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0944-7113/$ - se

doi:10.1016/j.ph

Abreviations:

steroid; BW, bo

extensor digitor�CorrespondE-mail addr

Phytomedicine 15 (2008) 691–698

www.elsevier.de/phymed

20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat

Noemi Totha, Andras Szaboc, Peter Kacsalab, Julia Hegerc, Erno Zadorc,�

aDepartment of Pharmacognosy, Szeged, HungarybFaculty of Pharmacy, Department of Pharmacodynamics and Biopharmacy, Szeged, HungarycFaculty of General Medicine, Institute of Biochemistry, University of Szeged, H-6720 Szeged, Hungary

Abstract

20-Hydroxyecdysone (20E) is an ecdysteroid hormone that regulates moulting in insects. Interestingly, 20E is alsofound most abundantly in plant species and has anabolic effects in vertebrates, i.e. increasing muscle size withoutandrogen influence. The effect of 20E on slow and fast fiber types of skeletal muscle has not been reported yet. Here wepresent that 20E affects the size (cross-sectional area, CSA) of the different fiber types in a muscle-specific manner. Theeffect on fiber size was modified by the distance from the site of the treatment and the presence of a regenerating soleusmuscle in the animal. Besides the fiber size, 20E also increased the myonuclear number in the fibers of normal andregenerating muscles, suggesting the activation of satellite cells. According to our results 20E may provide analternative for substitution of anabolic–androgenic steroids in therapeutic treatments against muscle atrophy.r 2008 Elsevier GmbH. All rights reserved.

Keywords: 20-Hydroxyecdysone; Muscle fiber size; Regeneration

Introduction

Ecdysteroids represent a large family of polyhydroxy-lated steroid invertebrate hormones that regulatemoulting, metamorphosis and reproduction of arthro-pods (Sehnal, 1989). These substances are also synthe-sized by 5–6% of the plant species (phytoecdysteroids)may be as a defence against phytophagus insects (Dinan,1995, 2001). Generally ecdysteroids are present in ahigher concentration in plants than in arthropods andare easier to extract from plants in order to investigatetheir pharmacological effect in mammals. The acutetoxicity of ecdysteroids is very low in mice: the LD50 of20E is 49 g/kg BW when given orally and 6.4 g/kg BW

e front matter r 2008 Elsevier GmbH. All rights reserved.

ymed.2008.04.015

20E, 20-hydroxyecdysone; AAS, anabolic–androgenic

dy weight; CSA, cross-sectional area; M. EDL, muscle

um longus; MyHC, myosin heavy chain.

ing author. Tel.: +3662 545 096; fax: +36 62 545 097.

ess: erno@biochem.szote.u-szeged.hu (E. Zador).

if injected intraperitoneally (Ogawa et al., 1974;Matsuda et al., 1970). Ecdysteroids have no side effectsin mammals, including humans (Slama and Lafont,1995). Moreover, some of the ecdysteroid-containingplants are included in the human diet and used in thetraditional medicine as well (Spinacia oleracea (Bathoriet al., 1982) or Rhaponticum carthamoides (Syrov andKurmukov, 1976)). Although ecdysteroids do not bindto the vertebrate steroid receptors and their mechanismof action is still unknown, many beneficial pharmaco-logical properties are attributed to them (reviewed bySlama and Lafont, 1995; Dinan and Lafont, 2006;Bathori et al. 2008). Increased body, organ and muscleweight and protein synthesis have been reported in caseof oral or intraperitoneal administration of ecdysteroidsin several animal species: Japanese quails (Slama et al.,1996), mice (Stopka et al., 1999), rats (Syrov, 2000) andpigs (Kratky et al., 1997). There is an importantdifference between the effect of ecdysteroids and

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Fig. 1. The chemical structure of 20-hydroxyecdysone (20E).

N. Toth et al. / Phytomedicine 15 (2008) 691–698692

anabolic steroids: castration in rats sharply decreases theprotein synthesis stimulating property of ecdysteroids,while steranabols (androgen steroids) still remaineffective under this condition (Syrov, 2000). The non-androgenic effect was suggested because the studiedphytoecdysteroids did not produce any significantchanges in the weight of ventral prostate and seminalvesicles in the tested animals (normal or castratedmature or immature rats, Syrov, 2000). Phytoecdyster-oids also showed an anabolic effect in female rats(Syrov, 2000).

While in the previous reports the muscle and bodyweight or the rate of protein synthesis have beenmeasured, a more sophisticated study has not beencarried out yet. It is not known whether 20E actspreferentially on fast or slow fiber types like the anabolicsteroids (Ustunel et al., 2003). Therefore, we examinedthe effect of 20E on the size of muscle fiber types and thenumber of myonuclei reflecting the accretion of myo-blasts into the fibers. We chose the soleus, a predomi-nantly slow and the extensor digitorum longus (EDL), apredominantly fast type muscle. We also investigatedthe effect of 20E on the regenerating fibers of the soleus.We found that 20E changes the size and the myonuclearnumber of the myosin fiber types in a muscle-specificmanner in the normal and regenerating soleus and thenormal EDL. This effect is influenced by the applieddose, the distance from the site of 20E injection and, incase of the normal muscles, by the presence of aregenerating soleus in the other hindlimb.

Materials and methods

20-Hydroxyecdysone

20E was isolated from Silene viridiflora by Toth andBathori (2008) in the University of Szeged, Faculty ofPharmacy, Department of Pharmacognosy, and pro-vided for the examination in a purity of 99%. Thephysical and spectroscopic data of the isolated com-pound are in accordance with those of the literature(Lafont et al., 2002). The chemical structure of 20E isshown in Fig. 1.

Animals and treatment

Animals were treated according to the regulations ofthe Ethical Committee of the University of Szeged.

Twenty male Wistar rats (304719 g) were dividedinto five groups, with four animals in each, and treatedaccording to the scheme shown in Fig. 2. The groupN20E received 5mg/kg BW of 20E daily as an sc.injection in the left thigh. On the 8th day the soleus andEDL muscles were dissected from the left and right

hindlimb and the animals were killed with an overdoseof chloralhydrate. Group C, the control of N20Ereceived 0.9% NaCl instead of 20E. Three groups(RC, R20E-1 and R20E-2) received snake venom(notexin) injection into the left soleus muscle. The ratswere narcotised and treated for induction of muscleregeneration as in Zador et al. (1998). From the 5th daygroup R20E-1 received 5mg/kg BW 20E, while groupR20E-2 received 0.5mg/kg BW of 20E in a dailysubcutaneous injection to the left leg for 7 days. GroupRC, the control received 0.9% NaCl instead of 20E. Onthe 12th day after the snake venom injection whichconstituted for the 8th day of new muscle formation, theregenerating soleus and the contralateral normal soleusand the EDL muscles were dissected and the animalswere killed with an overdose of chloralhydrate.

Collection of muscles and staining for HE or

immunocytochemistry

The dissected regenerated soleus, normal soleus andEDL muscles were frozen in isopentane cooled withliquid nitrogen and kept at �70 1C. In all, 15 mm thickcryosections were taken from the central part of eachfrozen muscles and stained with haematoxylin–eosin.Immunostaining was carried out as in Zador et al.(1998). We identified IIx fibers as non-stained ones witha combination of BA-D5 (mouse, 1:50), SC-71 (mouse,1:20) and BF-F3 (mouse, 1:10) antibodies.

Fiber cross-sectional area and myonuclear domain

The cross-sectional area (CSA) of 150 fibers of eachmuscle was measured by Olympus DP-soft, version 3.2program (Olympus, Hamburg, Germany) on haematox-ylin–eosin stained or on immunostained sections. Thenumber of myonuclei in the fibers (in more than 100fibers of each muscle) was counted on haematoxyli-n–eosin stained sections with the 40� objective of thelight microscope. The accuracy of this method wascontrolled by comparing the results of two independent

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Whistar rats

Weight 304 ±19.5 g

n = 20

Normal muscle model

Control group C

n = 4

20E (5 mg / kg BW) N20En = 4

Control group RC

n = 4

20E (5 mg / kg BW) R20E-1

n = 4

20E (0.5 mg / kg BW) R20E-2

n = 4

Regenerating muscle model

Fig. 2. The scheme of experimental design for the study of 20E effect on muscle fibers. See explanation in Materials and methods.

N. Toth et al. / Phytomedicine 15 (2008) 691–698 693

experienced persons. The myonuclear domains in eachmuscle were calculated from the average myonuclearnumber per fiber divided by the average fiber CSA. Inthe EDL the large fibers were counted for myonucleiand divided by the average CSA of the IIx and IIBfibers, as these fiber types were responsible for the sizeincrease in response to the 20E treatment. We alsocounted the number of myonuclei of the small fibers inEDL and divided by the average CSA of the type I andIIA fibers.

Statistics

The cumulative data of muscle fiber CSA’s obtainedfrom four muscles (150 from each) were comparedamong groups by using t-test for unpaired samples orone-way analysis of variance followed by Newman–Keuls post-hoc test. The number of myonuclei and thesize of myonuclear domain were also compared ascumulative data of four muscles (from 100 fibers ofeach). All tests were performed by using the GraphPadPrism version 3.00. Results were considered significantat po0.05. All data are expressed as means7SE.

Results

Body and muscle mass

The body and muscle mass increased significantly ingroup N20E compared to group C. However these twoparameters did not change in group R20E-1 and onlythe muscle mass of the right EDL increased in groupR20E-2 compared to group RC (Table 1).

CSA of MyHC fiber types

The size of muscle fibers increased significantly inresponse to the 20E treatment in the left soleus of groupN20E (5011767.44 mm2) compared to group C(4261749.68 mm2). The rat soleus muscle consists ofslow-oxidative type I (90%) and fast-oxidative type IIA(9%) fibers expressing MyHC1 and MyHC2a isoforms,respectively. The rate of IIx/d (mentioned as IIx) fibersis only 1%. The CSA of both type I and type IIA fibersin the soleus muscle of the treated left hindlimbincreased (po0.001) in group N20E compared tocontrol group C (Fig. 3A, black and empty columns).

20E also increased the size of muscle fibers in the EDL(3137756.98 vs. 2488748.07 mm2) and the fiber sizedistribution of the treated muscle showed a bimodularpattern, suggesting a distinct effect on the different fibertypes. In the EDL four fiber types (I, IIA, IIx and IIB)are present expressing correspondingly the MyHC1,MyHC2a MyHC2x and MyHC2b isoforms. However inthe left EDL the CSA of the type IIx fibers increased(po0.001), while the type I and type IIB fibers did notchange, but the CSA of type IIA fibers decreased(po0.001) compared to those of group C (Fig. 3B, blackand empty columns). This shows that 20E affects fibersize in a muscle-specific fashion instead of a fiber type-dependent manner.

The effect of 20E on the contralateral hindlimb

The fiber sizes in the treated (left) and non-treated(right) hindlimbs of group N20E were compared withthose of the corresponding hindlimbs in the control.Interestingly, the size of the same fiber types was

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Table 1. Body weight and muscle mass of the 20E-treated rats

Body weight (g) m. soleus (mg) m. EDL (mg) Regenerating m.

soleus (mg)Start of

treatment

End of

experiment

Change in

weight

Right Left Right Left

C

324.572.50

(318�330)

336.576.55

(320�352)

12.074.16

(2�22)

140.375.69

(128�155)

143.574.09

(135�151)

142.873.64

(136�152)

146.073.03

(137�150)

–

N20E

309.076.00

(304�316)

373.075.26

(364�388)

64.074.32***

(52�72)

160.074.77**

(150�170)

17577.14*

(160�194)

166.076.38*

(156�184)

172.079.58*

(154�197)

–

RC

285.373.82

(276�292)

353.877.96

(340�376)

68.5710.65

(49�94)

158.073

(150�160)

– 155.075

(150�170)

– 113.076

(100�130)

R20E-1

293.572.06

(290�298)

352.574.78

(340�360)

59.073.11

(50�64)

168.077.5

(150�180)

– 150.074.08

(140�160)

– 113.077.5

(100�130)

R20E-2

314.078.29

(296�336)

387.075.80

(376�402)

73.074.04

(66�80)

174.076.96

(158�189)

– 178.074.05*

(169�188)

– 116.0710.49

(90�136)

*, **, ***po0.05, 0.01, 0.001 compared to the correspondent control (0.9% NaCl and notexin+0.9% NaCl). The mass range is in parenthesis.

N. Toth et al. / Phytomedicine 15 (2008) 691–698694

different in the left than in the right hindlimb muscleseven in the control group (Fig. 3). In group N20E, thetype I fibers were larger (po0.05) in the right than in theleft soleus, while the IIA fibers became larger in the leftthan in the right soleus (po0.01) compared to those ingroup C (Fig. 3A). In the EDL, the CSA of type I fiberswere not different from the control, but the IIA fibersbecame smaller after the 20E treatment in the left thanin the right muscle. Moreover the size of IIx fibersincreased in the left (po0.001) but not in the right EDL,while the size of the IIB fibers was higher in the rightEDL of the control and it was decreased by 20E in theright but not in the left EDL compared to the controls(Fig. 3B). This shows that the distance from the site ofthe 20E treatment blunted the decrease of IIA fibers andthe increase of IIx fibers in the EDL.

20E stimulates the fiber growth in the regenerating

soleus

The soleus muscle consisted of entirely new myofibersafter 5 days of notexin treatment, later 98% of themexpressed slow myosin (Whalen et al. 1990). The size ofthe regenerating fibers has been increased (po0.001) by20E in group R20E-1 (Fig. 4A).

Injection of a 10 times lower dose of 20E (0.5mg/kgBW) for 7 days into rats (group R20E-2) increased thefiber size (po0.001) only in the regenerating soleus, butthis increase was less pronounced than in the animalstreated with 5mg/kg BW of 20E (group R20E-1)(Fig. 4B). This showed that the effect of 20E was dose

dependent and the 0.5mg/kg BW was still an effectivedose on the regenerating soleus but not on the fibers ofnormal muscles in the contralateral leg (data notshown).

The regenerating soleus modified the effect of 20E on

fiber size of normal muscles

The relative changes in CSA of fiber types in soleusand EDL of the right hindlimb of group R20E-1 andN20E were different compared to the correspondingcontrols, RC and C (Fig. 5). The increase of type I fibersizes in the right soleus was smaller (po0.001) in theR20E-1 than in the N20E group. A smaller but stillsignificant difference was also found between the relativechanges of the type IIA fibers of the right soleus musclesof the two groups (po0.05). In the right EDL the type Ifibers were not changed compared to controls. The IIAfibers were more decreased in R20E-1 than in the N20Egroup (po0.001). Interestingly, the size of IIx fibers wasincreased only in the R20E-1 group, while the size of IIBfibers increased in the R20E-1 group and decreased inthe N20E group. This showed that the presence of aregenerating soleus in the rat modifies the effect of 20Eon the size of fiber types.

Myonuclear number

The numbers of myonuclei followed the increase ofcross-sectional areas in most muscles after the 20Etreatment. This meant that the size of myonuclear

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m. soleus

0

2500

5000

7500

Fibe

r CSA

(µm

2 )

***

***

I IIA

C leftN20E left

***

C rightN20E right+

++

m. EDL

0

1000

2000

3000

4000

Fibe

r CSA

(µm

2 )

C leftN20E leftC rightN20E right

I IIA IIB

***

***

*

***+++

+++

++

IIx

Fig. 3. The size of MyHC fiber types in the left and right

soleus and EDL muscles of rats in group N20E treated with

20E in the left leg. Symbols *po0.05, ***po0.001 compared

to the control,+po0.05, ++po0.01, +++po0.001 com-

pared to the change in fiber size of the corresponding muscle in

the other hindlimb.

5 mg 20E / kg BW

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0

50

100

150

200

Fibe

r num

ber

RCR20E-1

0.5 mg 20E / kg BW

0

500

1000

1500

2000

2500

3000

3500

4000

0

50

100

150

200

Fibe

r num

ber

RCR20E-2

Fiber CSA (µm2)

Fiber CSA (µm2)

Fig. 4. Fiber size distribution of regenerating m. soleus after

administration of 5mg/kg BW and 0.5mg/kg BW 20E. Mean

fiber CSA of group RC is 1714728.34mm2. Mean fiber CSAs

of groups R20E-1 and R20E-2 are 2077724.58 and

1857721.40 mm2, respectively. Data are means7SE. Note

that the number of larger muscle fibers is increased by the 20E

treatment in a concentration-dependent manner in the

regenerating muscles.

N. Toth et al. / Phytomedicine 15 (2008) 691–698 695

domains did not change, except in group N20E when anincrease (po0.01) in the right soleus and decreases inlarge fibers (IIx and IIB) in EDL on both sides werefound (Table 2). The myonuclear domains also de-creased in small fibers of the EDL in R20E-1 group. Thepositions of myonuclei did not change in response to the20E treatment, i.e. they remained peripheral in normaland central in regenerating muscles.

Fiber type proportion

The relative fiber type proportion did not change inthe muscles of any of the groups. The 20E also did notinfluence the formation of predominantly slow typefibers in the regenerated soleus.

Discussion

Our study demonstrated that 20E affects the size offiber types in a different manner in the soleus than in the

EDL muscles. This suggests that this compound affectsfiber type size in a muscle-specific fashion. A muscle-specific effect on fiber size has also been reported in caseof anabolic–androgenic steroids (AAS). Treatment witha low (1.5mg/kg BW/week) and a high (7.5mg/kg BW/week) therapeutic dose of nandrolone decanoate over a5 weeks period increased the dimension of type IIx andIIB fibers in the diaphragm and of type IIa fibers in thegastrocnemius, while the other fiber types remainedunchanged (Bisschop et al., 1997). In human, where theMyHC2b is hardly expressed in type II fibers, a long-term AAS administration (power lifters taking anabolicsupplements for nearly 10 years) increased the CSA oftype I and type II fibers both in m. vastus lateralis andm. trapezius, while no alteration in the fiber typeproportions was found (Smerdu et al., 1994; Erikssonet al., 2005; Kadi et al., 1999). Similar observations weremade in healthy young men when the diameter of bothtype I and II fibers were increased after 20 weeks

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Right m. soleus

0

2500

5000

7500

Fibe

r CSA

(µm

2 )

I

***

*********

+++RCR20E-1CN20E

+

Right m. EDL

0

1000

2000

3000

4000

5000

Fibe

r CSA

(µm

2 )

IIBIIAI IIx

***

***

+++

***+++

+++RCR20E-1CN20E

***

*

IIA

Fig. 5. The presence of a regenerating soleus in the left

hindlimb modifies the effect of 20E in the contralateral normal

soleus and EDL. ***po0.001 compared to the corresponding

control.+po0.05, +++po0.001 compared to the change in

fiber size of the corresponding muscle of the other group of

rats with or without notexin treatment.

N. Toth et al. / Phytomedicine 15 (2008) 691–698696

supplementation of gonadotrop-releasing hormone ago-nist (to suppress endogenous testosterone release) and ofexogene testosterone (Sinha-Hikim et al., 2002).Although the effect of 20E on these muscles is to bedetermined, it appears that both anabolic steroids andecdysteroids are acting on the size of fibers in a multiple,muscle-specific fashion. However, it should be notedthat AAS are effective in a lower dose than 20E. It isanother difference that 20E did not alter the fiber typeproportion in the studied muscles as the anabolicsteroids (Holmang et al., 1990).

The fast anabolic effect of steroids in skeletal muscleis exerted through signal transduction pathways and notvia intracellular steroid receptors (Estrada et al. 2003).The overexpression of calcineurin, a central player insignalling in muscle, influences fiber phenotype anddifferentiation in a muscle-specific fashion (Talmadge etal. 2004). This is in line with the muscle-specific effects ofAAS and ecdysteroids on fiber types and it implies thatecdysteroids might act on skeletal muscle via signalpathways. In accordance with this, ecdysteroids havebeen shown to act on signal mechanisms of mammalianhematopoietic cells (Constantino et al. 2001).

The 20E showed a more pronounced effect in thetreated left hindlimb (on IIx and IIb fibers of the EDL)than in the untreated right hindlimb, probably becauseof the distance from the site of administration. Thisshows that the 20E acts locally and via the circulation.

20E increased fiber CSA in the regenerating soleusmuscle, suggesting that the 20E exerted a beneficialeffect on muscle regeneration. A similar effect of AAS(nandrolone) has been reported on the regeneratingsoleus but not on the EDL (Ferry et al., 1999). However,in our experiments, the regenerating soleus muscle in theleft hindlimb influenced the effect of 20E in thecontralateral hindlimb compared to the animals withoutregenerating soleus. This cannot be explained by theoverload of the right hindlimb (caused by the retaineduse of the left hindlimb), since overload increases thesize and the number of type I fibers (Zador et al., 1999),unlike it happened in the right hindlimb. It is more likelythat the regenerating soleus influences the growth factorenvironment which interacts with the effect of 20E onthe right EDL. In support of this, one example is knownwhen ecdysteroids altered the signalling by interleukin-3in mammalian hematopoetic cells (Constantino et al.,2001).

A 10 times lower dose (0.5mg/kg BW) of 20E induceda lower increase of the fiber’s CSA in the regeneratingsoleus but had no effect on the fibers in the contralateralnormal muscles. However the low dose of 20E increasedthe weight of the EDL, suggesting dose dependence anda difference in the mechanism of action of a low andhigh dose of 20E.

The 20E increased the myonuclear number in most ofthe affected muscles. Muscle fibers also grow in responseto AAS with increasing myonuclear number (Eriksson etal., 2005). The myonuclei derive from the accretion ofmyoblasts produced by the activated satellite cells(muscle resident mesenchymal stem cells), and help tomaintain gene expression within the fiber. The 20Eincreased the myonuclear number in proportion to thefiber growth, therefore maintained the size of themyonuclear domains (the sarcoplasm volume aroundthe myonuclei). This suggests that the 20E stimulationof fibers size involves the activation of satellite cellssimilar to AAS (reviewed by Chen et al., 2005).

In conclusion, 20E modifies muscle fiber size innormal and regenerating muscles even after 7 daysadministration in a slightly higher dose than theanabolic steroids. This effect is dose dependent and,similarly to that of anabolic steroids, it influences thesize of fiber types in a muscle-specific fashion. The 20Eprobably acts in concert with other growth factorsbecause its effect on normal muscles is modified by thepresence of a regenerating soleus. This suggests that 20Emay provide an opportunity for substitution of anabo-lic–androgenic steroids in therapeutic treatments againstmuscle atrophy.

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Table 2. Myonuclear numbers and domains in 20E-treated muscles

Myonuclei/fiber

m. soleus m. EDL Regenerating

m. soleus

Right Left Large fibers Small fibers

Right Left Right Left

C

0.8870.06 0.9170.03 1.59070.05 1.56370.03 0.4570.017 0.5270.013 –

N20E

0.8770.01 1.0970.03* 1.9670.07* 2.0470.08** 0.4470.02 0.4570.02 –

RC

0.9270.05 – 1.6770.05 – 0.4270.018 – 1.5770.06

R20E-1

0.9670.04 – 2.0070.13 – 0.5270.01** – 1.9170.06**

R20E-2

– – – – – – 1.8270.03**

Myonuclear domain (mm2)

C

4405781.73 44817179.9 1808796.41 1759755.42 30727197.6 27137168.4 –

N20E

55327306.0* 44937145.1 1447781.16* 1539742.02* 2860781.22 261376.4 –

RC

40117167.1 – 17747221.4 – 34497260.30 – 1234755.63

R20E-1

4378789.0 – 15967105.7 – 25907134.20* – 1082732.75

R20E-2

– – – – – – 10287104.2

*, **, ***po0.05, 0.01, 0.001 compared to the correspondent control (0.9% NaCl and notexin+0.9% NaCl).

N. Toth et al. / Phytomedicine 15 (2008) 691–698 697

Acknowledgements

This work was partially supported by the ETT 168/2003 grant from the Hungarian Ministry of Health.Thanks to professor Maria Bathori for the 20-hydro-xyecdysone. Thanks to Dr. Arpad Marki for technicalassistance in the initial phase of this study.

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