Research Article Effect of Electroacupuncture on the...

Research ArticleEffect of Electroacupuncture on the Expression ofGlycyl-tRNA Synthetase and Ultrastructure Changes inAtrophied Rat Peroneus Longus Muscle Induced bySciatic Nerve Injection Injury

Meng Wang1 Xiao Ming Zhang12 and Sheng Bo Yang1

1Department of Anatomy Zunyi Medical College Zunyi Guizhou 563000 China2Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City KS 66160 USA

Correspondence should be addressed to Sheng Bo Yang shengboyangzyhotmailcom

Received 13 January 2016 Revised 28 February 2016 Accepted 29 February 2016

Academic Editor Thomas Lundeberg

Copyright copy 2016 Meng Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Glycyl-tRNA synthetase (GlyRS) is one of the key enzymes involved in protein synthesis Its mutations have been reported tocause Charcot-Marie-Tooth disease which demonstratesmuscular atrophy in distal extremities particularlymanifested in peroneusmuscles In this situation the dysfunctions of mitochondria and sarcoplasmic reticulum (SR) affect energy supply and excitation-contraction coupling of muscle fibers therefore resulting in muscular atrophy Although the treatment of muscular atrophy is aglobal urgent problem it can be improved by electroacupuncture (EA) treatment To investigate the mechanism underlying EAtreatment improving muscular atrophy we focused on the perspective of protein synthesis by establishing a penicillin injection-induced sciatic nerve injury model In our model injured rats without treatment showed decreased sciatic functional index (SFI)decreased peroneus longusmuscle weight andmuscle fiber cross-sectional area aggregatedmitochondria with vacuoles appearingswollen SR and downregulatedmRNA and protein expression levels of GlyRS andmyosin heavy chain IIb (MHC-IIb)The injuredrats with EA treatment showed significant recovery These results indicated that EA stimulation can alleviate peroneus longusmuscular atrophy induced by iatrogenic sciatic nerve injury through promoting the recovery of GlyRS and muscle ultrastructureand increasing muscle protein synthesis

1 Introduction

The sciatic nerve injury can be caused by incorrect intra-muscular injection of medication in the gluteal region inclinic This is an iatrogenic injury which can be avoidedbut still exists especially in developing countries The injuryaffects mostly infants [1 2] The injury can lead to lower limbmuscle atrophy and even disability However the treatmentof muscular atrophy is a global urgent problem because theunderstanding of muscle atrophy mechanisms is not com-pletely clear The current understanding of the mechanismsfor muscle atrophy is attributed to an increase in proteindegradation andor a decrease in protein synthesis Theincrease in protein degradation ismainly due to the activationof the ubiquitin-proteasome pathway while the information

on the mechanism of the protein synthesis reduction islimited [3]

A key step in protein synthesis is the covalent linkageof tRNA with corresponding amino acids catalyzed by theaminoacyl-tRNA synthetases [4] GlyRS is one of the 20 vari-eties of aminoacyl-tRNA synthetases Charcot-Marie-Toothdisease is caused by the mutations of the GlyRS gene [5]The patients with this disease demonstrate muscular atrophyin distal extremities particularly manifested in peroneusmuscles Nevertheless can sciatic nerve injection injury causethe expression change of GlyRS in the peroneus musclesIs the muscle atrophy caused by corresponding proteinsynthesis It is assumed that the decrease of protein synthesisis caused by the change of GlyRS in muscle after the nerveinjury leading to muscle atrophy However we do not know

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7536234 10 pageshttpdxdoiorg10115520167536234

2 Evidence-Based Complementary and Alternative Medicine

which particular proteinrsquos synthesis is decreased Accordingto previous studies the most abundant protein expressionin muscle cells is myosin which forms myofibrillar thickfilaments After nerve injury the MHC-IIb demonstrates themost unstable feature and shows the most obvious decreasecausing the fastest muscle atrophy [6ndash8] Therefore in thisstudy we use MHC-IIb as an index to investigate the proteinexpression level change in muscle atrophy

In addition to providing energy in protein synthesismitochondria are also involved in cell cycle regulation andintracellular signal transduction Insulin-like growth factor-1phosphatidylinositol 3-kinaseprotein kinase Bglycogensynthase kinase 3 beta (IGF-1PI3KPKBGSK3120573) signalingpathway is an important pathway which regulates proteinsynthesis Upon activation of the pathway PKB is transferredinto mitochondria results in GSK3120573 phosphorylation torelieve the inhibition of eukaryotic initiation factor-2B andtherefore promotes protein synthesis [9 10] After denerva-tion the mitochondria swell and become vacuolated exhibitdysfunctions impair protein synthesis and aggravate muscleatrophy [11] Although SR without ribosomes is not directlyinvolved in protein synthesis it regulates the excitation-contraction coupling of muscle fibers as calcium storage andthereby is closely related to muscle atrophy [12 13]

Studies showed that electrostimulation can improveskeletal muscle atrophy through promoting axonal regener-ation of injured nerve and reinnervation of muscle [14ndash17]Does it affect the expression level of GlyRS and promotethe recovery of ultrastructural change of peroneus longusafter sciatic nerve injury Therefore in this study the effectof EA on muscular atrophy and the changes of GlyRS andultrastructure were observed The aim of this study wasto preliminarily investigate the mechanism underlying theimprovement brought about by EA of rats with muscularatrophy caused by sciatic nerve injury following penicillininjection We focus on the perspective of protein synthesishoping to afford new thinking on the treatment of muscularatrophy

2 Materials and Methods

21 Animal Care Grouping and Ethics Statement One hun-dred and eight Sprague-Dawley (SD) rats (meanweight 200plusmn50 g 7ndash9weeks old) were purchased fromLaboratory AnimalCenter Third Military Medical University (SCXK 2012-0005Chongqing China) eithermale or female randomly selectedThe animals were randomly divided into 4 groups namelycontrol (CON) sciatic nerve injury (SNI) CON+EA andSNI+EA Of these group SNI was administrated at 1 week2 weeks 4 weeks and 6 weeks after penicillin injection-induced sciatic nerve injury respectively group CON+EAwas administrated at 2 weeks and 4 weeks after EA stimula-tion ofHuantiao (30th Point ofGallbladderMeridian of Foot-Shaoyang (GB30) located at the posterior upper border ofthe hip joint of the hind limbs vertical needling to a depthof 6mm) and Zusanli (36th Point of Stomach Meridian ofFoot-Yangming (ST36) 5mm below the head of the fibulaunder the knee joint and 2mm lateral to the knee jointvertical needling to a depth of 6mm) respectively [18] group

SNI+EA was administrated at 2 weeks and 4 weeks after EAstimulation of GB30 and ST36 respectively 2 weeks afterpenicillin injection of sciatic nerve injury Totally there were9 animal groups and each group had 12 rats of which 6were used for histological observations and the other 6 wereused for molecular biological studies The rats were housedin individual cages and fed standard rat chow and water adlibitum All experiments were performed in accordance withthe guidelines of the China Animal Welfare Act This studywas approved by the Animal Care and Use Committee ofZunyi Medical College All the surgical steps were performedstrictly in accordance with the principles of aseptic surgeryset forth by Zunyi Medical College

22 Sciatic Nerve Injury Induced by Penicillin InjectionSeventy-two SD rats were taken for routine disinfection ofthe skin and then they were subjected to intraperitonealanesthesia with 10 chloral hydrate at 03mL100 g A 10 cmlongitudinal incision was applied on the right femoral areaand 05 cm horizontal incision under the third trochanterThe sciatic nerve was then exposed with blunt dissectionafter which 200000U that is 05mL penicillin sodium(Harbin Pharmaceutical Group Co Ltd General PharmFactory approved size A051134107 size 048 g08 millionunits) was injected with a No 4 needle at the outer side ofthe neural stem [7] The wound was then sutured layer bylayer and disinfected after surgery Among the 72 rats thatunderwent surgery with penicillin injection in the sciaticnerve 48were selected for group SNIThen 12were sacrificedat time points of 1 week 2 weeks 4 weeks and 6 weeksrespectively

23 EA Treatment In this experiment points of GB30and ST36 on rats were selected as a match pair based onthe principle of selecting two points on each site of theinjury Filiform needles were used to penetrate into GB30and ST36 points After acuesthesia the positive pole onthe G6805-II model electroacupuncture device (QingdaoXinsheng Industrial Co Ltd China) was connected to GB30point and the negative pole was connected to ST36 pointwith a 5Hz output frequency using continuous wave for 10minutes The stimulation intensity was regulated with 2mAand monitored by slight muscle tremor of the injured limbEA was performed on alternate days three times a weekand the entire course of treatment lasted 4 weeks [19] GroupCON+EA consisted of 24 normal rats receiving EA treatmentwithout surgery and nerve injury 12 of which received onecourse of treatment (group CON+EA at 2 weeks) and theother 12 received two courses of treatment (group CON+EAat 4 weeks) Of the 72 rats that were subjected to sciatic nerveinjury induced by penicillin injection 24 rats received EA at2 weeks after the injection 12 of which received one courseof treatment (group SNI+EA at 2 weeks) while the other12 received two courses of treatment (group SNI+EA at 4weeks)

24 Sciatic Functional Index (SFI) Testing After observingthe ratsrsquo gait at every time point of each group their soles were

Evidence-Based Complementary and Alternative Medicine 3

colored with ink and then rats were allowed to voluntarilywalk from one end to the other end of the self-made footprintbox leaving 4 to 5 clear footprints of each hind leg on eachside Print lengths (PL) of normal foot (N) experimental foot(E) toe spread (TS) and intermediary toe (IT) spread weremeasured respectively (Figure 1(b)) The above indexes weresubstituted into Bain equation to calculate the SFI in whichnormal SFI = 0 while SFI = minus100 indicates complete damageBain equation is as follows

SFI = 1095 (ETS minus NTS)NTS

minus383 (EPL minusNPL)

NPL

+133 (EIT minusNIT)

NITminus 88

(1)

(see [20])

25 Morphological Analysis The rats were sacrificed bycervical dislocation Peroneus longus muscle was removedquickly after observing the color and size of the muscleand was weighed and recorded Then partial muscle massin venter musculi was cut into the appropriate tissue blockaccording to the different experimental needs and was placedin formaldehyde or glutaraldehyde fixation for HampE stainingor electron microscope processing Some specimens werekept in freezer at minus80∘C for reverse transcriptase-polymerasechain reaction (RT-PCR) andWestern blot analysisThemus-cle mass for HampE staining was cross-sectioned at a thicknessof 8120583m After HampE staining the cross-sectional muscle fiberarea of peroneus longus muscle was measured using theOlympus DP26 and CellSens Standard 111 image analysissoftware (Olympus Corporation Japan) After being stainedwith uranyl acetate and lead citrate the muscle ultrathin sliceof the longitudinal section was observed using the HitachiH-7650 transmission electron microscope (Hitachi Co LtdJapan) and photographed

26 RT-PCR Total RNA was extracted from the peroneuslongus of SD rats using the TRIzol reagent (Sangon BiotechCo Ltd Shanghai China) One microliter of total RNA wasused for reverse transcription reaction using the FastQuantRT Kit (Tiangen Biotech Co Ltd Beijing China) Then the2x Taq PCR Master Mix kit (Tiangen Biotech Co Ltd) wasused for PCR amplification The PCR electrophoresis bandswere photographed in the gel imager and the optical densityvalues of the bands were measured using the Quantity Onesoftware (Bio-Rad Co Ltd California USA) The opticaldensity value of each objective gene band was divided by theoptical density value of 120573-actin which represents the mRNAexpression level of the housekeeping gene The sequences ofGlyRS MHC-IIb and 120573-actin primers which were designedand synthesized by Shanghai Sangon Biotech Co Ltd are asfollows

GlyRS-F 51015840-GGTCAGTGTGAAGAGATTCCAG-31015840GlyRS-R 51015840-AAGTCAATGGTGATGCCAAAC-31015840MHC-IIb-F 51015840-GGCATTGAGTGGGAGTTCAT-31015840MHC-IIb-R 51015840-GTCTTCAACCCGGACTTCTG-31015840

120573-actin-F 51015840-GAGAGGGAAATCGTGCGTGAC-31015840120573-actin-R 51015840-CATCTGCTGGAAGGTGGACA-31015840

27 Western Blot According to the procedure instructionsin the KeyGen total protein extraction kit (Nanjing KeyGenBiotech Co Ltd China) the muscle tissues were shearedinto small pieces and were placed in a glass homogenizerAfter adding the premade mixed cold lysis buffer the glasshomogenizer was manually operated 15 times followed by5min centrifugation of the tissue homogenate at 10000 rpmat 4∘C The total protein concentration of the supernatantwas measured according to the instructions of bicinchoninicacid protein concentration kit (Generay Biotech Co LtdShanghai China) Approximately 40120583g of the sample wasloaded in each lane for SDS-PAGEelectrophoretic separationand then the samples were transferred to a PVDFmembraneAfter the PVDF membrane was blocked with 5 skim milkpowder mouse anti-human GlyRS (dilution of 1 50) andmouse anti-chickenMHC-IIb (dilution of 1 2000) polyclonalantibodies (Santa Cruz Biotechnology Inc USA) were addedand incubated at 4∘C overnight Subsequently horseradishperoxidase- (HRP-) labeled goat anti-mouse secondary anti-body (Santa Cruz Biotechnology Inc USA) was added in1 2000 ratio and incubated at room temperature for 2 hGAPDHwas used as the housekeeping gene and the filmwasdeveloped using a chemiluminescent substrate Chemilumi-nescence Imaging System (Bio-Rad Co USA) was used totake photos for the bands The optical density values of thetarget proteinGAPDH were measured using the QuantityOne software (Bio-Rad Co USA) The ratio represented thespecific levels of protein expression

28 Statistical Analysis Data from this experiment were putinto SPSS170 software package (IBM SPSS Co USA) andthen processed using one-way ANOVA with test level 120572 =005

3 Results

31 General Observations Compared with the group CONthe hind legs of the group SNI rats became limp and had to bedragged One week after sciatic nerve injury rats developedclubfeet (Figure 1(a)) the surface of peroneus longus muscleslost glossiness and their sizes became smaller The crusbecame thinner and the muscle showed significant atrophyin the group SNI at 2 weeks and 4 weeks Compared with thegroup SNI at 2 weeks and 4 weeks there was no differenceunder the naked eye at 6 weeks However no such changeswere observed in the group CON+EA The above changesshowed obvious improvement in the group SNI+EA

32 SFI Findings The results of the SFI changes of the ratsin each experimental group are shown in Figure 1(d) In thegroup SNI SFI gradually reduced at 1 week and 2 weeks andthe SFI gradually recovered at 4 weeks and 6 weeks but notback to normal in comparison with the group CON (119901 lt005) No statistical significance was seen between the groupCON+EAand the groupCONComparedwith the group SNI


(a)

E

IT

PL TS

ITPL

TS

N

(b)

➂➁➀

(c)

minus100minus90minus80minus70minus60minus50minus40minus30minus20minus10

0

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Scia

tic fu

nctio

nal i

ndex

(SFI

)

998779 998779lowast

lowast

(d)

000

003

006

009

012

015

Mus

cle w

eigh

t (g)

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowast

(e)

0

40

80

120

160

200

240

280

320

360

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Cros

s-se

ctio

nal a

rea (

120583m

2)

998779

998779

lowast

(f)

Figure 1 Morphology and sciatic nerve functional index (SFI) measurements (a) Ratrsquos clubfeet on the side operated on (indicated by thewhite arrow) in the group SNI at 4 weeks (b) Ratrsquos footprints in the group SNI at 4 weeks N is the normal side and E is the experimentalside PL represents print length TS represents toe spread and IT represents intermediary toe spread (c) An HampE-stained cross section ofperoneus longus muscle from groups on a standard 50120583m scale It represents muscle cross sections from the group CONA the group SNIat 2 weeksB and the group SNI+EA at 4 weeksC (dndashf) show SFI (d) peroneus longus muscle weight (e) and muscle fiber cross-sectionalarea (f) changes respectively lowast119901 gt 005 versus the group CON 119901 lt 005 versus the group CON 998771119901 lt 005 versus the group SNI at 2 weeks998787119901 lt 005 versus the group SNI at 4 weeks or at 6 weeks


at 2 weeks SFI rebounded by 2388 and 3169 in the groupSNI+EA at 2 weeks and 4 weeks respectively however SFIincreased by only 1263 and 1895 in the group SNI at 4weeks and 6 weeks respectively Compared with the groupSNI at 4 weeks there was a significant difference in the groupSNI+EA at 2 weeks (119901 lt 005) Compared with the groupSNI at 6 weeks there was a significant difference in the groupSNI+EA at 4 weeks (119901 lt 005)

33 Weight Change in Peroneus Longus Muscle The findingsof peroneus longus muscle weight of SD rats in each groupare shown in Figure 1(e) Compared with the group CON themuscle weights in the group SNI were decreased by 12622793 3602 and 2107 at 1 week 2 weeks 4 weeks and6 weeks respectively (119901 lt 005) There was the greatestdecrease in the group SNI at 4 weeks But compared tothe group SNI at 4 weeks there was a slight recovery by685 at 6 weeks (119901 lt 005) The group CON+EA showedno difference in the muscle weight in comparison with thegroup CON (119901 gt 005) The group SNI+EA at 2 weeks and4 weeks showed increase of 1313 and 2184 in muscleweight compared with the group SNI at 2 weeks respectively(119901 lt 005) Compared with the group SNI at 4 weeks therewas a significant difference in the group SNI+EA at 2 weeks(119901 lt 005) Compared with the group SNI at 6 weeks therewas a significant difference in the group SNI+EA at 4 weeks(119901 lt 005)

34 Muscle Fiber Cross-Sectional Area Under the lightmicroscope the peroneus longus muscle cross section ofthe CON group rats was distributed as net shape with thecytoplasm being red and the cell nucleus blue and themusclecross-sectional area was 32668 plusmn 1921 120583m2 (Figure 1(c))Compared to the group CON the peroneus longus musclefiber in the group SNI became thinner in accordance withmuscle weight and the muscle cross-sectional areas werereduced by 2091 4481 5026 and 3892 at 1 week2 weeks 4 weeks and 6 weeks respectively No statisticaldifference was found between the group CON+EA and thegroup CON The group SNI+EA at 2 weeks and 4 weeksshowed 2127 and 3637 recovery respectively comparedwith the group SNI at 2 weeks Compared with the groupSNI at 4 weeks there was a significant difference in the groupSNI+EA at 2 weeks (119901 lt 005) Compared with the groupSNI at 6 weeks there was a significant difference in the groupSNI+EA at 4 weeks (119901 lt 005) These results are shown inFigures 1(c) and 1(f)

35 Ultrastructural Changes of the Peroneus Longus MuscleIn the group CON myofibrils arranged orderly sarcomereintegrity A band I band Z lines and M lines were clearlyvisible mitochondria with legible structure were distributedamong the myofibrils regularly the SR lay on myofibrilsaround by longitudinal distribution and were clearly visibleIn the group SNI at 1 week and 2 weeks part of myofibrilswere disordered A and I bands shortened M lines blurredand partial Z lines were not aligned and dissolved the swollenmitochondria were clustered and even vacuolized SR were

gradually swollen obviously In the group SNI at 4 weeks and6 weeks A and I bands were invisible Z lines were dissolvedbut still leave traces and M lines disappeared aggregatedmitochondria and vacuolization were seen in some areassome SR were swollen and showing abnormal structuresCompared to the group CON the ultrastructure in the groupCON+EA showed no significant difference A comparison ofthe group SNI+EAwith the group SNI at 2 weeks showed thatthe abovementioned structures gradually became clear andintact Since there was no difference in the group CON+EAat 2 weeks and 4 weeks there was just a different change ofdegree in the group SNI at 1 week and 2 weeks in the groupSNI at 4 weeks and 6 weeks and in the group SNI+EA at 2weeks and 4 weeks Therefore here we only put pictures ofthe group CON the group SNI at 2 weeks the group SNIat 6 weeks the group CON+EA at 4 weeks and the groupSNI+EA at 4 weeks in Figure 4

36 mRNA Expression Levels of GlyRS and MHC-IIb inPeroneus Longus Muscle The mRNA expression changes ofGlyRS and MHC-IIb in each experimental group are shownin Figures 2(a)ndash2(c) Compared with the group CON theoptical density value of mRNA expression of GlyRS in thegroup SNI was decreased by 5077 5972 5084 and2951 at 1 week 2 weeks 4 weeks and 6 weeks respectively(119901 lt 005) No statistical difference was found between thegroup CON+EA and the group CON Compared with thegroup SNI at 2 weeks the group SNI at 4 weeks and 6 weeksshowed that GlyRS expression only increased by 888 and3021 respectively (119901 lt 005) In the group SNI+EA at2 weeks and 4 weeks GlyRS expression levels increased by3486 and 5986 respectively compared to the group SNIat 2 weeks (119901 lt 005) The mRNA expression level of GlyRSof the group SNI+EA at 2 weeks was significantly differentfrom that of the group SNI at 4 weeks (119901 lt 005) Theexpression level of GlyRS of the group SNI+EA at 4 weekswas significantly different from the group SNI at 6 weeks (119901 lt005) The expression variation pattern of MHC-IIb mRNAwas the same as that of GlyRS with merely slight differencesin the expression values

37 GlyRS and MHC-IIb Protein Expression Changes inPeroneus LongusMuscle Protein expression levels are shownin Figures 3(a)ndash3(c) The variation patterns in the proteinexpression levels of GlyRS and MHC-IIb in peroneus longusmuscles were the same as their mRNA expression variationpatterns with differences only in the expression degree

4 Discussion

Gluteal region injection even with normal saline may endup with direct sciatic nerve damage or injury by regionalcompression The consequence is muscular atrophy or evendisability This brings about harm and even disaster to thepatient and family [21]Therefore the study of themechanismand the treatment of this muscle atrophy have been a popularconcern Although the mechanism study of an increase inprotein degradation had achieved great progress in skeletal


(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)

Figure 2 mRNA expression changes of GlyRS and MHC-IIb in peroneus longus muscle (a) RT-PCR products of GlyRS and MHC-IIb (topto bottom) M marker 120573 120573-actin CON control SNI 1 w SNI 2w SNI 4w and SNI 6w lanes correspond to rat groups that were sacrificed at1 week 2 weeks 4 weeks and 6 weeks respectively after sciatic nerve injury following penicillin injection CON+EA 2w and CON+EA 4wlanes correspond to normal rats who received EA after 2 weeks and 4 weeks respectively SNI+EA 2w and SNI+EA 4w lanes correspond torats that received EA after 2 weeks and 4 weeks respectively 2 weeks after sciatic nerve injury (b) and (c) are the relative mRNA expressionlevels ofGlyRS andMHC-IIb in peroneus longusmuscle in each experimental group respectively lowast119901 gt 005 versus the groupCON119901 lt 005versus the group CON 998771119901 lt 005 versus the group SNI at 2 weeks 998787119901 lt 005 versus the group SNI at 4 weeks or at 6 weeks

muscle atrophy caused by nerve injury [22] studies on thereduction of protein synthesis in muscle atrophy are poorlyunderstood At present there are a lot of reports on thetreatment of muscle atrophy especially the treatment usingacupuncture They all have obvious improvement in muscleatrophy but the mechanism of muscle atrophy is unknown[23 24] The probability of common peroneal nerve injury ishigher than that of the tibial nerve because of the lateral loca-tion of the former within the sciatic nerve starting from thegluteal region This feature makes the peroneal nerve morevulnerable for gluteal injection injury Thus in this study

we establish SD rat models with sciatic nerve injury-inducedperoneus longus muscular atrophy by intentionally injectingpenicillin which is extremely neurotoxic into the outer sideof sciatic nerve stem and then observe the improvement effectof muscle atrophy by EA The expression level of GlyRS andthe ultrastructural changes were detected before and afternerve injury or acupuncture treatment We tried to look intothe mechanism underlying the improvement brought aboutby EA from the perspective of protein synthesis

Muscle weight and muscle fiber cross-sectional area canreflect the extent of skeletal muscular atrophy [7 25]The SFI


GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)

Figure 3 Protein expression changes of GlyRS and MHC-IIb in peroneus longus muscles (a) Western blot results of GlyRS MHC-IIb andhousekeeping gene (top to bottom) (b) and (c) are the specific protein expression levels of GlyRS and MHC-IIb in peroneus longus musclein each experimental group respectively lowast119901 gt 005 versus the group CON 119901 lt 005 versus the group CON 998771119901 lt 005 versus the groupSNI at 2 weeks 998787119901 lt 005 versus the group SNI at 4 weeks or at 6 weeks

is an indicator of evaluating the sciatic nerve function [26]The motor function of the mice was significantly improvedby the treatment of EA after spinal cord transection [15] Ourresults showed that 1 week after sciatic nerve injection injurythe rats developed clubfeet and their hind legs became limpand had to be dragged The size of peroneus longus musclebecame smaller muscle weight and muscle fiber cross-sectional area decreased and SFI decreased significantlyMoreover themuscle weight andmuscle fiber cross-sectionalarea further reduced in the group SNI at 2 weeks Theseresults suggested that our muscular atrophy model of sciaticnerve injury induced by penicillin injection was successfulSFI showed themost significant decline in 2weeks after sciaticnerve injury The reason for this declination might be theacute nerve edema or degeneration caused by neurotoxiceffect of penicillin andor local compression to the nervestemThe SFI shows gradual restoration in the group SNI at 4weeks and 6weeksThismay be due to the gradual absorptionof penicillin and its toxicity release of local pressure andnerves regeneration

Themain component of the skeletalmuscle is protein andskeletal muscle atrophy depends on the balance of proteindegradation and synthesis However there are many factors

affecting protein synthesis One of them GlyRS is an essen-tial enzyme acylating tRNAGly with glycine involved in pro-tein synthesis [27] Protein synthesis is impaired when theaminoacylation of GlyRS is inhibited or damaged Currentlypeople understand more about the Charcot-Marie-Toothdisease type 2D The disease is a monogenic disease causedby GlyRS gene mutations and involves peripheral nervoussystem degeneration [28] One study found that the diseasehas impaired protein translation in motor and sensory neu-rons resulting in muscle atrophy and is unrelated to impairedaminoacylation of GlyRS [29] Our results show that theexpression levels of GlyRS and MHC-IIb were graduallyreduced at 1 week and 2 weeks after sciatic nerve injuryand the muscle weight muscle fiber cross-sectional areaand the expression level of MHC-IIb were also decreasedsuggesting that the sciatic nerve injury of penicillin injectionhad an influence on aminoacylation of GlyRS Howeverfurther studies are needed to investigate whether the nerveinjury is directly related to GlyRS One possibility is that thereduction of MHC-IIb expression is due to an increase inprotein degradation not a decrease in protein synthesis Ingroup SNI+EA the expression levels of GlyRS andMHC-IIbthe muscle weight muscle fiber cross-sectional area and SFI


(a) (b)

(c) (d)

(e)

Figure 4 Ultrastructural changes of the peroneus longus muscle after injection-induced sciatic nerve injury and EA treatment Bar = 2 120583m(a) is the group CON mitochondria with normal morphology (white boxes) are distributed among the myofibrils regularly the sarcoplasmicreticula (SR) (red arrow) are clearly visible around myofibrils with longitudinal distribution (b) and (c) are the group SNI at 2 weeks and 6weeks mitochondria (white boxes) and SR (red arrow) are swollen (b) aggregated and swollen mitochondria (white boxes) are seen in someareas some SR show abnormal structures (red arrow) (c) (d) is the group CON+EA at 4 weeks ultrastructures are similar to normal (e) isthe group SNI+EA at 4 weeks the structures of mitochondria (white boxes) and SR (red arrow) are almost normal and distributed regularly


showed obvious recovery compared with the group SNI at 2weeks while no statistical difference was shown in the groupCON+EA at 2 weeks and 4 weeks These results indicatethat the muscle atrophy can be improved by acupuncturetreatment and the possible mechanism is that acupuncturerestored the aminoacylation function of GlyRS in proteinsynthesis by promoting its expression after nerve injuryexcept for the normal individuals Compared with the groupSNI at 2 weeks the mild recoveries of the group SNI at 4weeks and 6 weeks might be due to the collaborative recoveryfunction of SD ratsrsquo own nerve regeneration ability

On the other hand the processing modification andtransport of protein synthesis are involved in endoplasmicreticulum Golgi apparatus and mitochondria with proteinsynthesis mainly carried out in the free ribosomes of cyto-plasm [30] Although SR without ribosomes in skeletal mus-cle is not directly involved in protein synthesis it regulates theexcitation-contraction coupling of muscle fibers as calciumstore and thereby it is closely related to muscle atrophyTheabundant mitochondria in muscle cells can provide lots ofenergy for the contraction of skeletal muscle In addition themitochondria are also involved in the regulation of cell cycleand cell signal transduction Sakakima et al put the sciaticnerves in a frozen medium and thawed them a few timesby liquid nitrogen and then observed the ultrastructuralchanges of soleus muscle with time At 1 day after freeze-thaw cycles myofibrils with their Z lines not in order wereonly partially observed At 3 days and 1 week disorganizedsarcomeres were gradually increased in number and size withtime At 2 weeks the range of these disorganized sarcomeressequentially increased Z lines were not disrupted but highlywavy A and I bands were not discernible and mitochondriawere clustered and enlarged At 3 weeks the range of thesedisorganized sarcomeres began to decrease and Z lines weredisrupted in many sarcomeres At 4 5 and 6 weeks theseabnormal conditions were gradually reduced and enlargedmitochondria were seen in clusters occasionally [31] Ourresults are shown in Figure 4 the ultrastructural changesin our study are similar to the above research only withdifferent observation time points These results imply thatthe acupuncture treatment can promote the recovery ofmitochondria after sciatic nerve injury thereby restoring thefunctions of mitochondria in protein synthesis and mito-chondrial regulation of cell signal transduction The resultsalso suggest that the acupuncture treatment can restore thefunction of the SR as calcium store during contraction of themuscle and then improve the muscle atrophy

In this study the expression levels of GlyRS andMHC-IIbwere in line with SFI changes after sciatic nerve injury butthere was slight delay in the recoveries of muscle weight andmuscle fiber cross-sectional areaThe possible reason for thisis that the restoration on the molecular level started earlierthan that on the morphological level

5 Conclusion

Peroneus longus muscle atrophy in rats can be improved byEA stimulation of GB30 and ST36 points after sciatic nerveinjury induced by penicillin injection The mechanism of

maintaining the normal protein synthesis may be to restorethe function of mitochondria and SR and upregulate theGlyRS expression The significance of this study is to inves-tigate the mechanism of improving muscle atrophy by EA inthe perspective of protein synthesis These findings point tothe design of a drug that can upregulate the GlyRS expressionandnutrient that can improve recoveries ofmitochondria andother organelles Nevertheless there are lots of limitationsin this research for example the upstream and downstreampathways associated with EA and GlyRS expression arenot investigated in this experiment the direct relationshipbetweenGlyRS andMHC-IIb is not clear the downregulationof MHC-IIb is due to protein synthesis reduction or degra-dation increase is not clear These are our future researchdirections

Abbreviations

GlyRS Glycyl-tRNA synthetaseMHC-IIb Myosin heavy chain IIbRT-PCR Reverse transcriptase-polymerase chain

reactionSD Sprague-DawleySFI Sciatic functional indexSR Sarcoplasmic reticulum

Competing Interests

The authors have declared that no competing interests exist

Acknowledgments

This work was supported by a grant from the Social Develop-ment Project of Guizhou (SY-[2012]3120)

References

[1] F C Sitati ENaddumba andT Beyeza ldquoInjection-induced sci-atic nerve injury in Ugandan childrenrdquo Tropical Doctor vol 40no 4 pp 223ndash224 2010

[2] M Mayer and O Romain ldquoSciatic nerve injury followingbuttock intramuscular injection in the child an ongoing riskfactorrdquo Archives de Pediatrie vol 8 no 3 pp 321ndash323 2001

[3] S B Yang N Yue Q Tang and M X Zhang ldquoExpression andregulation of MuRF-1 and Atrogin-1 are required for skeletalmuscle atrophyrdquo Austin Journal of Anatomy vol 2 no 1 article1028 2015

[4] G T Banks V Bros-Facer H P Williams et al ldquoMutant glycyl-tRNA synthetase (Gars) ameliorates SOD1G93A motor neurondegeneration phenotype but has little affect on Loa dyneinheavy chain mutant micerdquo PLoS ONE vol 4 no 7 Article IDe6218 2009

[5] N Kawakami K Komatsu H Yamashita et al ldquoA novel muta-tion in glycyl-tRNA synthetase caused Charcot-Marie-Toothdisease type 2Dwith facial and respiratorymuscle involvementrdquoClinical Neurology vol 54 no 11 pp 911ndash915 2014

[6] K A Huey and S C Bodine ldquoAltered expression of myosinmRNA and protein in rat soleus and tibialis anterior followingreinnervationrdquoTheAmerican Journal of Physiology vol 271 no6 part 1 pp C2016ndashC2026 1996


[7] S B Yang S Long XD Yi and J Q Yu ldquoEffects of acupunctureon gastrocnemius muscle after sciatic nerve injection injury inrabbitsrdquo Journal of Zunyi Medical University vol 37 no 1 pp62ndash66 2014

[8] V Cebasek L Kubınova J Janacek S Ribaric and I ErzenldquoAdaptation ofmuscle fibre types and capillary network to acutedenervation and shortlasting reinnervationrdquo Cell and TissueResearch vol 330 no 2 pp 279ndash289 2007

[9] H M McBride M Neuspiel and S Wasiak ldquoMitochondriamore than just a powerhouserdquo Current Biology vol 16 no 14pp R551ndashR560 2006

[10] C Rommel S C Bodine B A Clarke et al ldquoMediation of IGF-1-induced skeletal myotube hypertrophy by Pl(3)KAltmTORand Pl(3)KAktGSK3 pathwaysrdquoNature Cell Biology vol 3 no11 pp 1009ndash1013 2001

[11] P K Paul S K Gupta S Bhatnagar et al ldquoTargeted ablation ofTRAF6 inhibits skeletal muscle wasting in micerdquoThe Journal ofCell Biology vol 191 no 7 pp 1395ndash1411 2010

[12] G Avila and R T Dirksen ldquoFunctional impact of the ryanodinereceptor on the skeletal muscle L- type Ca2+ channelrdquo Journalof General Physiology vol 115 no 4 pp 467ndash479 2000

[13] K Montague B Malik A L Gray et al ldquoEndoplasmic retic-ulum stress in spinal and bulbar muscular atrophy a potentialtarget for therapyrdquo Brain vol 137 no 7 pp 1894ndash1906 2014

[14] A Onda Q Jiao Y Nagano et al ldquoAcupuncture amelioratedskeletal muscle atrophy induced by hindlimb suspension inmicerdquo Biochemical and Biophysical Research Communicationsvol 410 no 3 pp 434ndash439 2011

[15] F Liu Y Zou S Liu J Liu and T Wang ldquoElectro-acupuncturetreatment improves neurological function associated withdownregulation of PDGF and inhibition of astrogliosis in ratswith spinal cord transectionrdquo Journal ofMolecularNeurosciencevol 51 no 2 pp 629ndash635 2013

[16] L Hu J D Klein F Hassounah et al ldquoLow-frequency electricalstimulation attenuatesmuscle atrophy inCKD-a potential treat-ment strategyrdquo Journal of the American Society of Nephrologyvol 26 no 3 pp 626ndash635 2015

[17] K Elzinga N Tyreman A Ladak B Savaryn J Olson and TGordon ldquoBrief electrical stimulation improves nerve regenera-tion after delayed repair in Sprague Dawley ratsrdquo ExperimentalNeurology vol 269 pp 142ndash153 2015

[18] M F Wu S Q Zhang J B Liu Y Li Q S Zhu and R GuldquoNeuroprotective effects of electroacupuncture on early- andlate-stage spinal cord injuryrdquoNeural Regeneration Research vol10 no 10 pp 1628ndash1634 2015

[19] Q W Li Y Yisidatoulawo D M Masidabenhaqi G L LiF Wang and G J Wang ldquoEffects of electroacupuncture atdifferent frequencies on morphological changes of nervoustissues and electromyogram of skeletal muscles in the rat withinjury of sciatic nerverdquo Zhongguo Zhen Jiu vol 25 no 3 pp217ndash220 2005

[20] G Iohom G B Lan D P Diarra et al ldquoLong-term evaluationofmotor function following intraneural injection of ropivacaineusing walking track analysis in ratsrdquo British Journal of Anaesthe-sia vol 94 no 4 pp 524ndash529 2005

[21] E L Whitlock M J Brenner I K Fox A Moradzadeh D AHunter and S EMackinnon ldquoRopivacaine-induced peripheralnerve injection injury in the rodent modelrdquo Anesthesia ampAnalgesia vol 111 no 1 pp 214ndash220 2010

[22] S YangN Yue andX Zeng ldquoExpression ofMuRF1 andMAFbxin donor and recipient muscles after musculocutaneous nerve

transection and partial pectoralis major muscle transfer forreconstruction of elbow flexion in ratsrdquo International Journal ofMorphology vol 33 no 3 pp 975ndash982 2015

[23] N Agata N Sasai I-MMasumi et al ldquoRepetitive stretch supp-resses denervation-induced atrophy of soleus muscle in ratsrdquoMuscle amp Nerve vol 39 no 4 pp 456ndash462 2009

[24] J Huang Y Zhang L Lu X Hu and Z Luo ldquoElectrical stim-ulation accelerates nerve regeneration and functional recoveryin delayed peripheral nerve injury in ratsrdquo European Journal ofNeuroscience vol 38 no 12 pp 3691ndash3701 2013

[25] D Cai J D Frantz N E Tawa Jr et al ldquoIKK120573NF-120581B activationcauses severe muscle wasting in micerdquo Cell vol 119 no 2 pp285ndash298 2004

[26] M Kabiri S Oraee-Yazdani A Shafiee et al ldquoNeuroregen-erative effects of olfactory ensheathing cells transplanted ina multi-layered conductive nanofibrous conduit in peripheralnerve repair in ratsrdquo Journal of Biomedical Science vol 22 no 1article 35 2015

[27] M Adrian-Scotto and D Vasilescu ldquoQuantummolecular mod-eling of glycyl-adenylaterdquo Journal of Biomolecular Structure andDynamics vol 25 no 6 pp 697ndash708 2008

[28] A J Seo Y H Shin S J Lee et al ldquoA novel adenoviralvector-mediatedmousemodel of Charcot-Marie-Tooth type 2D(CMT2D)rdquo Journal ofMolecularHistology vol 45 no 2 pp 121ndash128 2014

[29] S Niehues J Bussmann G Steffes et al ldquoImpaired proteintranslation in Drosophila models for Charcot-Marie-Toothneuropathy caused by mutant tRNA synthetasesrdquo Nature Com-munications vol 6 p 7520 2015

[30] G Karp and N L Pruitt Cell and Molecular Biology Conceptsand Experiments JohnWileyamp SonsNewYorkNYUSA 2009

[31] H Sakakima S Kawamata S Kai J Ozawa and N MatsuuraldquoEffects of short-term denervation and subsequent reinnerva-tion on motor endplates and the soleus muscle in the ratrdquoArchives of Histology and Cytology vol 63 no 5 pp 495ndash5062000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


which particular proteinrsquos synthesis is decreased Accordingto previous studies the most abundant protein expressionin muscle cells is myosin which forms myofibrillar thickfilaments After nerve injury the MHC-IIb demonstrates themost unstable feature and shows the most obvious decreasecausing the fastest muscle atrophy [6ndash8] Therefore in thisstudy we use MHC-IIb as an index to investigate the proteinexpression level change in muscle atrophy

In addition to providing energy in protein synthesismitochondria are also involved in cell cycle regulation andintracellular signal transduction Insulin-like growth factor-1phosphatidylinositol 3-kinaseprotein kinase Bglycogensynthase kinase 3 beta (IGF-1PI3KPKBGSK3120573) signalingpathway is an important pathway which regulates proteinsynthesis Upon activation of the pathway PKB is transferredinto mitochondria results in GSK3120573 phosphorylation torelieve the inhibition of eukaryotic initiation factor-2B andtherefore promotes protein synthesis [9 10] After denerva-tion the mitochondria swell and become vacuolated exhibitdysfunctions impair protein synthesis and aggravate muscleatrophy [11] Although SR without ribosomes is not directlyinvolved in protein synthesis it regulates the excitation-contraction coupling of muscle fibers as calcium storage andthereby is closely related to muscle atrophy [12 13]

Studies showed that electrostimulation can improveskeletal muscle atrophy through promoting axonal regener-ation of injured nerve and reinnervation of muscle [14ndash17]Does it affect the expression level of GlyRS and promotethe recovery of ultrastructural change of peroneus longusafter sciatic nerve injury Therefore in this study the effectof EA on muscular atrophy and the changes of GlyRS andultrastructure were observed The aim of this study wasto preliminarily investigate the mechanism underlying theimprovement brought about by EA of rats with muscularatrophy caused by sciatic nerve injury following penicillininjection We focus on the perspective of protein synthesishoping to afford new thinking on the treatment of muscularatrophy

2 Materials and Methods

21 Animal Care Grouping and Ethics Statement One hun-dred and eight Sprague-Dawley (SD) rats (meanweight 200plusmn50 g 7ndash9weeks old) were purchased fromLaboratory AnimalCenter Third Military Medical University (SCXK 2012-0005Chongqing China) eithermale or female randomly selectedThe animals were randomly divided into 4 groups namelycontrol (CON) sciatic nerve injury (SNI) CON+EA andSNI+EA Of these group SNI was administrated at 1 week2 weeks 4 weeks and 6 weeks after penicillin injection-induced sciatic nerve injury respectively group CON+EAwas administrated at 2 weeks and 4 weeks after EA stimula-tion ofHuantiao (30th Point ofGallbladderMeridian of Foot-Shaoyang (GB30) located at the posterior upper border ofthe hip joint of the hind limbs vertical needling to a depthof 6mm) and Zusanli (36th Point of Stomach Meridian ofFoot-Yangming (ST36) 5mm below the head of the fibulaunder the knee joint and 2mm lateral to the knee jointvertical needling to a depth of 6mm) respectively [18] group

SNI+EA was administrated at 2 weeks and 4 weeks after EAstimulation of GB30 and ST36 respectively 2 weeks afterpenicillin injection of sciatic nerve injury Totally there were9 animal groups and each group had 12 rats of which 6were used for histological observations and the other 6 wereused for molecular biological studies The rats were housedin individual cages and fed standard rat chow and water adlibitum All experiments were performed in accordance withthe guidelines of the China Animal Welfare Act This studywas approved by the Animal Care and Use Committee ofZunyi Medical College All the surgical steps were performedstrictly in accordance with the principles of aseptic surgeryset forth by Zunyi Medical College

22 Sciatic Nerve Injury Induced by Penicillin InjectionSeventy-two SD rats were taken for routine disinfection ofthe skin and then they were subjected to intraperitonealanesthesia with 10 chloral hydrate at 03mL100 g A 10 cmlongitudinal incision was applied on the right femoral areaand 05 cm horizontal incision under the third trochanterThe sciatic nerve was then exposed with blunt dissectionafter which 200000U that is 05mL penicillin sodium(Harbin Pharmaceutical Group Co Ltd General PharmFactory approved size A051134107 size 048 g08 millionunits) was injected with a No 4 needle at the outer side ofthe neural stem [7] The wound was then sutured layer bylayer and disinfected after surgery Among the 72 rats thatunderwent surgery with penicillin injection in the sciaticnerve 48were selected for group SNIThen 12were sacrificedat time points of 1 week 2 weeks 4 weeks and 6 weeksrespectively

23 EA Treatment In this experiment points of GB30and ST36 on rats were selected as a match pair based onthe principle of selecting two points on each site of theinjury Filiform needles were used to penetrate into GB30and ST36 points After acuesthesia the positive pole onthe G6805-II model electroacupuncture device (QingdaoXinsheng Industrial Co Ltd China) was connected to GB30point and the negative pole was connected to ST36 pointwith a 5Hz output frequency using continuous wave for 10minutes The stimulation intensity was regulated with 2mAand monitored by slight muscle tremor of the injured limbEA was performed on alternate days three times a weekand the entire course of treatment lasted 4 weeks [19] GroupCON+EA consisted of 24 normal rats receiving EA treatmentwithout surgery and nerve injury 12 of which received onecourse of treatment (group CON+EA at 2 weeks) and theother 12 received two courses of treatment (group CON+EAat 4 weeks) Of the 72 rats that were subjected to sciatic nerveinjury induced by penicillin injection 24 rats received EA at2 weeks after the injection 12 of which received one courseof treatment (group SNI+EA at 2 weeks) while the other12 received two courses of treatment (group SNI+EA at 4weeks)

24 Sciatic Functional Index (SFI) Testing After observingthe ratsrsquo gait at every time point of each group their soles were





NPL

+133 (EIT minusNIT)

NITminus 88

(1)

(see [20])







3 Results




(a)

E

IT

PL TS

ITPL

TS

N

(b)

➂➁➀

(c)


0

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Scia

tic fu

nctio

nal i

ndex

(SFI

)

998779 998779lowast

lowast

(d)

000

003

006

009

012

015

Mus

cle w

eigh

t (g)

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowast

(e)

0

40

80

120

160

200

240

280

320

360

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Cros

s-se

ctio

nal a

rea (

120583m

2)

998779

998779

lowast

(f)










4 Discussion



(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)






GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















NPL

+133 (EIT minusNIT)

NITminus 88

(1)

(see [20])







3 Results




(a)

E

IT

PL TS

ITPL

TS

N

(b)

➂➁➀

(c)


0

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Scia

tic fu

nctio

nal i

ndex

(SFI

)

998779 998779lowast

lowast

(d)

000

003

006

009

012

015

Mus

cle w

eigh

t (g)

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowast

(e)

0

40

80

120

160

200

240

280

320

360

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Cros

s-se

ctio

nal a

rea (

120583m

2)

998779

998779

lowast

(f)










4 Discussion



(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)






GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

E

IT

PL TS

ITPL

TS

N

(b)

➂➁➀

(c)


0

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Scia

tic fu

nctio

nal i

ndex

(SFI

)

998779 998779lowast

lowast

(d)

000

003

006

009

012

015

Mus

cle w

eigh

t (g)

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowast

(e)

0

40

80

120

160

200

240

280

320

360

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

Cros

s-se

ctio

nal a

rea (

120583m

2)

998779

998779

lowast

(f)










4 Discussion



(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)






GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























4 Discussion



(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)






GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(bp)

GlyRS

120573-actin

120573-actin

600500400300

200

100

MHC-IIb

M600

400300

200

100

500

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

120573

(357bp)

(452bp)

(335bp)

(452bp)

(a)

00

02

04

06

08

10

Gly

RS m

RNA

expr

essio

n va

lue

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779998779

lowastlowast

(b)

00

02

04

06

08

10

12M

HC-

IIb

mRN

A ex

pres

sion

valu

e

998779

998779lowast

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

(c)






GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GlyRS

MHC-IIb

GAPDH

(kD)

200

36

75ndash80

SNI+

EA4

w

SNI+

EA2

w

CON+

EA4

w

CON+

EA2

w

SNI6

w

SNI4

w

SNI2

w

SNI1

w

CON

(a)

00

01

02

03

04

Gly

RS p

rote

in ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779lowast

(b)

00

02

04

06

MH

C-II

b pr

otei

n ex

pres

sion

valu

e

CONSNI

CON+EASNI+EA

Weeks0 1 2 4 6 2 4 2 4

998779

998779

lowastlowast

(c)






(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)






5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion



Abbreviations



Competing Interests


Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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