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Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemiareperfusion injury in mice Yaqin Xiong a, b, 1, Huan Yao c, 1, Yan Cheng d, Deying Gong d, Xin Liao e, Rurong Wang c, * a
Department of Anesthesiology, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China Key Laboratory of Birth Defects and Related Diseases of Women and Children, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China c Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China d Laboratory of Anesthesia and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China e Department of Operation Room, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2018 Accepted 16 October 2018 Available online xxx
Lung ischemia-reperfusion injury (LIRI) is a common and severe postoperative pathologic complication that often occurs when the oxygen supply disrupted to the lung tissue fallowed by reperfusion period, in most cases after lung transplantation and cardiopulmonary bypass. Endocannabinoids such as 2arachidonoylglycerol (2-AG) have very important role as regulators of inflammation. Monoacylglycerol lipase (MAGL) is the main 2-AG-degrading enzyme, and the downstream metabolites of 2-AG play a role in the inflammation. Ischemia reperfusion (IR) was induced by clamping the left pulmonary hilum for 60 min, followed by 120 min of reperfusion in male C57BL/6 mice. Effects of URB602, a MAGL inhibitor, were evaluated in a preventive or therapeutic regimen (5 min before ischemia or reperfusion, respectively). Oxygenation index, wet-to-dry weight ratio and lung injury score were analyzed. Endocannabinoids including 2-AG, anandamide (AEA) and arachidonic acid (AA) levels, metabolites such as Prostaglandin I2 (PGI2), Thromboxane B2 (TXB2) and Leukotrienes B4 (LTB4) and inflammatory markers (Interleukin 6 (IL-6) andTumor necrosis factor-a (TNF-a)) in lung tissues were measured by using mass spectrometry or ELISA analyses. We found that IR increased the wet-to-dry weight ratio of lung and lung injury score and decreased oxygenation index as compared to the sham group. Moreover, treatment with URB602 in preventive or therapeutic regimen reduced the wet-to-dry weight ratio and lung injury score while increased oxygenation index when compared with the IR group, with a more improvement in the preventive regimen group. In addition, treatment with URB602 before ischemia increased 2-AG level but decreased metabolites (AA, PGI2, TXB2, LTB4) and inflammatory markers (IL-6, TNF-a). Thus, our study demonstrated that a pretreatment with URB602 significantly reduced IR-induced lung injury and inflammation. URB602 inhibited LIRI and inflammation by increasing 2-AG level and reducing downstream metabolites from AA to PGI2, TXB2 and LTB4 in lung tissues. © 2018 Elsevier Inc. All rights reserved.
Keywords: Monoacylglycerol lipase Ischemia reperfusion Lung injury
1. Introduction Lung ischemia-reperfusion injury (LIRI) is a common and severe postoperative complication that often occurs after lung transplantation, cardiopulmonary bypass, cardiopulmonary resuscitation, pulmonary embolism, and sepsis [1,2]. Recently,
* Corresponding author. Department of Anesthesiology, West China Hospital of Sichuan University, No. 37 Guoxue Road, Chengdu 610041, Sichuan, PR China. E-mail address: [email protected] (R. Wang). 1 These authors contributed equally to this work.
developments in surgical technology and their subsequent wide application have resulted in an inevitable surge in LIRI. The mechanisms underlying LIRI have been investigated, and the pathogenesis has been found to primarily be due to oxidative stress, inflammation, cellular apoptosis, intracellular calcium overload, and neurogenic inflammatory pathways [3e6]. Unfortunately, these underlying mechanisms are often systematic and complicated and have not yet been completely elucidated. Cannabinoid receptors are abundant in human and rat pulmonary artery endothelial cells [7] and activation of cannabinoid receptors may inhibit oxidative stress and inflammatory response
https://doi.org/10.1016/j.bbrc.2018.10.098 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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[8e10]. Endogenous cannabinoid system consists of endogenous cannabinoids, cannabinoid receptors and endogenous cannabinoid degrading enzymes [11,12]. Arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) are the two most widely studied endocannabinoids, which can be degraded and rapidly inactivated by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively [13]. 2-AG is currently recognized as the most abundant and efficacious endocannabinoid. Although 2-AG might be metabolized by FAAH [14], MAGL plays the predominant role in catalyzing 2-AG hydrolysis. URB602 ([1,10 -biphenyl]-3-yl-carbamic acid, cyclohexyl ester) can specifically and noncompetitively inhibit MAGL activity [15] but increase 2-AG levels without alerting AEA level both in vitro and in vivo [16]. Numerous studies have suggested that the endocannabinoid system may modulate IR injury [17e20]. Although the seeking for the effects of endocannabinoids such as AEA and 2-AG on organ ischemia reperfusion injuries such as liver, heart, brain has received considerable research attention, the effects of MAGL inhibition on LIRI are still unclear. Identifying effective and safe methods to reduce the LIRI is still a hot research area. This work was designed to investigate the potential anti-inflammatory effect of MAGL inhibition in a murine model of LIRI using URB602 as a pharmacological tool. 2. Materials and methods
pentobarbital sodium (100 mg/kg, i.p.), and the left lung tissues were harvested. 2.4. Experimental design This study was conducted in two series of experiments in mice. The first series of experiments were performed in five groups to determine the effect of URB602 on LIRI in mice. Mice were randomly assigned into five groups, 8 mice were assigned to each group: (1) Sham group: After thoracotomy, mechanical ventilation was carried out for 3 h without ligation of hilum; (2) URB602 þ Sham group: 5 min before thoracotomy a quantity of 5 mg/kg URB602 was administered, the other is the same as Sham group; (3) Ischemia reperfusion group (IR group): after thoracotomy, left hilum was occluded for 1 h and then the occlusion was released for 2 h sustained reperfusion; (4) URB602 þ IR group: 5 min before occlusion, 5 mg/kg URB602 was administered, then left hilum was occluded for 1 h and released for 2 h sustained reperfusion; (5) IR þ URB602 group: 55min after the occlusion of left hilum, 5 mg/kg URB602 was administered, and left hilum continued to occlude for 5 min and release for 2 h. The second series of experiments were performed in four groups to determine the role of endocannabinoids metabolite pathway upon URB602 generated improvement of LIRI (n ¼ 8). The mice were assigned to the following groups: one group of sham (S), ischemia reperfusion (IR), URB602 þ sham, and URB602 þ IR.
2.1. Animals and treatments 2.5. Blood gas analyses All experimental procedures were carried out in accordance with the Institutional Animal Care and Use Committee of Sichuan University (No. 2016032A) and conformed to the guidelines of the National Institutes of Health (NIH), USA. Male C57BL/6 mice (Experimental Animal Center of Clinical Medical College of Sichuan University) weighing 20e25 g were housed under controlled temperature (22 ± 1 C), humidity (60 ± 10%) and light (12 h/day). All the animals were fasted for 8 h before the experiment. 2.2. Drugs URB602 (Sigma, America) was dissolved in Tween 80: DMSO: saline (1:1:18) to a concentration of 5 mg/ml. It was administered intraperitoneally (i.p.) to mice at 5 mg/kg. Animals in the control groups received a same volume of vehicle i.p.. The doses of URB602 used here were based on previous works in mice [14]. 2.3. Lung ischemia reperfusion model An in vivo mouse model of lung ischemia reperfusion was established based on previously published studies [21]. Mice were anesthetized by an intraperitoneal injection of 1.0% pentobarbital sodium (60 mg/kg). After endotracheal tube insertion, each mouse was ventilated with positive pressure using a HARVARD-Inspira ASVP rodent ventilator (Harvard Apparatus, Boston, MA) with an inspiratory oxygen fraction (FiO2) at 21% at a rate of 120 breaths/ min and a tidal volume of 6e8 mL/kg. Thoracotomy was performed at the anterior lateral side of the left fourth intercostal. Heparin (500U/kg) was given intraperitoneally for prevention of thrombus formation. Ischemia was induced by clamping the left pulmonary hilum (including the left main bronchus, artery and vein) with an artery clamp. After 1 h of ischemia, the occlusion was released for 2 h sustained reperfusion. The sham group underwent the left thoracotomy with hilum dissection and was ventilated for 3 h. During the experiment, the incision was covered with wet absorbent gauze to minimize evaporative losses. At the end of the experiment, the mice were euthanized with a large dose of
Arterial blood was collected from left ventricle of each mice immediately after the experiments. Blood gas analyses were performed and the ratio of PaO2 to FiO2 was obtained as an oxygenation index. The PaO2/FiO2 ratio is used to measure physiological dysfunction. 2.6. Wet-to-Dry weight ratio The left lungs were harvested for measurement of lung wet-todry weight (W/D) ratio, which is an estimation of lung tissue edema. The wet lung was weighed, and then placed in an oven at 80 C and weighed daily until its weight stabilizes (dry weight). 2.7. Histopathological examination Blocks of lung approximately 0.5 cm3 were taken from the left lung tissue. After processing and staining with hematoxylin-eosin, the lung samples were ranked by a pathologist who was blind to the experimental group. A modified acute lung injury score was used to determine the degree of lung damage: (a) Neutrophils in the alveolar space, (b) Neutrophils in the interstitial space, (c) Hyaline membranes, (d) Proteinaceous debris filling the airspaces, (e) Alveolar septal thickening [22]. 2.8. Endocannabinoid measurements Endocannabinoids including 2-AG, AEA and AA were measured using single-reaction monitoring-based liquid chromatographytandem mass spectrometry analysis, as previously described [23,24]. The lung tissue was cut in ice bath and 1 mL of pre-cooled 0.1% formic acid solution, 2 mg d8-AA, 3 ml of ethyl acetate-n-hexane mixed solution (9:1) was added. The lung tissue was ultrasonically homogenized for 1 min using an ultrasonic homogenizer, 8000 rate/min at 4 C and centrifuged for 15min. The upper organic phase was dried under nitrogen stream and the residue was dissolved in 200 ml of acetonitrile. 2-AG, AEA and AA contents were
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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determined by an Agilent 1260e6460 high performance liquid chromatography mass spectrometer (HPLC-MS) and an Agilent Extend C18 (3.0 100 mm, 3.5 mm) column. 2.9. Metabolite and cytokine analysis ELISA kits were used to measure Leukotrienes B4 (LTB4), Prostaglandin I2 (PGI2) and Thromboxane B2 (TXB2), Interleukin 6 (IL-6), Tumor necrosis factor-a (TNF-a) in lung tissue lysates. The assays were performed according to the manufactures’ instructions (Xinfan biotechnology company, Shanghai, China). 2.10. Statistical analysis SPSS 22 (SPSS Inc., Chicago, IL) was used for statistical analysis. The distribution of quantitative data was analyzed by the Kolmogorov-Smirnov test. Normally distributed variables were presented as mean ± standard deviation. Levene's test was used to evaluate the homogeneity of variances. Parametric data were analyzed by one-way ANOVA followed by LSD test for multiple comparisons. A value of P < 0.05 was defined as statistically significant. 3. Results 3.1. Effect of URB602 on blood gas exchange after lung ischemiareperfusion At the end of reperfusion, PaO2 and FiO2 were measured and the PaO2/FiO2 ratio was calculated. Administration of URB602 itself had no effect on the oxygenation ability of lung while ischemiareperfusion inhibited the blood gas exchange of lung compared to the sham group. Treatment by URB602 in either way, before the IR injury or after the ischemia of lung, effectively rescued the disturbed function of lung after IR (indicated by oxygenation index) with the fact that a less rescue extent was found in the IR þ URB602 group compared to URB602 þ IR group (Fig. 1A). 3.2. Effect of URB602 on lung wet-to-dry weight ratio The lung wet-to-dry (W/D) weight ratio was used as an index of lung water accumulation after the injury and the treatment with URB602. While URB602 alone had no effect on the W/D ratio, the ratio increased in the IR group, indicating that water content was elevated after IR injury, therefore suggesting the increased permeability of the alveolocapillary membrane after IR injury. This IR induced lung damage was prevented by administration of URB602 before IR was performed (URB602 þ IR group compared to IR group), demonstrating the protective function of URB602 on lung structure. In contrast, usage of URB602 right after the occurrence of ischemia showed no such protective effect (IR þ URB602 group compared to IR group), indicating the importance of time point of drug administration (Fig. 1B).
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and inflammation, while treatment with URB602 before IR statistically rescue the damage induced by IR. Since the protective effect of URB602 was always better if used before IR than used after IR in our study, only group of URB602 þ IR was used in the following studies to show the effect of URB602. 3.4. Effect of URB602 on endocannabinoid and eicosanoid Metabolism Consistent with previous reports [16e23], 2-AG and AEA levels increased in IR group compared to that in sham group. URB602 alone increased 2-AG level but not AEA level, which again validated the specificity of its inhibitory function on MAGL, the monoacylglycerol lipase that play predominant role on 2-AG hydrolysis (Fig. 2A and B). AA was a metabolite downstream of AEA and 2-AG [11]. The level of AA was increased in the URB602 alone group compared to sham group, which may suggest a compensatory mechanism was activated by usage of URB602 in the lungs of this group, catalyzing the conversion of AEA to AA. Administration of URB602 on top of IR efficiently decrease AA level compared to IR group (Fig. 2C), demonstrating that URB602 inhibited the hydrolysis of 2-AG to AA by inhibiting MAGL activity. AA was reported to be further metabolized by COX [25,26] and LOX [27] pathways and the metabolites were involved in the inflammation response. The representative metabolites of AA in these 2 pathways are LTB4, PGI2 and TXB2, whose were also measured in our experiment. Compared to sham group, IR induced the increase of all the 3 metabolites, which was expected since IR induced lung damage and inflammation. However, when mice were treated with URB602 alone, LTB4 and PGI2 levels were down-regulated but not TXB2 level. When URB602 was administrated upon IR injury, levels of all the 3 metabolites were down-regulated compared to that in IR group (Fig. 3DeF). This result indicated that URB602 probably have different effect extent on different metabolism pathway of AA. For example, less extent on COX pathway (indicated by TXB2 level) in our study. Noticeably, the mean level of TXB2 was still a little bit higher in URB602 þ IR group compared to sham group, while the mean level of LTB4 and PGI2 in URB602 þ IR group were lower than that in sham group, though there may not be a statistical significance. This result further indicated that URB602 affects different metabolism pathways of AA to a difference extent. 3.5. Effect of URB602 on cytokines Levels of IL-6 and TNF-a in the IR group increased significantly as compared with the Sham and URB602 þ Sham groups (P < 0.05), suggesting the inflammation induced by IR and URB602 alone would not induce inflammation When applied on top of IR, URB602 decreased IL-6 and TNF-a values significantly in the URB602 þ IR group compared to that in the IR group (P < 0.05) (Fig. 3A and B), indicating that URB602 significantly inhibited the inflammation induced by IR. 4. Discussion
3.3. Effect of URB602 on histopathology Pictures (Fig. 1C) demonstrated the representative histology of lung in each experimental group. URB602 alone didn't change the histology of lungs, obvious swelling of the lung walls, infiltration of inflammatory cells, and proteinaceous debris were observed after IR. Administration of URB602 could improve the histopathologic phenomena in lungs if used before IR occurrence but to a less extent after IR compared to the URB602 þ IR group. Excised lungs in each group were evaluated and the result was summarized (Fig. 1D). Consistent with our hypothesis, IR induced lung structure damage
In this study, we found that MAGL inhibitor URB602 reduced LIRI and inflammation through coordinately enhancing 2-AG level and downregulating eicosanoid pathways. A better protective effect was observed with a single dose of URB602 before ischemia than before reperfusion. It is well known that ischemia tissue produces and accumulates both 2-AG and AEA, whose protective role has been studied in heart, brain, and liver either by exogenous administration or by the inhibition of their metabolic enzymes [28e30]. MAGL is the main enzyme involved in 2-AG hydrolysis [31]. Specific MAGL inhibitors
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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Fig. 1. Oxygenation indexes changes and the results of W/D ratio, histopathologic evaluation, lung injury score. (A) URB602 improved Oxygenation indexes (PaO2/FiO2). (B) Only URB602 used before IR improved pulmonary edema. (C) Histopathologic evaluation for each group of mice. (D) Effects of IR and URB602 on lung injury score. Lung injury score was based on histopathologic evaluation result for each group. Results are expressed by means ± SD (n ¼ 8). IR increased lung injury score, while treatment with URB602 before IR statistically decreased the lung injury score. *p < 0.05 versus the IR group.
could block 2-AG deactivation and therefore amplify its intrinsic actions. URB602 is known to increase 2-AG levels and enhance 2AG mediated signaling [15,16], without affecting the activities of other lipid-metabolizing enzymes such as FAAH (which hydrolyzes AEA), diacylglycerol lipase, or COX-2 [16,32]. Thus, URB602 is a selective inhibitor of MAGL activity that may therefore regulate the
2-AG signaling. In the first part of this study, we established a model of LIRI in mice and applied URB602 at different time points. The development and progression of LIRI includes two stages. At first, long-term blocking of blood flow, lung collapse, atelectasis, ischemia and hypoxia would promote the release of inflammatory factors and
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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Fig. 2. Effect of URB602 on endocannabinoid and eicosanoid Metabolism. (A) 2-AG level. (B) AEA level. (C) AA level. (D) LTB4 level. (E) PGI2 level. (F) TXB2 level. *p < 0.05 versus the IR group; *p < 0.05 Sham Group versus URB602 þ Sham Group.
local acidic metabolites. Secondly, re-ventilation and blood flow reperfusion could increase free radical release and induce oxidative stress. Therefore, URB602 was administered at 5 min before ischemia (URB602 þ IR group) or 5 min before reperfusion (IR þ URB602 group). We demonstrated that administration of URB602 before ischemia and reperfusion both reduced LIRI. Other researches [23] have also reported similar results on hepatic ischemia reperfusion by administering MAGL inhibitor at various time points. Interestingly, injection of URB602 before ischemia produced better outcomes of lung injury score and lung W/D ratio as compared to before reperfusion. The possible reason may be that cell apoptosis induced by IR occurs mainly during the ischemia period, the use of URB602 after ischemia (just 5 min before reperfusion) is helpless to reverse the damage [33]. It is well known that LIRI is related to inflammatory response. In the second part of this study we also found IR increased the level of
IL-6 and TNF-a in the lung tissue and URB602 decreased the inflammatory factors, indicating URB602 reduced lung inflammation resulted from LIRI. AA is involved in the initiation of the inflammatory reaction through degradation by COX [25,26] and LOX [27] pathway, the metabolites include TXB2, PGI2, LTB4, LTC4, LTD4, LTE4, and so on. These metabolites play different roles in the inflammatory response. PGI2 is produced in vascular endothelial cells and can promote vasodilatation and inhibition of platelet aggregation [34]. TXB2 is produced in platelets and contracts blood vessels and promotes platelet aggregation [35]. LTB4 is derived from activated macrophages, neutrophils, platelets, mast cells, and has the effect of leukocyte activation, neutrophil adhesion and chemotaxis [36]. It has been reported that inhibition of COX2 and 5-LOX reduces lung injury by reducing related metabolites [37,38]. Maybe, URB602 can affect the production of downstream inflammatory metabolites by acting on the upstream biologically active substance of AA.
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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selective MAGL inhibitor, prevent and treat LIRI in mice in vivo. In addition, endogenous cannabinoid metabolism pathways play an important role in the URB602 attenuated LIRI and inflammation by increasing 2-AG levels in lung tissue and reducing the generation of downstream metabolites from AA to LTB4, PGI2 and TXB2. Conflicts of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.098. References
Fig. 3. Effect of URB602 on Cytokines. (A) IL-6 level. Levels of IL-6 in the IR group increased as compared with the Sham and URB602 þ Sham groups. When applied on top of IR, URB602 decreased IL-6 values significantly in the URB602 þ IR group. (B) TNF-a level. Levels of TNF-a in the IR group increased as compared with the Sham and URB602 þ Sham groups. When applied on top of IR, URB602 decreased TNF-a values significantly in the URB602 þ IR group. *p < 0.05 versus the IR group.
Furthermore, 2-AG can produce AA via hydrolysis mediated by MAGL. Thus, 2-AG-AA-PGI2, TXB2 and LTB4 constitute an important metabolic pathway of endocannabinoids. To ascertain the contribution of endocannabinoid signaling pathway in the protective effect of URB602, we tested the level of endocannabinoids and eicosanoids. We found that URB602 produced a pronounced 2-AG elevation without affecting AEA levels, confirming its selectively inhibitory activity at MAGL only, which is consistent with previous report showing the ability of this drug to elevate 2-AG only in mouse brain in vivo [39]. Both 2-AG and AEA can produce AA by hydrolysis [11]. On one hand, in agreement with previous study, the level of AA decreased after administration of URB602 due to its inhibitory activity at MAGL and the subsequently reduced the formation of AA [23]. On the other hand, the level of AA in the URB602 þ sham group was higher than that in the sham group, but less than that in the IR group, which probably indicates a compensatory increase in the hydrolysis of AEA after the hydrolysis of 2-AG is inhibited. Since 2-AG acts as potent endogenous ligand for both cannabinoid receptor subtypes, the systemic administration of URB602 at too high doses might enhance the 2-AG level in brain areas, thereby producing adverse psychoactive effects that could limit the therapeutic employment of such a compound [39]. A report by Comelli et al. [14] indicated that the systemic administration of URB602 5 mg/kg prevented the development of carrageenan-induced edema and did not show any cannabimimetic activity. Therefore, a single dose of 5 mg/kg were used in our study. In conclusion, this study provides evidence to that URB602, the
[1] G. Ailawadi, C.L. Lau, P.W. Smith, et al., Does reperfusion injury still cause significant mortality after lung transplantation? J. Thorac. Cardiovasc. Surg. 137 (2009) 688e694. [2] G.D. Rubenfeld, E. Caldwell, E. Peabody, et al., Incidence and outcomes of acute lung injury, N. Engl. J. Med. 353 (2005) 1685e1693. [3] M. de Perrot, M. Liu, T.K. Waddell, et al., Ischemia-reperfusion-induced lung injury, Am. J. Respir. Crit. Care Med. 167 (2003) 490e511. [4] P.D. Weyker, C.A. Webb, D. Kiamanesh, et al., Lung ischemia reperfusion injury: a bench-to-bedside review, Semin. CardioThorac. Vasc. Anesth. 17 (2013) 28e43. [5] F. Drakopanagiotakis, A. Xifteri, V. Polychronopoulos, et al., Apoptosis in lung injury and fibrosis, Eur. Respir. J. 32 (2008) 1631e1638. [6] T. Goto, A. Ishizaka, F. Kobayashi, et al., Importance of tumor necrosis factoralpha cleavage process in post-transplantation lung injury in rats, Am. J. Respir. Crit. Care Med. 170 (2004) 1239e1246. [7] A. Luchicchi, M. Pistis, Anandamide and 2-arachidonoylglycerol: pharmacological properties, functional features, and emerging specificities of the two major endocannabinoids, Mol. Neurobiol. 46 (2012) 374e392. [8] Y. Wei, X. Wang, L. Wang, Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells, Mol. Vis. 15 (2009) 1243e1251. [9] Y. Wei, X. Wang, F. Zhao, et al., Cannabinoid receptor 1 blockade protects human retinal pigment epithelial cells from oxidative injury, Mol. Vis. 19 (2013) 357e366. [10] C. Costola-de-Souza, A. Ribeiro, V. Ferraz-de-Paula, et al., Monoacylglycerol lipase (MAGL) inhibition attenuates acute lung injury in mice, PLoS One 8 (2013), e77706. [11] C. Turcotte, F. Chouinard, J.S. Lefebvre, et al., Regulation of inflammation by cannabinoids, the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their metabolites, J. Leukoc. Biol. 97 (2015) 1049e1070. [12] H.C. Lu, K. Mackie, An introduction to the endogenous cannabinoid system, Biol. Psychiatry 79 (2016) 516e525. [13] P. Pacher, G. Hasko, Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning, Br. J. Pharmacol. 153 (2008) 252e262. [14] F. Comelli, G. Giagnoni, I. Bettoni, et al., The inhibition of monoacylglycerol lipase by URB602 showed an anti-inflammatory and anti-nociceptive effect in a murine model of acute inflammation, Br. J. Pharmacol. 152 (2007) 87, 794. [15] J.K. Makara, M. Mor, D. Fegley, et al., Selective inhibition of 2-AG hydrolysis enhances endocannabinoid signaling in hippocampus, Nat. Neurosci. 8 (2005) 1139e1141. [16] A.G. Hohmann, R.L. Suplita, N.M. Bolton, et al., An endocannabinoid mechanism for stress-induced analgesia, Nature 435 (2005) 1108e1112. [17] C. Lagneux, D. Lamontagne, Involvement of cannabinoids in the cardioprotection induced by lipopolysaccharide, Br. J. Pharmacol. 132 (2001) 793e796. [18] L. Walter, N. Stella, Cannabinoids and neuroinflammation, Br. J. Pharmacol. 141 (2004) 775e785. [19] V.E. Mendizabal, E. Adler-Graschinsky, Cannabinoids as therapeutic agents in cardiovascular disease: a tale of passions and illusions, Br. J. Pharmacol. 151 (2007) 427e440. [20] B. Julien, P. Grenard, F. Teixeira-Clerc, et al., Antifibrogenic role of the cannabinoid receptor CB2 in the liver, Gastroenterology 128 (2005) 742e755. [21] Q. Zhao, W. Wang, R. Wang, et al., TRPV1 and neuropeptide receptor immunoreactivity and expression in the rat lung and brainstem after lung ischemiareperfusion injury, J. Surg. Res. 203 (2016) 183e192. [22] G. Matute-Bello, G. Downey, B.B. Moore, et al., An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals, Am. J. Respir. Cell Mol. Biol. 44 (2011) 725e738. [23] Z. Cao, M.M. Mulvihill, P. Mukhopadhyay, et al., Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice, Gastroenterology 144 (2013) 808e817, e15. [24] A. Ribeiro, V. Ferraz-de-Paula, M.L. Pinheiro, et al., Cannabidiol, a non-
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
Y. Xiong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[25]
[26]
[27]
[28]
[29]
[30]
[31]
psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: role for the adenosine A(2A) receptor, Eur. J. Pharmacol. 678 (2012) 78e85. X.P. Miao, J.S. Li, Q. Ouyang, et al., Tolerability of selective cyclooxygenase 2 inhibitors used for the treatment of rheumatological manifestations of inflammatory bowel disease, Cochrane Database Syst. Rev. 10 (2014). CD007744. J. Valles, A. Lago, A. Moscardo, et al., TXA2 synthesis and COX1-independent platelet reactivity in aspirin-treated patients soon after acute cerebral stroke or transient ischaemic attack, Thromb. Res. 132 (2013) 211e216. J. Jampilek, M. Dolezal, V. Opletalova, et al., 5-Lipoxygenase, leukotrienes biosynthesis and potential antileukotrienic agents, Curr. Med. Chem. 13 (2006) 117e129. D.E. Pellegrini-Giampietro, G. Mannaioni, G. Bagetta, Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death, FEBS J. 276 (2009) 2e12. M. Zhang, B.R. Martin, M.W. Adler, et al., Modulation of cannabinoid receptor activation as a neuroprotective strategy for EAE and stroke, J. Neuroimmune Pharmacol. 4 (2009) 249e259. D. Alonso-Alconada, F.J. Alvarez, A. Alvarez, et al., The cannabinoid receptor agonist WIN 55,212-2 reduces the initial cerebral damage after hypoxicischemic injury in fetal lambs, Brain Res. 1362 (2010) 150e159. T.P. Dinh, S. Kathuria, D. Piomelli, RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-
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arachidonoylglycerol, Mol. Pharmacol. 66 (2004) 1260e1264. [32] A.R. King, A. Duranti, A. Tontini, et al., URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices, Chem. Biol. 14 (2007) 1357e1365. [33] Q. Zhao, J. Wu, Z. Lin, et al., Resolvin D1 alleviates the lung ischemia reperfusion injury via complement, immunoglobulin, TLR4, and inflammatory factors in rats, Inflammation 39 (2016) 1319e1333. [34] S.L. Dorris, R.S. Peebles Jr., PGI2 as a regulator of inflammatory diseases, Mediat. Inflamm. 2012 (2012) 926968. [35] P. Fontana, A. Zufferey, Y. Daali, et al., Antiplatelet therapy: targeting the TxA2 pathway, J. Cardiovasc. Transl. Res. 7 (2014) 29e38. [36] T. Yokomizo, Leukotriene B4 receptors: novel roles in immunological regulations, Adv. Enzym. Regul. 51 (2011) 59e64. [37] R. Liu, J. Luo, J. Li, et al., Protective mechanisms of sevoflurane against onelung ventilation-induced acute lung injury: role of cyclooxygenase-2 and 5lipoxygenase pathways, Nan Fang Yi Ke Da Xue Xue Bao ¼ J. South. Med. Univ. 33 (2013) 625e630. [38] J.A. Robertson, D. Sauer, J.A. Gold, et al., The role of cyclooxygenase-2 in mechanical ventilation-induced lung injury, Am. J. Respir. Cell Mol. Biol. 47 (2012) 387e394. [39] S. Carloni, D. Alonso-Alconada, S. Girelli, et al., Pretreatment with the monoacylglycerol lipase inhibitor URB602 protects from the long-term consequences of neonatal hypoxic-ischemic brain injury in rats, Pediatr. Res. 72 (2012) 400e406.
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Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemiareperfusion injury in mice Yaqin Xiong a, b, 1, Huan Yao c, 1, Yan Cheng d, Deying Gong d, Xin Liao e, Rurong Wang c, * a
Department of Anesthesiology, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China Key Laboratory of Birth Defects and Related Diseases of Women and Children, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China c Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China d Laboratory of Anesthesia and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China e Department of Operation Room, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2018 Accepted 16 October 2018 Available online xxx
Lung ischemia-reperfusion injury (LIRI) is a common and severe postoperative pathologic complication that often occurs when the oxygen supply disrupted to the lung tissue fallowed by reperfusion period, in most cases after lung transplantation and cardiopulmonary bypass. Endocannabinoids such as 2arachidonoylglycerol (2-AG) have very important role as regulators of inflammation. Monoacylglycerol lipase (MAGL) is the main 2-AG-degrading enzyme, and the downstream metabolites of 2-AG play a role in the inflammation. Ischemia reperfusion (IR) was induced by clamping the left pulmonary hilum for 60 min, followed by 120 min of reperfusion in male C57BL/6 mice. Effects of URB602, a MAGL inhibitor, were evaluated in a preventive or therapeutic regimen (5 min before ischemia or reperfusion, respectively). Oxygenation index, wet-to-dry weight ratio and lung injury score were analyzed. Endocannabinoids including 2-AG, anandamide (AEA) and arachidonic acid (AA) levels, metabolites such as Prostaglandin I2 (PGI2), Thromboxane B2 (TXB2) and Leukotrienes B4 (LTB4) and inflammatory markers (Interleukin 6 (IL-6) andTumor necrosis factor-a (TNF-a)) in lung tissues were measured by using mass spectrometry or ELISA analyses. We found that IR increased the wet-to-dry weight ratio of lung and lung injury score and decreased oxygenation index as compared to the sham group. Moreover, treatment with URB602 in preventive or therapeutic regimen reduced the wet-to-dry weight ratio and lung injury score while increased oxygenation index when compared with the IR group, with a more improvement in the preventive regimen group. In addition, treatment with URB602 before ischemia increased 2-AG level but decreased metabolites (AA, PGI2, TXB2, LTB4) and inflammatory markers (IL-6, TNF-a). Thus, our study demonstrated that a pretreatment with URB602 significantly reduced IR-induced lung injury and inflammation. URB602 inhibited LIRI and inflammation by increasing 2-AG level and reducing downstream metabolites from AA to PGI2, TXB2 and LTB4 in lung tissues. © 2018 Elsevier Inc. All rights reserved.
Keywords: Monoacylglycerol lipase Ischemia reperfusion Lung injury
1. Introduction Lung ischemia-reperfusion injury (LIRI) is a common and severe postoperative complication that often occurs after lung transplantation, cardiopulmonary bypass, cardiopulmonary resuscitation, pulmonary embolism, and sepsis [1,2]. Recently,
* Corresponding author. Department of Anesthesiology, West China Hospital of Sichuan University, No. 37 Guoxue Road, Chengdu 610041, Sichuan, PR China. E-mail address: [email protected] (R. Wang). 1 These authors contributed equally to this work.
developments in surgical technology and their subsequent wide application have resulted in an inevitable surge in LIRI. The mechanisms underlying LIRI have been investigated, and the pathogenesis has been found to primarily be due to oxidative stress, inflammation, cellular apoptosis, intracellular calcium overload, and neurogenic inflammatory pathways [3e6]. Unfortunately, these underlying mechanisms are often systematic and complicated and have not yet been completely elucidated. Cannabinoid receptors are abundant in human and rat pulmonary artery endothelial cells [7] and activation of cannabinoid receptors may inhibit oxidative stress and inflammatory response
https://doi.org/10.1016/j.bbrc.2018.10.098 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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[8e10]. Endogenous cannabinoid system consists of endogenous cannabinoids, cannabinoid receptors and endogenous cannabinoid degrading enzymes [11,12]. Arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) are the two most widely studied endocannabinoids, which can be degraded and rapidly inactivated by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively [13]. 2-AG is currently recognized as the most abundant and efficacious endocannabinoid. Although 2-AG might be metabolized by FAAH [14], MAGL plays the predominant role in catalyzing 2-AG hydrolysis. URB602 ([1,10 -biphenyl]-3-yl-carbamic acid, cyclohexyl ester) can specifically and noncompetitively inhibit MAGL activity [15] but increase 2-AG levels without alerting AEA level both in vitro and in vivo [16]. Numerous studies have suggested that the endocannabinoid system may modulate IR injury [17e20]. Although the seeking for the effects of endocannabinoids such as AEA and 2-AG on organ ischemia reperfusion injuries such as liver, heart, brain has received considerable research attention, the effects of MAGL inhibition on LIRI are still unclear. Identifying effective and safe methods to reduce the LIRI is still a hot research area. This work was designed to investigate the potential anti-inflammatory effect of MAGL inhibition in a murine model of LIRI using URB602 as a pharmacological tool. 2. Materials and methods
pentobarbital sodium (100 mg/kg, i.p.), and the left lung tissues were harvested. 2.4. Experimental design This study was conducted in two series of experiments in mice. The first series of experiments were performed in five groups to determine the effect of URB602 on LIRI in mice. Mice were randomly assigned into five groups, 8 mice were assigned to each group: (1) Sham group: After thoracotomy, mechanical ventilation was carried out for 3 h without ligation of hilum; (2) URB602 þ Sham group: 5 min before thoracotomy a quantity of 5 mg/kg URB602 was administered, the other is the same as Sham group; (3) Ischemia reperfusion group (IR group): after thoracotomy, left hilum was occluded for 1 h and then the occlusion was released for 2 h sustained reperfusion; (4) URB602 þ IR group: 5 min before occlusion, 5 mg/kg URB602 was administered, then left hilum was occluded for 1 h and released for 2 h sustained reperfusion; (5) IR þ URB602 group: 55min after the occlusion of left hilum, 5 mg/kg URB602 was administered, and left hilum continued to occlude for 5 min and release for 2 h. The second series of experiments were performed in four groups to determine the role of endocannabinoids metabolite pathway upon URB602 generated improvement of LIRI (n ¼ 8). The mice were assigned to the following groups: one group of sham (S), ischemia reperfusion (IR), URB602 þ sham, and URB602 þ IR.
2.1. Animals and treatments 2.5. Blood gas analyses All experimental procedures were carried out in accordance with the Institutional Animal Care and Use Committee of Sichuan University (No. 2016032A) and conformed to the guidelines of the National Institutes of Health (NIH), USA. Male C57BL/6 mice (Experimental Animal Center of Clinical Medical College of Sichuan University) weighing 20e25 g were housed under controlled temperature (22 ± 1 C), humidity (60 ± 10%) and light (12 h/day). All the animals were fasted for 8 h before the experiment. 2.2. Drugs URB602 (Sigma, America) was dissolved in Tween 80: DMSO: saline (1:1:18) to a concentration of 5 mg/ml. It was administered intraperitoneally (i.p.) to mice at 5 mg/kg. Animals in the control groups received a same volume of vehicle i.p.. The doses of URB602 used here were based on previous works in mice [14]. 2.3. Lung ischemia reperfusion model An in vivo mouse model of lung ischemia reperfusion was established based on previously published studies [21]. Mice were anesthetized by an intraperitoneal injection of 1.0% pentobarbital sodium (60 mg/kg). After endotracheal tube insertion, each mouse was ventilated with positive pressure using a HARVARD-Inspira ASVP rodent ventilator (Harvard Apparatus, Boston, MA) with an inspiratory oxygen fraction (FiO2) at 21% at a rate of 120 breaths/ min and a tidal volume of 6e8 mL/kg. Thoracotomy was performed at the anterior lateral side of the left fourth intercostal. Heparin (500U/kg) was given intraperitoneally for prevention of thrombus formation. Ischemia was induced by clamping the left pulmonary hilum (including the left main bronchus, artery and vein) with an artery clamp. After 1 h of ischemia, the occlusion was released for 2 h sustained reperfusion. The sham group underwent the left thoracotomy with hilum dissection and was ventilated for 3 h. During the experiment, the incision was covered with wet absorbent gauze to minimize evaporative losses. At the end of the experiment, the mice were euthanized with a large dose of
Arterial blood was collected from left ventricle of each mice immediately after the experiments. Blood gas analyses were performed and the ratio of PaO2 to FiO2 was obtained as an oxygenation index. The PaO2/FiO2 ratio is used to measure physiological dysfunction. 2.6. Wet-to-Dry weight ratio The left lungs were harvested for measurement of lung wet-todry weight (W/D) ratio, which is an estimation of lung tissue edema. The wet lung was weighed, and then placed in an oven at 80 C and weighed daily until its weight stabilizes (dry weight). 2.7. Histopathological examination Blocks of lung approximately 0.5 cm3 were taken from the left lung tissue. After processing and staining with hematoxylin-eosin, the lung samples were ranked by a pathologist who was blind to the experimental group. A modified acute lung injury score was used to determine the degree of lung damage: (a) Neutrophils in the alveolar space, (b) Neutrophils in the interstitial space, (c) Hyaline membranes, (d) Proteinaceous debris filling the airspaces, (e) Alveolar septal thickening [22]. 2.8. Endocannabinoid measurements Endocannabinoids including 2-AG, AEA and AA were measured using single-reaction monitoring-based liquid chromatographytandem mass spectrometry analysis, as previously described [23,24]. The lung tissue was cut in ice bath and 1 mL of pre-cooled 0.1% formic acid solution, 2 mg d8-AA, 3 ml of ethyl acetate-n-hexane mixed solution (9:1) was added. The lung tissue was ultrasonically homogenized for 1 min using an ultrasonic homogenizer, 8000 rate/min at 4 C and centrifuged for 15min. The upper organic phase was dried under nitrogen stream and the residue was dissolved in 200 ml of acetonitrile. 2-AG, AEA and AA contents were
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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determined by an Agilent 1260e6460 high performance liquid chromatography mass spectrometer (HPLC-MS) and an Agilent Extend C18 (3.0 100 mm, 3.5 mm) column. 2.9. Metabolite and cytokine analysis ELISA kits were used to measure Leukotrienes B4 (LTB4), Prostaglandin I2 (PGI2) and Thromboxane B2 (TXB2), Interleukin 6 (IL-6), Tumor necrosis factor-a (TNF-a) in lung tissue lysates. The assays were performed according to the manufactures’ instructions (Xinfan biotechnology company, Shanghai, China). 2.10. Statistical analysis SPSS 22 (SPSS Inc., Chicago, IL) was used for statistical analysis. The distribution of quantitative data was analyzed by the Kolmogorov-Smirnov test. Normally distributed variables were presented as mean ± standard deviation. Levene's test was used to evaluate the homogeneity of variances. Parametric data were analyzed by one-way ANOVA followed by LSD test for multiple comparisons. A value of P < 0.05 was defined as statistically significant. 3. Results 3.1. Effect of URB602 on blood gas exchange after lung ischemiareperfusion At the end of reperfusion, PaO2 and FiO2 were measured and the PaO2/FiO2 ratio was calculated. Administration of URB602 itself had no effect on the oxygenation ability of lung while ischemiareperfusion inhibited the blood gas exchange of lung compared to the sham group. Treatment by URB602 in either way, before the IR injury or after the ischemia of lung, effectively rescued the disturbed function of lung after IR (indicated by oxygenation index) with the fact that a less rescue extent was found in the IR þ URB602 group compared to URB602 þ IR group (Fig. 1A). 3.2. Effect of URB602 on lung wet-to-dry weight ratio The lung wet-to-dry (W/D) weight ratio was used as an index of lung water accumulation after the injury and the treatment with URB602. While URB602 alone had no effect on the W/D ratio, the ratio increased in the IR group, indicating that water content was elevated after IR injury, therefore suggesting the increased permeability of the alveolocapillary membrane after IR injury. This IR induced lung damage was prevented by administration of URB602 before IR was performed (URB602 þ IR group compared to IR group), demonstrating the protective function of URB602 on lung structure. In contrast, usage of URB602 right after the occurrence of ischemia showed no such protective effect (IR þ URB602 group compared to IR group), indicating the importance of time point of drug administration (Fig. 1B).
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and inflammation, while treatment with URB602 before IR statistically rescue the damage induced by IR. Since the protective effect of URB602 was always better if used before IR than used after IR in our study, only group of URB602 þ IR was used in the following studies to show the effect of URB602. 3.4. Effect of URB602 on endocannabinoid and eicosanoid Metabolism Consistent with previous reports [16e23], 2-AG and AEA levels increased in IR group compared to that in sham group. URB602 alone increased 2-AG level but not AEA level, which again validated the specificity of its inhibitory function on MAGL, the monoacylglycerol lipase that play predominant role on 2-AG hydrolysis (Fig. 2A and B). AA was a metabolite downstream of AEA and 2-AG [11]. The level of AA was increased in the URB602 alone group compared to sham group, which may suggest a compensatory mechanism was activated by usage of URB602 in the lungs of this group, catalyzing the conversion of AEA to AA. Administration of URB602 on top of IR efficiently decrease AA level compared to IR group (Fig. 2C), demonstrating that URB602 inhibited the hydrolysis of 2-AG to AA by inhibiting MAGL activity. AA was reported to be further metabolized by COX [25,26] and LOX [27] pathways and the metabolites were involved in the inflammation response. The representative metabolites of AA in these 2 pathways are LTB4, PGI2 and TXB2, whose were also measured in our experiment. Compared to sham group, IR induced the increase of all the 3 metabolites, which was expected since IR induced lung damage and inflammation. However, when mice were treated with URB602 alone, LTB4 and PGI2 levels were down-regulated but not TXB2 level. When URB602 was administrated upon IR injury, levels of all the 3 metabolites were down-regulated compared to that in IR group (Fig. 3DeF). This result indicated that URB602 probably have different effect extent on different metabolism pathway of AA. For example, less extent on COX pathway (indicated by TXB2 level) in our study. Noticeably, the mean level of TXB2 was still a little bit higher in URB602 þ IR group compared to sham group, while the mean level of LTB4 and PGI2 in URB602 þ IR group were lower than that in sham group, though there may not be a statistical significance. This result further indicated that URB602 affects different metabolism pathways of AA to a difference extent. 3.5. Effect of URB602 on cytokines Levels of IL-6 and TNF-a in the IR group increased significantly as compared with the Sham and URB602 þ Sham groups (P < 0.05), suggesting the inflammation induced by IR and URB602 alone would not induce inflammation When applied on top of IR, URB602 decreased IL-6 and TNF-a values significantly in the URB602 þ IR group compared to that in the IR group (P < 0.05) (Fig. 3A and B), indicating that URB602 significantly inhibited the inflammation induced by IR. 4. Discussion
3.3. Effect of URB602 on histopathology Pictures (Fig. 1C) demonstrated the representative histology of lung in each experimental group. URB602 alone didn't change the histology of lungs, obvious swelling of the lung walls, infiltration of inflammatory cells, and proteinaceous debris were observed after IR. Administration of URB602 could improve the histopathologic phenomena in lungs if used before IR occurrence but to a less extent after IR compared to the URB602 þ IR group. Excised lungs in each group were evaluated and the result was summarized (Fig. 1D). Consistent with our hypothesis, IR induced lung structure damage
In this study, we found that MAGL inhibitor URB602 reduced LIRI and inflammation through coordinately enhancing 2-AG level and downregulating eicosanoid pathways. A better protective effect was observed with a single dose of URB602 before ischemia than before reperfusion. It is well known that ischemia tissue produces and accumulates both 2-AG and AEA, whose protective role has been studied in heart, brain, and liver either by exogenous administration or by the inhibition of their metabolic enzymes [28e30]. MAGL is the main enzyme involved in 2-AG hydrolysis [31]. Specific MAGL inhibitors
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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Fig. 1. Oxygenation indexes changes and the results of W/D ratio, histopathologic evaluation, lung injury score. (A) URB602 improved Oxygenation indexes (PaO2/FiO2). (B) Only URB602 used before IR improved pulmonary edema. (C) Histopathologic evaluation for each group of mice. (D) Effects of IR and URB602 on lung injury score. Lung injury score was based on histopathologic evaluation result for each group. Results are expressed by means ± SD (n ¼ 8). IR increased lung injury score, while treatment with URB602 before IR statistically decreased the lung injury score. *p < 0.05 versus the IR group.
could block 2-AG deactivation and therefore amplify its intrinsic actions. URB602 is known to increase 2-AG levels and enhance 2AG mediated signaling [15,16], without affecting the activities of other lipid-metabolizing enzymes such as FAAH (which hydrolyzes AEA), diacylglycerol lipase, or COX-2 [16,32]. Thus, URB602 is a selective inhibitor of MAGL activity that may therefore regulate the
2-AG signaling. In the first part of this study, we established a model of LIRI in mice and applied URB602 at different time points. The development and progression of LIRI includes two stages. At first, long-term blocking of blood flow, lung collapse, atelectasis, ischemia and hypoxia would promote the release of inflammatory factors and
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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Fig. 2. Effect of URB602 on endocannabinoid and eicosanoid Metabolism. (A) 2-AG level. (B) AEA level. (C) AA level. (D) LTB4 level. (E) PGI2 level. (F) TXB2 level. *p < 0.05 versus the IR group; *p < 0.05 Sham Group versus URB602 þ Sham Group.
local acidic metabolites. Secondly, re-ventilation and blood flow reperfusion could increase free radical release and induce oxidative stress. Therefore, URB602 was administered at 5 min before ischemia (URB602 þ IR group) or 5 min before reperfusion (IR þ URB602 group). We demonstrated that administration of URB602 before ischemia and reperfusion both reduced LIRI. Other researches [23] have also reported similar results on hepatic ischemia reperfusion by administering MAGL inhibitor at various time points. Interestingly, injection of URB602 before ischemia produced better outcomes of lung injury score and lung W/D ratio as compared to before reperfusion. The possible reason may be that cell apoptosis induced by IR occurs mainly during the ischemia period, the use of URB602 after ischemia (just 5 min before reperfusion) is helpless to reverse the damage [33]. It is well known that LIRI is related to inflammatory response. In the second part of this study we also found IR increased the level of
IL-6 and TNF-a in the lung tissue and URB602 decreased the inflammatory factors, indicating URB602 reduced lung inflammation resulted from LIRI. AA is involved in the initiation of the inflammatory reaction through degradation by COX [25,26] and LOX [27] pathway, the metabolites include TXB2, PGI2, LTB4, LTC4, LTD4, LTE4, and so on. These metabolites play different roles in the inflammatory response. PGI2 is produced in vascular endothelial cells and can promote vasodilatation and inhibition of platelet aggregation [34]. TXB2 is produced in platelets and contracts blood vessels and promotes platelet aggregation [35]. LTB4 is derived from activated macrophages, neutrophils, platelets, mast cells, and has the effect of leukocyte activation, neutrophil adhesion and chemotaxis [36]. It has been reported that inhibition of COX2 and 5-LOX reduces lung injury by reducing related metabolites [37,38]. Maybe, URB602 can affect the production of downstream inflammatory metabolites by acting on the upstream biologically active substance of AA.
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
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selective MAGL inhibitor, prevent and treat LIRI in mice in vivo. In addition, endogenous cannabinoid metabolism pathways play an important role in the URB602 attenuated LIRI and inflammation by increasing 2-AG levels in lung tissue and reducing the generation of downstream metabolites from AA to LTB4, PGI2 and TXB2. Conflicts of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.10.098. References
Fig. 3. Effect of URB602 on Cytokines. (A) IL-6 level. Levels of IL-6 in the IR group increased as compared with the Sham and URB602 þ Sham groups. When applied on top of IR, URB602 decreased IL-6 values significantly in the URB602 þ IR group. (B) TNF-a level. Levels of TNF-a in the IR group increased as compared with the Sham and URB602 þ Sham groups. When applied on top of IR, URB602 decreased TNF-a values significantly in the URB602 þ IR group. *p < 0.05 versus the IR group.
Furthermore, 2-AG can produce AA via hydrolysis mediated by MAGL. Thus, 2-AG-AA-PGI2, TXB2 and LTB4 constitute an important metabolic pathway of endocannabinoids. To ascertain the contribution of endocannabinoid signaling pathway in the protective effect of URB602, we tested the level of endocannabinoids and eicosanoids. We found that URB602 produced a pronounced 2-AG elevation without affecting AEA levels, confirming its selectively inhibitory activity at MAGL only, which is consistent with previous report showing the ability of this drug to elevate 2-AG only in mouse brain in vivo [39]. Both 2-AG and AEA can produce AA by hydrolysis [11]. On one hand, in agreement with previous study, the level of AA decreased after administration of URB602 due to its inhibitory activity at MAGL and the subsequently reduced the formation of AA [23]. On the other hand, the level of AA in the URB602 þ sham group was higher than that in the sham group, but less than that in the IR group, which probably indicates a compensatory increase in the hydrolysis of AEA after the hydrolysis of 2-AG is inhibited. Since 2-AG acts as potent endogenous ligand for both cannabinoid receptor subtypes, the systemic administration of URB602 at too high doses might enhance the 2-AG level in brain areas, thereby producing adverse psychoactive effects that could limit the therapeutic employment of such a compound [39]. A report by Comelli et al. [14] indicated that the systemic administration of URB602 5 mg/kg prevented the development of carrageenan-induced edema and did not show any cannabimimetic activity. Therefore, a single dose of 5 mg/kg were used in our study. In conclusion, this study provides evidence to that URB602, the
[1] G. Ailawadi, C.L. Lau, P.W. Smith, et al., Does reperfusion injury still cause significant mortality after lung transplantation? J. Thorac. Cardiovasc. Surg. 137 (2009) 688e694. [2] G.D. Rubenfeld, E. Caldwell, E. Peabody, et al., Incidence and outcomes of acute lung injury, N. Engl. J. Med. 353 (2005) 1685e1693. [3] M. de Perrot, M. Liu, T.K. Waddell, et al., Ischemia-reperfusion-induced lung injury, Am. J. Respir. Crit. Care Med. 167 (2003) 490e511. [4] P.D. Weyker, C.A. Webb, D. Kiamanesh, et al., Lung ischemia reperfusion injury: a bench-to-bedside review, Semin. CardioThorac. Vasc. Anesth. 17 (2013) 28e43. [5] F. Drakopanagiotakis, A. Xifteri, V. Polychronopoulos, et al., Apoptosis in lung injury and fibrosis, Eur. Respir. J. 32 (2008) 1631e1638. [6] T. Goto, A. Ishizaka, F. Kobayashi, et al., Importance of tumor necrosis factoralpha cleavage process in post-transplantation lung injury in rats, Am. J. Respir. Crit. Care Med. 170 (2004) 1239e1246. [7] A. Luchicchi, M. Pistis, Anandamide and 2-arachidonoylglycerol: pharmacological properties, functional features, and emerging specificities of the two major endocannabinoids, Mol. Neurobiol. 46 (2012) 374e392. [8] Y. Wei, X. Wang, L. Wang, Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells, Mol. Vis. 15 (2009) 1243e1251. [9] Y. Wei, X. Wang, F. Zhao, et al., Cannabinoid receptor 1 blockade protects human retinal pigment epithelial cells from oxidative injury, Mol. Vis. 19 (2013) 357e366. [10] C. Costola-de-Souza, A. Ribeiro, V. Ferraz-de-Paula, et al., Monoacylglycerol lipase (MAGL) inhibition attenuates acute lung injury in mice, PLoS One 8 (2013), e77706. [11] C. Turcotte, F. Chouinard, J.S. Lefebvre, et al., Regulation of inflammation by cannabinoids, the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their metabolites, J. Leukoc. Biol. 97 (2015) 1049e1070. [12] H.C. Lu, K. Mackie, An introduction to the endogenous cannabinoid system, Biol. Psychiatry 79 (2016) 516e525. [13] P. Pacher, G. Hasko, Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning, Br. J. Pharmacol. 153 (2008) 252e262. [14] F. Comelli, G. Giagnoni, I. Bettoni, et al., The inhibition of monoacylglycerol lipase by URB602 showed an anti-inflammatory and anti-nociceptive effect in a murine model of acute inflammation, Br. J. Pharmacol. 152 (2007) 87, 794. [15] J.K. Makara, M. Mor, D. Fegley, et al., Selective inhibition of 2-AG hydrolysis enhances endocannabinoid signaling in hippocampus, Nat. Neurosci. 8 (2005) 1139e1141. [16] A.G. Hohmann, R.L. Suplita, N.M. Bolton, et al., An endocannabinoid mechanism for stress-induced analgesia, Nature 435 (2005) 1108e1112. [17] C. Lagneux, D. Lamontagne, Involvement of cannabinoids in the cardioprotection induced by lipopolysaccharide, Br. J. Pharmacol. 132 (2001) 793e796. [18] L. Walter, N. Stella, Cannabinoids and neuroinflammation, Br. J. Pharmacol. 141 (2004) 775e785. [19] V.E. Mendizabal, E. Adler-Graschinsky, Cannabinoids as therapeutic agents in cardiovascular disease: a tale of passions and illusions, Br. J. Pharmacol. 151 (2007) 427e440. [20] B. Julien, P. Grenard, F. Teixeira-Clerc, et al., Antifibrogenic role of the cannabinoid receptor CB2 in the liver, Gastroenterology 128 (2005) 742e755. [21] Q. Zhao, W. Wang, R. Wang, et al., TRPV1 and neuropeptide receptor immunoreactivity and expression in the rat lung and brainstem after lung ischemiareperfusion injury, J. Surg. Res. 203 (2016) 183e192. [22] G. Matute-Bello, G. Downey, B.B. Moore, et al., An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals, Am. J. Respir. Cell Mol. Biol. 44 (2011) 725e738. [23] Z. Cao, M.M. Mulvihill, P. Mukhopadhyay, et al., Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice, Gastroenterology 144 (2013) 808e817, e15. [24] A. Ribeiro, V. Ferraz-de-Paula, M.L. Pinheiro, et al., Cannabidiol, a non-
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098
Y. Xiong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[25]
[26]
[27]
[28]
[29]
[30]
[31]
psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: role for the adenosine A(2A) receptor, Eur. J. Pharmacol. 678 (2012) 78e85. X.P. Miao, J.S. Li, Q. Ouyang, et al., Tolerability of selective cyclooxygenase 2 inhibitors used for the treatment of rheumatological manifestations of inflammatory bowel disease, Cochrane Database Syst. Rev. 10 (2014). CD007744. J. Valles, A. Lago, A. Moscardo, et al., TXA2 synthesis and COX1-independent platelet reactivity in aspirin-treated patients soon after acute cerebral stroke or transient ischaemic attack, Thromb. Res. 132 (2013) 211e216. J. Jampilek, M. Dolezal, V. Opletalova, et al., 5-Lipoxygenase, leukotrienes biosynthesis and potential antileukotrienic agents, Curr. Med. Chem. 13 (2006) 117e129. D.E. Pellegrini-Giampietro, G. Mannaioni, G. Bagetta, Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death, FEBS J. 276 (2009) 2e12. M. Zhang, B.R. Martin, M.W. Adler, et al., Modulation of cannabinoid receptor activation as a neuroprotective strategy for EAE and stroke, J. Neuroimmune Pharmacol. 4 (2009) 249e259. D. Alonso-Alconada, F.J. Alvarez, A. Alvarez, et al., The cannabinoid receptor agonist WIN 55,212-2 reduces the initial cerebral damage after hypoxicischemic injury in fetal lambs, Brain Res. 1362 (2010) 150e159. T.P. Dinh, S. Kathuria, D. Piomelli, RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-
7
arachidonoylglycerol, Mol. Pharmacol. 66 (2004) 1260e1264. [32] A.R. King, A. Duranti, A. Tontini, et al., URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices, Chem. Biol. 14 (2007) 1357e1365. [33] Q. Zhao, J. Wu, Z. Lin, et al., Resolvin D1 alleviates the lung ischemia reperfusion injury via complement, immunoglobulin, TLR4, and inflammatory factors in rats, Inflammation 39 (2016) 1319e1333. [34] S.L. Dorris, R.S. Peebles Jr., PGI2 as a regulator of inflammatory diseases, Mediat. Inflamm. 2012 (2012) 926968. [35] P. Fontana, A. Zufferey, Y. Daali, et al., Antiplatelet therapy: targeting the TxA2 pathway, J. Cardiovasc. Transl. Res. 7 (2014) 29e38. [36] T. Yokomizo, Leukotriene B4 receptors: novel roles in immunological regulations, Adv. Enzym. Regul. 51 (2011) 59e64. [37] R. Liu, J. Luo, J. Li, et al., Protective mechanisms of sevoflurane against onelung ventilation-induced acute lung injury: role of cyclooxygenase-2 and 5lipoxygenase pathways, Nan Fang Yi Ke Da Xue Xue Bao ¼ J. South. Med. Univ. 33 (2013) 625e630. [38] J.A. Robertson, D. Sauer, J.A. Gold, et al., The role of cyclooxygenase-2 in mechanical ventilation-induced lung injury, Am. J. Respir. Cell Mol. Biol. 47 (2012) 387e394. [39] S. Carloni, D. Alonso-Alconada, S. Girelli, et al., Pretreatment with the monoacylglycerol lipase inhibitor URB602 protects from the long-term consequences of neonatal hypoxic-ischemic brain injury in rats, Pediatr. Res. 72 (2012) 400e406.
Please cite this article as: Y. Xiong et al., Effects of monoacylglycerol lipase inhibitor URB602 on lung ischemia-reperfusion injury in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.10.098