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f zu{ } 18 í } 24 20140611 Çf Chinese Journal of Tissue Engineering Research June 11, 2014 Vol.18, No.24 Îöm q7³²pi3k/akt³ ùítî ŒqÃr iiºö ({œ ÁÞ 510630) s 1 šr öjîöm q 7 ùœt Î Œf³²Ãr²z Âr Œm kj ºxt ² Âr gdº² zøx鞈îöm q 7 à rrxtèé ùîöm q 7 ÃrqÉÍvŸÂ 2 Í üàoogö ² târ v ± Ímkg ±qt Î Œgt Î Œrºu{q oq 3 ÍžÊË ÃrqÎ ŒoxüÏ qîöm q 7 xö PI3K/Akt ³ ÍÏ LY294002 qké ŒÃrixö Akt ³ qÿ² q ä oîöm q 7 ³² PI3K/Akt ³ öºãr n²ÿº BAD-Caspase 9 Ãr²sÉqº ºÀ Î z zuíîöm q 7PI3K/Akt ³ tî ŒÃrž p-aktbad qcaspase 9 Œ qí² gè ŒÃrÎöm q 7 q ž «Ù dryºèr(2009b0308010952007a0300005) Îöm q 7 ù²tî Œq Œ«üà Âr² ² Ǻù Œ³²Î ŒÃrv ö² n º²qÉÍrŸ  rq Îöm q 7 ž ömãrqtî Œrmq nöº PI3K/Akt³ q É Îöm q 7 ÍtÎ ŒÃrqÉÍ ³² Pfirrmann É örºz² 12 Âr n ä tî Œý É ÍxÿƒzÉ 15% fžq»»qî Œ g{q ˆnž»» 48 h Ãr gâd žz³²ø üï qîöm q 7 xöüø PI3K/Akt ³ Ï LY294002 k ˆn Œü ŒÃri o p-akt Ÿ² qí² Aktp-AktBAD Caspase 9 }³ qqÿ² ä žãr Œ xãsîöm q 7 kéî ŒÃri ÂØ LY294002 ºü ný ŒÃri¼éÎ(P < 0.05)p-Akt o qí² ² ŸÃrÂd rø Îöm q 7 qí fp-akt Ÿ²ØºÃrrº q BADCaspase 9 qÿ²ã(p < 0.05) üø Akt ³ Ï LY294002 ýp-akt qÿ²  Ãrrº qqÿ²öír±îq(p < 0.05) Îöm q 7 ž qt Î ŒÃrf³² PI3K/Akt ³ o BAD-Caspase 9 rºq ŒÃr²zÍoÎ Œq² 1987 {œ xå Ôd vd {œ Á Þ 510630 doi:10.3969/j.issn.2095-4344. 2014.24.009 [http://www.crter.org] fé ú:r318 h u:a ú:2095-4344 (2014)24-03821-08 ÈÍ2014-05-16 iiºö.îöm q 7 ³² PI3K/Akt ³ ùítî ŒqÃr[J]. f zu{201418(24):3821-3828. Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway Yu Hao-tao, Xu Yi-chun, Wang Qi-you, Ou Ding-qiang, Zhou Wei (Department of Spinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China) Abstract BACKGROUND: Bone morphogenetic protein 7 (BMP-7) can promote extracellular matrix synthesis of human nucleus pulposus cells, and delay intervertebral disk degeneration. Recently scholars proposed that the above effects can be achieved through anti-apoaptosis, but further molecular mechanism remains poorly understood. OBJECTIVE: To observe the effects of BMP-7 on the apoptotic human nucleus pulposus cells under serum-free induction and the PI3K/Akt pathway, investigate the molecular mechanism underlying the anti-apoptosis effect of BMP-7. METHODS: Human nucleus pulposus tissue were harvested from 12 patients according to the modified Pfirrmann classification, and human nucleus pulposus cells were obtained from digestion of herniated nucleus pulposus tissue. The collected nucleus pulposus cells were divided into four groups. Blank group: cells were Yu Hao-tao, Master, Department of Spinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China Corresponding author: Xu Yi-chun, M.D., Department of Spiny Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China Accepted: 2014-05-16 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3821

cultured in normal culture medium containing 15% feral bovine serum; positive control group: cells were induced to apoptosis in the serum-free culture medium for 48 hours; treated group: cells were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7; antagonism group: cells were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7 and LY294002, an PI3K/Akt pathway inhibitor. Cellular apoptosis rate was quantified by flow cytometry. Immunoflurorescence was performed to observe p-akt expressions. Changes of Akt, p-akt, BAD, and Caspase 9 expression were detected with western blot analysis. RESULTS AND CONCLUSION: Results from flow cytometry showed that, the apoptosis rate of nucleus pulposus cells was significantly decreased as the increase of BMP-7 concentration, under the serum-free induction; after the cells were induced with LY294002, the apoptosis rate was increased again (P < 0.05). The p-akt immunoflurorescence and western blot analysis demonstrated that, compared with positive control group, the p-akt expression was significantly increased, while the expression of downstream apoptosis-related proteins such as BAD and Caspase 9, was significantly decreased in the cells treated with BMP-7 (P < 0.05). Furthermore, in the group treated with BMP-7 and LY294002, the p-akt expression was decreased, while the expression of apoptosis-related proteins was recovered to high levels (P < 0.05). BMP-7 activates the PI3K/Akt pathway, suppresses the downstream BAD and Caspase 9 signaling, and inhibits the apoptosis of human nucleus pulposus cells induced by serum-free situation. Subject headings: apoptosis; bone morphogenetic protein-7; protein kinase; serum Funding: Science and Technology Program of Guangdong Province, No. 2009B030801095, 2007A0300005 Yu HT, Xu YC, Wang QY, Ou DQ, Zhou W. Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(24):3821-3828. 0 Introduction Âr²Ÿ (intervertebral disc degeneration, IVDD) gâr²ÿooqokurðºok äqu{ýtyxoâr ̓¼Î ŽÎ Œy Œ n Œ«üà Œñ Éeq²z [1-5] Âr² ²q Íu{f ŒÃr²qr ²zrŸ¹ gâr²ÿ okíf q Éi [6] ²Â ³ Âr Œ³²r ƒ² ƒ²} ºÃr ú³ öãrâr ² sùéqº [7-10] ºp ú±²q u{xoâr ŒùŒâAkt³ Wnt³ MAPK³ }qrš³²q²±ãr öâr² [11-14] gívƒƒ¼y² Âr² ±oq ²qprrŠöËq ú±³ f PI3K/Akt³ º ƒ²q Š ö²öjº Îöm qqrp n o qº öjîöm qâr ŒfŒ Œ«ü ö ² n³²yùgöt ²  r Œfqu{ox Îöm q7ºf n qi [15-18] Îöm q7{ ³² y² Œ«ürŸÂ ͳ² o }Í Îöm q7t Î ŒÃrqÍ n² º t Î Œfÿ³²PI3K/Akt³ rºq ú± ÍöÃr nxî ŒÃr³ qu{ 1Materials and methods ŒƒÍ Âöp201211Ž20143f Â}çÃfÍ 3822 tƒî q p201211ž2013 11ÂfÂ}çÃŒyÂr{Ç oÿoq 12 Â1830nÐogƒö v Âr{Ço oº Âr PfirrmannÉ [19] àΞïo}uoo x ooöj ŒŒ~h{ öœçýçnöj Œ pœ oo >200 Àºwñ ²Ÿo Í fâ}çãkâ oox qu~ˆuüý ²Ÿ Îöm q³²3,.$nw³ ÍtÎ ŒÃrÍrº Ïöy Ïöy DMEM/F12» fž Œñ Œ q (ÿ/ÿ EDTA) Îöm q7 LY294002 taktp-aktbadcaspase 9GADPH È IgG tœñêsabc Ïr ¹ q ÏrBCA q ÏrECL ö ÏrAnnexin-V FITC/PIö Ïr À Œy ny ÁÁy Hyclone¹úˆ Gibco¹úˆ PROSPEC¹úxÊ vp¹ú mg¹ú ë¹ú êsæ¹ú Leica¹ú ˆBD¹ú äs¹¹ú ˆBeckman Coulter¹ú P.O. Box 10002, Shenyang 110180

tî ŒqÉyö»Žf ÇqÎ È r4 PBSqy~f15 min¼ Âú zã«nsyóò Î Óv 1 mm 1 mm 1 mmˆnƒzé 0.1%qŒñ 37 ~f²ä(12 h)200r ¾ˆ²yÄ ŒýØ ² ˆqÿ ƒzé15% fžqdmem/f12» í ñxî Œºw Œ g 1 10 8 L 1 Ø 25 cm 2 ² Œ»mf«ê» Œ»Ž70%80% üýoƒzé0.25%œ ä13~x~1xýµép6f»x²ÿ Œ ¹Í tî Œq¹ ˆÀÃý Î ŒmÀmÀŽ50%60%q~1 xî Œô6f»Ø 40 g/lšn Ø ƒzé30%²än 150 qü ¼²äg ýøƒzé5%bsa²ÿ žážáýø tœñêd Ø PBS4 ² ýüø ˆ IgGp37 ~ 1 hˆnsabc Ïr ndab Î ŒÃr ök ~1xÎ ŒmÀŽ 70%²ŸÍÃ{q qí PBSÀý nždmem/f12»»éìø ü Îöm q7xöly294002k ºƒÉ öèî {qd ÿƒzé15% fž»»48 h Âd opbs2ýø ÿžqdmem/f12»» 48 h ƒï Îöm q7 üâd kø ÿždmem/f12ü Ø Îöm q7ˆº ²Í50 μg/l»48 h fï Îöm q7 üâd kø ÿždmem/f12ü Ø Îöm q7ˆº ²Í100 μg/l»48 h ÎÏ Îöm q7 üâd kø ÿždmem/f12ü Ø Îöm q7ˆº ²Í200 μg/l»48 h LY294002 üfï Îöm q7 küø LY294002 ˆº ²Í50 μmol/l»48 h ŒAnnexin-Vö ŒÃrÀ ž Ãröüyk48 hýqî ŒˆnÿEDTA qœ ää PBSýx À Ø FITCPI ö15 minýˆnannexin-v FITC/PI ö Ïr³²FACS Calibur ŒyÃriq äàãr Œg(Annexin V + PI - )Ãr Œg(Annexin V + PI + ) ü Ãrià rºü p-akt o 6f»q~1xÎ Œ ŒmÀ²Í 70% ü²íé f{qd Âd fï Îöm q7 LY294002 º4 ²Ÿk48 hký² ŸÍØ 40 g/lšn Ø ƒzé30%² än 150 qü¼²äg ýøƒzé5%bsa²ÿžáø tp-aktn â 64 À~ ² ýüø ˆ IgG p37 ~ 1 hdapi 10 minopbsôýp À²ŸüÉ Œ¼p-Akt q Ÿ²d Œ q xöwestern blotãr 48 h öø rkýqü ŒˆnPBSýØ ¹ q ÏrfÈÁÿw ÍÏö q ÍÏ qq Œ À n ŒÍÄv Œö q ±zžy~f4 γy qnbca qÿ Ïr²Ÿü q«ÿ qý q«fø Àd10 minˆi ü Ÿ q ýˆn10%qéyœ5% Œ²ŸSDS-PAGE néy¼n±žpvdf Ãw äƒqˆ ntbstø5%bsaºwntbstøƒzé5%œœ ²ŸÁ1 hxβ-actin g¼õéìø ² r Ã(11 000) taktp-akt(ps 473) BADCaspase 9β-actinƒ4 ² TBST3 ýø xtbst13 000z qˆ IgG 30 minecl Ïr²Ÿö Œ fýˆnalpha±zk Érq g tî Œ ÀmÀ o 乜ñÿ² o Œ¼p-AktŸ ² ŒAnnexin V-FITCö ŒÃri qí²rºãr³ qqÿ² É «xx _ ±sÿxˆnspss 17.0 ±z²ÿ Éü  nê ÉöBonferroniee±ÍP < 0.05g i 2 Results 2.1 j ƒœñ ²ä qñxî Œùp»3 d¼«ñx Œ Ë«ý Œ{ ²À Œg gu { ±q Œ Žm¼ Œ²Í60%70% ürm Íu Œ² üºêýgg Ê~1x ŒmÀ²³»6 h¼í«œ¼vfœºvgu ±ñxr ÀÊôýn mxüüî ŒŒf Œù 1}üx h³qtî Œq [20-21] ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3823

A B C D 200 400 100 400 E F G H 200 400 100 400 1»qtÎ Œq( À) Figure 1 Morphology of human nucleus pulposus cells (optical microscope) f AB gñx»} 5 tî Œf CD gñx»} 9 tî Œf EF g~ 1 x»} 5 tî Œf GH g~ 1 x»} 9 tî ŒxüüÎ ŒŒf Œù A B C D 100 400 100 400 2 ~ 1 xtî ŒfŒñqŸ²( o ä) Figure 2 Collagen type II expression in subcultured human nucleus pulposus cells (immunocytochemical staining) f AB gtî ŒfŒñ o ä À uî ŒØf CD gw r À Âd À Œ¼²{ Ÿ 1 Œü Ãri ä Table 1 Apoptotic rate in each group as detected by flow cytometry Ì Ãri(%) {qd Âd ƒï Îöm q 7 fï Îöm q 7 ÎÏ Îöm q 7 LY294002 4.43±3.27 61.83±7.78 45.49±4.55 39.85±7.35 28.34±6.60 52.21±9.62 (x _ ±s) Ÿü Ãri ² Bonferroni ²Ÿee±Í i (P < 0.05) 2.2 ýê Ç Œñù Í uî ŒØ x ŒñŸ²}üÎ Œgüxw r À(PBS) gâd Œ¼²{ 2 2.3 öannexin V-FITCÆg û Í Îöm q7kýžjãsî öm q7ï Ø ŒÃriÂØ (P < 0.05)dØ PI3K/Akt³ ÏLY294002ý Îöm q7qãrã(ƒï Îöm q7 P < 0.05)12 Î ²Ÿ ÉÉü Ãri³²Bonferroniee ±  i(p < 0.05)² ºf q Ãr Âp312 ÂÊpŸ1ö4 2.4 p-akt Ϋ{qd Âd fp-akt Ÿ²mŽù Îöm q7 kqfï Îöm q7 ŒfÊù p-akt Ÿ²²yŸ²ÃsLY294002 Akt³ Ï LY294002qØ ÍÍ 4 2.5 Western blotî«western blot ²Í o ž Ãrp-Akt ú qqÿ² 5A ü ¼õβ-actinxöAktŸ²r}qÐ Âd ƒfîï Îöm q7 fãsî öm q7ï ØPI3K/Akt³ qp-aktÿ²ø(p < 0.05)ºÃrrº qbadcaspase 9 qÿ²ã (P < 0.05) LY294002 Ø Akt³ Ï LY294002ýp-AktŸ²Â BADCaspase 9Ÿ²ö Ír±Î(P < 0.05) 5BD 3824 P.O. Box 10002, Shenyang 110180

A B C UL 2.03 1.51 9.60 61.57 UR 5.03 3.73 211.13 107.38 LL 91.83 68.05 3.06 4.81 LR 1.11 0.82 91.10 10.60 UL 0.88 0.21 9.00 65.52 UR 52.21 12.14 250.33 106.07 LL 37.29 8.67 6.27 7.39 LR 9.62 2.24 176.97 18.89 D E F UL 0.11 0.02 13.46 34.29 UR 31.61 6.38 486.82 76.72 LL 45.63 9.22 7.27 6.91 LR 22.65 4.57 387.21 13.15 UL 0.65 0.19 10.02 63.87 UR 21.85 6.25 264.93 87.80 LL 70.15 20.07 5.55 6.27 LR 7.35 2.10 177.25 17.03 UL 1.27 0.37 10.78 71.93 UR 35.98 10.39 135.79 89.55 LL 54.26 15.67 5.50 7.45 LR 8.49 2.45 112.96 19.60 UL 0.80 0.26 9.95 65.25 UR 32.09 10.31 209.01 94.42 LL 59.33 19.07 5.97 6.18 LR 7.78 2.50 127.15 15.87 3 ŒÃriž ÃrkÎöm q 7 tî Œq Figure 3 Flow cytometry results of the anti-apoptosis effect of bone morphogenetic protein-7 in serum-free induced human nucleus pulposus cells x PI Ÿ²g FITC Ÿ²gÃr Œ(PI - )öãr Œ(PI + )É̃pú Â(UR)ú Â(LR) Ãriq gãrqé+ãrqéf AF ÉÌg{qd Âd ƒfîï Îöm q 7 (50 100200 μg/l) LY294002 ŒÃrÀ² 6 ŒÃri i A1 A2 A3 A4 {qd Âd fï Îöm q 7 LY294002 B1 B2 B3 B4 {qd Âd fï Îöm q 7 LY294002 C1 C2 C3 C4 {qd Âd fï Îöm q 7 LY294002 4 o Îöm q 7 kž tî ŒÃrý p-akt Ÿ²( 200) Figure 4 Immunofluorescence results of p-akt expression in bone morphogenetic protein-7 treated apoptotic human nucleus pulposus cells induced under serum-free condition ( 200) f A g DAPI B g p-akt C g DAPI p-akt øø ͈n t à p-akt öˆ IgG-FITC ²Ÿ p-akt DAPI ( )v Œƒˆr±p{qd Âd xö Ø Îöm q 7 qk f Œ¼ p-akt qÿ²( ~x) ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3825

A 1 2 3 4 5 6 M r B 1.2 Caspase 9 BAD p-akt 47 000 23 000 60 000 (p-akt/β-actin) 1.0 0.8 0.6 0.4 0.2 AKT 59 000 0 1 2 3 4 5 6 β-actin 42 000 C (BAD/β-Actin) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 D (Caspase 9/β-Actin) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 5 ž tî ŒÃrü qí²ü rºãr q Figure 5 Apoptotic relative protein expressions in serum-free induced human nucleus pulposus cells detected by western blot assay f A gž tî ŒÃrü qí²ü rºãr q f BD güí p-akt/bad/caspase 9 qí ² ¼õ β-actin i äâd ƒfîï Îöm q 7 fãsîöm q 7 Ï ØPI3K/Akt ³ q p-akt Ÿ²Ø(P < 0.05)ºÃrrº q BADCaspase 9 qÿ²ã(p < 0.05) LY294002 Ø Akt ³ Ï LY294002 ýp-akt Ÿ²Â BADCaspase 9 qÿ²öír±îq(p < 0.05)f 16 ÉÌg{qd Âd ƒfîï Îöm q 7 LY294002 3 Discussion pt Î Œƒ»qu{rÅgÙü qáèi} [16, 22] ³²Âr ²  r ²Ÿƒ» ŒmÀ qœñ} Œ«ÉÌ o Âr g² züºƒ»qëgí k Wei} [23] qu{rnpº qü târ ² zoxéé š ÍÎöm q7t ² Âr qî Œf qãrº ŒèÉxt go Âr Äu{Ä ± ü± iâqtr ³²t Âr ² q²²ÿoeqâíãío ÚÌ ooà}à³²pfirrmann É ÍÐ ÄŸÉÃqÂr²ŸÉ ² Âr² z± ±gžq 3826 gâ z ÍÐ qùüu{ PiffirrmannÉ šuvmri²ÿ ÊÉ ³²ƒ»É ºÂr qmg gsqº [24-25] o ÍqÂÍùŸ q ü ŒqmÀ³²ŒñqŸ² jç±qžíkr ²yг ² ŒüÍ ŒÃriq º Íq vöožz Ãrq Í fˆnîöm q7ýî ŒqÃriÃsÎ öm q7ï qø ²Â²y äº q ³² Œv oîöm q7tî ŒfqÃr ný² Îöm q7ãrqºƒéí²uºãr nq Ÿµº nñkqu{šr h³îöm q7î ŒÃr ú³ P.O. Box 10002, Shenyang 110180

rºqu{  öjoºâè Œ yq Œö p Œ¼q¹ y ŒqÉ äãrö n [26-31] Shimizu} [32] Îöm q 7È ŒÃrqu{ oîöm q4î öm q7µœpi3k² í³²ü ÃrºfÎöm q4 qpkc Î öm q7 qaktxoºùœpi3k/akt³ séqš xgu{ý Íq fø o ²Ÿ³²PI3K/Akt³ ÍÏLY294002Î öm q7ºü n ºÎöm q7 Ãr nqí oly294002í PI3K/Akt³ ýîöm q7qãr Ío qígoí Îöm q7î ŒÃr qí³²pi3k/akt³ ö ní³² o xöwestern blotpi3k/akt³ ú qp-akt² Ÿo xø Îöm q7qí fp-aktqÿ²véîo oîöm q7ºw äakt Ãr q³ qœ iaktýö³²qãr q ofãr² öpâr ŒÃr³ qu{x³ä fp~ qr ƒ² ƒ² ² pâmakperkaktwnt²}vršp ~ Ãr²iÂqfv ú²qu{º AKT ƒ²âqéº gí²v Âr ŒÃr ooq [12, 33-34] Ͳ o ƒ²fqãr qb Œ p/qžo2(b cell leukemia-2, Bcl-2) qf º qãrðƒbadxöà gqéœ q (caspase) qfqcaspase 9 q [35-36] ³² qí ²² Îöm q7w äaktý Ãr qq öjp-akt ØýBAD x Caspase 9e Ÿ² qçjozqâ Î ŒÃrqͺrºÎöm q7 PI3K/Akt³ ýbad-caspase 9rºq ƒ²rm oí n güåqñí ƒ²qºw qºw²qãrrº q²ÿ È qu{ foxx xt Î ŒÃrqÉÍxöÎ öm q7ãr² föq n iîöm q7ž qt Î ŒÃr²zfŒövqÃr n Œq öo ŒÃriÎöm q7qkï s rº dpi3k/akt³ ÍÏLY294002qnÎ öm q7ãrqíxoºùœ³²³ ö np-akt o y ú qqwestern BlotÊ²Í oîöm q7ký p-aktÿ²ébadcaspase 9}Ãrrº q Ÿ²ÂxoÎöm q7ùœ³²w äakt qpi3k/akt³ ÍBAD-Caspase 9 ƒ² rºãr³ o ŒÃr²zÍoÎ Œ q² «húeò ÂcÀ d ÌrÀ{ÀÀ íqd k üìh {œ ü컫d ~ n ~ n ÍŠÚ ÍŠÛ r dò Çd ukà íà d 4 õ h References [1] Kepler CK, Ponnappan RK, Tannoury CA, et al. The molecular basis of intervertebral disc degeneration. Spine J. 2013;13(3):318-330. [2] Vo NV, Hartman RA, Yurube T, et al. Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging anddegeneration. Spine J. 2013; 13(3):331-341. [3] Gopal D, Ho AL, Shah A, et al. Molecular basis of intervertebral disc degeneration. Adv Exp Med Biol. 2012; 760:114-33. [4] i,øë,i,}.² Ârmggu{²[J]. fu y,2013; 21(17):1738-1741. [5],«um.Âr²Ÿ qémg [J].f zu{,2012,7(39):7375-7379. [6] Ding F, Shao ZW, Xiong LM. Cell death in intervertebral disc degeneration. Apoptosis. 2013;18(7):777-785. [7] Park JB, Lee JK, Park EY, et al. Fas/FasL interaction of nucleus pulposus and cancer cells with the activation of caspases. Int Orthop. 2008;32(6):835-840. [8] Ding F, Shao ZW, Yang SH, et al. Role of mitochondrial pathway in compression-induced apoptosis of nucleus pulposus cells. Apoptosis. 2012;17(6):579-590. [9] Sudo H, Minami A. Caspase 3 as a therapeutic target for regulation of intervertebral disc degeneration in rabbits. Arthritis Rheum. 2011;63(6):1648-1657. [10] Ê,±,i,}.Âr² qéokíu{à[j]. fégç(nf),2013,(6):2664-2666. [11] Wang D, Hu Z, Hao J, et al. SIRT1 inhibits apoptosis of degenerative human disc nucleus pulposus cells through activation of Akt pathway. Age (Dordr). 2013;35(5): 1741-1753. [12] Liu G, Cao P, Chen H, et al. MiR-27a regulates apoptosis in nucleus pulposus cells by targeting PI3K. PLoS One. 2013; 8(9):e75251. [13] Niu CC, Lin SS, Yuan LJ, et al. Hyperbaric oxygen treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells. J Orthop Res. 2013;31(2):204-209. ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3827

[14] Hiyama A, Yokoyama K, Nukaga T, et al. A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells. Arthritis Res Ther. 2013;15(6):R189. [15] Than KD, Rahman SU, Vanaman MJ, et al. Bone morphogenetic proteins and degenerative disk disease. Neurosurgery. 2012;70(4):996-1002. [16] Wang Z, Hutton WC, Yoon ST. Bone morphogenetic protein-7 antagonizes tumor necrosis factor-α-induced activation of nuclear factor κb and up-regulation of the ADAMTS, leading to decreased degradation of disc matrix macromolecules aggrecan and collagen II. Spine J. 2014;14(3):505-512. [17] ±,,Âg.Îöm q7öºâr² nqu{²[j].fœœî,2011,21(5):416-418. [18] Imai Y, Miyamoto K, An HS, et al. Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells. Spine (Phila Pa 1976). 2007;32(12):1303-1309; discussion 1310. [19] Griffith JF, Wang YX, Antonio GE, et al. Modified Pfirrmann grading system for lumbar intervertebral disc degeneration. Spine (Phila Pa 1976). 2007;32(24):E708-E712. [20] Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J. 2011;11(10):947-960. [21] i, Âň,.t² ÂrÎ ŒƒÅ»q ö [J].fuy, 2013,21(13): 1346-1351. [22] Ha KY, Kim BG, Kim KW, et al. Apoptosis in the sequestrated nucleus pulposus compared to the remaining nucleus pulposusin the same patient. Spine (Phila Pa 1976). 2011; 36(9):683-689. [23] Wei A, Brisby H, Chung SA, et al. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J. 2008;8(3):466-474. [24] Yu LP, Qian WW, Yin GY, et al. MRI assessment of lumbar intervertebral disc degeneration with lumbar degenerative disease using the Pfirrmann grading systems. PLoS One. 2012;7(12):e48074. [25] Ü,~,ˆê,}.üPfirrmannÉ Âr¼Î Œm ggq±[j].œy,2011,9(4):244-248. [26] Kakoi H, Maeda S, Shinohara N, et al. Bone morphogenic protein (BMP) signaling up-regulates neutral sphingomyelinase 2 to suppress chondrocyte maturation via the akt protein signaling pathway as a negative feedback mechanism. J Biol Chem. 2014;289(12):8135-8150. [27] Zheng Y, Wang X, Wang H, et al. Bone morphogenetic protein 2 inhibits hepatocellular carcinoma growth and migration through downregulation of the PI3K/AKT pathway. Tumour Biol. 2014. [28] Lim AI, Chan LY, Tang SC, et al. BMP-7 represses albumin-induced chemokine synthesis in kidney tubular epithelial cells through destabilization of NF-κB-inducing kinase. Immunol Cell Biol. 2014. [29] Pei H, Cao D, Guo Z, et al. Bone morphogenetic protein-7 ameliorates cerebral ischemia and reperfusion injury via inhibiting oxidative stress and neuronal apoptosis. Int J Mol Sci. 2013;14(12):23441-23453. [30] Ciumas M, Eyries M, Poirier O, et al. Bone morphogenetic proteins protect pulmonary microvascular endothelial cells from apoptosis by upregulating α-b-crystallin. Arterioscler Thromb Vasc Biol. 2013;33(11):2577-2584. [31] Wang Z, Guo J. Mechanical induction of BMP-7 in osteocyte blocks glucocorticoid-induced apoptosis through PI3K/AKT/ GSK3β pathway. Cell Biochem Biophys. 2013;67(2):567-574. [32] Shimizu T, Kayamori T, Murayama C, et al. Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC. Biochem Biophys Res Commun. 2012;417(2):869-873. [33] Mehrkens A, Karim MZ, Kim S, et al. Canine notochordal cell-secreted factors protect murine and human nucleus pulposus cells from apoptosis by inhibition of activated caspase-9 and caspase-3/7. Evid Based Spine Care J. 2013; 4(2):154-156. [34] Hiyama A, Yokoyama K, Nukaga T, et al. A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells. Arthritis Res Ther. 2013;15(6):R189. [35] Ma Y, Qin H, Cui Y. MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway. Biochem Biophys Res Commun. 2013;441(4):958-963. [36] Iwasaki K, Sudo H, Yamada K, et al. Cytotoxic effects of the radiocontrast agent iotrolan and anesthetic agents bupivacaine and lidocaine in three-dimensional cultures of human intervertebral disc nucleus pulposus cells: identification of the apoptotic pathways. PLoS One. 2014; 9(3):e92442. 3828 P.O. Box 10002, Shenyang 110180