- Email: [email protected]
L-Arginine Modulates Intestinal Inflammation in Rats Submitted to Mesenteric Ischemia-Reperfusion Injury
L-Arginine Modulates Intestinal Inflammation in Rats Submitted to Mesenteric Ischemia-Reperfusion Injury M.O. Tahaa, J.V. de Oliveiraa, M. Dias Borgesa, F. de Lucca Meloa, F.G. Gualtieria, A.L. e Silva Aidara, R.L. Pachecoa, T. de Melo Alexandre e Silvab, R.K. Klajnerc, L.R. Iuamotod, L. Munhoz Torrese, B.J. Morais Mendes de Paulae, K. de Camposf, I. Souza de Oliveira Jra, and D.J. Fagundesa,* a Surgical Techniques and Experimental Surgery Division, Surgery Department, São Paulo Federal UniversityeUNIFESP, São Paulo-SP, Brazil; bMedical School of Anhembi Morumbi University, São Paulo-SP, Brazil; cUniversitary Center São Camilo, São Paulo-SP, Brazil; d University of São Paulo Medical SchooleUSP, São Paulo-SP, Brazil; eJundiaí Medical School, Jundiaí-SP, Brazil; and fMedical School of University of Mogi, Mogi das Cruzes-SP, Brazil
ABSTRACT Background. The goal of this study was to investigate whether exogenous offer of L-arginine (LARG) modulates the gene expression of intestinal dysfunction caused by ischemia and reperfusion. Methods. Eighteen Wistar-EPM1 male rats (250e300 g) were anesthetized and subjected to laparotomy. The superior mesenteric vessels were exposed, and the rats were randomized into 3 groups (n ¼ 6): the control group (CG), with no superior mesenteric artery interruption; the ischemia/reperfusion group (IRG), with 60 minutes of ischemia and 120 minutes of reperfusion and saline injections; and the L-arginine group (IRG þ LARG), with L-arginine injected in the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion. The total RNA was extracted and purified from samples of the small intestine. The concentration of each total RNA sample was determined by using spectrophotometry. The first-strand complementary DNA (cDNA) was synthesized in equal amounts of cDNA and the Master Mix SYBR Green qPCR Mastermix (SABiosciences, a Qiagen Company, Frederick, Md). Amounts of cDNA and Master Mix SYBR Green qPCR Mastermix were distributed to each well of the polymerase chain reaction microarray plate containing the predispensed gene-specific primer sets for Bax and Bcl2. Each sample was evaluated in triplicate, and the Student t test was applied to validate the homogeneity of each gene expression reaction (P < .05). Results. The gene expression of Bax in IRG (þ1.48) was significantly higher than in IRGLARG (þ9.69); the expression of Bcl2L1 in IRG (þ1.01) was significantly higher than IRG-LARG (þ22.89). Conclusions. The apoptotic cell pathway of 2 protagonists showed that LARG improves the gene expression of anti-apoptotic Bcl2l1 (Bcl2-like 1) more than the pro-apoptotic Bax (Bcl2-associated X protein).
I
NTESTINAL ISCHEMIA/REPERFUSION IS an important phenomenon in vascular surgery, small intestine and liver transplantation, hypovolemic and septic shock, cardiopulmonary bypass, strangulated hernias, and neonatal necrotizing enterocolitis. The ischemia followed by reperfusion triggers many biological responses. The local 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2015.12.063
512
This article was funded by a grant from CAPES of the Brazilian Ministry of Education. *Address correspondence to Djalma José Fagundes, PhD, Surgery Department, UNIFESP, São Paulo, SP, Brazil, Rua Sena Madureira, 1500-Vila Clementino, São Paulo-SP, 04021-001. E-mail: [email protected] ª 2016 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 48, 512e515 (2016)
L-ARGININE AND INTESTINAL INFLAMMATION
METHODS The study was designed as a randomized controlled trial with a blinded assessment of the outcome. It was approved by the Ethics Committee in Research of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil, according to the recommendations of international legislation on animal protection. Eighteen male Wistar-EPM1 rats weighing 250 to 300 g from the Center for the Development of Experimental Models for Medicine and Biology were housed under controlled temperature and light conditions with a 12-hour light/dark cycle. The rats had free access to water and standard pellet chow until 6 hours before the surgical procedures. After anesthesia (intramuscular injection of 80 mg/kg of ketamine and 10 mg/kg of xylazine), a median laparotomy was performed, exposing the superior mesenteric vessels. The animals were randomly divided into 3 groups:
1. Sham group (SG), n ¼ 6: no clamping of the superior mesenteric artery; 2. Ischemia and reperfusion group (IRG), n ¼ 6: clamping of superior mesenteric vessels for 60 minutes followed by 120 minutes of reperfusion; 0.9% saline solution was injected into the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion; 3. Ischemia and reperfusion and L-arginine group (IRG þ LARG), n ¼ 6: clamping of superior mesenteric vessels for 60 minutes followed by 120 minutes of reperfusion; LARG 100 mg/kg was injected in the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion. Intestinal segments (3 cm) were removed at a distance of 20 cm from the duodenum-jejunum flexure, opened longitudinally, gently washed in saline solution, wrapped in aluminum foil, and immediately frozen in liquid nitrogen. The tissue samples were prepared for assay by using Endothelial Cell Biology Rat RT2 Profiler PCR Array (SABiosciences, a Qiagen Company, Frederick, Md). Briefly, total RNA was extracted from tissues by using TRIzol reagent (Life Technologies, Carlsbad, Calif) and purified by using an RNeasy MiniKit (SABiosciences). Concentration of each total RNA sample was determined by using spectrophotometry, and the quality was assessed with electrophoresis on 2% agarose gels. The first strand of complementary DNA was synthesized by using 1 mg of total RNA and the RT2 First Strand Kit (SABiosciences). Equal amounts of complementary DNA and the Master Mix SYBR Green qPCR Mastermix (SABiosciences) were distributed to each well of the PCR microarray plate containing the predispensed gene-specific primer sets. PCR was performed according to the manufacturer’s instructions in 96-well plates to detect the expression of 84 genes related to oxidative stress, 5 housekeeping genes (ACTB, Gapdh, Hsp90ab1, Hprt1, and Gusb) used for normalizing the PCR microarray data, and 1 negative control for genomic DNA contamination. The negative control primer set specifically detects nontranscribed, repetitive genomic DNA with a high level of sensitivity. Three wells of reverse transcription control samples were used to verify the efficiency of the reverse transcription reaction with a quantitative PCR assay that specifically detects template synthesized from the RNA control of the first-strand synthesis kit. The replicate positive PCR control samples were used to determine the efficiency of the PCR itself. These controls use a predispensed artificial DNA sequence and a specific primer set to detect it. The 2 sets of replicate control wells (reverse transcription control samples and positive PCR control samples) also test for inter-well and intraplate consistency. The instrument’s software (MxPro Equipment Real Time Systems, Stratagene [San Diego, Calif]/GE Healthcare, Little Chalfont, United Kingdom) calculates the cycle threshold (Ct) values for the 2 genes in the array. Finally, it performs pair-wise comparisons by calculating fold changes in gene expression from the raw Ct data by using the DDCt method. The method used is contained in the spreadsheet for PCR Array Data Analysis version 3.3 (SABiosciences) [10].
Statistical Analysis Gene expression data for each sample were evaluated in triplicate. The Student t test was applied to validate the homogeneity of the reaction of expression of each gene (P < .05). The software calculated the variation in the Ct in the study group (IRG) compared with the Ct of the control group (SG), and this outcome was expressed as a logarithmic base (2) value according to the formula [2 (DDCt)]. The gene expression results are presented as ˇ
inflammatory response, as well as the remote injury, causes multiple organ failure, followed by death in critically ill patients [1,2]. A variety of strategies have been implemented to eliminate or reduce reperfusion injury in animal models of intestinal ischemia/reperfusion injury (IRI). Research is focused on the application of drugs such as L-arginine, which, as a precursor of nitric oxide (NO), may have a role in the treatment of some conditions in which oxidative stress is present. Arginine plays an important role in cell division and healing of wounds, and it acts by reducing wound healing time and accelerating damaged tissue repair time [3,4]. It is well known that reactive oxygen species (ROS) are produced by all aerobic cells and are believed to play a crucial role in the ischemia followed by reperfusion phenomenon. The identification of genes and pathways affected by specific oxidizing led to the assumption that ROS serve as messenger transduction pathways and gene regulatory signals. Furthermore, antioxidants, scavengers, and preconditioning procedures can activate numerous genes and biochemical pathways [5,6]. Use of real-time polymerase chain reaction (PCR) allows analysis of the expression of a focused panel of genes related to the oxidative stress and apoptosis. Among the genes involved in the oxidative stress are Bax and Bcl2. Bcl2 has two functions in relation anti-apoptotic: kidnapping and inhibiting the activation of pro-apoptotic proteases, and directly preventing the release of cytochrome c. However, the Bax gene has three homology domains BCL2s, which form homodimers and heterodimers with Bcl2. It promotes apoptosis, not just due to the release of cytochrome c but also because of the increasing permeability of the mitochondrial membranes, which leads to activation of caspase [7,8]. When Bax predominates, programmed cell death is accelerated, and the repressor activity of Bcl2 apoptosis is inhibited. This scenario suggests a model in which the ratio of Bax and Bcl2 determines survival or apoptotic death after the stimulus [9]. The goal of the present study was to investigate whether exogenous offer of L-arginine modulates the gene expression of intestinal dysfunction caused by ischemia and reperfusion.
513
514
TAHA, DE OLIVEIRA, DIAS BORGES ET AL
positive/up-regulation expression (IRG > GC) or negative/downregulation expression (IRG < CG). The numbers represent how many folds each gene was expressed above (þ) or below (). The software established the results 3-fold above (overexpression) or 3-fold below (hypoexpression). The threshold allowed by the algorithm [2 (DDCt)] are biologically relevant [11,12]. The same procedure was performed between the IRG þ LARG and the SG groups. ˇ
RESULTS
Table 1 displays the expression levels of genes Bax (Bcl2associated X protein) and Bcl2l1 (Bcl2-like 1) related to rat enteric endothelial cell biology from the SG, IRG, and IRG þ LARG groups. DISCUSSION
This study evaluated the modulation effect of L-arginine in a rat model of intestinal IRI. We found that L-arginine promotes overexpression of Bcl2L1, which has an important role in the protection or acceleration of intestinal apoptosis after ischemia and/or reperfusion. When the Bcl2L1 levels decrease, it triggers a caspase cascade, ultimately leading to cell apoptosis. In contrast, increased Bcl2L1 levels can inhibit apoptosis [13]. Conversely, our results illustrate the overexpression of Bax that is expressed during stress conditions and acts to accelerate programmed cell death, which encodes a protein that is responsible for triggering apoptosis. This protein forms a heterodimer with BCL2 and interacts with mitochondrial membrane, which leads to the release of cytochrome C [2]. It also activates caspase-3, which is related to apoptosis. During ischemia and after reperfusion, formation of ROS occurs, which is responsible for the cascade of deleterious cellular responses, and it induces cell death [14e16]. Administration of exogenous LARG has been show to protect tissues against toxic or ischemic injury and ameliorate the oxidative stress secondary to IRI [17]. LARG acts by increasing NO bioavailability, promoting vasodilation of the microvasculature, inhibition of aggregation, and platelet adhesion. Operating in the interaction between endothelial surface and leukocytes, NO can mitigate inflammatory activity [18e20]. Moreover, NO configures as a molecule with significant capacity to down-regulate the inflammatory response Table 1. Expression of Bax and Bcl21 Genes Related to Enteric Endothelial Cell Biology From Rats in the SG, IRG, and IRG D LARG Groups No.
1 2
Bank
Symbol
IRG
IRG þ LARG
P
NM_017059 NM_031535
Bax Bcl2L1
þ1.48 þ1.01
D9.69 D22.89
.000002* .000002*
Significant values (in boldface) of fold up-regulation (þ) or down-regulation [2 (DDCt)]. Abbreviations: IRG, ischemia and reperfusion group; IRG þ LARG, ischemic and reperfusion plus L-arginine group; SG, sham group. *P .05.
caused by ischemia to eliminate the ROS. It is a key regulator of gene expression, capable of playing either positive or negative roles, which means it can be cytotoxic as well as beneficial during IRI. The ambiguous role of NO during intestinal IRI is still unknown. It may act both as a protector of the lesion due to its vasodilator effect, antiplatelet and scavenger of ROS, and as a deleterious agent by interaction with superoxide radicals, forming the radical peroxynitrite [19,20]. The NO produced by endothelial cells would act to modulate IRI through its vasodilator activity and counterbalancing of the endothelin activity [18]. Cellular responses to oxidative stress vary from growth arrest to cell death depending on the stress stimuli, duration of exposure, cell type, and surrounding cell environment. ROS-induced injury endothelial dysfunction is considered an important cellular mechanism [21]. Our hypothesis was that during intestinal IRI, ROS causes cellular changes (particularly cell membrane damage), which results in modifications on gene expression of 2 proteins from the Bcl2 cluster as Bax (pro-apoptotic) and Bcl2L1 (antiapoptotic). In addition, it is expected that L-arginine can promote modulating effects on the gene expression that encode these 2 proteins [22]. Although mixed apoptotic and necrotic cell death occurs, abundant findings implicate the Bcl-2 family as mediators of the hyperoxic cell death response. The Bcl-2 family proteins act directly on the mitochondrial outer membrane permeabilization, often a critical early step in apoptosis [22]. Regulation of cytochrome C released by both pro-apoptotic and antiapoptotic members of the Bcl-2 family of proteins has been well documented [23]. The historical method of determining the function of a protein involves extensive genetic and biochemical analyses, unless the amino acid sequence of the protein resembles another whose function is known [24]. With complete genome sequences and total messenger RNA expression patterns, new strategies have become available. The fully sequenced genomes of numerous organisms offer large amounts of information about cellular biology. It is a central challenge of bioinformatics to use this information in discovering the function of proteins. Functional assignments of genes originate primarily from biochemical experimentation, which can be extended by matching recently sequenced proteins to those that have already been characterized. To establish the real meaning of gene expression is challenging. This research, therefore, offers an option that can be followed to determine the association of gene expression as a primary and reliable response in the biochemical pathways of the inflammatory response to intestinal IRI. CONCLUSIONS
Our results demonstrate that exogenous LARG was associated with antiapoptotic effects on the rat intestine IRI by up-regulated expression of gene Bcl2L1. Furthermore, LARG was associated with significantly lower expression of
ˇ
L-ARGININE AND INTESTINAL INFLAMMATION
the pro-apoptotic gene Bax. These results suggest that LARG would be useful in the treatment of intestinal IRI, but other experimental studies must be conducted before clinical application is possible.
REFERENCES [1] Abu-Amara M, Yang SY, Tapuria N, Fuller B, Davidson B, Seifalian A. Liver ischemia/reperfusion injury: processes in inflammatory networks. A review. Liver Transpl 2010;16:1016e32. [2] Maoa Y, Zheng X, Cai J, You X, Deng X, Zhang JH, et al. Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats. Biochem Biophys Res Com 2009;381: 602e5. [3] Acquaviva R, Lanteri R, Destri GL, Caltabiano R, Vanella L, Lanzafame S, et al. Beneficial effects of rutin and L-arginine coadministration in a rat model of liver ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 2009;296:G664e70. [4] Guan Y, Worrell RT, Pritts TA, Montrose MH. Intestinal ischemia-reperfusion injury: reversible and irreversible damage imaged in vivo. Am J Physiol Gastrointest Liver Physiol 2009;297: G187e96. [5] Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012;48:158e67. [6] Silachev DN, Isaev NK, Pevzner IB, Zorova LD, Stelmashook EV, Novikova SV, et al. The mitochondria-targeted antioxidants and remote kidney preconditioning ameliorate brain damage through kidney-to-brain cross talk. PLoS One 2012;7: e51553. [7] Hotchkiss RS, Strasser A, McDunn JE, Swansin PE. Mechanisms of disease cell death. N Engl J Med 2009;361:1570e83. [8] Gregocá K, Cikos S, Czikková S, Rabajdová MB, Urban P, Veselá J. Effects of intestinal ischemia reperfusion injury on the level of specific genes in rats. J Ecol Health 2013;17:141e6. [9] PCR Array Data Analysis v3.3, https://www.qiagen.com/br/ products/genes%20and%20pathways/data-analysis-center-overviewpage/ [10] Deepak SA, Kottapalli KR, Rakwal R, Oros G, Rangappa KS, Iwahashi H, et al. Real-time PCR: revolutionizing detection and expression analysis of genes. Curr Genomics 2007;8:234e51. [11] Ikejiri AT, Somaio Neto F, Chaves JC, Bertoletto PR, Teruya R, Bertoletto ER, et al. Gene expression profile of oxidative
515 stress in the lung of inbred mice after intestinal ischemia/reperfusion injury. Acta Cir Bras 2014;293:186e92. [12] Somaio Neto F, Ikejiri AT, Chaves JC, Bertoletto PR, Teruya R, Bertoletto ER, et al. Gene expression related to oxidative stress in the heart of mice after intestinal ischemia. Arq Bras Cardiol 2014;102:142e9. [13] Bcl2l1 Bcl2-like 1 [Rattus norvegicus (Norway rat), http:// www.ncbi.nlm.nih.gov/gene/24888 [accessed 9/29/15]. [14] UniProtKB-Q63690 (BAX_RAT), http://www.uniprot.org/ uniprot/Q63690/ [accessed 9/29/15]. [15] UniProtKB-Q07812 (BAX_HUMAN), http://www.uniprot. org/uniprot/Q07812/ [accessed 9/29/15]. [16] NCBI. BAX BCL2-associated X protein [Homo sapiens (human)], http://www.ncbi.nlm.nih.gov/gene?cmd¼Retrieve&dopt¼ full_report&list_uids¼581/ [accessed 9/29/15]. [17] Taha O, Caricati-Neto A, Ferreira RM, Simões MJ, Monteiro HP, Fagundes DJ. L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury. Acta Cir Bras 2012;27:616e23. [18] Lucas ML, Rhoden CR, Rhoden EL, Zettler CG, Mattos AA. Effects of L-arginine and L-NAME on ischemiareperfusion in rat liver. Acta Cir Bras 2015;30:345e52. [19] Senbel AM, Omar AG, Abdel-Moneim LM, Mohamed HF, Daabees TT. Evaluation of l-arginine on kidney function and vascular reactivity following ischemic injury in rats: protective effects and potential interactions. Pharmacol Rep 2014;66:976e83. [20] Cao B, Li N, Wang Y, Li J. Protective effect of L-arginine preconditioning on ischemia and reperfusion injury associated with rat small bowel transplantation. World J Gastroenterol 2005;21: 2994e7. [21] Zhou Y, Wang Q, Evers BM, Chung DH. Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis. Pediatr Res 2005;58:1192e7. [22] Taha MO, Ferreira RM, Taha NSA, Monteiro HP, CaricatiNeto A, Fagundes DJ. Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats. Acta Cir Bras 2014;29:445e9. [23] Vitiello PF, Staversky RJ, Keng PC, O’Reilly MA. Puma inactivation protects against oxidative stress through p21/Bcl-xl inhibition of Bax death. Free Radic Biol Med 2008;44:367e74. [24] Yasuda O, Fukuo K, Sun X, et al. Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis. J Biol Chem 2006;281:23899e907.
ABSTRACT Background. The goal of this study was to investigate whether exogenous offer of L-arginine (LARG) modulates the gene expression of intestinal dysfunction caused by ischemia and reperfusion. Methods. Eighteen Wistar-EPM1 male rats (250e300 g) were anesthetized and subjected to laparotomy. The superior mesenteric vessels were exposed, and the rats were randomized into 3 groups (n ¼ 6): the control group (CG), with no superior mesenteric artery interruption; the ischemia/reperfusion group (IRG), with 60 minutes of ischemia and 120 minutes of reperfusion and saline injections; and the L-arginine group (IRG þ LARG), with L-arginine injected in the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion. The total RNA was extracted and purified from samples of the small intestine. The concentration of each total RNA sample was determined by using spectrophotometry. The first-strand complementary DNA (cDNA) was synthesized in equal amounts of cDNA and the Master Mix SYBR Green qPCR Mastermix (SABiosciences, a Qiagen Company, Frederick, Md). Amounts of cDNA and Master Mix SYBR Green qPCR Mastermix were distributed to each well of the polymerase chain reaction microarray plate containing the predispensed gene-specific primer sets for Bax and Bcl2. Each sample was evaluated in triplicate, and the Student t test was applied to validate the homogeneity of each gene expression reaction (P < .05). Results. The gene expression of Bax in IRG (þ1.48) was significantly higher than in IRGLARG (þ9.69); the expression of Bcl2L1 in IRG (þ1.01) was significantly higher than IRG-LARG (þ22.89). Conclusions. The apoptotic cell pathway of 2 protagonists showed that LARG improves the gene expression of anti-apoptotic Bcl2l1 (Bcl2-like 1) more than the pro-apoptotic Bax (Bcl2-associated X protein).
I
NTESTINAL ISCHEMIA/REPERFUSION IS an important phenomenon in vascular surgery, small intestine and liver transplantation, hypovolemic and septic shock, cardiopulmonary bypass, strangulated hernias, and neonatal necrotizing enterocolitis. The ischemia followed by reperfusion triggers many biological responses. The local 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2015.12.063
512
This article was funded by a grant from CAPES of the Brazilian Ministry of Education. *Address correspondence to Djalma José Fagundes, PhD, Surgery Department, UNIFESP, São Paulo, SP, Brazil, Rua Sena Madureira, 1500-Vila Clementino, São Paulo-SP, 04021-001. E-mail: [email protected] ª 2016 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 48, 512e515 (2016)
L-ARGININE AND INTESTINAL INFLAMMATION
METHODS The study was designed as a randomized controlled trial with a blinded assessment of the outcome. It was approved by the Ethics Committee in Research of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil, according to the recommendations of international legislation on animal protection. Eighteen male Wistar-EPM1 rats weighing 250 to 300 g from the Center for the Development of Experimental Models for Medicine and Biology were housed under controlled temperature and light conditions with a 12-hour light/dark cycle. The rats had free access to water and standard pellet chow until 6 hours before the surgical procedures. After anesthesia (intramuscular injection of 80 mg/kg of ketamine and 10 mg/kg of xylazine), a median laparotomy was performed, exposing the superior mesenteric vessels. The animals were randomly divided into 3 groups:
1. Sham group (SG), n ¼ 6: no clamping of the superior mesenteric artery; 2. Ischemia and reperfusion group (IRG), n ¼ 6: clamping of superior mesenteric vessels for 60 minutes followed by 120 minutes of reperfusion; 0.9% saline solution was injected into the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion; 3. Ischemia and reperfusion and L-arginine group (IRG þ LARG), n ¼ 6: clamping of superior mesenteric vessels for 60 minutes followed by 120 minutes of reperfusion; LARG 100 mg/kg was injected in the femoral vein 5 minutes before ischemia, 5 minutes after reperfusion, and after 55 minutes of reperfusion. Intestinal segments (3 cm) were removed at a distance of 20 cm from the duodenum-jejunum flexure, opened longitudinally, gently washed in saline solution, wrapped in aluminum foil, and immediately frozen in liquid nitrogen. The tissue samples were prepared for assay by using Endothelial Cell Biology Rat RT2 Profiler PCR Array (SABiosciences, a Qiagen Company, Frederick, Md). Briefly, total RNA was extracted from tissues by using TRIzol reagent (Life Technologies, Carlsbad, Calif) and purified by using an RNeasy MiniKit (SABiosciences). Concentration of each total RNA sample was determined by using spectrophotometry, and the quality was assessed with electrophoresis on 2% agarose gels. The first strand of complementary DNA was synthesized by using 1 mg of total RNA and the RT2 First Strand Kit (SABiosciences). Equal amounts of complementary DNA and the Master Mix SYBR Green qPCR Mastermix (SABiosciences) were distributed to each well of the PCR microarray plate containing the predispensed gene-specific primer sets. PCR was performed according to the manufacturer’s instructions in 96-well plates to detect the expression of 84 genes related to oxidative stress, 5 housekeeping genes (ACTB, Gapdh, Hsp90ab1, Hprt1, and Gusb) used for normalizing the PCR microarray data, and 1 negative control for genomic DNA contamination. The negative control primer set specifically detects nontranscribed, repetitive genomic DNA with a high level of sensitivity. Three wells of reverse transcription control samples were used to verify the efficiency of the reverse transcription reaction with a quantitative PCR assay that specifically detects template synthesized from the RNA control of the first-strand synthesis kit. The replicate positive PCR control samples were used to determine the efficiency of the PCR itself. These controls use a predispensed artificial DNA sequence and a specific primer set to detect it. The 2 sets of replicate control wells (reverse transcription control samples and positive PCR control samples) also test for inter-well and intraplate consistency. The instrument’s software (MxPro Equipment Real Time Systems, Stratagene [San Diego, Calif]/GE Healthcare, Little Chalfont, United Kingdom) calculates the cycle threshold (Ct) values for the 2 genes in the array. Finally, it performs pair-wise comparisons by calculating fold changes in gene expression from the raw Ct data by using the DDCt method. The method used is contained in the spreadsheet for PCR Array Data Analysis version 3.3 (SABiosciences) [10].
Statistical Analysis Gene expression data for each sample were evaluated in triplicate. The Student t test was applied to validate the homogeneity of the reaction of expression of each gene (P < .05). The software calculated the variation in the Ct in the study group (IRG) compared with the Ct of the control group (SG), and this outcome was expressed as a logarithmic base (2) value according to the formula [2 (DDCt)]. The gene expression results are presented as ˇ
inflammatory response, as well as the remote injury, causes multiple organ failure, followed by death in critically ill patients [1,2]. A variety of strategies have been implemented to eliminate or reduce reperfusion injury in animal models of intestinal ischemia/reperfusion injury (IRI). Research is focused on the application of drugs such as L-arginine, which, as a precursor of nitric oxide (NO), may have a role in the treatment of some conditions in which oxidative stress is present. Arginine plays an important role in cell division and healing of wounds, and it acts by reducing wound healing time and accelerating damaged tissue repair time [3,4]. It is well known that reactive oxygen species (ROS) are produced by all aerobic cells and are believed to play a crucial role in the ischemia followed by reperfusion phenomenon. The identification of genes and pathways affected by specific oxidizing led to the assumption that ROS serve as messenger transduction pathways and gene regulatory signals. Furthermore, antioxidants, scavengers, and preconditioning procedures can activate numerous genes and biochemical pathways [5,6]. Use of real-time polymerase chain reaction (PCR) allows analysis of the expression of a focused panel of genes related to the oxidative stress and apoptosis. Among the genes involved in the oxidative stress are Bax and Bcl2. Bcl2 has two functions in relation anti-apoptotic: kidnapping and inhibiting the activation of pro-apoptotic proteases, and directly preventing the release of cytochrome c. However, the Bax gene has three homology domains BCL2s, which form homodimers and heterodimers with Bcl2. It promotes apoptosis, not just due to the release of cytochrome c but also because of the increasing permeability of the mitochondrial membranes, which leads to activation of caspase [7,8]. When Bax predominates, programmed cell death is accelerated, and the repressor activity of Bcl2 apoptosis is inhibited. This scenario suggests a model in which the ratio of Bax and Bcl2 determines survival or apoptotic death after the stimulus [9]. The goal of the present study was to investigate whether exogenous offer of L-arginine modulates the gene expression of intestinal dysfunction caused by ischemia and reperfusion.
513
514
TAHA, DE OLIVEIRA, DIAS BORGES ET AL
positive/up-regulation expression (IRG > GC) or negative/downregulation expression (IRG < CG). The numbers represent how many folds each gene was expressed above (þ) or below (). The software established the results 3-fold above (overexpression) or 3-fold below (hypoexpression). The threshold allowed by the algorithm [2 (DDCt)] are biologically relevant [11,12]. The same procedure was performed between the IRG þ LARG and the SG groups. ˇ
RESULTS
Table 1 displays the expression levels of genes Bax (Bcl2associated X protein) and Bcl2l1 (Bcl2-like 1) related to rat enteric endothelial cell biology from the SG, IRG, and IRG þ LARG groups. DISCUSSION
This study evaluated the modulation effect of L-arginine in a rat model of intestinal IRI. We found that L-arginine promotes overexpression of Bcl2L1, which has an important role in the protection or acceleration of intestinal apoptosis after ischemia and/or reperfusion. When the Bcl2L1 levels decrease, it triggers a caspase cascade, ultimately leading to cell apoptosis. In contrast, increased Bcl2L1 levels can inhibit apoptosis [13]. Conversely, our results illustrate the overexpression of Bax that is expressed during stress conditions and acts to accelerate programmed cell death, which encodes a protein that is responsible for triggering apoptosis. This protein forms a heterodimer with BCL2 and interacts with mitochondrial membrane, which leads to the release of cytochrome C [2]. It also activates caspase-3, which is related to apoptosis. During ischemia and after reperfusion, formation of ROS occurs, which is responsible for the cascade of deleterious cellular responses, and it induces cell death [14e16]. Administration of exogenous LARG has been show to protect tissues against toxic or ischemic injury and ameliorate the oxidative stress secondary to IRI [17]. LARG acts by increasing NO bioavailability, promoting vasodilation of the microvasculature, inhibition of aggregation, and platelet adhesion. Operating in the interaction between endothelial surface and leukocytes, NO can mitigate inflammatory activity [18e20]. Moreover, NO configures as a molecule with significant capacity to down-regulate the inflammatory response Table 1. Expression of Bax and Bcl21 Genes Related to Enteric Endothelial Cell Biology From Rats in the SG, IRG, and IRG D LARG Groups No.
1 2
Bank
Symbol
IRG
IRG þ LARG
P
NM_017059 NM_031535
Bax Bcl2L1
þ1.48 þ1.01
D9.69 D22.89
.000002* .000002*
Significant values (in boldface) of fold up-regulation (þ) or down-regulation [2 (DDCt)]. Abbreviations: IRG, ischemia and reperfusion group; IRG þ LARG, ischemic and reperfusion plus L-arginine group; SG, sham group. *P .05.
caused by ischemia to eliminate the ROS. It is a key regulator of gene expression, capable of playing either positive or negative roles, which means it can be cytotoxic as well as beneficial during IRI. The ambiguous role of NO during intestinal IRI is still unknown. It may act both as a protector of the lesion due to its vasodilator effect, antiplatelet and scavenger of ROS, and as a deleterious agent by interaction with superoxide radicals, forming the radical peroxynitrite [19,20]. The NO produced by endothelial cells would act to modulate IRI through its vasodilator activity and counterbalancing of the endothelin activity [18]. Cellular responses to oxidative stress vary from growth arrest to cell death depending on the stress stimuli, duration of exposure, cell type, and surrounding cell environment. ROS-induced injury endothelial dysfunction is considered an important cellular mechanism [21]. Our hypothesis was that during intestinal IRI, ROS causes cellular changes (particularly cell membrane damage), which results in modifications on gene expression of 2 proteins from the Bcl2 cluster as Bax (pro-apoptotic) and Bcl2L1 (antiapoptotic). In addition, it is expected that L-arginine can promote modulating effects on the gene expression that encode these 2 proteins [22]. Although mixed apoptotic and necrotic cell death occurs, abundant findings implicate the Bcl-2 family as mediators of the hyperoxic cell death response. The Bcl-2 family proteins act directly on the mitochondrial outer membrane permeabilization, often a critical early step in apoptosis [22]. Regulation of cytochrome C released by both pro-apoptotic and antiapoptotic members of the Bcl-2 family of proteins has been well documented [23]. The historical method of determining the function of a protein involves extensive genetic and biochemical analyses, unless the amino acid sequence of the protein resembles another whose function is known [24]. With complete genome sequences and total messenger RNA expression patterns, new strategies have become available. The fully sequenced genomes of numerous organisms offer large amounts of information about cellular biology. It is a central challenge of bioinformatics to use this information in discovering the function of proteins. Functional assignments of genes originate primarily from biochemical experimentation, which can be extended by matching recently sequenced proteins to those that have already been characterized. To establish the real meaning of gene expression is challenging. This research, therefore, offers an option that can be followed to determine the association of gene expression as a primary and reliable response in the biochemical pathways of the inflammatory response to intestinal IRI. CONCLUSIONS
Our results demonstrate that exogenous LARG was associated with antiapoptotic effects on the rat intestine IRI by up-regulated expression of gene Bcl2L1. Furthermore, LARG was associated with significantly lower expression of
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L-ARGININE AND INTESTINAL INFLAMMATION
the pro-apoptotic gene Bax. These results suggest that LARG would be useful in the treatment of intestinal IRI, but other experimental studies must be conducted before clinical application is possible.
REFERENCES [1] Abu-Amara M, Yang SY, Tapuria N, Fuller B, Davidson B, Seifalian A. Liver ischemia/reperfusion injury: processes in inflammatory networks. A review. Liver Transpl 2010;16:1016e32. [2] Maoa Y, Zheng X, Cai J, You X, Deng X, Zhang JH, et al. Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats. Biochem Biophys Res Com 2009;381: 602e5. [3] Acquaviva R, Lanteri R, Destri GL, Caltabiano R, Vanella L, Lanzafame S, et al. Beneficial effects of rutin and L-arginine coadministration in a rat model of liver ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 2009;296:G664e70. [4] Guan Y, Worrell RT, Pritts TA, Montrose MH. Intestinal ischemia-reperfusion injury: reversible and irreversible damage imaged in vivo. Am J Physiol Gastrointest Liver Physiol 2009;297: G187e96. [5] Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012;48:158e67. [6] Silachev DN, Isaev NK, Pevzner IB, Zorova LD, Stelmashook EV, Novikova SV, et al. The mitochondria-targeted antioxidants and remote kidney preconditioning ameliorate brain damage through kidney-to-brain cross talk. PLoS One 2012;7: e51553. [7] Hotchkiss RS, Strasser A, McDunn JE, Swansin PE. Mechanisms of disease cell death. N Engl J Med 2009;361:1570e83. [8] Gregocá K, Cikos S, Czikková S, Rabajdová MB, Urban P, Veselá J. Effects of intestinal ischemia reperfusion injury on the level of specific genes in rats. J Ecol Health 2013;17:141e6. [9] PCR Array Data Analysis v3.3, https://www.qiagen.com/br/ products/genes%20and%20pathways/data-analysis-center-overviewpage/ [10] Deepak SA, Kottapalli KR, Rakwal R, Oros G, Rangappa KS, Iwahashi H, et al. Real-time PCR: revolutionizing detection and expression analysis of genes. Curr Genomics 2007;8:234e51. [11] Ikejiri AT, Somaio Neto F, Chaves JC, Bertoletto PR, Teruya R, Bertoletto ER, et al. Gene expression profile of oxidative
515 stress in the lung of inbred mice after intestinal ischemia/reperfusion injury. Acta Cir Bras 2014;293:186e92. [12] Somaio Neto F, Ikejiri AT, Chaves JC, Bertoletto PR, Teruya R, Bertoletto ER, et al. Gene expression related to oxidative stress in the heart of mice after intestinal ischemia. Arq Bras Cardiol 2014;102:142e9. [13] Bcl2l1 Bcl2-like 1 [Rattus norvegicus (Norway rat), http:// www.ncbi.nlm.nih.gov/gene/24888 [accessed 9/29/15]. [14] UniProtKB-Q63690 (BAX_RAT), http://www.uniprot.org/ uniprot/Q63690/ [accessed 9/29/15]. [15] UniProtKB-Q07812 (BAX_HUMAN), http://www.uniprot. org/uniprot/Q07812/ [accessed 9/29/15]. [16] NCBI. BAX BCL2-associated X protein [Homo sapiens (human)], http://www.ncbi.nlm.nih.gov/gene?cmd¼Retrieve&dopt¼ full_report&list_uids¼581/ [accessed 9/29/15]. [17] Taha O, Caricati-Neto A, Ferreira RM, Simões MJ, Monteiro HP, Fagundes DJ. L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury. Acta Cir Bras 2012;27:616e23. [18] Lucas ML, Rhoden CR, Rhoden EL, Zettler CG, Mattos AA. Effects of L-arginine and L-NAME on ischemiareperfusion in rat liver. Acta Cir Bras 2015;30:345e52. [19] Senbel AM, Omar AG, Abdel-Moneim LM, Mohamed HF, Daabees TT. Evaluation of l-arginine on kidney function and vascular reactivity following ischemic injury in rats: protective effects and potential interactions. Pharmacol Rep 2014;66:976e83. [20] Cao B, Li N, Wang Y, Li J. Protective effect of L-arginine preconditioning on ischemia and reperfusion injury associated with rat small bowel transplantation. World J Gastroenterol 2005;21: 2994e7. [21] Zhou Y, Wang Q, Evers BM, Chung DH. Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis. Pediatr Res 2005;58:1192e7. [22] Taha MO, Ferreira RM, Taha NSA, Monteiro HP, CaricatiNeto A, Fagundes DJ. Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats. Acta Cir Bras 2014;29:445e9. [23] Vitiello PF, Staversky RJ, Keng PC, O’Reilly MA. Puma inactivation protects against oxidative stress through p21/Bcl-xl inhibition of Bax death. Free Radic Biol Med 2008;44:367e74. [24] Yasuda O, Fukuo K, Sun X, et al. Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis. J Biol Chem 2006;281:23899e907.