- Email: [email protected]
Niacin Pretreatment Attenuates Lung Ischemia and Reperfusion-Induced Pulmonary Barrier Function Impairment by Reducing Oxidative Stress and Activating SIRT1 in an Isolated-Perfused Rat Lung Model
Niacin Pretreatment Attenuates Lung Ischemia and Reperfusion-Induced Pulmonary Barrier Function Impairment by Reducing Oxidative Stress and Activating SIRT1 in an Isolated-Perfused Rat Lung Model N.C. Wua,c and J.J. Wangb,* a Division of Cardiovascular Surgery, Chi-Mei Foundation Hospital, Tainan, Taiwan; bSchool of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan; and cDepartment of Hospital and Health Care Administration, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
ABSTRACT Purpose. Alveolar-capillary barrier dysfunction, characterized by alveolar protein leak and lung edema, is a common scenario following cardiopulmonary surgery and thoracic organ transplantation. Reactive oxygen species generated through lung ischemia and reperfusion (I/R) injury during surgery plays a crucial role. Niacin, also known as vitamin B3, has been demonstrated to possess antioxidative and anti-inflammatory capacity. In this study, we examine the pulmonary barrier function via capillary filtration coefficient (Kfc) following lung I/R injury with and without niacin treatment. Methods. Studies were conducted on male Sprague-Dawley rats in 3 groups: shamoperated, lung I/R injury, and niacin-pretreated lung I/R injury group. Rats were subjected to isolated perfused lung preparation. Lung ischemia was established by continuous perfusion and stopping ventilation for 60 minutes, followed by 60 minutes of ventilation. We assessed the Kfc, lung water content, and protein concentration in the lung lavage; pulmonary oxidative stress and lung inflammation were assessed by leukocyte counts, tissue level of tumor nercrosis factor alpha (TNF-a), and tissue content of malondialdehyde (MDA), respectively. We also assessed the tissue protein level of sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1). Results. Lungs subjected to I/R injury significantly increased Kfc, pulmonary oxidative stress, lung water content, and lavage leukocyte count and protein concentration (P < .05). Rats treated with niacin of 100 mg/kg/day for 4 days increased lung SIRT1 (P < .05) and attenuated lung I/R injury-induced pulmonary oxidative stress and inflammation and also improved Kfc. Conclusions. Niacin pretreatment protects lungs against I/R injury-induced barrier function impairment through the activation of SIRT1 and reduced pulmonary oxidative stress and lung inflammation.
L
ung ischemia and reperfusion injury, manifested by the impairment of alveolar-capillary barrier function, characterized by alveolar protein leak and lung edema, is the major cause of graft failure subsequent to lung transplantation and cardiopulmonary bypass surgery [1]. Lung ischemia, induced by clamping the pulmonary blood vessels during the operation, causes oxygen deprivation and imbalance between the metabolic demand and supply that
0041-1345/18 https://doi.org/10.1016/j.transproceed.2018.04.047
2834
triggers a series of events, resulting in different intensities of cellular damage and subsequent activation of cytotoxic enzymes that ultimately culminate in cell death [2]. Lung *Address correspondence to Jiun-Jr Wang, PhD, School of Medicine, Fu Jen Catholic University, No. 510, Zhongzheng Road, Xinzhuang Dist., New Taipei City, 24205, Taiwan. Tel: 886-229053498. Fax: 886-2-29053496. E-mail: [email protected] ª 2018 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 50, 2834e2838 (2018)
NIACIN REDUCES KFC THROUGH SIRT1 ACTIVATION
reperfusion, albeit necessary to revitalize ischemic lungs, often caused chains of multifaceted consequences leading to further lung damages, known as pulmonary ischemia and reperfusion (I/R) injury. Although the exact mechanism responsible for pulmonary I/R induced lung injury has yet to be fully illustrated, excessive production of reactive oxygen species (ROS), such as superoxide and peroxides, infiltration of polymorphonuclear leukocytes (PMN), and segregation of macrophages in the lungs, all play important roles in lung injury [2]. Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1), nicotinamide adenine dinucleotide (NADþ)-dependent deacetylase sirtuin-1, is an enzyme that deacetylates proteins associated with cellular responses to stressors and longevity [3]. NADþ, which is biosynthetically converted by niacin (vitamin B3), is essential in all living cells [3]. Niacin treatment both increases the substrate of SIRT-1, for example, NADþ, and the activity of SIRT-1 that in turn stimulate antioxidant protection [3] and inhibit tumor necrosis factor alpha (TNF-a) induced inflammation [4]. In this study, we aim to investigate the protective efficacy of niacin pretreatment against lung I/R-induced pulmonary barrier function impairment. The severity of lung injury was assessed by lung water content, via lung-weight-to-bodyweight ratio (LW/BW) and lung wet-weight-to-dry-weight ratio (W/D), protein concentration in the lung lavage, and capillary filtration coefficient (Kfc). The degree of lung inflammation was assessed by the lavage leukocyte count and TNF-a level. The level of pulmonary oxidative stress was measured by lavage malondialdehyde (MDA) concentration. We also assessed the tissue protein level of SIRT1 via an Enzyme-Linked ImmunoSorbent Assay (ELISA) kit. MATERIALS AND METHODS Experimental Design The study was performed on 8-week-old male Sprague-Dawley rats (250e300 g; Biolasco Co. Taipei, Taiwan) in 3 groups: shamoperated (sham; n ¼ 6), lung I/R (I/R; n ¼ 6), and a group treated intraperitoneally with niacin 4 consecutive days before the study (niacin; 100 mg/kg/day; n ¼ 6). In the I/R and niacin group, 60 minutes of ischemia was followed by 60 minutes of reperfusion. In the sham group, 2 hours of normal ventilation was conducted. The dose of 100 mg/kg of intraperitoneal niacin was previously adopted by Nash et al [5] to treat poloxamer 407-induced hyperlipidemia in rats, by Richman et al [6] to activate ERK 1/2 mitogen-activated protein kinase in mice, and by Sarjana et al [7] to promote DNA repair, and maintenance of genomic stability and to inhibit carcinogenesis in mice.
Preparation of Isolated-Perfused Rat Lung and Calculation of Kfc Pulmonary Kfc was assessed using a model of in situ isolatedperfused rat lung. The procedure has been described previously [2]. In brief, rats were anaesthetized with Zoletil 50 (50 mg/kg, intraperitoneal). A midline thoracotomy was operated. The heart was exposed and 1 U/g of heparin was injected in the right ventricle. Five minutes of circulation was allowed for proper mixture of heparin and blood, and 10 mL of blood was drawn from the left
2835 ventricle, mixing with 5 mL of Hanks’ solution to form the perfusate. The perfusate was pre-warmed to 37 C before use. The pulmonary artery (PA) and the left atrium (LA) were cannulated to be connected to a peristaltic pump (MiniPuls III, Gilson Inc., Middleton, WI, USA) and a collecting reservoir, respectively. Lungs were ventilated with a mixture of 95% room air and 5% CO2. The lung was perfused at a constant rate of 30 mL/min/kg of the body weight. The PA and LA pressures were constantly monitored through pressure sensors (Deltran, Utah Medical Inc., Midvale, UT, USA) at side branches. Ischemia was induced by stopping the ventilator for 60 minutes followed by reperfusion through turning ventilator for 60 minutes. Rats were placed on a precision electronic scale, and the lung weight change was measured and electronically transmitted to a desktop computer at a rate of 2 Hz. Assessments were performed as lungs were ventilated and perfused. Recordings started when the weight reached the isogravimetric state, and PLA was raised rapidly by 10 cmH2O for a period of 7 minutes, followed by quickly returned to the baseline. Two phases of weight gain were observed following the rapid increase in PLA, namely a phase of fast weight gain during the first 1 minute, primarily due to pulmonary vascular recruitment, followed by a phase of slow weight gain, due mainly to the filtration (eg, fluid moving across the alveolar capillary). Kfc was calculated as the rate of slow weight gain (DW/Dt) using a linear regression on data collected between 3 and 7 minutes, with a unit of grams per minute per unit change in PLA (cmH2O) per 100 grams of lungs.
Measurements of LW and the Lung W/D rRatio Lungs and trachea were removed and weighed immediately at the end of the study. The left lung was prepared for the bronchoalveolar lavage acquisition, and the right lung was used for lung water content measurement and protein assessments. The right main bronchus was firmly tied. The upper lobe of the right lung was dissected and measured for the wet weight, and then placed in an oven at 70oC for 7 days to determine the dry weight.
Protein Concentration in Broncho-Alveolar Lavage Fluid Broncho-alveolar lavage fluid (BALF) was acquired by slowly flushing the right lung (excluding the upper right lobe) three times with saline (1.25 mL). The volume extracted was 0.95 0.08 mL. BALF was centrifuged at 1500 g for 10 minutes. Albumin level was assessed in the top supernatant of BALF by a spectrophotometry (Multiskan, Thermo Fisher, NC, US) at 630 nm.
Differential Neutrophil and Macrophage Cell Counts in BALF Ten microliters of cell pellets of centrifuged BALF were placed on a slide and stained with Liu’s stain. Leukocytes were counted and categorized using an optical microscope (X1000, Olympus CX31), where 100 cells were counted on each slide. Neutrophil and macrophage count were presented as a percentage of total leukocytes.
Assessments of Pulmonary Oxidative Stress and Lung Inflammation by MDA and TNF-a in the Lung Lavage Lung oxidative stress and inflammation were assessed by MDA and TNF-a levels in the supernatant of lung lavage, using ELISA kits (ab46070 and ab11897, Abcam, Cambridge, MA, US) following manufacturer’s guidance.
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WU AND WANG
lavage MDA and TNF-a levels were lower as compared to the untreated I/R group (*, P < .05 as compared with I/R; #, P < .05 as compared with sham). Figure 2 shows that as compared to the sham group (left), 60 minutes of ischemia followed by 60 minutes of reperfusion (middle) notably increased the LW/BW ratio (A), the W/D ratio (B), alveolar-capillary protein leakage (PCBAL) (C), lung weight gain with respect to time (D), pulmonary Kfc (E), lavage neutrophil and macrophage differential count (F), lavage contents of MDA (G), and TNF-a (H), but decreased SIRT1 concentration in lung tissue (I). In contrast, niacin treatment (right) averted the detrimental effects of I/R injury, by reducing pulmonary oxidative stress and lung inflammation, improving Kfc, lung protein leakage and lung water contents, and that was positively associated with the increased tissue SIRT1 protein expression. (*P < .05, #P < .01)
Assessment of Lung Tissue SIRT1 A section of the lower lobe of the right lung tissues (w200 mg) was homogenized in lysis buffer. The homogenates were transferred to 1.5 mL Eppendorf tubes, centrifuged at 15,000 g for 10 minutes. The level of SIRT1 was assessed in the supernatant using a commercial ELISA kit (ab206983, Abcam) following manufacturer’s guidance.
Data Analysis Data were presented as mean SEM. Comparisons across multiple groups were analyzed using one-way analysis of variance, with a Tukey post hoc test. P < .05 was considered statistically significant.
RESULTS
Figure 1 shows the dose-dependence study. In addition to 100 mg/kg of niacin adopted in this study, the doses of 50 and 200 mg/kg of intraperitoneal niacin were each examined in 4 8-week-old Sprague-Dawley male rats. No statistically significant differences were observed in lung weight gain (A), Kfc (B) and the lavage MDA (C) and TNF-a (D) levels, between rats pretreated with 200 mg/kg and those pretreated with 100 mg/kg of niacin. Though pretreatment of 50 mg/kg of niacin did not significantly improve Kfc, the
SIRT1 is a NAD-dependent class III protein deacetylase. SIRT1 plays a critical role in cell survival through deacetylating histones and transcription factors involving autophagy and ROS productions [3]. In response to oxidative stress,
B I/R I/R+niacin 100 mg/kg Sham I/R+niacin 50 mg/kg I/R+niacin 200 mg/kg
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Fig 1. The dose-dependence study. In addition to 100 mg/kg of niacin, the doses of 50 and 200 mg/kg of intraperitoneal niacin were each examined in 4 8-week-old Sprague-Dawley male rats. No statistically significant differences were observed in lung weight gain (A), Kfc (B) and the lavage MDA (C) and TNF-a (D) levels, between rats pretreated with 200 mg/kg and those pretreated with 100 mg/kg of niacin. Though pretreatment of 50 mg/kg of niacin did not significantly improve Kfc, the lavage MDA and TNF-a levels were lower as compared to the untreated I/R group. (*, P < .05 as compared with I/R; #, P < .05 as compared with sham.)
0.018
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Lung Weight/Body Weight (LW/BW)
NIACIN REDUCES KFC THROUGH SIRT1 ACTIVATION
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Fig 2. As compared to the sham group (left), 60 min of ischemia followed by 60 minutes of reperfusion (middle) notably increased the LW/BW ratio (A), the W/D ratio (B), PCBAL (C), lung weight gain with respect to time (D), pulmonary capillary filtration coefficient (Kfc) (E), lavage neutrophil and macrophage differential count (F), lavage contents of MDA (G), and TNF-a (H), but decreased SIRT1 concentration in lung tissue (I). Niacin treatment (right) averted the detrimental effects of I/R injury, by reducing pulmonary oxidative stress and lung inflammation, improving Kfc, lung protein leakage, and lung water contents, and that was positively associated with the increased tissue SIRT1 protein expression. (*P < .05, #P < .01.)
2838
SIRT1 modulates chromatin dynamics and transcriptional factors regulating cellular cycles and glucose homeostasis [8]. Besides, SIRT1 activation suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)driven immune responses [9]. Recent evidence revealed crosstalk between SIRT1 and oxidative stress; while SIRT1 tackles oxidative stress, ROS also inhibit SIRT1 activity through oxidative modifications on cysteine residues [3]. SIRT1 can be activated by NADþ, which is converted from niacin [10] Niacin is an antioxidant vitamin. As a therapeutic agent, the interests of niacin was focused mostly on cardiovascular protective effectiveness. Niacin has been demonstrated to attenuate myocardial injury and limit infarct size through lowering the cytosolic the ratio of NADH to NADþ and improve cellular glycolysis, as well as neutralizing intracellular oxygen free radicals [11]. In a large-scale multiple-center study, Brown et al [12] also showed that niacin supplements reduced coronary diseases in wide spectrum patients. In a large systemic meta analyses Lavigne and Karas [13] demonstrated that niacin reduced cardiovascular events and through a mechanism not related to changes in high-density lipoprotein cholesterol concentration, the known purpose for niacin prescription, and that was likely associated with reducing oxidative stress and inflammation and improving mitochondrial viability and eventually regulating energy homeostasis. In a rat model of kidney I/R injury, Tai et al [14] reported that niacin treatment reduced oxidative stress and improved ventricular dysfunction. Eppinger et al [15] demonstrated that pulmonary I/R injury induces hyperpolarization of inner mitochondrial membrane potential and impairs respiratory chain function complexes, promoting myeloperoxidase activity and lung edema. In this isolated perfused rat lung model, 60 minutes of ischemia followed by 60 minutes of reperfusion markedly increased pulmonary oxidative stress (increased MDA) while decreasing SIRT1 level, along with increased lung inflammation (increased lavage neutrophil count and TNFa), impaired pulmonary barrier function (increased Kfc, lung water content and PCBAL). Wang et al [16] reported that SIRT1 activation through miR-34a-59 inhibition attenuates acute lung injury induced by intestinal I/R injury. Very recently, Liu et al [17] reported that activation of SIRT1 through overexpression of heat shock protein 70 protected against pulmonary I/R injury. Consistently, we showed that niacin pretreatment increased SIRT1 and decreased pulmonary oxidative stress and averted pulmonary barrier function impairment and lung inflammation. The protective efficacy of niacin treatment against I/R induced pulmonary barrier function impairment and lung inflammation is most likely associated with both increasing NADþ level and niacin’s antioxidative capacity. In summary, lung I/R lung injury involves oxidative stress and reduction of pulmonary SIRT1 level, while niacin pretreatment reduces pulmonary oxidative stress and activates
WU AND WANG
SIRT1 and thus protect pulmonary barrier function and reduces lung inflammation. ACKNOWLEDGMENTS The study was supported by operating grants (104-CMFJ-07) from the Chi-Mei Foundation Hospital.
REFERENCES [1] Palazzo R, Hamvas A, Shuman T, et al. Injury in nonischemic lung after unilateral pulmonary ischemia with reperfusion. J Appl Physiol (1985) 1992;72(2):612e20. [2] Yeh DY, Fu YH, Yang YC, et al. Resveratrol alleviates lung ischemia and reperfusion-induced pulmonary capillary injury through modulating pulmonary mitochondrial metabolism. Transplant Proc 2014;46(4):1131e4. [3] Salminen A, Kaarniranta K, Kauppinen A. Crosstalk between Oxidative Stress and SIRT1: Impact on the Aging Process. Int J Mol Sci 2013;14(2):3834e59. [4] Zhu X, Liu Q, Wang M, et al. Activation of Sirt1 by resveratrol inhibits TNF-alpha induced inflammation in fibroblasts. PLoS One 2011;6(11):e27081. [5] Nash VJ, Johnston TP, Palmer WK. Effects of nicotinic acid on poloxamer 407-induced hyperlipidemia. Pharmacotherapy 1996;16(1):10e5. [6] Richman JG, Kanemitsu-Parks M, Gaidarov I, et al. Nicotinic acid receptor agonists differentially activate downstream effectors. J Biol Chem 2007;282(25):18028e36. [7] Surjana D, Halliday GM, Damian DL. Role of nicotinamide in DNA damage, mutagenesis, and DNA repair. J Nucleic Acids 2010. Volume 2010, Article ID 157591, 1-13. [8] Rajendran R, Garva R, Krstic-Demonacos M, Demonacos C. Sirtuins: molecular traffic lights in the crossroad of oxidative stress, chromatin remodeling, and transcription. J Biomed Biotechnol 2011;2011:368276. [9] Salminen A, Kauppinen A, Suuronen T, Kaarniranta K. SIRT1 longevity factor suppresses NF-kappaB -driven immune responses: regulation of aging via NF-kappaB acetylation? Bioessays 2008;30(10):939e42. [10] Sauve AA. NADþ and vitamin B3: from metabolism to therapies. J Pharmacol Exp Ther 2008;324(3):883e93. [11] Trueblood NA, Ramasamy R, Wang LF, et al. Niacin protects the isolated heart from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2000;279(2):H764e71. [12] Brown BG, Zhao XQ, Chait A, et al. Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med 2001;345(22):1583e92. [13] Lavigne PM, Karas RH. The current state of niacin in cardiovascular disease prevention: a systematic review and metaregression. J Am Coll Cardiol 2013;61(4):440e6. [14] Tai ST, Fu YH, Yang YC, et al. Niacin ameliorates kidney warm ischemia and reperfusion injury-induced ventricular dysfunction and oxidative stress and disturbance in mitochondrial metabolism in rats. Transplant Proc 2015;47(4):1079e82. [15] Eppinger MJ, Deeb GM, Bolling SF, et al. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997;150(5): 1773e84. [16] Wang G, Yao J, Li Z, et al. miR-34a-5p inhibition alleviates intestinal ischemia/reperfusion-induced reactive oxygen species accumulation and apoptosis via activation of SIRT1 signaling. Antioxid Redox Signal 2016;24(17):961e73. [17] Liu S, Xu J, Fang C, et al. Over-expression of heat shock protein 70 protects mice against lung ischemia/reperfusion injury through SIRT1/AMPK/eNOS pathway. Am J Transl Res 2016;8(10):4394e404.
ABSTRACT Purpose. Alveolar-capillary barrier dysfunction, characterized by alveolar protein leak and lung edema, is a common scenario following cardiopulmonary surgery and thoracic organ transplantation. Reactive oxygen species generated through lung ischemia and reperfusion (I/R) injury during surgery plays a crucial role. Niacin, also known as vitamin B3, has been demonstrated to possess antioxidative and anti-inflammatory capacity. In this study, we examine the pulmonary barrier function via capillary filtration coefficient (Kfc) following lung I/R injury with and without niacin treatment. Methods. Studies were conducted on male Sprague-Dawley rats in 3 groups: shamoperated, lung I/R injury, and niacin-pretreated lung I/R injury group. Rats were subjected to isolated perfused lung preparation. Lung ischemia was established by continuous perfusion and stopping ventilation for 60 minutes, followed by 60 minutes of ventilation. We assessed the Kfc, lung water content, and protein concentration in the lung lavage; pulmonary oxidative stress and lung inflammation were assessed by leukocyte counts, tissue level of tumor nercrosis factor alpha (TNF-a), and tissue content of malondialdehyde (MDA), respectively. We also assessed the tissue protein level of sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1). Results. Lungs subjected to I/R injury significantly increased Kfc, pulmonary oxidative stress, lung water content, and lavage leukocyte count and protein concentration (P < .05). Rats treated with niacin of 100 mg/kg/day for 4 days increased lung SIRT1 (P < .05) and attenuated lung I/R injury-induced pulmonary oxidative stress and inflammation and also improved Kfc. Conclusions. Niacin pretreatment protects lungs against I/R injury-induced barrier function impairment through the activation of SIRT1 and reduced pulmonary oxidative stress and lung inflammation.
L
ung ischemia and reperfusion injury, manifested by the impairment of alveolar-capillary barrier function, characterized by alveolar protein leak and lung edema, is the major cause of graft failure subsequent to lung transplantation and cardiopulmonary bypass surgery [1]. Lung ischemia, induced by clamping the pulmonary blood vessels during the operation, causes oxygen deprivation and imbalance between the metabolic demand and supply that
0041-1345/18 https://doi.org/10.1016/j.transproceed.2018.04.047
2834
triggers a series of events, resulting in different intensities of cellular damage and subsequent activation of cytotoxic enzymes that ultimately culminate in cell death [2]. Lung *Address correspondence to Jiun-Jr Wang, PhD, School of Medicine, Fu Jen Catholic University, No. 510, Zhongzheng Road, Xinzhuang Dist., New Taipei City, 24205, Taiwan. Tel: 886-229053498. Fax: 886-2-29053496. E-mail: [email protected] ª 2018 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 50, 2834e2838 (2018)
NIACIN REDUCES KFC THROUGH SIRT1 ACTIVATION
reperfusion, albeit necessary to revitalize ischemic lungs, often caused chains of multifaceted consequences leading to further lung damages, known as pulmonary ischemia and reperfusion (I/R) injury. Although the exact mechanism responsible for pulmonary I/R induced lung injury has yet to be fully illustrated, excessive production of reactive oxygen species (ROS), such as superoxide and peroxides, infiltration of polymorphonuclear leukocytes (PMN), and segregation of macrophages in the lungs, all play important roles in lung injury [2]. Sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1), nicotinamide adenine dinucleotide (NADþ)-dependent deacetylase sirtuin-1, is an enzyme that deacetylates proteins associated with cellular responses to stressors and longevity [3]. NADþ, which is biosynthetically converted by niacin (vitamin B3), is essential in all living cells [3]. Niacin treatment both increases the substrate of SIRT-1, for example, NADþ, and the activity of SIRT-1 that in turn stimulate antioxidant protection [3] and inhibit tumor necrosis factor alpha (TNF-a) induced inflammation [4]. In this study, we aim to investigate the protective efficacy of niacin pretreatment against lung I/R-induced pulmonary barrier function impairment. The severity of lung injury was assessed by lung water content, via lung-weight-to-bodyweight ratio (LW/BW) and lung wet-weight-to-dry-weight ratio (W/D), protein concentration in the lung lavage, and capillary filtration coefficient (Kfc). The degree of lung inflammation was assessed by the lavage leukocyte count and TNF-a level. The level of pulmonary oxidative stress was measured by lavage malondialdehyde (MDA) concentration. We also assessed the tissue protein level of SIRT1 via an Enzyme-Linked ImmunoSorbent Assay (ELISA) kit. MATERIALS AND METHODS Experimental Design The study was performed on 8-week-old male Sprague-Dawley rats (250e300 g; Biolasco Co. Taipei, Taiwan) in 3 groups: shamoperated (sham; n ¼ 6), lung I/R (I/R; n ¼ 6), and a group treated intraperitoneally with niacin 4 consecutive days before the study (niacin; 100 mg/kg/day; n ¼ 6). In the I/R and niacin group, 60 minutes of ischemia was followed by 60 minutes of reperfusion. In the sham group, 2 hours of normal ventilation was conducted. The dose of 100 mg/kg of intraperitoneal niacin was previously adopted by Nash et al [5] to treat poloxamer 407-induced hyperlipidemia in rats, by Richman et al [6] to activate ERK 1/2 mitogen-activated protein kinase in mice, and by Sarjana et al [7] to promote DNA repair, and maintenance of genomic stability and to inhibit carcinogenesis in mice.
Preparation of Isolated-Perfused Rat Lung and Calculation of Kfc Pulmonary Kfc was assessed using a model of in situ isolatedperfused rat lung. The procedure has been described previously [2]. In brief, rats were anaesthetized with Zoletil 50 (50 mg/kg, intraperitoneal). A midline thoracotomy was operated. The heart was exposed and 1 U/g of heparin was injected in the right ventricle. Five minutes of circulation was allowed for proper mixture of heparin and blood, and 10 mL of blood was drawn from the left
2835 ventricle, mixing with 5 mL of Hanks’ solution to form the perfusate. The perfusate was pre-warmed to 37 C before use. The pulmonary artery (PA) and the left atrium (LA) were cannulated to be connected to a peristaltic pump (MiniPuls III, Gilson Inc., Middleton, WI, USA) and a collecting reservoir, respectively. Lungs were ventilated with a mixture of 95% room air and 5% CO2. The lung was perfused at a constant rate of 30 mL/min/kg of the body weight. The PA and LA pressures were constantly monitored through pressure sensors (Deltran, Utah Medical Inc., Midvale, UT, USA) at side branches. Ischemia was induced by stopping the ventilator for 60 minutes followed by reperfusion through turning ventilator for 60 minutes. Rats were placed on a precision electronic scale, and the lung weight change was measured and electronically transmitted to a desktop computer at a rate of 2 Hz. Assessments were performed as lungs were ventilated and perfused. Recordings started when the weight reached the isogravimetric state, and PLA was raised rapidly by 10 cmH2O for a period of 7 minutes, followed by quickly returned to the baseline. Two phases of weight gain were observed following the rapid increase in PLA, namely a phase of fast weight gain during the first 1 minute, primarily due to pulmonary vascular recruitment, followed by a phase of slow weight gain, due mainly to the filtration (eg, fluid moving across the alveolar capillary). Kfc was calculated as the rate of slow weight gain (DW/Dt) using a linear regression on data collected between 3 and 7 minutes, with a unit of grams per minute per unit change in PLA (cmH2O) per 100 grams of lungs.
Measurements of LW and the Lung W/D rRatio Lungs and trachea were removed and weighed immediately at the end of the study. The left lung was prepared for the bronchoalveolar lavage acquisition, and the right lung was used for lung water content measurement and protein assessments. The right main bronchus was firmly tied. The upper lobe of the right lung was dissected and measured for the wet weight, and then placed in an oven at 70oC for 7 days to determine the dry weight.
Protein Concentration in Broncho-Alveolar Lavage Fluid Broncho-alveolar lavage fluid (BALF) was acquired by slowly flushing the right lung (excluding the upper right lobe) three times with saline (1.25 mL). The volume extracted was 0.95 0.08 mL. BALF was centrifuged at 1500 g for 10 minutes. Albumin level was assessed in the top supernatant of BALF by a spectrophotometry (Multiskan, Thermo Fisher, NC, US) at 630 nm.
Differential Neutrophil and Macrophage Cell Counts in BALF Ten microliters of cell pellets of centrifuged BALF were placed on a slide and stained with Liu’s stain. Leukocytes were counted and categorized using an optical microscope (X1000, Olympus CX31), where 100 cells were counted on each slide. Neutrophil and macrophage count were presented as a percentage of total leukocytes.
Assessments of Pulmonary Oxidative Stress and Lung Inflammation by MDA and TNF-a in the Lung Lavage Lung oxidative stress and inflammation were assessed by MDA and TNF-a levels in the supernatant of lung lavage, using ELISA kits (ab46070 and ab11897, Abcam, Cambridge, MA, US) following manufacturer’s guidance.
2836
WU AND WANG
lavage MDA and TNF-a levels were lower as compared to the untreated I/R group (*, P < .05 as compared with I/R; #, P < .05 as compared with sham). Figure 2 shows that as compared to the sham group (left), 60 minutes of ischemia followed by 60 minutes of reperfusion (middle) notably increased the LW/BW ratio (A), the W/D ratio (B), alveolar-capillary protein leakage (PCBAL) (C), lung weight gain with respect to time (D), pulmonary Kfc (E), lavage neutrophil and macrophage differential count (F), lavage contents of MDA (G), and TNF-a (H), but decreased SIRT1 concentration in lung tissue (I). In contrast, niacin treatment (right) averted the detrimental effects of I/R injury, by reducing pulmonary oxidative stress and lung inflammation, improving Kfc, lung protein leakage and lung water contents, and that was positively associated with the increased tissue SIRT1 protein expression. (*P < .05, #P < .01)
Assessment of Lung Tissue SIRT1 A section of the lower lobe of the right lung tissues (w200 mg) was homogenized in lysis buffer. The homogenates were transferred to 1.5 mL Eppendorf tubes, centrifuged at 15,000 g for 10 minutes. The level of SIRT1 was assessed in the supernatant using a commercial ELISA kit (ab206983, Abcam) following manufacturer’s guidance.
Data Analysis Data were presented as mean SEM. Comparisons across multiple groups were analyzed using one-way analysis of variance, with a Tukey post hoc test. P < .05 was considered statistically significant.
RESULTS
Figure 1 shows the dose-dependence study. In addition to 100 mg/kg of niacin adopted in this study, the doses of 50 and 200 mg/kg of intraperitoneal niacin were each examined in 4 8-week-old Sprague-Dawley male rats. No statistically significant differences were observed in lung weight gain (A), Kfc (B) and the lavage MDA (C) and TNF-a (D) levels, between rats pretreated with 200 mg/kg and those pretreated with 100 mg/kg of niacin. Though pretreatment of 50 mg/kg of niacin did not significantly improve Kfc, the
SIRT1 is a NAD-dependent class III protein deacetylase. SIRT1 plays a critical role in cell survival through deacetylating histones and transcription factors involving autophagy and ROS productions [3]. In response to oxidative stress,
B I/R I/R+niacin 100 mg/kg Sham I/R+niacin 50 mg/kg I/R+niacin 200 mg/kg
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DISCUSSION
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I/R /kg /kg /kg mg 0 mg 0 mg 0 5 10 20
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Fig 1. The dose-dependence study. In addition to 100 mg/kg of niacin, the doses of 50 and 200 mg/kg of intraperitoneal niacin were each examined in 4 8-week-old Sprague-Dawley male rats. No statistically significant differences were observed in lung weight gain (A), Kfc (B) and the lavage MDA (C) and TNF-a (D) levels, between rats pretreated with 200 mg/kg and those pretreated with 100 mg/kg of niacin. Though pretreatment of 50 mg/kg of niacin did not significantly improve Kfc, the lavage MDA and TNF-a levels were lower as compared to the untreated I/R group. (*, P < .05 as compared with I/R; #, P < .05 as compared with sham.)
0.018
0.014 0.012 0.010 0.000
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2837
I/R+niacin
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*
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Lung Weight/Body Weight (LW/BW)
NIACIN REDUCES KFC THROUGH SIRT1 ACTIVATION
Col 4 Col 7
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Fig 2. As compared to the sham group (left), 60 min of ischemia followed by 60 minutes of reperfusion (middle) notably increased the LW/BW ratio (A), the W/D ratio (B), PCBAL (C), lung weight gain with respect to time (D), pulmonary capillary filtration coefficient (Kfc) (E), lavage neutrophil and macrophage differential count (F), lavage contents of MDA (G), and TNF-a (H), but decreased SIRT1 concentration in lung tissue (I). Niacin treatment (right) averted the detrimental effects of I/R injury, by reducing pulmonary oxidative stress and lung inflammation, improving Kfc, lung protein leakage, and lung water contents, and that was positively associated with the increased tissue SIRT1 protein expression. (*P < .05, #P < .01.)
2838
SIRT1 modulates chromatin dynamics and transcriptional factors regulating cellular cycles and glucose homeostasis [8]. Besides, SIRT1 activation suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)driven immune responses [9]. Recent evidence revealed crosstalk between SIRT1 and oxidative stress; while SIRT1 tackles oxidative stress, ROS also inhibit SIRT1 activity through oxidative modifications on cysteine residues [3]. SIRT1 can be activated by NADþ, which is converted from niacin [10] Niacin is an antioxidant vitamin. As a therapeutic agent, the interests of niacin was focused mostly on cardiovascular protective effectiveness. Niacin has been demonstrated to attenuate myocardial injury and limit infarct size through lowering the cytosolic the ratio of NADH to NADþ and improve cellular glycolysis, as well as neutralizing intracellular oxygen free radicals [11]. In a large-scale multiple-center study, Brown et al [12] also showed that niacin supplements reduced coronary diseases in wide spectrum patients. In a large systemic meta analyses Lavigne and Karas [13] demonstrated that niacin reduced cardiovascular events and through a mechanism not related to changes in high-density lipoprotein cholesterol concentration, the known purpose for niacin prescription, and that was likely associated with reducing oxidative stress and inflammation and improving mitochondrial viability and eventually regulating energy homeostasis. In a rat model of kidney I/R injury, Tai et al [14] reported that niacin treatment reduced oxidative stress and improved ventricular dysfunction. Eppinger et al [15] demonstrated that pulmonary I/R injury induces hyperpolarization of inner mitochondrial membrane potential and impairs respiratory chain function complexes, promoting myeloperoxidase activity and lung edema. In this isolated perfused rat lung model, 60 minutes of ischemia followed by 60 minutes of reperfusion markedly increased pulmonary oxidative stress (increased MDA) while decreasing SIRT1 level, along with increased lung inflammation (increased lavage neutrophil count and TNFa), impaired pulmonary barrier function (increased Kfc, lung water content and PCBAL). Wang et al [16] reported that SIRT1 activation through miR-34a-59 inhibition attenuates acute lung injury induced by intestinal I/R injury. Very recently, Liu et al [17] reported that activation of SIRT1 through overexpression of heat shock protein 70 protected against pulmonary I/R injury. Consistently, we showed that niacin pretreatment increased SIRT1 and decreased pulmonary oxidative stress and averted pulmonary barrier function impairment and lung inflammation. The protective efficacy of niacin treatment against I/R induced pulmonary barrier function impairment and lung inflammation is most likely associated with both increasing NADþ level and niacin’s antioxidative capacity. In summary, lung I/R lung injury involves oxidative stress and reduction of pulmonary SIRT1 level, while niacin pretreatment reduces pulmonary oxidative stress and activates
WU AND WANG
SIRT1 and thus protect pulmonary barrier function and reduces lung inflammation. ACKNOWLEDGMENTS The study was supported by operating grants (104-CMFJ-07) from the Chi-Mei Foundation Hospital.
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