Protective mechanisms of sevoflurane against one-lung ventilation-induced acute lung injury: role of cyclooxygenase-2 and 5-lipoxygenase pathways

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1 J South Med Univ, 13, 33(5): 5-3 doi 1.399/j.issn Original Article Protective mechanisms of sevoflurane against one-lung ventilation-induced acute lung injury: role of cyclooxygenase- and 5-lipoxygenase pathways LIU Rui 1,, LUO Jing 3, LI Jiang, MA Qingjie, LI Yanhua, WANG Dianhua 1 1 School of Pharmacy and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 531, China; Department of Anesthesiology, 3 Department of Pain Management, First People's Hospital of Yunnan Province, Kunming 53, China Abstract: Objective To explore the protective mechanisms of sevoflurane against acute lung injury (ALI) induced by one-lung ventilation (OLV) in view of cyclooxygenase- (COX) and 5-lipoxygenase (5-LOX) pathways. Method Eighteen healthy Japanese white rabbits were randomized into sham-operated group (S group), OLV group (O group) and OLV + sevoflurane group (OS group). COX and 5-LOX protein and mrna expressions in the lungs were detected by Western blotting and real-time PCR, respectively. Prostaglandin I (PGI), thromboxane A (TXA) and leukotrienes B (LTB) in the lung tissues were quantified with ELISA. Histological scores and lung wet/dry weight (W/D) ratios were determined for lung injury assessment. Results COX and 5-LOX protein and mrna expressions and the contents of LTB, TXA and PGI in the lungs, lung W/D ratio and histological scores were significantly higher while PGI/TXA ratio was significantly lower in O group and OS group than in S group (P<.5). Compared with those in O group, COX and 5-LOX expressions, pulmonary contents of LTB, TXA and PGI, and lung W/D ratio all decreased significantly but PGI/TXA ratio was significantly elevated in OS group (P<.5). Conclusion OLV may activate COX and 5-LOX pathways to result in increased production of arachidonic acid metabolites. Sevoflurane protects against OLV-induced ALI probably by reducing AA metabolites and regulating PGI/TXA ratio through inhibitions of COX and 5-LOX pathways. Key words: one-lung ventilation; sevoflurane; cyclooxygenase-; 5-lipoxygenase INTRODUCTION Studies have shown that one-lung ventilation (OLV) can cause pulmonary inflammation responses [1-]. Specific eicosanoids, lipid mediators derived from arachidonic acid (AA), are known to contribute to the initiation of inflammation [3]. Blocking the pathways of AA metabolism, therefore, may alleviate the lung injury induced by OLV. In mammals, AA can be metabolized mainly by two pathways, namely the cyclooxygenase (COX) pathway which generates classical prostaglandins (PGs), prostacyclin (PGI ) and thromboxane (TXA ), and the lipoxygenase (LOX) pathway which generates leukotrienes (LTs) and lipid peroxides. There are two different COX isoforms, COX 1 and COX, and the latter is an inducible isoform that is found and expressed mainly in inflammation responses []. Among the various end products derived from the sequential metabolism of AA via COX, PGI, which is a potent vasodilator and an inhibitor of platelet aggregation produced mainly by Received: Accepted: Supported by National Natural Science Foundation of China (383). LIU Rui and LUO Jing contributed equally to this work. Corresponding author: WANG Dianhua, vascular endothelial cells, has been implicated as the PGs most responsible for inflammation []. As another product of the sequential metabolism of AA, TXA is generated mainly by platelet COX 1 pathway and executes the functions of both a potent vasoconstrictor and a stimulator of platelet aggregation []. In physiological conditions, the levels of TXA and PGI maintain a relative equilibrium state, and breaching this balance between them results in disease just as the lessons learned from the specific COX blocking agent that led to cardiovascular complications [5]. This suggests that maintaining the equilibrium state of TXA and PGI is more important than simply manipulating either of them in the disease process. Therefore, it is more reasonable to use the ratio between PGI and TXA to reflect the severity of the disease than use either of them as a single indicator. Leucotrienes synthesized via the 5-LOX pathway play a major part in the inflammatory process []. The final and biologically active metabolites of the 5-LOX cascade are LTC, LTD, LTE and LTB ; the first three mainly cause bronchospasm and mucosal secretion, and LTB is a potent stimulator of leucocytes activation, allows these cells to adhere to the vascular endothelium, and elicits chemokinetic and chemotactic responses [7-9]. During a brief exposure to LTB, polymorphonuclear

2 J South Med Univ, 13, 33(5): leucocytes (PMN) are predominantly recruited [9]. Robertson et al [1] and Caironi et al [11] showed that inhibition of COX and 5-LOX expressions exerted an anti-inflammatory effect in ventilator-induced lung injury. Although some studies showed that sevoflurane, a commonly used anesthetic in clinical practice, played an anti-inflammatory role in OLV [1-], the effects of sevoflurane on COX and 5-LOX in rabbit lung tissues with OLV-induced injuries are still unclear. We hypothesize that sevoflurane may also protect against OLV-induced acute lung injury (ALI) by modulating AA metabolism through COX and 5-LOX pathways. MATERIALS AND METHODS Animals and grouping Eighteen healthy Japanese white rabbits of either sex weighing.-.5 kg were purchased from the Experimental Animal Center of Kunming Medical University [Animal Certificate of Conformity: 9; animal license number: scxk (Yunnan) 11-]. The rabbits were randomized equally into 3 groups, namely the sham-operated group (S group), OLV group (O group), and OLV plus sevoflurane group (OS group). In S group, the rabbits received sham operations by exposing the trachea, left common carotid artery and right external jugular vein, and placement of endotracheal tube (inner diameter of. mm) through tracheotomy between and 3 tracheal rings. In O group, OLV was achieved through advancing the tracheotomy tube into the right main bronchus in rabbits, and mechanical ventilation was performed by OLV for h followed by two-lung ventilation (TLV) for 1 h. The ventilator settings (Datex Ohmeda, Aestiva/5 79, USA) in OLV and TLV were identical with an inspired oxygen fraction (FiO ) of 1., tidal volume (VT) of ml/kg, respiration rate (RR) of 3 min -1, and inspiratory/ expiratory (I/E) ratio of 1. In OS group,.5% sevoflurane was administered during mechanical ventilation with the same settings as in O group. Anesthesia and intraoperative detections All the rabbits were anesthetized with pentobarbital sodium (3 mg/kg) via the ear marginal vein during the sham operations. Maintenance of anesthesia in O group and OS group was achieved by continuous infusion of remifentanil at a rate of 1 μg kg -1 min -1 and intermittent administration of vecuronium (.1 mg/kg per 3 min). Invasive blood pressure, end tidal CO and sevoflurane concentrations were continuously monitored and maintained within the normal ranges or at the preset concentration. Determining pulmonary contents of LTB, PGI -keto-pgf 1α, TXA, and TXB Due to the short tissue half-life of TXA (3 s) and PGI (3 min) [], the quantities of their stable metabolites, TXB and -keto-pgf1α, respectively, were determined to represent their values. Enzyme-linked immunosorbent assay (ELISA) kits (Biosynthesis Biothechnology Co., LTD, China) were used to detect the amounts of LTB, -keto-pgf1α and TXB in the lungs of the rabbits according to the manufacturer's instructions. Western blotting for determining pulmonary COX and 5-LOX protein contents The right middle lobes of the rabbit lungs were sampled (weighing about 1 mg) and quickly placed on ice. One milliliter of pre-cooled RIPA lysis buffer (Beyotime Institute of Biotechnology, P13C) and 15 μl protease inhibitor cocktail (Roche, 93131) were added. The soluble proteins were isolated by centrifugation at 13 r/min for 3 min, and the protein concentrations in the supernatant were determined using BCA kit (Beyotime Institute of Biotechnology, P1). For Western blotting, the samples were boiled in loading buffer, and the proteins were separated by SDS-PAGE and transferred to PVDF membranes (.5 μm, Millipore). The membranes were then blocked with 5% bovine serum albumin (BSA) at room temperature for min, rinsed with TBST for 3 times, incubated overnight at with the primary antibodies of COX (Biosynthesis Biotechnology Co. Ltd, bs-73r, dilution 1 3) and 5-LOX (Biosynthesis Biotechnology Co. Ltd, bs-5r, dilution 1 ), and then at room temperature with the secondary antibody (Abmart, M13, dilution 1 3) for h. The proteins were detected by enhanced chemiluminescence (BIO-RAD), and the band intensities were quantified using Image J software. Quantitative real-time PCR for COX and 5-LOX mrna expressions Under sterile conditions, the total RNA was extracted from the right middle lobe tissues (each sample weighing about mg) using Trizol RNA extraction kit (Roche, 8981), and the concentrations and purities of the total RNA were measured. The first strand cdnas were synthesized using RevertAid TM First Strand cdna Synthesis Kit (Fermentas, K11) according to the manufacturer's instructions. Real-time RT-PCR amplifications of the samples in triplicate were performed using the SYBR Green PCR Master Mix (Applied Biosystems) in a total reaction volume of 5 μl containing distilled water (9.5 μl), SYBR Green Master (1.5 μl) (Roche, ), 1 μmol/l upper-stream primer (1 μl), 1 μmol/l downstream primer (1 μl) and template cdna (1 μl). All the PCR assays were run in the ABI Prism 73 Sequence Detection System at 95 for 3 min, 95 for 15 s, for 3 s, 7 for 3 s, followed by cycles of 95 for 15 s, for 3 s, and 7 for 3 s, with a final extension at 7 for 5 min. The primers used for PCR amplification were synthesized by Invitrogen Corporation (Tab.1).

3 J South Med Univ, 13, 33(5): Tab.1 Primers for PCR amplification of COX, 5-LOX and β-actin cdna and the expected product lengths Gene Oligonucleotide sequence Tm ( C) bp β-actin F: 5'-CATCCTGACGCTCAAGTA -3' S: 5'-GTTGTAGAAGGTGTGGTG-3' COX F: 5'-ACATCGTCAATAGCATTC-3' S: 5'-TAGTAGGAGAGGTTAGAGA-3' LOX F: 5'-GCTACATCTTCGGGAAAC-3' S: 5'-ATGAGGCAATAGTTGAGTATG-3' Lung histological scores A sample of the right upper lobe was sectioned for histological examination. The sections were fixed in 1% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. The slides were viewed by a blinded pathologist under light microscopy for histological evaluation of the lung injury according to the criteria proposed by Webb et al [1]. Lung wet/dry weight (W/D) ratio At the end of the experiments, the right lower lobe was excised. The wet weight was recorded, and the lung was desiccated at 8 for 7 h for measurement of the dry weight to calculate the W/D ratio. Statistical analysis The quantitative data are presented as Mean ± SE. One-way analysis of variance (ANOVA) with repeated measures followed by Fisher's least significant difference post hoc test was used as indicated for comparison between the groups. A P value less than.5 was considered to indicate a statistically significant difference. RESULTS Pulmonary expressions of COX and 5-LOX In both O group and OS group, COX and 5-LOX expressions (at both the protein and mrna levels if not indicated otherwise) were significantly increased as compared with those in S group (P<.5). COX and 5-LOX expressions were significantly lower in OS group than in O group (P<.5, Fig.1). Pulmonary contents of LTB, -K-PGF 1α (PGI ), TXB (TXA ) and -K-PGF 1α/TXB Compared with those in S group, the pulmonary contents of LTB, TXB and -K-PGF 1α increased while -K-PGF 1α/TXB ratios decreased significantly in O and OS groups (P<.5). The contents of LTB, TXB and -K-PGF 1α were lower but -K-PGF 1α/TXB ratio was higher significantly in OS group than in O group (P<.5, Fig.). Lung W/D ratio and histological scores In both O group and OS group, lung W/D ratios and histological scores were significantly increased as compared with those in S group (P<.5). The lung W/D ratio and histological scores were significantly lower in OS group than in O group (P<.5, Fig.3). Lung histology under light microscopy No significant pathological changes were observed in S group except for mild inflammations and capillary dilatation in some areas of the lung tissues (Fig.A1-). Serious hyperemia and hemorrhage in the lung tissues, thickening and exudation of the alveolar wall, marked red blood cell and inflammatory cell infiltration in the alveolar space were found in O group (Fig.B1-). These pulmonary histopathological changes were alleviated significantly in OS group (Fig.C1-). DISCUSSION Our data showed that COX and 5-LOX expressions increased significantly along with LTB, TXA and PGI in the rabbit lungs after OLV, which led to serious lung injuries. These results suggest that OLV activates the COX and 5-LOX pathways and then results in increased metabolites of the two pathways. We also found a significantly decreased PGI /TXA ratio in the lung tissue after OLV, which can be the results of multiple factors. LTB generated through the activation of 5-LOX pathway induced by OLV can activate a large amount of inflammatory cells, which adhere to the vascular endothelium and cause vascular endothelial cell damages. The vascular endothelial damages lead to platelet aggregation and activation, and then the production of numerous TXA. The generated TXA, in return, causes further platelet aggregation and activation, thus completing a vicious cycle to lead to drastically increased TXA contents in the lung tissue. Conversely, OLV-induced damages of the vascular endothelial cells, which are the major source of PGI synthesis, cause a reduction in PGI production. Therefore, even if OLV increases COX expression, PGI production is in disadvantage as compared with TXA to result in a significantly decreased PGI /TXA ratio in the lung tissue. We observed that administration of sevoflurane

4 LTB (ng/g) -K-PCF1α (ng/mg) -K-PGF1α/TXB ratio TXB (ng/mg) COX mrna expression relative copy number 5-LOX mrna expression relative copy number COX/GAPDH 5-LOX/GAPDH 8 J South Med Univ, 13, 33(5): A1 O S OS B1 O S OS COX 5 5-LOX 78 GAPDH 37 GAPDH 37 A.7 C B 1. Fig.1 Expressions of COX protein (A1, A), 5-LOX protein (B1, B), COX mrna (C) and 5-LOX mrna (D) in rabbit lungs in different groups. P<.5 vs S group; P<.5 vs O group (Mean±SE, n=). D S O OS A B C D S O OS Fig. Contents of LTB (A), PGI(-K-PGF1α)(B), -K-PGF1α/TXB ratio(c) and TXA (TXB) (D) in rabbit lungs in different groups. P<.5 vs S group; P<.5 vs O group (Mean±SE, n=). down-regulated the expression levels of COX and 5-LOX as well as the pulmonary contents of LTB, TXA and PGI, and alleviated lung injuries induced by OLV. These results indicate that the protective effects of sevoflurane against OLV-induced ALI are probably mediated by inhibiting COX and 5-LOX pathways. However, the specific molecular mechanisms of such effects remain to be further studied. Another finding in this study was that sevoflurane can increase the lung PGI /TXA ratio in OLV induced ALI. This effect might be attributed to the inhibition of the vicious cycle of TXA generation by sevoflurane through down-regulating 5-LOX expression, and to the greater sensitivity of COX 1 to sevoflurane than COX.

5 Histologioal score J South Med Univ, 13, 33(5): A Lung w/d ratio Fig.3 B Lung W/D ratio (A) and histological scores (B) of rabbit lungs in different groups. P<.5 vs S group; P<.5 vs O group (Mean±SE, n=) S O OS This hypothesis is supported by evidences from other studies. First, studies in vivo and in vitro showed that sevoflurane has strong anti-aggregatory effects by inhibiting TXA formation and suppressing cycloo-xygenase activity, even at a subanesthetic concentration of.5% [13-1]. Wacker and colleagues showed that AA-induced platelet aggregation could be inhibited by inhalation of low- concentration sevoflurane (less than 1% end-tidal) [15]. As a potent stimulator of platelet aggregation, TXA is the predominant product of COX in platelets [] which contain only COX 1 but not COX. A1 A B1 B C1 C Fig. Lung histology under light microscopy in different groups. A: S group; B: O group; C: OS group (HE staining, original magnification: 1 for A1-C1 and for A-C). Such studies indicated that the anti-aggregatory effects of sevoflurane were mediated by suppressing COX 1, even at low concentrations. Secondly, the study of constant-flow perfused lungs of Japanese white rabbits showed that cyclooxygenase products did not mediate the inhibition of hypoxic pulmonary vasoconstriction (HPV) by 1. MAC (approximately % ) sevoflurane [1]. The in vitro study of human feto-placental vasculature showed that at the concentrations of % and 8%, sevoflurane-mediated vasodilation was cyclooxygenaseindependent [17]. Like- wise, Heindl et al found that sevoflurane at MAC, but not at 1 MAC, could reduce stimulated endothelial PGI production by about 5% [18]. PGI is a potent vasodilator generated mainly by COX

6 3 J South Med Univ, 13, 33(5): 5-3 pathway in vascular endothelial cells, and sevofluraneinduced vasodilation (cyclooxygenase-independent) may be caused by the weak effects of sevoflurane on COX. Based on these studies, we speculate that the sensitivity differences of COX1 and COX to sevoflurane may be one of the reasons for sevoflurane-induced up-regulation of the lung PGI/TXA ratio in OLV-induced ALI. In summary, this study demonstrates for the first time that OLV may activate COX and 5-LOX pathways to result in increased generation of AA metabolities, and sevoflurane can produce protective effects against OLV-induced ALI possibly by reducing AA metabolites and regulating PGI/TXA ratio via inhibition of COX and 5-LOX pathways. REFERENCES 1 Sugasawa Y, Yamaguchi K, Kumakura S, et al. Effects of sevoflurane and propofol on pulmonary inflammatory responses during lung resection J. J Anesth, 1, (1): -9. Schilling T, Kozian A, Senturk M, et al. Effects of volatile and intravenous anesthesia on the alveolar and systemic inflammatory response in thoracic surgical patients J. Anesthesiology, 11, 115 (1): Samuelsson B. Arachidonic acid metabolism: role in inflammation J. Z Rheumatol, 1991, 5 Suppl 1: 3-. Sharma JN, Jawad NM. Adverse effects of COX- inhibitors J. Sci World J, 5, 5: Drazen JM. COX- inhibitors-a lesson in unexpected problems J. N Engl J Med, 5, 35: Sala A, Zarini S, Bolla M. Leukotrienes: lipid bioeffectors of inflammatory reactions J. Biochemistry (Mosc), 1998, 3: Martel-Pelletier J, Lajeunesse D, Reboul P, et al. Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal anti-inflammatory drugs J. Ann Rheum Dis, 3, (): Lewis RA, Ansten KF, Soberman RJ. Leukotrienes and other products of the 5-lipoxigenase pathway. Biochemistry and relation to pathobiology in human diseases J. N Engl J Med,, 19: Bray MA, Ford-Hutchinson AW, Smith MJ. Leukotriene B: an inflammatory mediator in vivo J. Prostaglandins, 1981, : Robertson JA, Sauer D, Gold JA, et al. The role of cyclooxygenase- in mechanical ventilation-induced lung injury J. Am J Respir Cell Mol Biol, 1, 7(3): Caironi P, Ichinose F, Liu R, et al. 5-Lipoxygenase deficiency prevents respiratory failure during ventilator-induced lung injury J. Am J Respir Crit Care Med, 5, 17(3): Webb HH, Tierney DF. Experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures. Protection by positive end-expiratory pressure J. Am Rev Respir Dis, 197, 11: Lee A, Chui PT, Aun CS, Possible interaction between sevoflurane and Aloe vera J. Ann Pharmacother,, 38(1): Hirakata H, Ushikubi F, Toda H, et al. Sevoflurane inhibits human platelet aggregation and thromboxane A formation, possibly by suppression of cyclooxygenase activity J. Anesthesiology, 199, 85 (): Wacker J, Lucchinetti E, Jamnicki M, et al. Delayed inhibition of agonist-induced granulocyte-platelet aggregation after low-dose sevoflurane inhalation in humans J. Anesth Analg, 8, 1(): Ishibe Y, Gui X, Uno H, et al. Effect of sevoflurane on hypoxic pulmonary vasoconstriction in the perfused rabbit lung J. Anesthesiology, 1993, 79(): Farragher R, Maharaj CH, Higgins BD, et al. Sevoflurane and the feto-placental vasculature: the role of nitric oxide and vasoactive eicosanoids J. Anesth Analg, 8, 17(1): Heindl B, Reichle F, Becker BF. Sevoflurane but not isoflurane can reduce prostacyclin production of endothelial cells J. Eur J Anaesthesiol, 3, (): 从 CO COX X 和 5-LOX 途径探讨七氟醚抗单肺通气致急性肺损伤的 作用机 作用 机制 刘 睿 1 罗 静 3 李 江 马庆杰 李艳华 王殿华 1 昆明医科大学 1 药学院暨云南省天然药物药理重点实验室 云南 昆明 531 云南省第一人民医院 麻醉科 3 疼痛科 云南 昆明 53 摘要 目的 从COX 和5-LOX途径探讨七氟醚抗单肺通气致急性肺损伤作用机制 方法 18只健康日本大耳白兔随机分为假手 术组 S 组 单肺通气组 O 组 单肺通气+七氟醚组 OS 组 每组 只 用 Western blotting 和定量 RT-PCR 分别检测肺组织环 氧化酶- COX 5-脂氧化酶 5-LOX 蛋白和 mrna 表达水平 ELISA 检测肺组织白细胞三烯 B LTB 血栓素 A TXA 和 前列腺素 I PGI 含量 以肺湿/干 W/D 比值和肺组织学评分评价肺损伤的严重程度 结果 与 S 组相比 O 组和 OS 组动物肺 组织 COX 5-LOX 蛋白和 mrna 表达水平 LTB TXA PGI 含量 肺 W/D 比值及肺组织学评分均明显升高 P<.5 而肺组 织 PGI/TXA 比值明显降低 P<.5 与 O 组相比 OS 组肺组织 PGI/TXA 比值明显增高 P<.5 而其它各项指标均明显降 低 P<.5 结论 本研究首次证实单肺通气可使实验动物肺组织 COX 5-LOX 的蛋白和 mrna 表达水平增高 七氟醚具有 抗单肺通气致急性肺损伤的作用 其机制可能与下调肺组织COX 和5-LOX的表达水平 减少肺组织PGI TXA 和LTB 生成及 调控PGI/TXA 比值有关 关键词 单肺通气 七氟醚 环氧化酶- 5-脂氧化酶 收稿日期 基金项目 国家自然科学基金 383 作者简介 刘 睿 在读博士研究生 主治医师 liur13@hotmail.com 罗 静 学士 主治医师 @qq.com 刘 睿 罗 静共 同为第一作者 通信作者 王殿华 教授 博士生导师 wangdianhuakm@1.com