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Endothelium-dependent and -independent relaxation of rat aorta induced by extract of Schizophyllum commune.

ABSTRACT

Schizophyllum commune (SC) is widely consumed by Chinese, especially in southern part of China. The aim of the present study was to assess the extract of SC on vascular tone and the mechanisms involved. Experiments were performed on aorta of 18-week-old male Sprague-Dawley rats. Dried SC was extracted with 50% ethanol, 90% ethanol and deionized water, respectively. The effects of SC on the isometric tension of rat aortic rings were measured. Protein expression for the endothelial nitric oxide synthase (eNOS) was also determined in the primarily cultured rat aortic arterial endothelial cells (RAECs). The results showed that the water extract of SC induced a marked relaxation in aortic rings with or without endothelium. After the pretreatments of [N.sup.[omega]]-nitro-L-arginine methyl ester, indomethacin, RP-cAMP, and methylene blue, the SC-induced relaxation was significantly decreased. In addition, the contraction due to [Ca.sup.2+] influx and intracellular [Ca.sup.2+] release was also inhibited by SC. Furthermore, expression of the eNOS protein was significantly elevated in RAECs after treatment of SC. In conclusion, the water extract of SC induces an endothelium-dependent and -independent relaxation in rat aorta. The relaxing effect of SC involves the modulation of NO-cGMP-dependent pathways, [PGI.sub.2]-cAMP-depedent pathways, [Ca.sup.2+] influx though calcium channels and intracellular [Ca.sup.2+] release.

Keywords:

Schizophyllum commune

Vasorelaxation

Rat aorta

Introduction

Schizophyllum commune (SC) belongs to the genus Schizophyllum of the family Schizophyllaceae in the Agaricales order (Kirk et al., 2008). Using wood as a substrate, SC is the most common member of the genus Schizophyllum, probably the most widespread fungus in existence. SC is proved edible and consumed widely in South China for a long historic time. It embraces the value of health care by keeping body strong and treating neurasthenia. Recent investigations suggest that the extract of SC may exert numerous medicinal effects because of its antioxidant (Wong and Chye, 2009) and anti-tumor activities (Patel and Goyal, 2012). The potential therapeutic effects of SC on cardiovascular diseases have also been suggested by a study showing that the water extract of SC inhibited the angiotensin-converting enzyme (ACE) activities (Abdullah et al., 2012). Furthermore, SC contains many biologically active ingredients such as schizophyllan (SPG) (Kumari et al., 2008), cerebrosides (Kawai and Ikeda, 1985) and lectins (Chumkhunthod et al., 2006), which are known to possess cardiovascular effects. For instance, SPG, a [beta]-1,6-branched 1,3-[beta]-D-glucan, is a neutral extracellular polysaccharide (Rop et al., 2009). Administration of SPG can significantly decrease the vascular permeability in mice (Miura et al., 2000). Cerebrosides inhibits the calcium-activated chloride channels in rat arterial smooth muscle cells (Gao et al., 2007), which theoretically may lead to relaxation (Nilius and Droogmans, 2003). Intravenous administration of lectins also causes a mean arterial blood pressure reduction in rat, probably through the activation of adenosine A2 receptors and/or nitric oxide (NO) production (Wang et al., 1996). Because of the abundance of these active ingredients in SC, we hypothesize that SC may exert certain effects on blood vessel tone. In the present study, the endothelium-dependent and -independent relaxing effects of SC on rat aorta were assessed, and the mechanisms involved were examined.

Materials and methods

Chemicals and drugs

[N.sup.[omega]]-nitro-L-arginine methyl ester (l-NAME), methylene blue (MB), indomethacin (INDO), Rp-Adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP), acetylcholine (ACh), phenylephrine (PE), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). The other reagents were of analytical purity.

Preparation of the extract of SC

Dried SC used in the present study was purchased at local medicinal material market (Kunming, Yunnan Province, China).The plant was identified at the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, China, where a voucher specimen, MZYS0001, was deposited. Five hundred grams of pulverized SC was extracted twice with 50% ethanol, 90% ethanol and deionized water at 90[degrees]C for 1.5 h, respectively. The extracts were filtered and the filtrate was concentrated under reduced pressure using rotary evaporator at 40[degrees]C until extraction solvent was completely dried. The extracts were stored in the refrigerator at 4[degrees]C for further use. They were dissolved in a vehicle containing 50% ethanol, 90% ethanol or deionized water before studies were carried out.

Chromatographic analysis

The high performance liquid chromatography (HPLC, Agilent 1100 series, Agilent Technologies, USA) equipped with Ultimate[TM] XB-C18 column (4.6 mm i.d. x 250 mm, 5 [micro]m particle size, Welch, Shanghai, China) and a diode-array detector (DAD, Agilent G 1315B) was used for analysis of water extract of the fruiting bodies of SC. Methanol (solvent A) and Milli-Q water (solvent B) were used as mobile phase. Samples were eluted in gradient mode. The gradient elution program is shown in Table 1. The detection wavelength was set at 200-400 nm according to the absorption peaks of the characteristic spectrum. The flow rate was 0.8 ml/min. The sample injection volume was 5 [micro]l and the column temperature was maintained at 30[degrees]C. The solvents used for analysis were purchased from Fisher Scientific (Pittsburgh, PA, USA).

Animals and tissue preparation

Male Sprague-Dawley rats (250-300 g) were supplied from the Laboratory Animal Unit of Kunming Medical University (Kunming, China). All experiments performed in this study were approved by the Committee on the Use of Live Animals in Teaching and Research of Yunnan Minzu University. After anesthetized with pentobarbitone sodium (50 mg/kg, i.p.), the rats were killed by stunning and cervical dislocation, and the thoracic aorta was dissected out. The aorta was cleaned of adhering fat and connective tissue and cut into 3 mm wide rings. Care was taken to avoid abrading the intimal surface in order to maintain the integrity of the endothelial layer. In certain experiments, endothelium was removed by gently rubbing the intimal space with a cotton swab.

Isometric tension measurement

The aortic rings were immediately immersed in Krebs solution (mM: 118 NaCl, 4.7 KCl, 2.5 Ca[Cl.sub.2], 1.2 MgS[O.sub.4], 1.2 K[H.sub.2]P[O.sub.4], 25 NaHC[O.sub.3], and 11.1 glucose, at 37[degrees]C), aerated with 5% C[O.sub.2]/95% [O.sub.2] and connected to a force transducer (Powerlab model ML785 and ML119; AD Instruments, Incorporated, Colorado Spring, CO, USA). The rings were stretched progressively to their optimal resting tensions (2.0 g; determined in preliminary experiments) and allowed to equilibrate for 60 min. All changes in tension were expressed as a percentage decreased in the contraction to phenylephrine (PE; 1 [micro]M). The absence of relaxation responses to ACh was taken as evidence that vessel segments were functionally denuded of endothelium.

Protocol 1: relaxing effects of SC on rat aorta

PE was used to induce steady contraction in aortic rings followed by cumulative addition of SC (0-1000 mg/l). In order to study whether or not NO, 3',5'-cyclic guanosine monophosphate (cGMP), 3',5'-cyclic adenosine monophosphate (cAMP) and prostacyclin ([PGI.sub.2]) were involved in the relaxing effect of SC, endotheliumintact rings were incubated with L-NAME (a NO synthase inhibitor; 100 [micro]M), MB (a soluble guanylyl cyclase inhibitor; 10 [micro]M), Rp-cAMP (a cAMP antagonist; 10 [micro]M), or Indomethacin (INDO; a non-selective inhibitor of cyclooxygenase, 10 [micro]M) for 60 min prior to the application of PE, respectively. SC was then cumulatively added to the tissue.

Protocol 2: effects of SC on calcium influx and intracellular calcium store

To verify whether or not [Ca.sup.2+] influx was involved in the SC-induced relaxation, rat aortic rings were washed with [Ca.sup.2+]-free Krebs solution four to five times before PE (1 p.M) was applied. Then, [Ca.sup.2+] was added cumulatively to obtain a concentration-response curve (0.01-3 mM). SC (100 mg/l and 1000 mg/l) was added 10 min before the addition of [Ca.sup.2+].

To study whether or not the SC-induced relaxation was related to the inhibition of intracellular [Ca.sup.2+] release, the aortic rings were exposed to [Ca.sup.2+]-free solution with 50 [micro]M EGTA for 15 min before the application of PE (1 [micro]M) to induce the first transient contraction (con1). The rings were then washed three times and incubated with normal Krebs solution for at least 40min to refill the intracellular [Ca.sup.2+] stores. Subsequently, the normal Krebs solution was rapidly replaced with [Ca.sup.2+]-free solution and the rings were incubated for another 15 min. The second contraction (con2) was then induced by PE (1 [micro]M) in the absence or presence of 100 mg/l SC or 1000 mg/l, which was added 10 min before PE application. The ratio of the second contraction to the first contraction (con2/con1) was calculated.

Culture of rat aortic arterial endothelial cells (RAECs)

RAECs were isolated from the aorta ofSD rats, using the previous protocol with slight modification (Yang et al., 2006). Briefly, after SD rats were anesthetized with pentobarbitone sodium (50 mg/kg, i.p.), the aortas were removed and placed in sterilized dishes. The fat and connective tissues were cleared, and the vessels were opened longitudinally and cut into 2 [mm.sup.2] pieces. The tissues were incubated at 37[degrees]C (5% C[O.sub.2]/95% air) in RPM1 1640 containing 10% fetal calf serum (Gibco, Invitrogen, California, USA), 100 U/ml G-penicillin (Gibco) and 100|xg/ml streptomycin (Gibco). After 3-4 days, the tissues were removed and the cells were harvested. The identity of RAECs was confirmed by immunostaining using the goat anti-rat Factor VIII (an endothelial cell marker), goat anti-rat [alpha]-actin antibody (a vascular smooth muscle cell marker).

Proteins extractions

Proteins were extracted from RAECs treated without (control) or with SC (100 mg/l or 1000 mg/l) for 72 h. Briefly, RAECs were homogenized in ice-cold lysis buffer (containing 20 mM Tris-HCl, 1% Triton X-100,150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2.5 mM sodium pyrophosphate, 1 mM [beta]-glycerophosphate, 1 mM sodium orthovanadate) supplemented with a cocktail of protease inhibitors. The mixture was centrifuged at 5000 rpm for three minutes at 4[degrees]C and the supernatant was kept at 80[degrees]C until use. The protein concentration was determined spectrophotometrically using the Bradford protein assay (Bio-rad, Hercules, CA, USA).

Western blot analysis

Twenty [micro]g of proteins were separated by SDS-PAGE (7.5%) at 200 V, 300 mA for 50 min. The proteins were transferred onto polyvinylidene fluoride membranes at 200 V, 300 mA for 45 min. The membranes were blocked by 5% dry milk at room temperature for 1 h, followed by incubation with polyclonal rabbit anti-rat endothelial NO synthase (eNOS) antibody (1:1000, Cell Signaling Technology, Inc., Beverly, Mass, USA) in TBS overnight at 4[degrees]C. Then, the membranes were incubated with HRP-conjugated anti-rabbit antibody (1:2000 in TBS, room temperature, 1 h, Amersham Biosciences, Piscataway, NJ, USA). Bound secondary antibody was detected by chemiluminiscence (Amersham Biosciences) and exposed to X-ray film. Optical density values of eNOS band were normalized to those of [beta]-actin.

Statistical analysis

The changes in isometric tension were expressed as the percentage decreased in the contraction to PE. In western blotting assay, changes in protein levels were described as the ratio to [beta]-actin. All data were expressed as means [+ or -] S.E.M. Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL). Comparison between two groups was analyzed using Student's t-test. Comparison among three or more groups was analyzed using one-way ANOVA, p < 0.05 was considered statistically significant.

Results

The HPLC chromatogram of SC

The HPLC chromatogram of the water extract of the fruiting bodies of SC at 254 and 306 nm is presented in Fig. 1A. The Ultraviolet spectrum of main peaks in the figure is shown in Fig. 1B-F. Peaks on the HPLC chromatogram show apparent absorptions from 230 to 300 nm. These findings indicate that the water extract of SC may contain phenolic constituents. Fig. 1G shows the typical appearance of dried SC.

The endothelium-dependent and -independent relaxation induced by SC

Fig. 2 shows that the water extract of SC induced a relaxation (87.26 [+ or -] 4.10% at 600 mg/l), which was significantly higher than the relaxation caused by the 50% or 90% ethanol extracts (62.46 [+ or -] 4.43% and 12.19 [+ or -] 8.12%, respectively, p < 0.05). Therefore, the water extract of SC were used in the subsequent experiments. Fig. 3 shows that the relaxing effect of SC were significantly reduced but not abolished when endothelium was removed from aorta (81.50 [+ or -] 7.57% vs. 36.57 [+ or -] 12.11% for aorta with and without endothelium, respectively. SC at 600 mg/L, p < 0.05).

Mechanisms underlying the SC-induced relaxation

Fig. 4 shows that the relaxing effect of SC (1000 mg/l) was significantly reduced by l-NAME (from 85.96 [+ or -] 4.91 % to 45.04 [+ or -] 6.93%; p < 0.05), INDO (from 85.96 [+ or -]4.91% to 30.51 [+ or -] 4.60%; p< 0.05), Rp-cAMP (from 85.96 [+ or -] 4.91% to 25.82 [+ or -] 2.65%; p < 0.05) and MB (from 85.96 [+ or -] 4.91% to 1.61 [+ or -] 2.50%; p < 0.01).

Effect of SC on [Ca.sup.2+]influx and intracellular [Ca.sup.2+] release

To study the effect of SC on [Ca.sup.2+] channels, studies were performed in [Ca.sup.2+]-free preparations. Priming with PE(1 [micro]M) produced small increase in resting tone, and subsequent cumulative addition of Ca[Cl.sub.2] ([10.sup.-5] to 3 x [10.sup.-3] M) caused stepwise constriction. Fig. 5 shows that the [Ca.sup.2+]-dependent contraction was inhibited when the aortic rings were preincubated with SC (100 mg/l) for 30 min before PE was applied. The [Ca.sup.2+]-dependent contraction was further inhibited when a higher concentration of SC (1000 mg/l) was used.

To study the effect of SC on intracellular [Ca.sup.2+] release, first contraction was induced by PE (1 p,M) in [Ca.sup.2+]-free buffer with EGTA (50 [micro]M). The intracellular [Ca.sup.2+] was then refilled by incubating the tissue in normal Krebs solution. The second contraction was induced by PE in [Ca.sup.2+]-free solution again. Fig. 6 shows that the ratio of second contraction to first contraction was significantly decreased when the tissue was pretreated with SC (in both 100 mg/l and 1000 mg/l, p < 0.05) before second contraction.

Effect of SC on the expression of eNOS in RAECs

Fig. 7 shows that the protein expression level of eNOS in RAECs was significantly increased after the cells were pretreated with SC for 72 h. The increased expression of eNOS by SC was concentration-dependent.

Discussion

The effect of SC on blood pressure has never been reported but a recent study has shown that the water exact of SC inhibits ACE activity, with an [IC.sub.50] value of 320.00 mg/l (Abdullah et al., 2012). Our study provides the first evidence that water extract of SC can directly induce the relaxation of blood vessels. The [EC.sub.50] value of the SC-induced relaxation of rat aorta was 70.56 mg/l, which is much lower than that for ACE inhibition. The relaxing responses to SC in aorta with endothelium were greater than the aorta without endothelium, suggesting that the relaxing effect of SC was mainly mediated through an endothelium-dependent mechanism whereas a small part of relaxation was due to the direct effect on vascular smooth muscle. The endothelium-dependent relaxation was abolished by MB, indicating that soluble guanylyl cyclase is target site for SC. Only 80% but not all of the endothelium-dependent relaxation was inhibited by L-NAME. It implies that SC mainly induced the release of NO from endothelial cells but there was an NO-independent mechanism that could stimulate the soluble guanylyl cyclase. Further experiments are required to study if SC may directly activate soluble guanylyl cyclase in vascular smooth muscle cells. SC up-regulated the protein expression of eNOS, which adds weight to the hypothesis that SC induces relaxation by increased production of NO. Apart from NO, [PGI.sub.2] is another important endothelium-derived relaxing factor so the effect of SC on [PGI.sub.2]-cAMP-depedent relaxation was also studied. The relaxing effect of SC could be inhibited by INDO and Rp-cAMP by as high as 80%, which was close to the inhibition by L-NAME. Such a high degree of inhibition was unexpected since the contribution of [PGI.sub.2]-cAMP mechanism to relaxation of blood vessels is used to be much smaller than that of NO-cGMP mechanism. One of the possible explanations is that cyclooxygenase may be required for the activation of certain ingredients in SC. Besides, we cannot exclude the possibility that there may be a linkage between SC and cAMP-responsive element binding (CREB) protein, which may increase or decrease the transcription of the downstream genes related to NO. To support this notion, a few studies have demonstrated that the expressions of NOS and tetrahydrobiopterin (a NOS cofactor) are regulated by CREB protein-dependent mechanisms (Boissel et al., 2004; Sun et al., 2009).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The chemical components of SC have hitherto been well identified. Some components in SC such as cerebrosides (Kawai and Ikeda, 1985), SPG (Kumari et al., 2008) and lectins (Chumkhunthod et al., 2006) have potential vasodilatory effects which attract more and more attention. Cerebrosides were identified in many kinds of fungi (Barreto-Bergter et al., 2004) and herbal medicines (Gao et al., 2007). They can induce the production of NO in plant cells but its actions on animal cells remained obscured (Wang et al., 2007). SPG is a water-soluble polysaccharide isolated from fruiting body, mycelium or their fermentation broth of SC. It belongs to [beta]-D-glucans with combination bonds (1-3) and (1-6) (Rop et al., 2009). The administration of (l-3)-[beta]-D-glucan protected the rat myocardium from the ischemia/reperfusion injury though the modulation of Toll-like receptor (Li et al., 2004). In addition, SPG can modulate endothelium-dependent vasodilation in mice (Harrington et al., 2011). It has also been previously reported that [beta]-D-glucans play considerable roles in increased production of NO (Novak and Vetvicka, 2009). Lectins have also been purified and characterized f SC (Chumkhunthod et al., 2006). Lectins isolated from Pisum arvense seeds exert relaxing effects on isolated rat aorta rings through the increase in NO synthesis (Assreuy et al., 2011). In vivo studies have shown that the lectin isolated from Tricholoma mongolicum can directly reduce the mean arterial blood pressure in rat (Wang et al., 1996). Therefore, it will be of interest to study whether or not the endothelium-dependent relaxation induced by SC as observed in the present study is related to SPG and/or lectins.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

By performing HPLC chromatogram, we found that the water extract of SC might contain phenolic constituents. This finding is consistent with the results of previous studies which show the phenolic compounds are bioactive components in SC (Abugri and McElhenney, 2013; Mirfat et al., 2010). In addition to the antioxidant activity, it has also been documented that phenolic compounds from medicinal plants may account for their antihypertensive effect (Kumar et al., 2011; Ranilla et al., 2010). Therefore, it is reasonable to presume that the phenolic compounds from SC may also play important roles in the regulation of tension of the blood vessel and fluctuation of blood pressure.

[FIGURE 7 OMITTED]

The mechanisms involved in SC-induced endothelium-independent relaxation were closely associated with the inhibitions of [Ca.sup.2+] influx and intracellular [Ca.sup.2+] release. [Ca.sup.2+] is a critical factor in excitation-contraction coupling in smooth muscle cells (Lohn et al., 2000; Wellman and Nelson, 2003). There are two types of [Ca.sup.2+] channels on the plasma membranes of vascular smooth muscle cells: receptor-operated [Ca.sup.2+] channels (ROCCs) and voltage-dependent [Ca.sup.2+] channels (VDCCs) (Xiong and Sperelakis, 1995). Influx of [Ca.sup.2+] through ROCCs and VDCCs and the release of [Ca.sup.2+] from sarcoplasmic reticulum by activation of 1,4,5-triphosphate inositol and ryanodine receptors (Imtiaz et al., 2006; Thorneloe and Nelson, 2005) result in the increased intracellular [Ca.sup.2+], which causes vascular smooth muscle contraction. Based on our results, we believed that SC inhibited the [Ca.sup.2+] influx through VDCC and attenuated the intracellular [Ca.sup.2+] release in vascular smooth muscle cells, thereby leading to a fall in intracellular [Ca.sup.2+] level and hence causing relaxation. Other mechanisms cannot be completely ruled out when the endothelium-independent relaxation is concerned. Cerebrosides are present in SC and they are able to block calcium-activated chloride channels in rat pulmonary artery smooth muscle cells (Gao et al., 2007). It is well known that calcium-activated chloride channels are crucial regulators of in vascular tone by regulating the resting membrane potential of vascular smooth muscle cells. Although relevant experiment was not carried in our current study, it is not impossible that SC may inhibit calcium-activated chloride channels on vascular smooth muscle cells, which subsequently cause the membrane hyperpolarization and relaxation.

In conclusion, our study has demonstrated that SC induces both endothelium-dependent and -independent relaxation in rat thoracic aorta. The endothelium-dependent relaxation is predominantly mediated by NO-cGMP-dependent pathway but the [PGI.sub.2]-cAMP-dependent mechanism may be partly involved. The endothelium-independent relaxation may be related to the inhibitions of voltage-dependent [Ca.sup.2+] channels and intracellular [Ca.sup.2+] release. Our findings may constitute the basis for further functional studies which is going to reveal of the identities of active ingredients in SC which are responsible to the above mechanisms.

http://dx.doi.org/10.1016/j.phymed.2014.06.008

ARTICLE INFO

Article history:

Received 3 January 2014

Received in revised form 3 May 2014

Accepted 19 June 2014

Acknowledgements

This work was supported by grants from the Natural Science Foundation of China (No. 81160404) and Natural Scientific Fund of Yunnan Province (No. 2012FB171 and No. 2012FD039).

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Haiyun Chen (a), Sujuan Li (a), Peng Wang (b), Saimei Yan (a), Lin Hu (a), Xiaoxia Pan (a), Cui Yang (a,c), *, George Pakheng Leung (c), **

(a) Ethnic Drug Screening & Pharmacology Center, Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650500, PR China

(b) School of Pharmaceutical Science & Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 650500, PR China

(c) Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, PR China

* Corresponding author at: Ethnic Drug Screening & Pharmacology Center. Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650500, PR China. Tel.: +86 871 5910017; fax: +86 871 5910017.

** Corresponding author at: Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, PR China. Tel.: +852 28192861; fax: +852 28170859.

E-mail addresses: yangynni@163.com (C. Yang), gphleung@hkucc.hku.hk (G.P. Leung).
Table 1
HPLC elution profile program.

Time (min)   A%    B%   Flow (ml/min)

0             5    95        0.8
15           15    85        0.8
25           50    50        0.8
55           100   0         0.8
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Author:Chen, Haiyun; Li, Sujuan; Wang, Peng; Yan, Saimei; Hu, Lin; Pan, Xiaoxia; Yang, Cui; Leung, George P
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
Words:4810
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