in each group. * p < 0.05 compared with NS groups.
ing an automated chemistry analyzer. The results showed that after reperfusion for 24 h, serum TP and ALB levels in the Gln group were significantly higher than in the NS group (p < 0.05; fig. 3a, b). The serum TBIL, DBIL and UDBIL levels in the Gln group were mostly lower than those seen in the corresponding NS groups, depending on the duration of reperfusion: TBIL and DBIL levels were significantly reduced at 1 and 24 h of reperfusion compared with the NS groups (p < 0.05), and slightly reduced at 6 h of reperfusion. The serum UDBIL level in the Gln group
was similar to that in the NS group at 1 h of perfusion, and reduced at both 6 and 24 h of reperfusion, compared with the NS groups (p < 0.05; fig. 3c–e). The serum AST, ALT and LDH levels in the Gln group were all significantly lower than those in the NS groups (p < 0.05; fig. 3f–h).
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Gln Reduces Inflammatory Cytokine Levels of ICAM-1, IL-1β and TNF-α in Hepatic Tissue after I/R Injury Inflammatory mediators play an important role in the pathogenesis of I/R injury. ICAM-1-positive cells were Xu/Dai/Peng/Zhao/Jia/Xu
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Fig. 6. Tissue levels of GSH peroxidase (a), MDA (b) and MPO (c) as detected by ELISA in rats pretreated with NS or Gln after 1, 6 and 24 h of reperfusion. * p < 0.05 compared with NS groups.
Gln Regulates GSH, MDA and MPO Levels in Hepatic Tissue Levels of GSH, MDA and MPO in the liver were detected using commercially available kits. The results showed that, at all time points, GSH levels in hepatic tissue from rats treated with Gln were significantly increased compared with those in the NS-treated groups (p < 0.05). Both MDA and MPO levels significantly decreased in the Gln groups compared with the NS groups at different time points of reperfusion (p < 0.05; fig. 6).
stained with antibodies against bcl-2 and bax. The results showed that the bcl-2-positive cells in liver sections from the Gln group drastically increased (fig. 7a, c). Bax-positive cells decreased after I/R injury at all 3 time points (fig. 7b, d). In the meantime, relative caspase 3 mRNA levels were quantified by RT-PCR. They were significantly lower in the Gln group than in the NS group (p < 0.05; fig. 8).
Discussion
Gln Modulates Apoptotic Protein Expression in Hepatic Tissue after I/R Injury To test the effects of Gln on the expression of apoptotic molecules in hepatic tissue, liver sections were
The present study shows that Gln pretreatment improves the intestinal mucosal architecture and its barrier function, which is in line with previous reports [21–25]. Bacterial translocation and oxidative stress resulting from obstructive jaundice cause intestinal mucosal atrophy and barrier dysfunction [24]. Previous studies showed that administration of Gln protected the intestinal mucosa from damage by reducing intestinal bacterial translocation and intestinal endotoxemia [24, 25]. Importantly, our study reveals that in rats Gln pretreatment significantly improves hepatic structure and function after I/R injury caused by
Gln Protects against Hepatic Injury in Rats
Pharmacology 2014;93:155–165 DOI: 10.1159/000360181
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strongly downregulated in hepatic tissue from the Gln group compared with that from the NS group (fig. 4) after reperfusion for 1, 6 and 24 h (p < 0.05). Cytokine IL1β and TNF-α levels were detected by ELISA. The study revealed that IL-1β and TNF-α levels were significantly reduced in the Gln group compared with those in the NS groups (p < 0.05) at all time points after I/R injury (fig. 5).
NS-reperfusion 1 h
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Fig. 7. Hepatic levels of apoptotic proteins
obstructive jaundice. This is evidenced by the improvement in hepatic and intestinal morphology, increases in serum TP and ALB levels, reduction in liver enzymes (markers of hepatocyte damage) and decreases in oxidative and inflammatory stress and apoptosis. The protective effects of Gln against hepatic I/R in rats are consistent with those found in previous studies [17, 18]. Zhang et al. [17] reported that Gln preconditioning effectively protected against hepatic I/R injury. These protective effects were related to the dose of Gln and due to the reduction in intracellular calcium overload and improvements in the activity of Na+/K+ ATPase and SOD. Schuster et al. [18] showed that Gln protected rats against I/R-induced hepatic injury by attenuating inflammatory response. 162
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a
Gln plays a critical role in cell growth, proliferation and metabolism. It is not only an essential component in cell structure but also important precursor material for the synthesis of GSH, a key antioxidant molecule. Moreover, it is an important energy resource and a source of nitrogen for the synthesis of other amino acids and proteins [26]. The intestinal tract is the main organ consuming Gln, especially the small intestine, which plays a key role in Gln metabolism. Enhancement of Gln metabolism promotes gluconeogenesis and liver glycogen synthesis [27]. Therefore, supplementation of Gln promotes liver glycogen synthesis and increases the ability of cells to resist oxidative stress in the liver. Xu/Dai/Peng/Zhao/Jia/Xu
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as detected in rats pretreated with NS or Gln after 1, 6 and 24 h of reperfusion. a Proapoptotic protein bcl-2. ×400.
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Gln Protects against Hepatic Injury in Rats
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Bcl-2 (average integrated optical density)
Fig. 7. Hepatic levels of apoptotic proteins
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Fig. 8. Hepatic mRNA levels of caspase 3 as detected by semi-quantitative RT-PCR in rats pretreated with NS or Gln after 1, 6 and 24 h of reperfusion. a PCR products resolved in 1% agarose gel. b Optical density values of
caspase 3 in each group. * p < 0.05 compared with NS groups.
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ates epithelial cell repair. Studies showed (1) that Gln affects the composition of the intestinal bacterial flora by restraining the growth of Gram-negative bacteria [28] and (2) that Gln promotes the secretion of IgA in intestinal mucosal lymphoid tissue and reduces the attachment and colonization of bacteria in the intestinal mucosa [29]. The reduced bacterial translocation and endotoxins in the liver system inactivate hepatic Kupffer cells, inhibit the release of TNF-α, IL-1 and other cytokines, and reduce the production of oxygen free radicals, therefore protecting the liver from these sources of damage [30]. Apoptosis plays an important role in the pathophysiology of obstructive jaundice. Apoptotic cell death can trigger inflammatory cell transmigration, worsening apoptotic cell injury. Caspase 3, a cell death protease, is a key enzyme inducing apoptosis in mammalian cells. Activation of caspase 3 activity is mediated by two signaling pathways: the death receptor-dependent pathway (i.e. extracellular pathway) involving Fas/TNF-α and the mitochondrial pathway (i.e. intracellular pathway) involving the release of cytochrome c [31]. The activation of caspase 3 has effects on a variety of cellular activities and leads to cell apoptosis and cell death. Apoptotic signaling is delicately regulated: it depends on the balance between proand antiapoptotic factors [32]. Bcl-2 is one of the most important antiapoptotic proteins; it is located on the membrane, endoplasmic reticulum and mitochondrial membrane, and prevents apoptosis caused by various stimuli. Bax is a proapoptotic protein which was discovered in recent years; it exhibits 21% homology with the bcl-2 gene [33]. The present study revealed that Gln regulates apoptotic protein expression in liver tissue. Gln inhibits caspase 3 mRNA levels in hepatic tissue, downregulates hepatic bax expression and upregulates bcl-2 exXu/Dai/Peng/Zhao/Jia/Xu
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Increased oxidative stress in tissues is very common in the pathophysiology of obstructive jaundice. Hyperbilirubinemia resulting from obstructive jaundice reduces the content of bile salt in the intestinal lumen, resulting in dysfunction of the intestinal barrier and induction of enterotoxemia. The deterioration in intestinal function and development of enterotoxemia induce the production of free oxygen radicals and impair the antioxidant systems. Increased oxidative stress causes tissue injury and organ malfunction [27]. GSH, MPO and MDA are key markers for evaluating oxidative stress in cells. Our study showed that Gln significantly increased the GSH level and reduced MPO and MDA levels in liver tissue. This suggests that Gln improves liver function and protects the liver from I/R injury by elevating the level of GSH in the liver tissue, eliminating ROS. Another mechanism by which Gln exerts a beneficial effect is that it protects the intestinal mucosa, reduces bacterial translocation and eliminates endotoxemia, therefore attenuating the inflammation of, and injury to, the liver. Our study showed that Gln significantly reduces ICAM-1, TNF-α and IL-1β production in liver tissue. ICAM-1 (CD54) is an endothelium- and leukocyte-associated transmembrane protein. It stabilizes cell-cell interactions, facilitates leukocyte endothelial transmigration and mediates inflammatory response due to a variety of stress stimuli. ICAM-1 is induced by cytokines TNF-α and IL-1β. Bile duct ligation reduces intestinal barrier function, increases permeability, and induces bacterial translocation and organ damage. Supplementation with Gln provides an energy substrate to the intestinal metabolism, increases the antioxidant capacity of the intestine, promotes intestinal mucosal cell proliferation and acceler-
pression. This implies that the protective effect of Gln against hepatic injury induced by obstructive jaundice might be mediated by the inhibition of apoptosis. The antioxidant effect of Gln is mediated by neutralizing the production of ROS during ischemia, hypoxia and reperfusion injury; thus it significantly reduces the oxidative injury to hepatocytes and endothelial cells and stabilizes cell membrane structure and function. This stabilization helps to maintain a balance between anti- and proapoptotic protein activities. The ability of Gln to increase the production of ATP also helps to stabilize the cell membrane and reduce the calcium overload. A study showed that a shortage of GSH decreases the expression of bcl-2, induces apoptosis and increases tissue injury [34].
In conclusion, the current study demonstrates that in rats with obstructive jaundice, Gln effectively protects hepatocytes from I/R-induced injury. The protective effects are evidenced by improved hepatic and intestinal histology and organ function. Inhibition of ROS and inflammatory cytokines, as well an increased imbalance between pro- and postapoptotic protein activities, all contribute to the protective effect of Gln in rats.
Acknowledgment The study was supported by the Liaoning Province Natural Science Fund (2013021068).
References
Gln Protects against Hepatic Injury in Rats
13
14
15
16
17
18
19
20 21 22
23
injury. Asian Cardiovasc Thorac Ann 2007; 15:463–467. San-Miguel B, et al: Glutamine prevents fibrosis development in rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid. J Nutr 2010;140:1065–1071. Cetinbas F, Yelken B, Gulbas Z: Role of glutamine administration on cellular immunity after total parenteral nutrition enriched with glutamine in patients with systemic inflammatory response syndrome. J Crit Care 2010; 25:661e1–e6. Mondello S, et al: Glutamine treatment attenuates the development of ischaemia/reperfusion injury of the gut. Eur J Pharmacol 2010; 643:304–315. Hu YM, et al: Glutamine administration ameliorates sepsis-induced kidney injury by downregulating the high-mobility group box protein-1-mediated pathway in mice. Am J Physiol Renal Physiol 2012;302:F150–F158. Zhang WX, et al: Protective effects of glutamine preconditioning on ischemia-reperfusion injury in rats. Hepatobiliary Pancreat Dis Int 2011;10:78–82. Schuster H, et al: Protective effects of glutamine dipeptide and α-tocopherol against ischemia-reperfusion injury in the isolated rat liver. Clin Nutr 2009;28:331–337. Wu GH, et al: Glutamine supplemented parenteral nutrition prevents intestinal ischemia-reperfusion injury in rats. World J Gastroenterol 2004;10:2592–2594. Stangl R, et al: Reduction of liver ischemiareperfusion injury via glutamine pretreatment. J Surg Res 2011;166:95–103. Owari M, et al: Glutamine prevents intestinal mucosal injury induced by cyclophosphamide in rats. Pediatr Surg Int 2012;28:299–303. Li Y, et al: Protective effect of glutamine-enriched early enteral nutrition on intestinal mucosal barrier injury after liver transplantation in rats. Am J Surg 2010;199:35–42. Marks SL, et al: Effects of glutamine supplementation of an amino acid-based purified
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diet on intestinal mucosal integrity in cats with methotrexate-induced enteritis. Am J Vet Res 1999;60:755–763. Margaritis VG, et al: Effect of oral glutamine administration on bacterial translocation, endotoxemia, liver and ileal morphology, and apoptosis in rats with obstructive jaundice. World J Surg 2005;29:1329–1334. Assimakopoulos SF, et al: Beneficial effects of glutamine on intestinal barrier function in obstructive jaundice. World J Surg 2005; 29: 935–936. Hall JC, Heel K, McCauley R: Glutamine. Br J Surg 1996;83:305–312. Ziegler TR, et al: Glutamine and the gastrointestinal tract. Curr Opin Clin Nutr Metab Care 2000;3:355–362. Denno R, et al: Glutamine-enriched total parenteral nutrition enhances plasma glutathione in the resting state. J Surg Res 1996; 61: 35–38. Zhang W, et al: Glutamine reduces bacterial translocation after small bowel transplantation in cyclosporine-treated rats. J Surg Res 1995;58:159–164. Noguchi Y, et al: Increased glutamine consumption in small intestine epithelial cells during sepsis in rats. Am J Surg 1997; 173: 199–205. Fumarola C, Guidotti GG: Stress-induced apoptosis: toward a symmetry with receptor-mediated cell death. Apoptosis 2004; 9: 77–82. Li SM, et al: Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia. World J Gastroenterol 2003; 9: 2579– 2582. Sheen-Chen SM, et al: Effect of glutamine and bile acid on hepatocyte apoptosis after bile duct ligation in the rat. World J Surg 2004;28: 457–460. Celli A, et al: Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes. Am J Physiol 1998;275:G749–G757.
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1 Li C, Jackson RM: Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002; 282:C227–C241. 2 Jaeschke H: Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15–G26. 3 Galaris D, Barbouti A, Korantzopoulos P: Oxidative stress in hepatic ischemia-reperfusion injury: the role of antioxidants and iron chelating compounds. Curr Pharm Des 2006;12: 2875–2890. 4 Elias-Miró M, et al: Current knowledge on oxidative stress in hepatic ischemia/reperfusion. Free Radic Res 2013;47:555–568. 5 Mates JM, et al: Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death. Int J Biochem Cell Biol 2002;34:439–458. 6 Liu D, Chen Z: The regulatory effects of glutamine on illness and health. Protein Pept Lett 2011;18:658–662. 7 Peng HC, et al: Effects of glutamine administration on inflammatory responses in chronic ethanol-fed rats. J Nutr Biochem 2011; 22: 282–288. 8 Stachowicz-Stencel T, Synakiewicz A: Glutamine as a supplemental treatment in pediatric and adult oncology patients. Expert Opin Investig Drugs 2012;21:1861–1871. 9 Kim HA, et al: Glutamine inhibits plateletactivating factor-mediated pulmonary tumour metastasis. Eur J Cancer 2012;48:1730– 1738. 10 Zhang SC, et al: Tissue-protective effect of glutamine on hepatic ischemia-reperfusion injury via induction of heme oxygenase-1. Pharmacology 2013;91:59–68. 11 Araújo Júnior RJ, et al: Preconditioning with L-alanyl-glutamine reduces hepatic ischemiareperfusion injury in rats. Acta Cir Bras 2011; 26(suppl 1):8–13. 12 Bolotin G, et al: Glutamine improves myocardial function following ischemia-reperfusion
Received: November 5, 2013 Accepted after revision: January 31, 2014 Published online: April 30, 2014
Preconditioning with Glutamine Protects against Ischemia/Reperfusion-Induced Hepatic Injury in Rats with Obstructive Jaundice Feng Xu Chao-Liu Dai Song-Lin Peng Yang Zhao Chang-Jun Jia Yong-Qing Xu Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital, China Medical University, Shenyang, China
Abstract Objective: To ascertain whether glutamine (Gln) pretreatment protects rats with obstructive jaundice from hepatic ischemia/reperfusion (I/R) injury and to determine the underlying molecular mechanisms. Methods: An obstructive jaundice rat model was developed by bile duct ligation. On the first day after the operation, all rats were randomized into two groups and received oral Gln or normal saline (NS) daily for 7 days. Then both groups underwent a 15-min liver ischemia via the Pringle maneuver. Blood samples as well as liver and intestinal tissues were harvested and measured after 1, 6 and 24 h of reperfusion. Results: The results showed that the histological morphology of the liver and intestinal tissues significantly improved in the Gln group after I/R injury compared with the NS group. Serum proteins and enzymes associated with hepatic function also significantly improved in the Gln group. The level of glutathione increased and the levels of malondialdehyde and myeloperoxidase
© 2014 S. Karger AG, Basel 0031–7012/14/0934–0155$39.50/0 E-Mail [email protected] www.karger.com/pha
decreased in the Gln group. The levels of interleukin-1β and tumor necrosis factor-α decreased in the Gln group. Moreover, bcl-2 protein expression was upregulated and intercellular adhesion molecule 1 and bax protein expression downregulated in the Gln group; the caspase 3 mRNA level significantly increased in the Gln group. Conclusions: The study demonstrates that preconditioning with Gln significantly improves hepatic structure and function after I/R injury in rats with obstructive jaundice. The protective effect of Gln was mediated by the inhibition of reactive oxygen species and inflammation as well as a reduction in hepatocyte apoptosis. © 2014 S. Karger AG, Basel
Introduction
Most of the patients with malignant tumors at the liver hilum present with obstructive jaundice due to extrahepatic biliary obstruction. Radical surgical resection, i.e. major liver resection, remains the best treatment option for these people. To prevent massive blood loss during the hepatectomy, intermittent or continuous total hepatic inflow occlusion (the Pringle maneuver) is widely used as Chao-Liu Dai, MD, PhD Department of Hepatobiliary and Splenic Surgery General Surgery, Shengjing Hospital, China Medical University 36 Sanhao Street, Heping District, Shenyang 110004 (China) E-Mail daichaoliusjh @ 126.com
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Key Words Obstructive jaundice · Reactive oxygen species · Liver · Ischemia/reperfusion injury · Glutamine · Inflammation · Apoptosis
Materials and Methods Animals Male Wistar rats (weighing 250–300 g) were housed on standard laboratory diet and tap water ad libitum, and in an environmentally controlled room under a 12-hour light-dark cycle. The protocol was conducted according to ethical standards and approved by the local animal care and use committee.
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Development of Hepatic I/R Injury Model in Rats with Obstructive Jaundice All procedures were performed under general anesthesia induced by intraperitoneal injection of 10% chloral hydrate (3 ml/ kg). Extrahepatic biliary obstruction was induced in the rats by common bile duct ligation. Briefly, their abdomen was shaved and disinfected with 1% antiseptic povidone-iodine. After an epigastric midline laparotomy of 1.5–2 cm, the common bile duct was ligated and obstructive jaundice induced within 1 week. The abdominal incisions were closed in two layers using a 5-0 silk suture. The rats were randomly allocated to two main groups: (1) a normal saline (NS)-treated group (n = 18) and (2) a Gln-treated group (n = 18). In each group, the rats were further divided into three subgroups (n = 6 in each group) depending on different reperfusion times: (a) 1 h, (b) 6 h and (c) 24 h. Gln granules (Chongqing Yaoyou Pharmaceutical Co., Ltd., China; 0.5 mg/kg body weight) dissolved in 3 ml of NS were given to the rats by oral gavage once on the first day after bile duct ligation; 3 ml of NS were administered to the control rats. The treatment lasted 7 days. After this, the animals were anesthetized again, followed by laparotomy at the original incision site, as described above. The dilated common bile duct was freed after severing the peripheral hepatic ligaments. Warm ischemia of 15 min duration was induced by total hepatic inflow occlusion (Pringle maneuver) using an atraumatic arterial clip; then the artery clamp was removed to restore the liver’s blood supply. A wet cotton swab was used to loosen the liver hilum. An epidural catheter (diameter: 0.9 mm) was inserted into the common bile duct and ligated with a 2-0 silk suture. The other side of the catheter was inserted into the duodenum about 1.5–2 cm deep and buried with a purse-string suture. After liver reperfusion for 1, 6 or 24 h, 3 ml of blood were drawn from the abdominal aorta with sterilized pyrogen-free syringes. Then the blood was placed on ice for 20 min, and centrifuged at 3,000 rpm for 10 min. The serum was collected and stored at –20 ° C for later assays of serum total protein (TP), albumin (ALB), biochemical enzyme and bilirubin levels. The right lobe of the liver was harvested and stored at –80 ° C for later ELISA and RT-PCR assays. The left lateral lobe of the liver (1 cm3) and the terminal ileal segment (2 cm) were harvested and fixed in 10% formaldehyde for later histological staining. Another 2-cm ileal segment was fixed in 2.5% glutaraldehyde solution for electron microscopic scanning.
Histology Samples from the liver and a segment of the ileum were embedded in paraffin and then sections measuring 5 μm in thickness were cut. The specimens were stained with hematoxylin-eosin and examined under a light microscope. Electron Microscopy Biofilm samples from the segment of the ileum were prepared for high-resolution transmission electron microscopy by drying small segments of the ileum on copper mesh-supported grids coated with Formvar. The samples were then coated with gold and examined with a Hitachi S-450 Field Emission Scanning Electron Microscope (3 kV). Assessment of Hepatocellular Injury Blood samples were collected from the animals after 1, 6 and 24 h of reperfusion. Serum levels of TP, ALB, total bilirubin (TBIL),
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an efficient method, but this causes hepatic warm ischemia. Liver ischemia/reperfusion (I/R) injury is inevitable upon restoring the inflow blood supply. Many factors including proinflammatory cytokines, reactive oxygen species (ROS), lipid mediators, intracellular calcium overload, activated leukocytes and platelet aggregation are involved in the pathophysiology of hepatic I/R injury. Cholestasis has been identified as a major risk factor for oxidative stress and renders these livers more susceptible to I/R than any normal liver. ROS play an important role in the pathophysiology of hepatic I/R injury [1–4]. Glutathione (GSH) is one of key enzymatic defense mechanisms of eliminating ROS in rodents and the human body. Three amino acids are responsible for GSH synthesis: glycine, cysteine and glutamate (derived from glutamine, Gln). Decreased Gln levels are closely correlated with reduction in intracellular GSH [5, 6]. Gln is a conditionally essential amino acid. It does not possess an antioxidant effect on its own. However, it has been shown to protect cells and tissues against a variety of stressful stimuli including inflammation [7– 9], ischemia [10–12], infection, fibrosis and cancer [13– 16]. In recent years, the protective effects of Gln against I/R-induced organ injury have attracted more and more attention in the scientific field [10–12]. For rodents, some reports have demonstrated that pretreatment with Gln helps to reduce I/R injury in a variety of organs by using different I/R animal models [11, 17–20]. Nevertheless, few studies have been carried out on this topic using the obstructive jaundice hepatic I/R model. Our current study is designed to ascertain whether or not Gln pretreatment protects rats with obstructive jaundice from I/R-induced hepatic injury and to determine the underlying molecular mechanisms. It shows that pretreatment of rats with Gln significantly reduces I/R-induced hepatic injury, which is evidenced by improved morphology, reduced mortality, induction of proapoptotic proteins and inhibition of inflammatory chemokines. It demonstrates that Gln protects rats with obstructive jaundice against I/R-induced hepatic injury.
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direct bilirubin (DBIL), indirect bilirubin (i.e. unconjugated BIL, UDBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were determined by standard spectrophotometry using a clinical automated chemistry analyzer. Immunohistochemistry The paraffin-embedded sections were cleared and immunohistochemically stained with antibodies against intercellular adhesion molecule 1 (ICAM-1; 1: 100), bcl-2 (1: 75) and bax (1: 75; all purchased from Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA) using the standard immunohistochemical protocol. Briefly, following antigen retrieval using 0.01 mol/l EDTA, pH 9.0, and inactivation of endogenous peroxidase using 3% hydrogen peroxide, sections were blocked for 10 min in 10% goat serum. The sections were first incubated with specific primary antibody for 1 h,
Gln Protects against Hepatic Injury in Rats
then with SP-9000 (Beijing Zhongshan Biological Technology Co., Ltd., Beijing, China) for 30 min at 37 ° C, after which they were developed with DAB (Beijing Zhongshan Biological Technology) for 5 min and counterstained with hematoxylin. Images were obtained using an Olympus BX5 microscope (Olympus, Tokyo, Japan). Bcl2- and bax-positive cells were referred to the cells with brown granules in the cytoplasm. ICAM-1-positive cells were referred to the cells with brown granules in both the cytoplasm and cell membrane. The integral optical densities of ICAM-1, bcl-2 and bax expression were calculated by image analysis software (integrated optical density average).
Assays Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the rat liver were determined by ELISA according to the manufacturer’s protocols. Tissue levels of GSH, myeloperoxidase (MPO)
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different reperfusion time points after bile duct ligation in rats pretreated with NS or Gln. ×400.
a
NS-reperfusion 24 h (×200)
Gln-reperfusion 24 h (×100)
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Fig. 2. Morphological changes to the ileum shown 24 h after bile duct ligation in rats pretreated with NS or Gln. a Light microscopic images. b Electron microscopic images.
and malondialdehyde (MDA) were detected using GSH, MDA or MPO detection kits, which were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The TNF-α and IL-1β ELISA kits were purchased from Shanghai Senxiong Technology Industry Co., Ltd. (Shanghai, China). Three independent experiments were performed. RNA Extraction, Reverse Transcription and Semi-Quantitative RT-PCR Amplification Approximately 500 mg of frozen liver tissue was homogenized in 1 ml TRIzol reagent and total RNA was prepared according to the procedures suggested by the manufacturer. First-strand cDNA was synthetized by using an RT-PCR kit [TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China] using 5 μg total RNA as a template in a GeneAmp PCR System 9700. Amplification of caspase 3 was performed by using the following primers: forward – 5′-CTG GAC TGC GGT ATT GAG-3′; reverse – 5′-GGG TGC GGT AGA GTA AGC3′. β-Actin was used as an internal reference. The primers for β-actin were: forward – 5′-CAC CCT GTG CTG CTC ACC GAG GCC-3′; reverse – 5′-CCA CAC AGA TGA CTT GCG CTC AGG-3′. Statistical Analysis The data in all the figures are expressed as means ± SD. Data analysis was performed with a statistical software package (SPSS version 11.5). Student’s t test was used to compare differences between groups. p < 0.05 was considered statistically significant.
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Normal ileum without pretreatment (×200)
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Results
Gln Significantly Improves the Histological Morphology of the Liver after I/R Injury Study under the light microscope revealed that at all 3 time points after reperfusion, the hepatic morphology was significantly improved in the Gln group compared with that seen in the NS group, as evidenced by the reduced swelling of liver cells in hematoxylin-eosin-stained sections. The architectures of the liver cell cord and hepatic sinusoid were well organized, accompanied by an expanded hepatic sinusoid. The structure was abnormal in the NS group. The swelling and necrosis of hepatocytes around the central veins were obvious, especially 6 h after hepatic reperfusion (fig. 1). Gln Significantly Improves the Histological Morphology of the Intestine after I/R Injury Observation by light microscopy revealed that the number of small intestinal villi had significantly decreased in the NS group. Distorted and deformed structures were visible, accompanied by hemorrhage, necrosis and shedXu/Dai/Peng/Zhao/Jia/Xu
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ding, damage to intestinal crypt epithelial cells and mucosa, and lamina propria fibrosis. Compared with the NS group, the number of intestinal villi significantly increased in the Gln group, and they had a more organized structure and small gaps between the villi. The villi were thick and long. The mucosal hyperemia and edema significantly receded (fig. 2a). Study by electron microscopy showed that microvilli in the normal ileum are well organized with tight junctions. The intestinal microvilli in the rats treated with NS lost their normal structure and were sparsely ar-
Gln Significantly Improves Hepatic Function after I/R Injury To test whether Gln could improve hepatic function after I/R-induced injury, the serum levels of proteins and enzymes associated with liver function were assayed by us-
Gln Protects against Hepatic Injury in Rats
Pharmacology 2014;93:155–165 DOI: 10.1159/000360181
ranged with big gaps in between. In the Gln group, the intestinal microvilli were tied closely together, with small gaps and an enlarged surface. These changes are similar to what can be seen in the normal ileum (fig. 2b).
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*
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× 400 b The average integral optical density of ICAM-1 proteins in each group. * p < 0.05 compared with NS groups.
ing an automated chemistry analyzer. The results showed that after reperfusion for 24 h, serum TP and ALB levels in the Gln group were significantly higher than in the NS group (p < 0.05; fig. 3a, b). The serum TBIL, DBIL and UDBIL levels in the Gln group were mostly lower than those seen in the corresponding NS groups, depending on the duration of reperfusion: TBIL and DBIL levels were significantly reduced at 1 and 24 h of reperfusion compared with the NS groups (p < 0.05), and slightly reduced at 6 h of reperfusion. The serum UDBIL level in the Gln group
was similar to that in the NS group at 1 h of perfusion, and reduced at both 6 and 24 h of reperfusion, compared with the NS groups (p < 0.05; fig. 3c–e). The serum AST, ALT and LDH levels in the Gln group were all significantly lower than those in the NS groups (p < 0.05; fig. 3f–h).
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Gln Reduces Inflammatory Cytokine Levels of ICAM-1, IL-1β and TNF-α in Hepatic Tissue after I/R Injury Inflammatory mediators play an important role in the pathogenesis of I/R injury. ICAM-1-positive cells were Xu/Dai/Peng/Zhao/Jia/Xu
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Gln Regulates GSH, MDA and MPO Levels in Hepatic Tissue Levels of GSH, MDA and MPO in the liver were detected using commercially available kits. The results showed that, at all time points, GSH levels in hepatic tissue from rats treated with Gln were significantly increased compared with those in the NS-treated groups (p < 0.05). Both MDA and MPO levels significantly decreased in the Gln groups compared with the NS groups at different time points of reperfusion (p < 0.05; fig. 6).
stained with antibodies against bcl-2 and bax. The results showed that the bcl-2-positive cells in liver sections from the Gln group drastically increased (fig. 7a, c). Bax-positive cells decreased after I/R injury at all 3 time points (fig. 7b, d). In the meantime, relative caspase 3 mRNA levels were quantified by RT-PCR. They were significantly lower in the Gln group than in the NS group (p < 0.05; fig. 8).
Discussion
Gln Modulates Apoptotic Protein Expression in Hepatic Tissue after I/R Injury To test the effects of Gln on the expression of apoptotic molecules in hepatic tissue, liver sections were
The present study shows that Gln pretreatment improves the intestinal mucosal architecture and its barrier function, which is in line with previous reports [21–25]. Bacterial translocation and oxidative stress resulting from obstructive jaundice cause intestinal mucosal atrophy and barrier dysfunction [24]. Previous studies showed that administration of Gln protected the intestinal mucosa from damage by reducing intestinal bacterial translocation and intestinal endotoxemia [24, 25]. Importantly, our study reveals that in rats Gln pretreatment significantly improves hepatic structure and function after I/R injury caused by
Gln Protects against Hepatic Injury in Rats
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strongly downregulated in hepatic tissue from the Gln group compared with that from the NS group (fig. 4) after reperfusion for 1, 6 and 24 h (p < 0.05). Cytokine IL1β and TNF-α levels were detected by ELISA. The study revealed that IL-1β and TNF-α levels were significantly reduced in the Gln group compared with those in the NS groups (p < 0.05) at all time points after I/R injury (fig. 5).
NS-reperfusion 1 h
Gln-reperfusion 1 h
NS-reperfusion 6 h
Gln-reperfusion 6 h
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Fig. 7. Hepatic levels of apoptotic proteins
obstructive jaundice. This is evidenced by the improvement in hepatic and intestinal morphology, increases in serum TP and ALB levels, reduction in liver enzymes (markers of hepatocyte damage) and decreases in oxidative and inflammatory stress and apoptosis. The protective effects of Gln against hepatic I/R in rats are consistent with those found in previous studies [17, 18]. Zhang et al. [17] reported that Gln preconditioning effectively protected against hepatic I/R injury. These protective effects were related to the dose of Gln and due to the reduction in intracellular calcium overload and improvements in the activity of Na+/K+ ATPase and SOD. Schuster et al. [18] showed that Gln protected rats against I/R-induced hepatic injury by attenuating inflammatory response. 162
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a
Gln plays a critical role in cell growth, proliferation and metabolism. It is not only an essential component in cell structure but also important precursor material for the synthesis of GSH, a key antioxidant molecule. Moreover, it is an important energy resource and a source of nitrogen for the synthesis of other amino acids and proteins [26]. The intestinal tract is the main organ consuming Gln, especially the small intestine, which plays a key role in Gln metabolism. Enhancement of Gln metabolism promotes gluconeogenesis and liver glycogen synthesis [27]. Therefore, supplementation of Gln promotes liver glycogen synthesis and increases the ability of cells to resist oxidative stress in the liver. Xu/Dai/Peng/Zhao/Jia/Xu
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as detected in rats pretreated with NS or Gln after 1, 6 and 24 h of reperfusion. a Proapoptotic protein bcl-2. ×400.
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Gln Protects against Hepatic Injury in Rats
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caspase 3 in each group. * p < 0.05 compared with NS groups.
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ates epithelial cell repair. Studies showed (1) that Gln affects the composition of the intestinal bacterial flora by restraining the growth of Gram-negative bacteria [28] and (2) that Gln promotes the secretion of IgA in intestinal mucosal lymphoid tissue and reduces the attachment and colonization of bacteria in the intestinal mucosa [29]. The reduced bacterial translocation and endotoxins in the liver system inactivate hepatic Kupffer cells, inhibit the release of TNF-α, IL-1 and other cytokines, and reduce the production of oxygen free radicals, therefore protecting the liver from these sources of damage [30]. Apoptosis plays an important role in the pathophysiology of obstructive jaundice. Apoptotic cell death can trigger inflammatory cell transmigration, worsening apoptotic cell injury. Caspase 3, a cell death protease, is a key enzyme inducing apoptosis in mammalian cells. Activation of caspase 3 activity is mediated by two signaling pathways: the death receptor-dependent pathway (i.e. extracellular pathway) involving Fas/TNF-α and the mitochondrial pathway (i.e. intracellular pathway) involving the release of cytochrome c [31]. The activation of caspase 3 has effects on a variety of cellular activities and leads to cell apoptosis and cell death. Apoptotic signaling is delicately regulated: it depends on the balance between proand antiapoptotic factors [32]. Bcl-2 is one of the most important antiapoptotic proteins; it is located on the membrane, endoplasmic reticulum and mitochondrial membrane, and prevents apoptosis caused by various stimuli. Bax is a proapoptotic protein which was discovered in recent years; it exhibits 21% homology with the bcl-2 gene [33]. The present study revealed that Gln regulates apoptotic protein expression in liver tissue. Gln inhibits caspase 3 mRNA levels in hepatic tissue, downregulates hepatic bax expression and upregulates bcl-2 exXu/Dai/Peng/Zhao/Jia/Xu
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Increased oxidative stress in tissues is very common in the pathophysiology of obstructive jaundice. Hyperbilirubinemia resulting from obstructive jaundice reduces the content of bile salt in the intestinal lumen, resulting in dysfunction of the intestinal barrier and induction of enterotoxemia. The deterioration in intestinal function and development of enterotoxemia induce the production of free oxygen radicals and impair the antioxidant systems. Increased oxidative stress causes tissue injury and organ malfunction [27]. GSH, MPO and MDA are key markers for evaluating oxidative stress in cells. Our study showed that Gln significantly increased the GSH level and reduced MPO and MDA levels in liver tissue. This suggests that Gln improves liver function and protects the liver from I/R injury by elevating the level of GSH in the liver tissue, eliminating ROS. Another mechanism by which Gln exerts a beneficial effect is that it protects the intestinal mucosa, reduces bacterial translocation and eliminates endotoxemia, therefore attenuating the inflammation of, and injury to, the liver. Our study showed that Gln significantly reduces ICAM-1, TNF-α and IL-1β production in liver tissue. ICAM-1 (CD54) is an endothelium- and leukocyte-associated transmembrane protein. It stabilizes cell-cell interactions, facilitates leukocyte endothelial transmigration and mediates inflammatory response due to a variety of stress stimuli. ICAM-1 is induced by cytokines TNF-α and IL-1β. Bile duct ligation reduces intestinal barrier function, increases permeability, and induces bacterial translocation and organ damage. Supplementation with Gln provides an energy substrate to the intestinal metabolism, increases the antioxidant capacity of the intestine, promotes intestinal mucosal cell proliferation and acceler-
pression. This implies that the protective effect of Gln against hepatic injury induced by obstructive jaundice might be mediated by the inhibition of apoptosis. The antioxidant effect of Gln is mediated by neutralizing the production of ROS during ischemia, hypoxia and reperfusion injury; thus it significantly reduces the oxidative injury to hepatocytes and endothelial cells and stabilizes cell membrane structure and function. This stabilization helps to maintain a balance between anti- and proapoptotic protein activities. The ability of Gln to increase the production of ATP also helps to stabilize the cell membrane and reduce the calcium overload. A study showed that a shortage of GSH decreases the expression of bcl-2, induces apoptosis and increases tissue injury [34].
In conclusion, the current study demonstrates that in rats with obstructive jaundice, Gln effectively protects hepatocytes from I/R-induced injury. The protective effects are evidenced by improved hepatic and intestinal histology and organ function. Inhibition of ROS and inflammatory cytokines, as well an increased imbalance between pro- and postapoptotic protein activities, all contribute to the protective effect of Gln in rats.
Acknowledgment The study was supported by the Liaoning Province Natural Science Fund (2013021068).
References
Gln Protects against Hepatic Injury in Rats
13
14
15
16
17
18
19
20 21 22
23
injury. Asian Cardiovasc Thorac Ann 2007; 15:463–467. San-Miguel B, et al: Glutamine prevents fibrosis development in rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid. J Nutr 2010;140:1065–1071. Cetinbas F, Yelken B, Gulbas Z: Role of glutamine administration on cellular immunity after total parenteral nutrition enriched with glutamine in patients with systemic inflammatory response syndrome. J Crit Care 2010; 25:661e1–e6. Mondello S, et al: Glutamine treatment attenuates the development of ischaemia/reperfusion injury of the gut. Eur J Pharmacol 2010; 643:304–315. Hu YM, et al: Glutamine administration ameliorates sepsis-induced kidney injury by downregulating the high-mobility group box protein-1-mediated pathway in mice. Am J Physiol Renal Physiol 2012;302:F150–F158. Zhang WX, et al: Protective effects of glutamine preconditioning on ischemia-reperfusion injury in rats. Hepatobiliary Pancreat Dis Int 2011;10:78–82. Schuster H, et al: Protective effects of glutamine dipeptide and α-tocopherol against ischemia-reperfusion injury in the isolated rat liver. Clin Nutr 2009;28:331–337. Wu GH, et al: Glutamine supplemented parenteral nutrition prevents intestinal ischemia-reperfusion injury in rats. World J Gastroenterol 2004;10:2592–2594. Stangl R, et al: Reduction of liver ischemiareperfusion injury via glutamine pretreatment. J Surg Res 2011;166:95–103. Owari M, et al: Glutamine prevents intestinal mucosal injury induced by cyclophosphamide in rats. Pediatr Surg Int 2012;28:299–303. Li Y, et al: Protective effect of glutamine-enriched early enteral nutrition on intestinal mucosal barrier injury after liver transplantation in rats. Am J Surg 2010;199:35–42. Marks SL, et al: Effects of glutamine supplementation of an amino acid-based purified
24
25
26 27 28
29
30
31
32
33
34
diet on intestinal mucosal integrity in cats with methotrexate-induced enteritis. Am J Vet Res 1999;60:755–763. Margaritis VG, et al: Effect of oral glutamine administration on bacterial translocation, endotoxemia, liver and ileal morphology, and apoptosis in rats with obstructive jaundice. World J Surg 2005;29:1329–1334. Assimakopoulos SF, et al: Beneficial effects of glutamine on intestinal barrier function in obstructive jaundice. World J Surg 2005; 29: 935–936. Hall JC, Heel K, McCauley R: Glutamine. Br J Surg 1996;83:305–312. Ziegler TR, et al: Glutamine and the gastrointestinal tract. Curr Opin Clin Nutr Metab Care 2000;3:355–362. Denno R, et al: Glutamine-enriched total parenteral nutrition enhances plasma glutathione in the resting state. J Surg Res 1996; 61: 35–38. Zhang W, et al: Glutamine reduces bacterial translocation after small bowel transplantation in cyclosporine-treated rats. J Surg Res 1995;58:159–164. Noguchi Y, et al: Increased glutamine consumption in small intestine epithelial cells during sepsis in rats. Am J Surg 1997; 173: 199–205. Fumarola C, Guidotti GG: Stress-induced apoptosis: toward a symmetry with receptor-mediated cell death. Apoptosis 2004; 9: 77–82. Li SM, et al: Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia. World J Gastroenterol 2003; 9: 2579– 2582. Sheen-Chen SM, et al: Effect of glutamine and bile acid on hepatocyte apoptosis after bile duct ligation in the rat. World J Surg 2004;28: 457–460. Celli A, et al: Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes. Am J Physiol 1998;275:G749–G757.
Pharmacology 2014;93:155–165 DOI: 10.1159/000360181
165
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1 Li C, Jackson RM: Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002; 282:C227–C241. 2 Jaeschke H: Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003;284:G15–G26. 3 Galaris D, Barbouti A, Korantzopoulos P: Oxidative stress in hepatic ischemia-reperfusion injury: the role of antioxidants and iron chelating compounds. Curr Pharm Des 2006;12: 2875–2890. 4 Elias-Miró M, et al: Current knowledge on oxidative stress in hepatic ischemia/reperfusion. Free Radic Res 2013;47:555–568. 5 Mates JM, et al: Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death. Int J Biochem Cell Biol 2002;34:439–458. 6 Liu D, Chen Z: The regulatory effects of glutamine on illness and health. Protein Pept Lett 2011;18:658–662. 7 Peng HC, et al: Effects of glutamine administration on inflammatory responses in chronic ethanol-fed rats. J Nutr Biochem 2011; 22: 282–288. 8 Stachowicz-Stencel T, Synakiewicz A: Glutamine as a supplemental treatment in pediatric and adult oncology patients. Expert Opin Investig Drugs 2012;21:1861–1871. 9 Kim HA, et al: Glutamine inhibits plateletactivating factor-mediated pulmonary tumour metastasis. Eur J Cancer 2012;48:1730– 1738. 10 Zhang SC, et al: Tissue-protective effect of glutamine on hepatic ischemia-reperfusion injury via induction of heme oxygenase-1. Pharmacology 2013;91:59–68. 11 Araújo Júnior RJ, et al: Preconditioning with L-alanyl-glutamine reduces hepatic ischemiareperfusion injury in rats. Acta Cir Bras 2011; 26(suppl 1):8–13. 12 Bolotin G, et al: Glutamine improves myocardial function following ischemia-reperfusion
