Research Article Effect of Acupotomy on FAK-PI3K Signaling Pathways in KOA Rabbit Articular ...

12 Pages·2017·8.69 MB·English

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Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 4535326, 11 pages
https://doi.org/10.1155/2017/4535326
Research Article
Effect of Acupotomy on FAK-PI3K Signaling Pathways in
KOA Rabbit Articular Cartilages
Shi-NingMa,1Zhan-guoXie,2YanGuo,3Jia-NiYu,4JuanLu,1WeiZhang,1
Li-JuanWang,1JingXu,1Rui-LiZhao,1ShuaiZhou,1andChang-QingGuo1
1SchoolofAcupuncture-MoxibustionandTuina,BeijingUniversityofChineseMedicine,Beijing100029,China
2TheFirstPeople’sHospitalofDongchengDistrict,Beijing100075,China
3FirstHospitalAffiliatedtoTianjinCollegeofTraditionalChineseMedicine,Tianjin300193,China
4TheSecondAffiliatedHospitalofGuangdongUniversityofTraditionalChineseMedicine,Guangdong510120,China
CorrespondenceshouldbeaddressedtoChang-QingGuo;[email protected]
Received 16 March 2017; Accepted 28 August 2017; Published 12 October 2017
AcademicEditor:AntonellaFioravanti
Copyright©2017Shi-NingMaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Objective.Byobservingtheneedle-knifeofKOArabbitmorphology,kneejointcartilagep-FAK,p-PI3K,Aggrecangene,andprotein
expression,tostudytheeffectofneedle-knifetopromotecartilagecellsynthesismetabolismmechanism.Method.49maleNew
Zealand rabbits, randomly divided into normal group (Z), model group (M), model-inhibitors (MP), needle-knife group (D),
needle-knifeinhibitorsgroup(DP),electroacupuncturegroup(E),andelectroacupunctureinhibitors(EP).RT-PCRandWestern
Blotwereusedtotesteachanimalcartilagep-FAK,p-PI3K,andAggrecangeneandproteinexpressionlevel.Results.Compared
withNgroup,p-FAKandp-PI3KproteinandmRNAexpressionofMgroup,Dgroup,andEgroupincreased(P<0.05),whilethe
proteinandmRNAexpressionofAggrecanreduced(P<0.05).ComparedwithMgroup,p-FAK,p-PI3K,Aggrecanprotein,and
mRNAofEandDgroupincreased(P<0.05).ComparedwithEgroup,p-FAK,p-PI3K,Aggrecanprotein,andmRNAexpression
ofDgroupincreased(P<0.05);afteraddinginhibitors,p-FAK,p-PI3K,Aggrecanprotein,andmRNAexpressionreduced(P<
0.05).Conclusion.Needle-knifetherapycanpromotetherepairmentofcartilagecellsbyactivatingFAK-PI3Ksignalingpathways,
promotingthesynthesisofcartilagecellmetabolism.
1.Introduction mortality [6–13]. While genetics, aging, obesity, injury, and
biomechanicalstressareconsideredasthemainriskfactors
Knee osteoarthritis (Knee Osteoarthritis KOA) takes pain, involved in the pathogenesis of OA, obesity is the primary
stiffness and functional disorder as the main clinical mani- preventable risk factor for OA [14–18]. Obesity increases
festations,andcartilagedegenerationastypicalpathological the risk of developing OA in both weight-bearing joints
change [1]. Often called wear-and-tear arthritis, KOA is a (especially the knee) and non-weight-bearing joints (the
kneejointdegenerativediseasethataffectssofttissuesaround hand) [15], indicating that obesity-related mechanical and
thekneeincludingthemuscle,tendonandjointcapsule[2]. nonmechanicalfactorsincreasetheriskofOA.Inaddition,
According to epidemiological survey, for middle-aged and theKOAismorecommoninwomenthaninmen,implying
elderly people in china, the incidence rate of KOA is about that differences in sex hormones modulate the disease, and
20%[3,4].KOAaffectsnearly8millionpeopleintheUnited the effect of oestrogen replacement therapy may protect
Kingdom and about 27 million people in the United States againstincidentofOAinpostmenopausalwomen[19–21].
[5]. Asadiseaseoftheentirekneejoint,KOAcanoriginate
In serious condition, it could lead to joint deformities notonlyfromdegenerativechangesofcartilagesandbones
andeventhelossofjointfunction,therebyaffectingpatients’ (such as cartilage wearing, subchondral bone lesions, and
life quality and mobility and it is linked with an excess osteophytes)butalsofromtearsandsubluxationofmenisci,
2 Evidence-BasedComplementaryandAlternativeMedicine
sprainofligaments,synovitis,etc.[17,22–24].Itisbecoming 2.2.TheExperimentalMaterials. Theexperimentalmaterials
clear that articular tissues other than cartilage play an are GuBao resin bandage (specification: 15cm × 180cm,
importantroleintheprocessofOA. ZhuHaiLiZhuMedicalBiologicalMaterialCo.,Ltd.);macro-
Because the causes behind OA development and pro- molecule resin bandage (specification: 7.5cm × 360cm,
gressioncontinuetoremainlargelyundefined,understand- Suzhou Knight Medical Technology Co., Ltd.); HZ series
ing the molecular pathogenesis of the disease remains a disposable needle-knife (specification: 0.4 × 40mm, Bei-
priority [25]. Experimental researches proved that inflam- jing Outstanding Huayou Medical Instrument Co., Ltd.);
matory cytokines, free radical, chondrocyte apoptosis and Zhongyan Taihe disposable acupuncture needle (specifica-
metabolism,protease,andinhibitorwereallinvolvedinthe tion: 0.25 × 25mm, Beijing Research Taihe Pharmaceutical
pathogenyofKOA[26]. Co.,Ltd.).
Besides well-known molecules of mediating articular
cartilagedestruction,suchastumornecrosisfactor-a(TNF- 2.3. The Instrument. The instruments are as follows: HANS
a),interleukin-1(IL-1),andinterleukin-6(IL-6),thehuman acupoint nerve stimulator, LH202H type, Beijing Huawei
cartilage glycoprotein chitinase 3-like-1 (CHI3L1), lubricin, IndustryDevelopmentcompany;theelectrophoresisofpro-
and 𝛽-defensins-4 are also introduced into the pathophysi- teinandthemembranesystem,theUSABio-Radcompany;
ological process (repairing) of OA recently [27–29]. Recent full-wavelengthmultifunctionalenzymemark,TecanAustria
studies have shown that the levels of CHI3L1 may be Co.,Ltd.,models:InfinitetheM1000;gelimagingsystem,the
particularly involved in the pathogenesis of OA. Produced Alpha Innotech(cid:2) company, models: Fluor Chem FC2; PCR
by articular chondrocytes, synoviocytes, and macrophages, instrument type 5020, Finland thermoelectric; PCR C1000,
CHI3L1isassociatedwiththecartilagedamageandmediators the United States bole; ultraviolet spectrophotometer, UV-
ofinflammation[30–33].Lubricinhasbeenfoundinseveral 2600, the German Eppendorf company; scanning electron
tissuesincludingthesynovialmembranesandSF,thesuper- microscope(sem):Quanta250,Czechrepublic,FEI;manual
ficial zone of articular cartilage, tendon and ligament, disc, magnetronsputteringcoatingmachine:SC7620,Britain,the
andmeniscus.Maintainingjointintegrity,lubricinprevents Quorum;criticalpointdrying apparatus:K850, Britain,the
cartilage wear and synovial cell adhesion and proliferation Quorum.
andreducestheamountoffrictionofthearticularcartilage
surface [34]. Recently studies demonstrated that the lack
2.4. The Reagent. The reagents are as follows: PF-562271,
of lubricin secretion may be involved in the pathology of
the item number: S2890, Selleck company; P-FAK rat rab-
OA [35]. It is also reported that CHI3L1 and lubricin are
bit monoclonal antibody, the item number: FM1211, ECM
two glycoproteins that are functionally associated with the
Bioscience companies in the United States; P-PI3K sheep
developmentofOAandinparticularwithgrade2/3ofOA
rabbitpolyclonalantibody,theitemnumber:sc-12929,Santa
[25].
Cruz companies in the United States; Aggrecan rat rabbit
However,asthecorepathologicalchangeofKOA,carti- monoclonalantibody,theitemnumber:NB600-504,Novus
lagedegradationhasalwaysbeenthefocusofKOAresearch companies in the United States; TRIzol reagent, the item
[36]. Many clinical studies have demonstrated that needle- number:15596-026,Invitrogen(cid:3)|ThermoFisherScientific;
knifeissimple,inexpensive,effective,andsafeintreatmentof reverse transcription cDNA kits, the item number: k1622,
KOA[37–40].Inourpreviousanimalexperiments,wefound Invitrogen | Thermo Fisher Scientific; Power SYBR Green,
thatneedle-knifeinterventioncanimprovegeneandprotein the item number: 4367659, ABI; PCR primers, Invitrogen |
expression of cartilage integrin beta 1 (integrin beta 1) and ThermoFisherScientificsynthesis;glutaraldehyde,SPIcom-
repaircartilage[41].Throughanalyzingneedle-knife’seffect panies in the United States; 4% paraformaldehyde: Beijing
onmorphologyandgeneandproteinexpressionofp-FAK,p- Chemicalscompany;15%EDTA,BeijingChemicalReagents
PI3K,andAggrecaninkneearticularcartilageofKOArabbit, company; osmic acid, SPI companies in the United States;
this study explores the mediating pathway through which acetone,BeijingEastHairLuePetroleumChemicalIndustry
needle-kniferepairsthecartilageandtofurtherimprovethe Co., Ltd.; iso-amyl acetate, Shanghai Vibration Spectrum
theoreticalandexperimentalbasisofthistherapyforKOA. Biological Technology Co., Ltd; ethanol, Shengyang Moist
(Beijing)TechnologyCo.,Ltd.;liquidcarbondioxide,Beijing
YanglilaiChemicalGasCo.,Ltd.;0.1mol/LPH7.2phosphate
2.Material
buffer, Beijing KYKY instrument Technology Development
Co.,Ltd.
2.1.AnimalsPreparation. 49NewZealandwhiterabbitsaged
6 months, weight 2.5 ± 0.54kg, bought from Beijing Jin
Muyang Laboratory Animal Technology Co., Ltd., animal 3.Methods
batch number: SCXK Beijing 2015-0005, raised in Beijing
militaryregiongeneralhospitalorthopedicexperimentcen- 3.1. Animal Grouping. 49 New Zealand rabbits were ran-
ter, the experimental animals housed separately in single domlydividedintoblankcontrolgroup,modelgroup,model-
cage,whicharefedandwateredfreelyandwhichareprovided inhibitor group, needle-knife group, needle-knife inhibitor
with natural light. The temperature in the room remained group, electroacupuncture group, and electroacupuncture
at 22 ± 2∘C, and the humidity is 40∼60%, disinfecting the inhibitorgroupbyrandomnumbertablemethod,7rabbits
laboratorybyultravioletonaregularbasis. in each group. Six rabbits were used for the final index
Evidence-BasedComplementaryandAlternativeMedicine 3
detection,andonewasusedtodeterminesuccessofmodel ofneedle-knifeparalleltothetendon.Afterreleasingtothe
preparation with morphological detection. In the course of directionoftendonsandbonejunction,theneedle-knifewas
theexperiment,theanimalsaredisposedstrictlyaccordingto withdrawnwiththepointpressedforawhile.(4)Muscular
the Ethical Committees on Animal Committees on Animal fibertrabsandnodules:tosearchandlocatefibertrabsand
Research (Animal Experimental Ethical Inspection Form nodulesaroundkneejointbypalpation.Andtheninsertthe
Number Kj-dw-18-20150604-01), with approval in National needle-knife perpendicularly with the edge of the needle-
NaturalScienceFoundation(number8157150725). knife parallel to the fiber trabs or nodules. After releasing
along the fiber trabs, the needle-knife was withdrawn with
the point pressed for a while. D E group: one week after
3.2. Model Preparation. Animals except those of the blank
the removal of fixation, Liang qiu (ST 34), Xue hai (SP
control group were prepared as KOA models by improved
10), Nei xi yan (EX-LE4), and Wai xi yan (EX-LE5) were
Videman’smethod,whichfixedthelefthindlimbinextension
selectedforneedling.Afterinsertionofneedles,rarefaction-
position:rabbitsintendedformodelpreparationwerefasted
dense wave was applied with a frequency of 2/100Hz and
10–16hoursandanesthetizedwith3%pentobarbitalsodium
current of 3mA by Han’s acupoint nerve stimulator. Two
(30mg/kg)throughearedgeveininjection.Afteranesthesia,
circuits were formed by connecting Liang qiu (ST 34) with
therabbitswerefixedontheworkbenchinsupineposition,
Xue hai (SP 10) and Nei xi yan (EX-LE4) with Wai xi yan
with left hind limb in full extension. The left hind limb
(EX-LE5). The treatment lasted for 20mins each time, 3
was fixed from the groin to toe by softened resin bandage timesperweekand4weeksinall.EInthesethreegroups,
(softened by hot water at 65–85 degrees centigrade), with
model-inhibitor group, needle-knife inhibitor group, and
absorbentcottonontheinnersideofthebandage.Theankle
electroacupuncture inhibitor group, PFK-specific inhibitor
jointwasdorsiflexedto60degrees,andkneejointwasfixed PF-562271 (200𝜇M, 0.5ml) was injected intra-articularly 2
instraightposition.Resinbandagewasfixedbythemedical
hours before each treatment, and other operations are the
gauze and then wrapped with two layers of macromolecule
same with the corresponding model group, needle-knife
resinbandage.Silkstockingwaspulledonthebandageafter
group,andelectroacupuncturegroup.
the bandage was set to protect it from animal bite. The
stockingwasfixedwithmedicaltapeandpartlycuttoexpose
thetoesforobservingbloodsupplyandswelling.Motionof 3.4. SampleObtaining. Alltheanimalswerethensacrificed
animals’lefthindlimbwasconstrainedbythebandagesand byoverdoseofanesthesia.Kneejointcavityofrighthindlimb
stocking for 5 weeks for model preparation. After that, one wasopened.Andligamentandothersofttissuesaroundthe
rabbitineachgroupwassacrificedandthearticularcartilage kneejoint,includinganteriorandanteriorcruciateligament,
was taken for pathological examination. The bandage and werecutoff.Meniscuswascutofftoexposethecartilageof
stocking of all animals were removed after we proved the distalfemurandproximaltibia.A1∗1cmcartilagespecimen
successofmodelpreparation. ofthemedialcondyleofthefemurwasobtainedwithrongeur.
Sampleswereflushedwithsalinetowashawayblood,mucus,
and tissue debris and then fixed in 4% poly formaldehyde
3.3. Interventions. A Blank control group: there was no
solution for 72h for subsequent index detection. Adequate
intervention. B Model group: there was no intervention
cartilage tissues were cut by surgical blade, collected in
aftermodelpreparation.CNeedle-knifegroup:needle-knife freezing tube, and stored in −80∘C refrigerator for Western
interventionwasappliedoneweekaftermodelpreparation.
BlotandPCRreal-timedetectionafterbeingquick-frozenin
Afterroutineskinpreparationanddisinfection,needle-knife
liquidnitrogen.
was pierced and then withdrawn, with the point pressed to
stopbleeding.Theinterventionwasgiven2timesperweek
andlastedfor4weeks.Thespecificoperationoneachpiercing 3.5.IndexDetection
pointisasfollows:(1)insertionofneedle-knifeintendonsof
3.5.1. Scanning Electron Microscope Observation. Sample
medialvastusmuscleandmusculusvastuslateralis:torelease
preparation:fixedspecimenswererinsedby0.1molnatrium
tendon extension of medial vastus muscle and musculus
cacodylicumbuffersolutionandfixedin1%osmiumtetrox-
vastuslateralis.Inserttheneedle-knifeperpendiculartothe
idefortwohours.Thentheywererinsedby0.1molnatrium
muscle belly with the edge of needle-knife parallel to the
cacodylicumbuffersolutionanddehydratedbyethanol,then
tendon.Afterreleasingtothedirectionoftendonsandbone
keptinisoamylacetateforonenight.Afterdyingbycarbon
connection,theneedle-knifewaswithdrawnwiththepoint
dioxidecriticalpointdyingmethod,thespecimensadhered
pressed for a while. (2) Insertion of needle-knife in tendon
toobjectivetable,weresprayedwithAUcoatingbyvacuum
of rectus femoris: to release upper edge of patella, where
sputter,andwereobservedandphotographedwithscanning
there is the junction of tendon of rectus femoris and the
electronmicroscope.
patella.Insert theneedle-knifeperpendiculartothemuscle
belly with the edge of needle-knife parallel to the tendon.
Afterreleasingtothedirectionoftendonsandbonejunction, 3.5.2. Detection of p-FAK, p-PI3K, and Aggrecan Gene
needle-knife was withdrawn with the point pressed for a ExpressionwithRT-PCR. TotalRNAwasextractedfromthe
while. (3) Insertion of needle-knife in tendon of biceps crushedcartilagetissuewithTRIzolreagent.RNAconcentra-
femoris: to release the tendon of biceps femoris. Insert the tionwascalculatedaccordingtotheODvalueoftheextracts.
needle-knifeperpendiculartothemusclebellywiththeedge Sample solution containing 5𝜇l RNA stock solution was
4 Evidence-BasedComplementaryandAlternativeMedicine
mixedwith1𝜇lOligodT,4𝜇l5xreactionbuffer,1𝜇lRiboLock gelatinoussubstancecanbeseenonthesurfaceofthesample
RNase inhibitor, 2𝜇l 10mM dNTP Mix, and 1𝜇l RevertAid material. Collagen fibers reduced also (Figure1(c) (×2000),
M-Mul V RT to perform reverse transcription and acquire Figure1(c)(×5000),andFigure1(c)(×10000)).
cDNA. Then 2𝜇l cDNA was mixed with 0.2𝜇l upstream Surface of knee articular cartilage of needle-knife rab-
primer and 0.2𝜇l downstream primer, 7.2𝜇l RNAse-free bits was smooth. The furrows got deeper and were regu-
water, and 10𝜇l Power SYBR Green for amplification. The larly arranged. However, no obviously parallel arrangement
obtaineddatawasanalyzedbyrelativequantitative(2−ΔΔCT) betweenfurrowswasobserved.Cartilagesurfacewasevenly
method. ΔCT = Ct value of target gene minus Ct value covered by amorphous substance. But small cracks of the
of control gene (GAPDH) of the same sample. −ΔΔCT = amorphous substance can be observed all over the car-
ΔCT minus the mean ΔCT value of the blank group. The tilage surface. Exposed collagenous fiber can occasionally
expression of target gene of each sample was calculated as be observed (Figure1(d) (×2000), Figure1(d) (×5000), and
2−ΔΔCT. Figure1(d)(×10000)).
Scanning electron microscope showed that the surface
of knee articular cartilage of needle-knife-inhibitor group
3.5.3. Western Blot Method for Detection of Protein Expres-
rabbitswasnotsmooth.Thefurrowsgotdeeperandwerereg-
sion of p-FAK, p-PI3K, and Aggrecan. The total protein of
ularlyarranged.However,noobviouslyparallelarrangement
cartilage tissue was extracted by cell lysis method. Protein
betweenfurrowswasobserved.Collagenfiberswereturned
concentrationwastestedbyBCAmethod.AfterSDS-PAGE
upwithenlargementofthesampledistribution(Figure1(e)
electrophoresis and nitrocellulose filter transfer, antigen- (×2000),Figure1(e)(×5000),andFigure1(e)(×10000)).
antibodyreactionbyprimaryantibodyandsecondaryanti-
Knee articular cartilage surface of electroacupuncture
body, and color rendering by ECL, Alpha Innotech Fluor
group rabbits was of rough surface. And the furrows were
ChemFC2gelimagingsystemwasusedtoacquireimageand
gettingdeeperandincreasedinnumber,noobviouslyparallel
ImageJsoftwarewasappliedforanalyzingtheopticaldensity
arrangement. Collagen fibers were turned up (Figure1(f)
valueofeachtargetprotein.Theratioofopticaldensityvalue (×2000),Figure1(f)(×5000),andFigure1(f)(×10000)).
ofeachtargetproteintoopticaldensityofbeta-actinwasused
Scanningelectronmicroscopeshowedthatthesurfaceof
forstatisticalanalysis.
kneearticularcartilageofEA-inhibitorgrouprabbitswasof
roughsurface.Thesmallupliftsweredeepenedandwidened,
3.6.StatisticalAnalysis. ThedatawereanalyzedbySPSS21.0 with a rock sample appearance. There are visible fibrinoid
statisticalsoftware.Forthedatawithnormaldistributionand structure and irregular collagen fibers (Figure1(g) (×2000),
homogeneity of variance, single-factor analysis of variance Figure1(g)(×5000),andFigure1(g)(×10000)).
wasusedtocomparethedataamonggroups.LSD-𝑡-testwas
usedtocomparethetwogroups.Forthedataaccordedwith
4.2.GeneandProteinExpressionofp-FAK,p-PI3K,andAggre-
normal distribution, rather than homogeneity of variance,
caninCartilage. Aftermodelestablishment,geneexpression
Tamhane’sT2methodwasusedtocomparethedataamong
ofp-FAKandp-PI3Kincreasedinmodelgroup,needle-knife
gmreoaunps±. Thstaensdaamrdplededvaitaatifoonr(e𝑥ach±g𝑠r)o,uwpitihs e𝑃xp<ress0e.d05byfotrhae group,andEgroup(𝑃∗ <0.05or𝑃∗∗ <0.01);comparedwith
significant difference and 𝑃 < 0.01 for a very significant maftoedretlregartomupe,ngteinneneexepdrlees-skinonifeofgpro-FuApKanadndEpg-rPouI3pK(𝑃in#cr<ea0s.0ed5
difference. or𝑃## < 0.01);comparedwithEgroup,geneexpressionof
p-PI3Kincreasedinneedle-knifegroup(𝑃󳵳 < 0.05);when
4.Results
inhibitorwasapplied,geneexpressionofp-FAKandp-PI3K
decreased(𝑃Δ < 0.05or𝑃ΔΔ < 0.01).Meanwhile,compared
4.1.ObservationofCartilagebyScanningElectronMicroscope.
withcontrolgroup,geneexpressionofAggrecandecreasedin
Scanning electron microscope showed that the surface of modelgroup,needle-knifegroup,andEgroup(𝑃∗ <0.05or
kneearticularcartilageofnormalgrouprabbitswassmooth. 𝑃∗∗ <0.01);comparedwithmodelgroup,geneexpressionof
Therearesmallupliftsonsurfaceofthecartilagewithfurrows
Aggrecanincreasedaftertreatmentinneedle-knifegroupand
paralleled to it. The surface of cartilage was evenly covered Egroup(𝑃# < 0.05or𝑃## < 0.01);comparedwithEgroup,
withamorphoussubstance,indicatingcompletestructureof
geneexpressionofAggrecanincreasedinneedle-knifegroup
tahnedsFuipgeurrfiec1ia(al)la(y×e1r0(0F0ig0u))r.e1(a)(×2000),Figure1(a)(×5000), (𝑃󳵳󳵳 <0.01);wheninhibitorwasapplied,geneexpressionof
Aggrecandecreased(𝑃Δ <0.05or𝑃ΔΔ <0.01).
Scanning electron microscope showed that the surface
of knee articular cartilage of model group rabbits was not
smooth.Thestructureofsmallupliftsonsurfaceofthecarti- 4.3. Protein Expression of p-FAK, p-PI3K, and Aggrecan in
lagewithfurrowsdisappears,withcollagenfibersinsublayer Cartilage. Comparedwithnormalgroup,proteinexpression
enlarged,inthebranchesofthesampledistribution,irregular of p-FAK, p-PI3K, and Aggrecan increased in different
arrangement (Figure1(b) (×2000), Figure1(b) (×5000), and degreesaftertreatmentinmodelgroupandinneedle-knife
Figure1(b)(×10000)). group and E group (𝑃∗ < 0.05, 𝑃∗∗ < 0.01); compared
Surface of knee articular cartilage of model inhibitor withmodelgroup,proteinexpressionofp-FAKandp-PI3K
grouprabbitswasseriouslydamaged.Therearemaybecracks increased in needle-knife group and E group (𝑃# < 0.05,
on the surface of the sample. Serious desquamation of 𝑃## < 0.01); compared with E group, protein expression of
Evidence-BasedComplementaryandAlternativeMedicine 5
(×2000) (×5000) (×10000)
(a) SEMofthenormalgroupofKOArabbitarticularcartilages
(×2000) (×5000) (×10000)
(b) SEMofthemodelgroupofKOArabbitarticularcartilages
(×2000) (×5000) (×10000)
(c) SEMofthemodel+PF562271groupofKOArabbitarticularcartilages
(×2000) (×5000) (×10000)
(d) SEMoftheneedle-knifegroupofKOArabbitarticularcartilage
Figure1:Continued.
6 Evidence-BasedComplementaryandAlternativeMedicine
(×2000) (×5000) (×10000)
(e) SEMoftheneedle-knife+PF562271groupofKOArabbitarticularcartilages
(×2000) (×5000) (×10000)
(f) SEMoftheelectroacupuncturegroupofKOArabbitarticularcartilages
(×2000) (×5000) (×10000)
(g) SEMoftheelectroacupuncture+PF562271groupofKOArabbitarticularcartilages
Figure1
p-FAKandp-PI3Kincreasedinneedle-knifegroup(𝑃󳵳󳵳 < ofAggrecanincreasedinneedle-knifegroup(𝑃󳵳󳵳 < 0.01);
0.01); when inhibitor was applied, protein expression of wheninhibitorwasapplied,proteinexpressionofAggrecan
p-FAK and p-PI3K decreased and compared with their decreasedandcomparedwiththeirrespectivenoninhibitors
respective noninhibitors group was statistically significant group was statistically significant (𝑃Δ < 0.05, 𝑃ΔΔ < 0.01)
(𝑃Δ < 0.05). Meanwhile, compared with control group, (Figure2,Tables1and2).
protein expression of Aggrecan decreased in model group,
needle-knife group, and E group (𝑃∗∗ < 0.01); compared 5.Discussion
withmodelgroup,proteinexpressionofAggrecanincreased
aftertreatmentinneedle-knifegroupandEgroup(𝑃# <0.05, KOA takes cartilage damage as the core of its pathological
𝑃## < 0.01); compared with E group, protein expression change[42,43].Cartilageisaviscoelasticstructurecomposed
Evidence-BasedComplementaryandAlternativeMedicine 7
Table1:GeneexpressionofFAK,PI3K,andAggrecanofKOArabbitarticularcartilages(𝑥±𝑠).
Group 𝑁 GeneexpressionofFAK GeneexpressionofPI3K GeneexpressionofAggrecan
Normal 6 1.01±0.19 1.02±0.21 1.01±0.17
Model 6 1.30±0.10∗ 1.22±0.21∗ 0.55±0.07∗∗
Model-inhibitor 6 1.05±0.08Δ 1.09±0.08 0.38±0.05Δ
Needleknife 6 1.57±0.20∗∗# 1.46±0.17∗∗#󳵳 0.76±0.07∗∗##󳵳󳵳
Needleknife-inhibitor 6 1.25±0.13ΔΔ 0.98±0.11ΔΔ 0.51±0.07ΔΔ
Electroacupuncture 6 1.47±0.33∗∗ 1.25±0.24∗ 0.61±0.06∗∗#
Electroacupunctureinhibitor 6 1.06±0.10ΔΔ 0.84±0.12ΔΔ 0.43±0.08ΔΔ
Versusnormalgroup:𝑃∗ <0.05and𝑃∗∗ <0.01;versusmodelgroup:𝑃# <0.05and𝑃## <0.01;versuselectroacupuncturegroup:𝑃󳵳 <0.05and𝑃󳵳󳵳 <
0.01;versusnoninhibitorgroup:𝑃Δ<0.05and𝑃ΔΔ<0.01.
Table2:ProteinexpressionofFAK,PI3K,andAggrecanofKOArabbitarticularcartilages(𝑥±𝑠).
Group 𝑁 ProteinexpressionofFAK Proteinexpressionofp-PI3K ProteinexpressionofAggrecan
Control 6 0.06±0.01 0.14±0.02 1.54±0.16
Model 6 0.45±0.13∗∗ 0.41±0.18∗∗ 0.59±0.11∗∗
Model-inhibitor 6 0.15±0.15Δ 0.22±0.09Δ 0.19±0.05ΔΔ
Needleknife 6 2.32±0.32∗∗##󳵳󳵳 0.83±0.19∗∗##󳵳󳵳 1.11±0.16∗∗##󳵳󳵳
Needleknife-inhibitor 6 1.95±0.35Δ 0.65±0.18Δ 0.69±0.08ΔΔ
Electroacupuncture 6 1.25±0.28∗∗## 0.60±0.17∗∗# 0.76±0.14∗∗#
Electroacupunctureinhibitor 6 0.92±0.23Δ 0.41±0.15Δ 0.37±0.13ΔΔ
Versusnormalgroup:𝑃∗∗ <0.01;versusmodelgroup:𝑃# <0.05and𝑃## <0.01;versuselectroacupuncturegroup:𝑃󳵳󳵳 <0.01;versusnoninhibitorgroup:
𝑃Δ<0.05and𝑃ΔΔ<0.01.
Z M MP D DP E EP of the important factors for maintenance of the health of
cartilageduetoitsspecialphysiologicalstructure[46].
P-FAK
In recent years, studies have found that moderate
dynamic mechanical load is a potential method for the
GAPDH
preventionandtreatmentofKOA,althoughbothinsufficient
and excessive stress of the knee joint will induce cartilage
P-PI3K degradation. Its therapeutic effects of inhibiting inflam-
matory reaction and maintaining balance of chondrocyte
GAPDH metabolism may be achieved through some mechanical
transduction pathways that are not yet clear [47]. Studies
haveshownthatmoderatedynamicmechanicalloadscould
Aggrecan
obviously reduce inflammation and protect ECM. They
inhibitexpressionofproinflammatorycytokinessuchasIL-
GAPDH 1𝛽, IL-6, and TNF-𝛼 and inflammatory mediators such as
COX-2, PGE2, and NO and promote expression of anti-
Figure2:ProteinexpressionstripeofFAK,PI3K,andAggrecan.
inflammatory cytokines such as IL-4 and IL-10 to exert
anti-inflammatory effects. Meanwhile, they protect ECM
by suppressing the expression of MMPs, disintegrin, and
ofcartilagematrixandcartilagecells[44].Itbearsmechanical ADAMTS [48]. The special mechanical receptor on the
load and absorbs and cushions stress. The functions of surface of chondrocytes and the mechanical transduction
cartilage mainly depend on the cartilage matrix. Cartilage pathwayinducedbyitplayedakeyroleintheaboveprocess.
cellssynthesizeandsecretetheessentialcomponentswhich O’Conoretal.provedthatdirectmoderatestressstimulation
maintaintheintegrityofcartilagematrix,suchasproteogly- tothechondrocytescanpromotesynthesisofchondrocytes
can (Aggrecan). Under physiological conditions, there is a andalleviatedegradationofcartilagematrix[49].Therefore,
dynamic balance between the synthesis and decomposition benign stress can not only maintain the metabolic balance
ofchondrocytes,whichisthebasicguaranteeofthenormal ofhealthycartilagebutalsopromotetherecoveryofinjured
structure of cartilage matrix [45]. When suffering from cartilage.Inpreviousstudies,weconfirmedthatneedle-knife
KOA,decompositionofchondrocytesdominatesmetabolism treatmentcanpromotethesynthesisofchondrocytestosome
of chondrocytes and breaks the homeostasis. As a result, extentandrepairthedamageofcartilage.Afterneedle-knife
degradation of cartilage finally occurred. The stress is one treatment,theexpressionofAggrecanproteinofextracellular
8 Evidence-BasedComplementaryandAlternativeMedicine
matrixofKOArabbits’kneecartilageincreased,togetherwith compared with that of the model group, and needle-knife
the upregulationof protein expression of integrin beta one, treatment has a better effect. This result is consistent with
whichisthemostimportantmechanicalreceptoroncartilage testresultsofproteinandgeneexpression.Theaboveresults
[50].Therefore,themechanismofcartilagerepairbyneedle- showedthatbothneedle-knifeandEAcanactivateFAK-PI3K
knifeiscloselyrelatedtotheimprovementofstressbalance pathway,promotethesynthesisofchondrocytes,increasethe
of knee joint. However, the underlying signal pathway is expressionofAggrecan,andfinallypromotetherecoveryof
unknown. cartilage.Needle-knifehasabettereffectthanEA.Webelieve
Integrinisoneofthemechanicalreceptorsoncellsurface. thatthiseffectisaresultofactivationofFAK-PI3Kmechan-
The mechanical receptors on the surface of chondrocyte ical pathway, which plays an important mediating role in
are mainly integrin, a kind of transmembrane protein that the recovery of cartilage. Furthermore, the mechanism of
consistsofalphaandbetasubunits[51].Withitsextracellular cartilagerecoverybyneedle-knifeisrelatedtotherestoration
domain connecting to matrix ligand and its intracellular ofbenignstressofkneejoint.
domaininteractingwithkinasesofintracellularpathwaysto In this study although the PROM and motor function
inducecascadereaction,theintegrintransducesextracellular wereimproved,westillcloudnotexplainhowthecompres-
signal into the cell to complete the process of transduction sivestressofcartilagesurfacechanges.Henceitisadvisable
from mechanics to chemical function, thereby regulating to do cartilage compressive stress and related mechanical
the proliferation, differentiation, survival, and migration of experiments of knee joint cartilage in order to improve the
chondrocytesandmorphogenesisandremodelingoftissues. theoreticalresearchonKOAbiomechanicalmechanismwith
Inthetransductionprocess,focaladhesionkinase(FAK)isa needle-knife.
keymaterialofsignalpathwaymediatedbyintegrin.Itworks Clinically,ontheearlystageofKOA,patientsareusually
likeanintegrateddevicethatreceivesandintegratesthesignal treated by acupuncture and functional exercises by one of
carried by integrin and then amplifies it within the cell. As thefirstauthors(Shi-NingMa).Withthepathologicaldevel-
aresult,itactivatesmanydownstreamsignalpathways,like opmentofKOA,theneedle-knifeisnecessaryforloosening
phosphatidylcholine-3kinase(PI3K),MAPKpathway,andso tissue adhesion mechanically when organic injuries (such
on[52]. as tissue hyperplasia, hypertrophy, and fibrosis) take place.
FAKisacytoplasmicnonreceptorproteintyrosinekinase Generally,theKOApatients’subjectivesatisfactionandsub-
[53].Itisamemberoffocaladhesioncomplexesfamilyand jectivefunctionalimprovementpresentapositivecorrelation
consists of 1028 amino acids [54]. The structure of FAK in the needle-knife treatment. Sometimes, subjective and
can be divided into three functional areas including N- objectiveevaluationsdo notentirelychimewitheach other
end function area, C-end function area, and kinase region [59]. Usually, a few patients who could perform the daily
[50].N-endfunctionareamediatestheinteractionbetween living activity (such as walking, stair activity) originally
FAK and integrin protein and the kinase region is the before treatment are not very satisfied, though there are
catalytic site of FAK activation. C-end function area can improvementsonobjectivescoresaftertreatment.However,
makeFAKadheretofocaladhesion,formingfocaladhesion therearestillsomepatientsthatareextremelypleasedwith
complexestomakeFAKplayitsrole[55].Whentheintegrin thefunctionalrecoveryofdailylivingactivitydespitelimited
was stimulated by extracellular mechanical stimulation, its scoreimprovementaftertreatment.
configuration changed followed by the recruitment of FAK Theasepticoperationiscrucialintheclinicaltreatment,
to intracellular region of the integrin and the initiation of and both acupuncture and needle-knife are invasive oper-
self-phosphorylationofTyr397.Tyr397self-phosphorylation ations. No matter how small the incision is, even the per-
induces its combination with SH2 structural domain of pendicular needling of acupuncture operation, there is still
intracellulartyrosinekinase(Src)andtheformationofSrc- a certain potential risk of infection. The risks of secondary
FAKcomplex,leadingtotheactivationofSrc.Theactivated infectionafteroperationwillincreasesignificantlyespecially
SrccancatalyzethephosphorylationofTyr576/577ofFAK, ontheweakelderly,theimmunocompromisedsubjects,the
thereby completely activatingFAK. The fully activated FAK infectious subjects, and the patients with chronic diseases
willfurtheractivatePI3KbybindingtoitsP58subunitwith [60].Becausetheacupotomyistheresultofcombinationof
site Tyr397 [56, 57]. The activated PI3K can produce the TraditionalChineseMedicine(TCM)andWesternMedicine,
secondmessengers,phosphatidylinositol3,4,5-triphosphate, strict sterile manipulation is the basic requirement which
to alleviate the degradation of cartilage by synthetizing couldavoidtheriskofinfectiontotheutmostextent.
and secreting important components of cartilage, such as Clinical painless operation is another important issue
proteoglycan[58]. that we should be pay attention to [60, 61]. With a similar
In our study, protein and gene expression of p-FAK, puncturing method and surgical approach to traditional
p-PI3K, and Aggrecan increased in different degrees after acupuncture, the pain stimulus produced by the special
treatmentin needle-knife group and E groupbut obviously needling instrument of acupotomy is acceptable for most
decreased when specific inhibitor for FAK was applied. patients. Therefore, communicating well with patients in
Furthermore,theeffectofneedle-knifeonproteinexpression order to eliminate patients’ fear to most is necessary before
of p-FAK, p-PI3K, and Aggrecan is better than that of EA operation.
treatment. Meanwhile, the result of cartilage scanning elec- Actually, the needle-knife is not the real meaning of
tron microscope showed that cartilage condition of rabbits “knife.”Thedifferencebetweenneedleandknifeisnotonlyin
inbothneedle-knifeandEgroupimprovedaftertreatment, thedifferenceofshapebutalsointhedifferenceoffunction.
Evidence-BasedComplementaryandAlternativeMedicine 9
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ConflictsofInterest
[13] A.Mobasheri,C.Matta,R.Za´ka´ny,andG.Musumeci,“Chon-
drosenescence:definition,hallmarksandpotentialroleinthe
The authors declare that they have no financial conflicts of
pathogenesisofosteoarthritis,”Maturitas,vol.80,no.3,pp.237–
interest. 244,2015.
[14] G.Musumeci,F.C.Aiello,M.A.Szychlinska,M.DiRosa,P.
Authors’Contributions Castrogiovanni,andA.Mobasheri,“OsteoarthritisintheXXIst
century:riskfactorsandbehavioursthatinfluencediseaseonset
Shi-NingMaisthefirstauthor.Zhan-GuoXieistheco-first and progression,” International Journal of Molecular Sciences,
authorandhecontributedtothisworkequallyalso. vol.16,no.3,pp.6093–6112,2015.
[15] E. Yusuf, R. G. Nelissen, A. Ioan-Facsinay et al., “Association
betweenweightorbodymassindexandhandosteoarthritis:a
Acknowledgments
systematicreview,”AnnalsoftheRheumaticDiseases,vol.69,no.
4,pp.761–765,2010.
The programme was funded by National Natural Science
[16] N. Yoshimura, S. Muraki, H. Oka et al., “Accumulation of
FoundationofChina(no.8157150725).
metabolic risk factors such as overweight, hypertension, dys-
lipidaemia, and impaired glucose tolerance raises the risk of
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