detorsion-induced testicular injury in rats

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FTY720 mitigates torsion/detorsion-induced testicular injury in rats Hung-Jen Shih, MD,a,b Jiin-Cherng Yen, PhD,b Allen W. Chiu, MD, PhD,c Yung-Chiong Chow, MD, PhD,d,e Wynn H.T. Pan, PhD,b Tao-Yeuan Wang, MD,f and Chun-Jen Huang, MD, PhDg,h,* a

Division of Urology, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan c School of Medicine, National Yang-Ming University, Taipei, Taiwan d Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan e Mackay Medical College, Taipei, Taiwan f Department of Pathology, Mackay Memorial Hospital, Taipei, Taiwan g Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan h School of Medicine, Tzu Chi University, Hualien, Taiwan b

article info

abstract

Article history:

Background: FTY720, a sphingosine-1-phosphate (S1P) receptor agonist, possesses potent

Received 3 October 2014

anti-inflammation capacity. We evaluated the therapeutic potentials of FTY720 against

Received in revised form

testicular injury induced by testicular torsion and/or detorsion (T/D).

12 January 2015

Materials and methods: Young adult male SpragueeDawley rats were allocated to receive T/D

Accepted 10 March 2015

(the T/D group) and T/D plus FTY720 (4 mg/kg, the T/D-FTY group, n ¼ 6 in each group). To

Available online 16 March 2015

investigate the possible roles of the S1P receptors, another group of rats received T/D plus FTY720 plus the potent S1P receptor antagonist VPC23019 (1 mg/kg, the T/D-FTY-VPC

Keywords:

group, n ¼ 6). FTY720 was administered immediately before testicular detorsion, and

Sphingosine-1-phosphate

VPC23019 was administered 30 min before FTY720. Another set of rats that received sham

Testis

operation, immediately followed by injection of normal saline, FTY720, or FTY720 plus

Torsion

VPC23019, served as control groups. Sham control groups were run simultaneously. After

Ischemia

euthanization, levels of testicular injury were measured.

Reperfusion

Results: Histologic findings revealed severe testicular injury changes in both the T/D and T/D-FTY-VPC groups and moderate testicular injury changes in the T/D-FTY group. In addition, malondialdehyde activity (oxidative status), concentration of interleukin-1b (inflammation index), myeloperoxidase activity (neutrophil infiltration index), and wet-todry weight ratio (tissue edema index) of both the T/D and T/D-FTY-VPC groups were significantly higher than those of the T/D-FTY group. These data confirmed the protective effects of FTY720 against testicular T/D. Moreover, antagonizing the S1P receptors could reverse the protective effects of FTY720. Conclusions: FTY720 significantly mitigated testicular injury induced by testicular T/D. The mechanisms may involve activating the S1P receptors. ª 2015 Elsevier Inc. All rights reserved.

* Corresponding author. Department of Anesthesiology, Taipei Tzu Chi Hospital, 289, Jianguo Road, Sindian District, New Taipei City 231, Taiwan. Tel.: þ886 2 66289779x2639; fax: þ886 2 66289009. E-mail address: [email protected] (C.-J. Huang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.03.014

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1.

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Introduction

Testicular torsion is a urological emergency that requires early testicular detorsion to prevent testicular injury [1,2]. Although restoration of blood flow to the testis is important to prevent irreversible injury, reperfusion may exacerbate testicular injury [1]. Testicular torsion and/or detorsion (T/D)induced inflammation has been shown to be a key mechanism of tissue injury [2e4]. Notably, germ cells are subject to the influence of testicular T/D [5]. Diminished spermatogenesis after testicular torsion has been reported in several human studies even after detorsion or orchiectomy [6]. Consequently, testicular T/D is considered to be a typical form of ischemiaereperfusion injury [1,2]. Many drugs, such as glibenclamide [7], edaravone [8], sivelestat [9], NG-nitro-Larginine methyl ester [10], hemin [11], and simvastatin [12], had been shown to reduce the testicular injury in an experimental testicular torsion animal model. However, there are currently no effective treatments to minimize injury after testicular T/D in humans. Sphigosine-1-phosphate (S1P) is a bioactive signaling molecule that regulates cell survival, angiogenesis, lymphocyte trafficking, and endothelial barrier function [13,14]. S1P is synthesized de novo from serine and palmitoyl coenzyme A or by activation of sphingomyelinase [13,14]. The level of S1P is regulated by the sphingosine kinase (for S1P production), S1P phosphatase, and S1P lyase (for reversible and irreversible S1P degradation, respectively) [13,14]. Dynamic balance of S1P plays important roles in regulating cell movement, differentiation, survival, and immunity [15]. Different stimuli such as hypoxia, growth factor, or interleukin (IL) can result in significant increases in S1P levels [16]. Much of these diversity effects of S1P are mediated via five S1P receptors (i.e., the S1P1, S1P2, S1P3, S1P4, and S1P5 receptors) [13,14]. The S1P receptors are widely expressed in adult mammal tissues, including the brain, heart, lung, kidney, and testis [17]. FTY720 is an analog of the important bioactive signaling molecule S1P [18e21]. Previous data revealed that administration of FTY720 could exert potent anti-inflammatory effects [18e21]. FTY720 is converted to its active form FTY720-phosphate in vivo by sphingosine kinase 2 [22]. FTY720-phosphate then can bind to the S1P receptors and subsequently activate the S1P receptors to exert antiinflammatory effects [23,24]. The therapeutic potential of S1P receptor activation was further confirmed by previous data reporting that FTY720 could inhibit inflammation and mitigate organ dysfunction in septic rats [25]. However, the effect of FTY720 on mitigating testicular T/D-induced testicular injury remains unclear. Therefore, we conducted this rodent study with the hypothesis that FTY720 could mitigate testicular T/D-induced testicular injury in rats.

2.

Material and methods

2.1.

Animals

Young adult male SpragueeDawley rats (BioLASCO, Taipei, Taiwan) weighing 200e250 g (7e8-wk-old) were used. The

Institutional Animal Care and Use Committee of Changhua Christian Hospital in Taiwan approved all animal experiments (CCH-AE-99034), and all animal care and handling was performed in accordance with the United States National Institutes of Health guidelines.

2.2.

Drug administration

FTY720 (4 mg/kg, intraperitoneal [IP]; BioVision Inc, Milpitas, CA) and VPC23019 (1 mg/kg, IP; Tocris Bioscience, Bristol, United Kingdom) were used to activate and antagonize S1P receptors, respectively. The doses of FTY720 and VPC23019 were determined according to the preliminary data of IL-1b derived from a pilot study performed in our laboratory. Our preliminary data revealed that FTY720 at the dose of 4 mg/kg (but not 1 and 2 mg/kg) could significantly mitigate T/Dinduced upregulation of IL-1b in twisted testicular tissues (P ¼ 0.002, Fig. 1A). Our preliminary data also revealed that the previously mentioned effects of 4 mg/kg FTY720 could be significantly offset by 1 mg/kg of VPC23019 (P ¼ 0.002, Fig. 1B). This study thus determined to use 4 mg/kg of FTY720 and 1 mg/kg of VPC23019 to facilitate further investigation.

2.3.

FTY720 conversion to FTY720-phopshate

To elucidate the kinetics of the conversion of FTY720 to FTY720-phosphate, four rats were euthanized at different time points (1, 2, 6, and 24 h) after FTY720 administration (4 mg/kg, IP). Plasma and testicular tissues samples were collected, as reported previously [11,12]. Then, the concentrations of FTY720 and FTY720-phosphate in plasma and testicular tissues were determined using liquid chromatographyetandem mass spectrometry, as described previously [26].

2.4.

FTY720 binding to the S1P receptors

Immunofluorescence staining assay was performed to investigate binding of FTY720/FTY720-phosphate to the S1P1 receptor in the testicular tissues. To facilitate investigation, biotin-FTY720 (Cayman Chemical, Ann Arbor, MI) was used. According to the manufacturer, the target of phosphorylation (i.e., the hydroxymethyl side chain) of biotin-FTY is retained and thus biotin-FTY720 can be phosphorylated after administration. Three rats received biotin-FTY720 administration (4 mg/kg, IP) and maintained for 24 h. Then, the rats were euthanized, and the testicular tissues were collected and immersed in Bouin solution. Sections of testicular tissues were incubated overnight with primary antibody against the S1P1 receptor (1: 500 dilution, polyclonal anti-S1P1 antibody; Abcam, Cambridge, MA) followed by incubation with fluorescent rhodamine isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Inc, West Grove, PA) for 1 h to facilitate detecting the S1P1 receptor. Next, the sections were incubated for 1 h with anti-biotin antibody (1:500 dilution, polyclonal anti-biotin fluorescein isothiocyanate [FITC] antibody; Abcam) to facilitate detecting FTY720/FTY720-phosphate. The immunofluorescencestained sections were evaluated using confocal microscope

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Fig. 1 e (A) Preliminary IL-1b concentration in ipsilateral twisted testicular tissues from the testicular T/D plus normal saline (N/S), the T/D plus FTY720 (1 mg/kg; FTY [1]), the T/D plus FTY720 (2 mg/kg; FTY [2]), and the T/D plus FTY720 (4 mg/kg; FTY [4]) groups. Data were means ± standard errors. *P < 0.05 versus the T/D group. (B) Preliminary IL-1b concentration in ipsilateral twisted testicular tissues from the T/D plus FTY (4), the T/D plus FTY (4) plus VPC23019 (0.5 mg/kg; VPC [0.5]), and the T/D plus FTY (4) plus VPC23019 (1 mg/kg; VPC [1]) groups. Data were means ± standard errors. #P < 0.05 versus the T/D plus FTY (4) group.

(TCS SP5 AOBS; Leica Microsystems CMS GmbH, Mannheim, Germany).

2.5.

Ipsilateral testicular T/D model

Rats were anesthetized using an IP injection of sodium pentobarbital (50 mg/kg) and were placed in a supine position on a heating pad. Ipsilateral testicular T/D was induced as described previously [11,12]. In brief, the left testis was rotated 720 in the clockwise direction and fixed for 2 h; it was then untwisted. Sham-operated animals underwent a similar surgical procedure, except that the left testicle was not twisted.

2.6. Effect of FTY720 on mitigating testicular injury induced by testicular T/D 2.6.1.

Study design

plasma, and the plasma samples were stored at 80 C for subsequent analysis.

2.6.3.

Histopathology

Testicular tissues immersed in Bouin solution were sectioned and then stained using hematoxylin and eosin and were evaluated microscopically by a pathologist blinded to the experimental conditions. Each characteristic, including hemorrhage, edema, vascular congestion, polymorphonuclear leukocyte (PMN) infiltration, and germ cell ischemia, was scored on a scale from 0e3 (0, normal; 3, severe). The score of germ cell ischemia was according to a previous report [27]. The score of hemorrhage, edema, vascular congestion, and PMN infiltration was according to the area of influence on the examination section (1: <20%; 2: 20%e50%; and 3: >50%). Overall testicular injury was further graded according to the total scores (0e5, normal to mild injury; 6e10, moderate injury; and 11e15, severe injury).

The rats were randomly allocated into 1 of 6 groups (n ¼ 6 in each group) as follows: (1) sham operation (sham), (2) sham plus FTY720 (sham-FTY), (3) sham plus FTY720 plus VPC23019 (sham-FTY-VPC), (4) T/D, (5) T/D plus FTY720 (T/D-FTY), and (6) T/D plus FTY720 plus VPC23019 (T/D-FTY-VPC). FTY720 was administered immediately before testicular detorsion, and VPC23019 was administered 30 min before FTY720. FTY720 and VPC23019 were administered at comparable time points in the sham groups. To control for vehicle effects, rats in the sham and T/D groups also received normal saline (0.5 mL, IP). Twenty-four hours after testicular detorsion or at a comparable time point in the sham groups, all rats were sacrificed using a high dose of pentobarbital (300 mg/kg, IP).

The indices of lipid oxidation status (malondialdehyde [MDA] activity) and PMN infiltration (myeloperoxidase [MPO] activity) were measured as reported previously [11,12]. The concentration of the inflammatory cytokine IL-1b was determined using an enzyme-linked immunosorbent assay (colorimetric IL-1b ELISA kit; Pierce Biotechnology, Inc, Rockford, IL), as reported previously [12]. In addition, freshly harvested testes were weighed, dried for 24 h at 60 C, and then weighed again to determine the wet-to-dry weight ratio (an index of tissue edema).

2.6.2.

2.7.

Tissue sample collection

Tissue samples were collected as reported previously [11,12]. In brief, bilateral testes were longitudinally divided into two halves. One half was snap-frozen in liquid nitrogen. The upper portion of the other half was immersed in Bouin solution, and the lower portion was used to assay the wet-to-dry weight ratio. In addition, blood samples were centrifuged to separate

2.6.4. Assays of malondialdehyde activity, IL-1b, myeloperoxidase activity, and ratio of wet-to-dry weight

Statistical analysis

Statistical analyses were performed using one-way analysis of variance with Tukey post hoc test. Data are presented as means and standard errors. The significance level was set at 0.05. A commercial software package (SPSS 11.5 for Windows; IBM Corp, Armonk, NY) was used for all data analyses.

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3.

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Results

3.1. Rapid conversion of FTY720 to FTY720-phosphate after administration Liquid chromatographyetandem mass spectrometry (Fig. 2A) revealed that >80% of FTY720 was converted to FTY720phosphate within 1 h of administration in plasma (Fig. 2B) and within 2 h in testicular tissues (Fig. 2C).

3.2. Binding of FTY720 and the S1P receptors in testicular tissues Immunofluorescent staining assay revealed positive expression of the S1P1 receptor in the testicular tissues (Fig. 2D). Our data also revealed that the S1P1 receptor mainly located in the germ cells of the testicular tissues (Fig. 2D). Moreover, signal of FTY720/FTY720-phosphate was noted mainly in the S1P1 receptor-positive cells (Fig. 2D), indicating the binding of FTY720 to the S1P receptors in testicular tissues.

3.3. FTY720 mitigated testicular T/D-induced testicular injury Representative microscopic images from the testicular T/D revealed five pathological characteristics (Fig. 3A). Histologic findings revealed normal to mild testicular injury in ipsilateral twisted testicular tissues in the sham group (Fig. 3B). Similar changes were observed in the sham-FTY and sham-FTY-VPC groups (data not shown). In contrast, histologic analysis of the ipsilateral twisted testicular tissues revealed severe testicular injury in the T/D group (Fig. 3C) and moderate testicular injury in the T/D-FTY group (Fig. 3D). Severe testicular injury was also observed in ipsilateral twisted testicular

tissues in the T/D-FTY-VPC group (Fig. 3E). Moreover, histologic analysis of contralateral testicular tissues in all groups revealed normal to mild testicular injury (data not shown). In addition, the injury scores (Fig. 3F) paralleled the histologic findings.

3.4. FTY720 mitigated testicular T/D-induced lipid oxidation, inflammation, PMN infiltration, and tissue edema Figure 4AeD showed the results of MDA activity, IL-1b concentration, MPO activity, and wet-to-dry weight ratio, respectively. MDA activity, IL-1b concentration, MPO activity, and wet-to-dry weight ratio were low in the twisted testes from the sham, sham-FTY, and sham-FTY-VPC groups. As expected, MDA activity (P < 0.001), IL-1b concentration (P < 0.001), MPO activity (P ¼ 0.014), and wet-to-dry weight ratio (P ¼ 0.010) were significantly higher in ipsilateral twisted testes in the T/D group compared with those in the sham group. Moreover, MDA activity (P < 0.001; P ¼ 0.005), IL-1b concentration (P < 0.001; P < 0.001), MPO activity (P ¼ 0.008; P ¼ 0.010), and wet-to-dry weight ratio (P ¼ 0.003; P ¼ 0.010) were significantly lower in ipsilateral twisted testes in the T/D-FTY group compared with those in the T/D and T/D-FDYVPC groups, respectively. In addition, differences in all four parameters in the contralateral testes were not significant among the groups.

4.

Discussion

Data obtained in the present study demonstrated that testicular T/D caused significant testicular injury [1,2] and confirmed our hypothesis that FTY720 could mitigate testicular T/D-induced testicular injury. These data support the concept that incorporating FTY720 as a therapeutic adjunct

Fig. 2 e (A) Liquid chromatographyetandem mass spectrometry analysis of FTY720 and FTY720-phosphate concentrations. The ratio of FTY720/FTY720-phosphate (FTY/FTY-p) in (B) plasma and (C) testicular tissues was measured at 1, 2, 6, and 24 h after FTY720 administration. Black bar/gray bar: FTY/FTY-p. (D) Immunofluorescence assay of FTY720 binding to the S1P1 receptor in the testicular tissues. Biotin-FTY720 and the anti-biotin fluorescein isothiocyanate antibody were used for FTY720/FTY720-phosphate (FTY/FTY-p) staining. The S1P1 receptor was stained with fluorescent rhodamine isothiocyanate-conjugated antibody. The sections were imaged using a confocal microscope. (Color version of the figure is available online.)

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Fig. 3 e FTY720 mitigates testicular T/D-induced testicular injury. Microscopy was used to visualize ipsilateral twisted testicular tissues stained with hematoxylin and eosin. (A) Representative microscopic images from the testicular T/D revealed five pathologic characteristics (340). (B) Representative microscopic images from the sham group revealed normal to mild testicular injury (3100). (C) Representative microscopic images from the testicular T/D group revealed severe testicular injury (3100). (D) Representative microscopic images from the T/D plus FTY720 (T/D-FTY) group revealed moderate testicular injury (3100). (E) Representative microscopic images of the T/D plus FTY720 plus VPC23019 (T/D-FTYVPC) group revealed severe testicular injury (3100). (F) The testicular injury scores in bilateral testicular tissues from the sham-operated (sham), sham plus FTY720 (sham-F), sham plus FTY720 plus VPC23019 (sham-F-V), T/D, T/D-F, and T/D-F-V groups. Data were means ± standard errors. Black bar: ipsilateral twisted testis. Gray bar: contralateral testis. *P < 0.05 versus the sham group. yP < 0.05 versus the T/D group. zP < 0.05, the T/D-F-V group versus the T/D-F group. (Color version of the figure is available online.)

could be a novel and effective therapeutic strategy against testicular T/D. Because FTY720 is already approved for clinical use [28,29], data from this study could have profound clinical implications; therefore, our findings warrant further investigation. Reperfusion injury is inevitable after detorsion of the testis [1,2,5]. The critical pathophysiology involved in reperfusion injury may include leukocyte activation as well as immune system activation induced by oxidative stress and the subsequent development of inflammatory response, including PMN infiltration and upregulation of inflammatory molecules [3,4]. Using a kidney ischemiaereperfusion model, previous data indicated that ischemiaereperfusion could also upregulate chemokine expression and subsequently induce an early and transient infiltration of T cells into the kidney [30]. Being a typical ischemiaereperfusion, testicular T/D can also act through these previously mentioned mechanisms to cause testicular tissue damage [4]. This concept is confirmed by the present study because our data revealed that testicular T/D caused significant increases in lipid oxidation, IL-1b expression, and PMN infiltration in testicular tissues. Endothelial

barrier failure is another important consequence of ischemic reperfusion injury [31]. Vascular hypoxia during tissue ischemia and microvascular dysfunction caused by infiltrated leukocytes and their active products after reperfusion can lead to increased vascular permeability [32,33]. With endothelial barrier failure, interstitial edema can occur because of excessive filtration of proteins and fluid [32,33]. This concept is supported by the present study, which revealed significant tissue edema in testicles after T/D. Together, these data indicated that FTY720 could mitigate testicular T/D-induced testicular injury and the mechanisms may involve abrogating the effects of testicular T/D on increasing lipid oxidation, IL-1b expression, PMN infiltration, and endothelial barrier failure. S1P is a bioactive sphingolipid metabolite, which plays an important role in many physiological and pathologic processes [13,14]. The multiple effects of S1P are mostly dependent on the S1P receptor activation [13,14]. FTY720 is a potent agonist on all S1P receptors except S1P2 [28]. As aforementioned, FTY720 could activate the S1P receptors and exert significant anti-inflammatory effects in the kidney and lungs

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Fig. 4 e FTY720 mitigates testicular T/D-induced lipid oxidation, inflammation, PMN infiltration, and edema in testicular tissues. (A) MDA activity (oxidative status index), (B) IL-1b concentration (inflammatory index), (C) myeloperoxidase activity (neutrophil infiltration index), and (D) wet-to-dry weight ratio (tissue edema index) in bilateral testicular tissues from the sham-operated (sham), sham plus FTY720 (sham-FTY), sham plus FTY720 plus VPC23019 (sham-FTY-VPC), testicular T/D (T/D), T/D plus FTY720 (T/D-FTY), and T/D plus FTY720 plus VPC23019 (T/D-FTY-VPC) groups. Data were means ± standard errors. Black bar: ipsilateral twisted testis. Gray bar: contralateral testis. *P < 0.05 versus the sham group. yP < 0.05 versus the T/D group. zP < 0.05, the T/D-FTY-VPC group versus the T/D-FTY group.

[23e25]. Previous data also revealed that activation of the S1P receptors by FTY720 can exert protective effects in the brain [34]. Moreover, previous rodent data further demonstrated that FTY720-induced activation of the S1P receptors could mitigate acute kidney injury induced by kidney ischemiaereperfusion [20,23]. Of note, the mechanisms may involve inhibiting PMN infiltration and enhancing mitochondria function in the renal tissue [30,35]. Moreover, previous data also revealed that the previously mentioned effects of FTY720 against kidney ischemiaereperfusion could be reversed by antagonizing the S1P receptors [20]. These data highlighted the crucial roles of the S1P receptors in mediating the protective effects of FTY720 observed in kidney ischemiaereperfusion. In line with this notion, we thus speculated that the protective effects of FTY720 observed in this study may also involve the S1P receptors. Data from the present study demonstrated that FTY720 could bind to the S1P receptors on the germ cells in testicular tissues. Our data further demonstrated that the protective effects of FTY720 on mitigating testicular injury induced by testicular T/D could be offset by antagonizing the S1P receptors. Collectively, these data provide clear evidence to support the concept that FTY720 may act through activating the S1P receptors to exert its protective effects against injury induced by testicular T/D. Certain study limitations exist. First, this is a rodent study. Therefore, cautions should be used if further data interpretation is intended. Second, we measured the data only at 24 h after testicular detorsion. The long-term effects (e.g., spermatogenesis) of FTY720 against testicular T/D remain to be elucidated. Third, previous data indicated significant S1P receptor expression (i.e., the S1P5 receptor) in young adult (i.e., 7-wk-old) but not in prepubertal (i.e., 3-wk-old) rodent testicular tissues [36]. We thus chose to use young adult male

rats in this study to facilitate investigation. The question of whether FTY720 could exert similar protective effects in prepubertal or pubertal rats remains unstudied.

5.

Conclusions

FTY720 significantly mitigated testicular injury induced by testicular T/D in rats.

Acknowledgment The authors express their appreciation to Ms Ming-Cheng Lee, Taipei Tzu Chi Hospital, for her excellent technical support in LC-MS/MS analysis. Authors’ contributions: H.-J.S. and C.-J.H. contributed to the conception and design. H.-J.S., J.-C.Y., A.W.C., Y.-C.C., W.H.T.P., T.-Y.W., and C.-J.H. did the analysis and interpretation, data collection, and writing of the article. C.-J.H. did the critical revision of the article and obtained the funding. This work was supported by a grant from the National Science Council, Taiwan (NSC 100-2314-B-371-002) awarded to H.-J.S. and C.-J.H.

Disclosure None of the authors have conflict of interest to be disclosed on the publication of this article.

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