Hindawi 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;Guochangqing5901@163.com 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

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