Effect of Ermiao Fang with Xixin (Herba Asari Mandshurici) on bone marrow stem cell directional homing to a focal zone in an osteoarthritis

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1 Online Sumissions: J Trit Chin Me 2014 August 15; 34(4): info@journltm.om SSN JTCM. All rights reserve. EXPERMENTAL STUDY TOPC Effet of Ermio Fng with Xixin (Her Asri Mnshurii) on one mrrow stem ell iretionl homing to fol zone in n osteorthritis rt moel Ying Xu, Guojing Di, Qin Liu, Hongwei Zhu,Weiheng Chen, Ping Zhng,Tiejun Zho, N Lin Ying Xu, Guojing Di, Qin Liu, N Lin, the Center for Theory of Tritionl Chinese Meiine, nstitute of Chinese Mteri Mei, Chin Aemy of Chinese Meil Sienes, Beijing , Chin Hongwei Zhu, Weiheng Chen, Ping Zhng, Tiejun Zho, Deprtment of Bone n Joint, Wngjing Hospitl, Chin Aemy of Chinese Meil Sienes, Beijing , Chin Supporte y Grnts from the Ntionl Nturl Siene Fountion of Chin Projet of Guiing Tritionl Chinese Meiine nue Bone Mrrow Stem Cell Diretionl Homing to Fol Zone for the Tretment of Osteorthritis (No ) Corresponene to: Prof. N Lin, nstitute of Chinese Mteri Mei, Chin Aemy of Chinese Meil Sienes, Beijing , Chin. linn888@163.om Telephone: ; Aepte: Novemer 6, 2013 Astrt OBJECTVE: To investigte the effets of Ermio Fng (EM) with meil guie Xixin (Her Asri Mnshurii) (HAM) on one mrrow stem ell migrtion to fol zone in osteorthritis (OA) rts. METHODS: OA rts were inue y rthretomy n ssigne to shm-operte, moel, EM, or EM plus HAM groups. All rts were injete with reominnt humn grnuloyte olony-stimulting ftor 30 μg kg -1-1 for 7 ys n trete with EM or EM plus HAM t 1.6 or 1.9 g kg -1-1 for 3 or 6 weeks, respetively. Chonroyte poptosis n rtilge mtrix omponents were teste y trnsferse-meite eoxyuriine triphosphte-iotin nik en leling ssy n speil stining. Levels of interleukin-1 et (L-1β) tumor nerosis ftor lph (TNF-α) nitri oxie (NO), n inuile nitri oxie synthse (inos) in serum were etete y enzyme-linke immunosorent ssy or rioimmunossy. Mtrix metlloproteinses (MMPs)-13, tissue inhiitors of metlloproteinses (TMPs)-1, Bromoeoxyuriine (BrU), luster of ifferentition 34 (CD34), n stroml ell-erive ftor 1 (SDF-1) were mesure y immunohistohemil ssy. RESULTS: The EM n EM plus HAM groups h signifintly less rtilge mge n synovium inflmmtion the moel group. Moreover, the EM n EM plus HAM groups h less honroyte poptosis n more proteoglyn n ollgen ontent thn the moel group. The EM n EM plus HAM groups h oviously higher MMPs-13 n TMPs-1 expression in the rtilge thn the moel group. Moreover, the two formul groups h less relese of L-1β, TNF-α, NO, n inos thn moel group. mportntly, the expressions of BrU, CD34, n SDF-1 in rtilge were signifintly higher in the EM n EM plus HAM-Metrete rts thn moel group. Notly, the EM plus HAM tretment seeme to hve the gretest effets. CONCLUSON: HAM improves the therpeuti effets of EM on OA rts y enhning BMSC iretionl homing to the fol zone JTCM. All rights reserve. Key wors: Osteorthritis; Myeloi progenitor ells; Homing; Ermio Fng; Xixin(HerAsriMnshurii) JTCM www. journltm. om 477

2 Xu Y et l. / Experimentl Stuy NTRODUCTON Osteorthritis (OA) is egenertive joint isese hrterize y honroyte funtion loss n extrellulr mtrix estrution. 1 Surgil n phrmeutil interventions re iffiult for restortion of norml rtilge funtion. 2 Bone mrrow stroml ells (BMSCs) hve honroyte ifferentite poteny 3 n re inue with trnsforming growth ftor et3 (TGF-β 3) to stimulte proteoglyn n ollgen type Ⅱ synthesis. 4,5 This synthesis ppers to e iel in therpeuti use for rtilge regenertion n is new fiel of stuy in the pplition of stem ells. BMSCs n e summone y inflmmtory irrittion of lesions. However, the effet of BMSC homing to fol zone is limite. 6 Therefore, evising n gent or metho tht n promote BMSC homing to pthologi tissues is importnt. Ermio Fng (EM) is ompoun presription ompose of Hungi (Cortex Phelloenri Amurensis) n Cngzhu (Rhizom Atrtylois Lnee) in speifie rtio. 7 EM hs een use s tretment for rthrlgi synrome (Bi synrome) in Tritionl Chinese Meiine sine the Ming Dynsty to ler het n remove mpness. Toy in lini, EM is lso si presription for treting OA. 8 Moreover, vrious meil guie hers le to etter urtive effets. For exmple, Xixin (Her Asri Mnshurii) (HAM), s meil guie, is use in the tretment for rthrlgi. 9 Although meiinl guie hs n ovious effet-ireting funtion, its link to the pplition of meiinl guie n homing of BMSCs hs een unhrterize. This stuy is esigne to employ grnuloyte olony-stimulting ftor (G-CSF) to moilize BMSCs to peripherl loo n then oserve the influene of EM formul plus HAM, meil guie, on moilize BMSC homing to n osteorthriti mge re. MATERALS AND METHODS Chemils n regents Mtrix metlloproteinse (MMP)-13 ntioy (275628) n tissue inhiitors of metlloproteinse (TMP)-1 ntioy were purhse from Am (Cmrige, UK). Tunel kit (0811), Bru ntioy (u330201), CD34 ntioy (10m61), stroml ell erive ftor (SDF) -1 ntioy (Y-0729), n type ollgen ntioy (10 m222) were purhse from Boster (Wuhn, Chin). All other hemils were of nlytil gre. Herl preprtion EM ws prepre s esrie in the Chinese Phrmopei of Briefly, the three ingreients, Cngzhu (Rhizom Atrtylois Lnee) (500 g, frie with slt) n Hungi (Cortex Phelloenri Amurensis) 500 g, n Xixin (Her Asri Mnshurii) (HAM, 200 g) were pulverize to fine power, suspene in istille wter to onentrtion of 0.16 g/ml (EM) n 0.19 g/ml (EM + HAM), respetively, n mixe well efore ministrtion. OA niml moel n tretment Forty-eight mle Sprgue-Dwley rts of len gre, 4-months-ol, weighing ( ) g, were purhse from Experimentl Animl Center of Aemy Of Militry Meil Sienes (Certifite No. SCXK [jun] ) n were nesthetize with hlothne. After eing shve n isinfete, the right knee joint ws expose with meil pr ptellr pproh. The ptell ws islote lterlly n the knee ple in full flexion, followe y nterior ruite ligment trnsetion n meil menisus resetion (ACLT + MMx) with miro-sissors. Shm-rthrotomize nimls were negtive ontrols. The rts were rnomly ivie into four groups with rnom numer tle metho: ACLT+MMx without tretment (Moel, n=12), shm operte (Shm, n=12), ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (EM, n= 12), n trete with EM (1.6 g/kg) plus HAM (0.3 g/ kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (EM plus HAM, n=12). Dose lultions followe guielines orrelting ose equivlents etween humns n lortory nimls, on the sis of rtios of oy surfe re. 10 The ose equivlents 1.6 or 1.9 g/kg re se on reommene oses of 0.25 or 0.3 g/kg in humns, respetively. Untrete ontrol rts inue y ACLT+MMx n shm rts reeive istille wter only. Menwhile, ll nimls were suutneously injete with reominnt humn G-CSF (rhg-csf) t ose of 30 μg kg -1-1 for 7 ys fter rthretomy. To lel BMSCs, ll rts were intrperitonelly injete with BrU (50 mg/kg) for 3 ys efore smpling t the en of the 3r n 6th week. All proeures for onsiertion of niml welfre were reviewe n pprove y the ethil ommittee of Chin Aemy of Tritionl Chinese Meiine. Count of peripherl white loo ells Before (0 y) n 3, 5, n 7 ys fter rhg-csf injetion, 20 μl of loo were tken from the oritl venous plexus. White loo ells (WBC) in peripherl loo were teste y n utomti iohemil nlyzer (Hithi Lt., Tokyo, Jpn). Histologil nlysis Animls were srifie fter 3 or 6 weeks of tretment, the tii n femur were issete, fixe in 4% prformlehye for 24 h, elifie in 10% ethylene imine tetreti i-2n for elifition, n emee in prffin. Tissue setions (4 μm) were mounte on ommon slies for stining with hemtoxylin n eosin (HE), toluiine lue, n Msson's trihrome, s esrie. 11,12 Crtilge histopthologil fetures were nlyze using the soring system y Mnkin et l 13 (sore rnging from 0-12 for norml to omplete isorgniztion n hypoellulrity). Synovium histopthology ws evlute oring to Yoshi- JTCM www. journltm. om 478

3 Xu Y et l. / Experimentl Stuy mi's histologil gring (sore rnging 0-18 for norml to most severe retion). 14 mge-pro Plus 6.0 System (Mei Cyernetis n., Rokville, MD, USA) imge nlysis system ws use for quntittive nlysis. The positive inex ws lulte s integrte optil ensity (OD). All setions were rnomize n evlute y trine oserver who ws line to the tretment groups. Terminl eoxynuleotie trnsferse-meite UTP nik-en leling (TUNEL) stining Apoptoti ells in speimens were reognize using in situ ell poptosis etetion kits (Boster) oring to the mnufturer's instrutions. 15 Briefly, eprffinize setions were permeilize in 0.1% Triton X-100, inute in fluoresein-lele UTP n terminl eoxynuleotiyl trnsferse (TT) mixture, then proe using nti-fluoresein ntioy onjugte with lkline phosphtse. Setions were evelope using sustrte solution ontining fst re. Setions without primry TT were use s negtive ontrols for the TUNEL stining. TUNEL-positive ells in three ifferent res were ounte uner mirosope. mge-pro Plus 6.0 System (Mei Cyernetis n., Rokville, MD, USA) imge nlysis system ws use for quntittive nlysis. mmunolotting nlysis Prffin setions (4 μm) of joint tissues were mounte on poly-l-lysine-ote slies. Prffin setions were routinely ewxe n inute for 10 min with 3% H 2O 2. Eh setion ws inute with loking serum (Vetstin ABC Kit, Vetor Lortories, Burlingme, CA, USA) t room temperture for 30 min n then with primry rit monolonl ntioy ginst type Ⅱ ollgen (ilution 1 30), rit polylonl ntioy ginst MMP-13 (ilution 1 50), rit monolonl ntioy ginst TMP-1 (ilution 1 80), mouse monolonl ntioy ginst Bru (ilution 1 100), rit polylonl ntioy ginst CD34 (ilution 1 100), n rit polylonl ntioy ginst SDF-1 (ilution 1 50) overnight t 4. Setions inute in phosphte-uffer sline (PBS) without ntioy serve s negtive ontrols. Then, slies were inute with iotinylte seonry ntioy n then inute with viin-iotin omplex regent ontining horserish peroxise for 30 min. Setions were then stine with 3,3'-iminoenziine (DAB) (Sigm, St. Louis, MO, USA). mge-pro Plus 6.0 System (Mei Cyernetis n., Rokville, MD, USA) imge nlysis system ws use for quntittive nlysis. Serum n nlysis Animl loo ws ollete from the ominl ort n levels of serum interleukin-1β (L-1β), tumor nerosis ftor-α (TNF-α), one morphogeneti protein-2 (BMP-2), trnsforming growth ftor et 1 (TGF-β1) n inuile nitri oxie synthse (inos) were nlyze y enzyme-linke immunosorent ssy or rioimmunossy ssy. Nitri oxie (NO) ws etete y ssy kit (Beijing 4A Bioteh Co., Lt., Beijing, Chin). Sttistis nlysis All t re expresse s men±stnr evition n were lulte using two tile stuent's t-test, two-smple ssuming unequl vrine, within the Mirosoft Exel 2007 softwre pkge from Mirosoft (Remon, WA, USA) Vlues were onsiere signifintly ifferent if P<0.05. RESULTS Moiliztion of BMSCs After rhg-gsf injetion, WBC ount in peripherl loo inrese, n WBC ount inrese until rehing its pek on the 7th y. However, there were no signifint ifferenes mong the four groups (ll P> 0.05) (Figure 1). This suggeste tht the pplition of 9 (ys) Figure 1 Comprison of WBC ount mong groups efore n fter rhg-csf njetion Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). WBC: white loo ells; rhg-csf: reominnt humn grnuloyte olony-stimulting ftor; EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. JTCM www. journltm. om 479

4 Xu Y et l. / Experimentl Stuy A B C D E F G H J K L Figure 2 Comprison of effets on rtilge histology n Mnkin's sore A-H: histopthologi hnges of rtilge in weeks 3 n 6 (HE stining 200). A-D: histopthologi hnges of Shm, Moel, EM n EM plus HAM in week 3; E-H: histopthologi hnges of Shm, Moel, EM n EM plus HAM in week 6. -L: the Mkin's sore of rtilge, the sores of struture, ell n tiemrk in weeks 3 n 6. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n= 12). HE: hemtoxylin n eosin; EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.01, ompre with the shm group. P<0.01 n P<0.05, ompre with the moel group. P<0.05, ompre with the EM group. rhg-csf ws effetive in moilizing BMSCs to peripherl loo. Pthologi hnges of rtiulr rtilge n synovium Figure 2 shows in the shm group, HE stining showe smooth surfe of the rtiulr rtilge with norml ellulrity. n ontrst, ACLT + MMx rts h signifint histopthologil hnges with signifintly inrese overll Mnkin sore ompre with shm rts (P<0.01), s inite y surfe irregulrity, isorgniztion of rtiulr rtilge with pprent loning of honroytes in the trnsitionl n ril zones, n isrupte tiemrk. These histomorphologil hnges in the rtilge were reue in the EM n EM plus HAM- Metrete rts. Menwhile, the overll Mnkin sores of the two Tritionl Chinese Meiine groups were signifintly lowere s ompre with those of the moel group t 3 n 6 weeks tretment (P<0.01 or P<0.05). Notly, EM plus HAM signifintly lowere the overll Mnkin sores ompre with EM t 6 weeks (P<0.05). Untrete ACLT + MMx rts h more hyperplsi of synovil lining ells, hypertrophy of synovil lining lyer, n growth of the infiltrtion of inflmmtory ells in synovil tissue ompre with shm rts. EM n EM plus HAM rts h less hypertrophy of the synovil lining lyer, inflmmtory ellulr infiltrtion, n h signifintly lower histologil severity sores ompre with untrete ACLT+MMx rts fter 6 weeks of tretment (P<0.05 or 0.01). The lrgest inflmmtion suppression ws foun in the EM plus HAM JTCM www. journltm. om 480

5 Xu Y et l. / Experimentl Stuy group. However, there were no signifint ifferenes etween the EM n EM plus HAM groups. (Figure 3) 0.05) (Figure 5). Expressions of MMP-13 n TMP-1 in rtiulr rtilge Figure 6 shows tht ACLT + MMx inue signifint inrese in MMP-13 expressions n ler erese of TMP-1 expressions in the joints in weeks 3 or 6. EM n EM plus HAM tretment showe signifint ifferenes fter 6 weeks of tretment. EM plus HAM h 15.9% less positive MMP-13 expression n 26.9% more TMP-1 expression ompre with the EM group in week 6 (ll P<0.05). MMP-13 n TMP-1 were expresse in the honroytes of the knee joint in ll groups. The positive stining ws preominntly ytoplsmi or ytosoli in the honroytes. Chonroyte poptosis Figure 4 shows tht there were mny poptoti honroytes in the rtilge of untrete ACLT+MMx rts in weeks 3 or 6. EM n EM plus HAM rts h signifintly lower perentges of poptoti honroytes in weeks 3 or 6. The EM plus HAM group h 35% fewer poptoti honroytes ompre with EM tretment in week 6 (P<0.01). Crtilge mtrix repir Untrete ACLT+MMx rts h signifintly less proteoglyn, ollgen, n type Ⅱ ollgen in weeks 3 or 6. The EM n EM plus HAM groups h signifintly higher levels of proteoglyn n ollgen n more expression of type Ⅱ ollgen in week 6 ompre with the moel group. EM plus HAM group h 1.2-, 1.3- n 1.3-fol inrese in respetive proteoglyn, ollgen n type Ⅱ ollgen expressions, ompre with the EM tretment group in week 6 (ll P< Expressions of BrU, CD34, n SDF-1 on honroyte in rtiulr rtilge Compre with the shm-operte group, the numer of BrU-positive ells in the moel group ws higher oth in weeks 3 n 6, ut there were no sttistil ifferenes (P>0.05). Compre with the untrete A B C D E F G H J JTCM www. journltm. om Figure 3 Comprison of effets on synovil histology n Mnkin's K sore A-D: synovium histopthologi hnges of Shm, Moel, EM n EM plus HAM in week 3 (HE stining, 200); E-H: the synovium histopthologi hnges of Shm, Moel, EM n EM plus HAM in week 6 (HE stining, 200). -K: the totl Mkin's sore of synovium, the sores of synovil lining lyern susynovil tissue in weeks 3 n 6. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/ kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). HE: hemtoxylin n eosin; EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT + MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.01, ompre with the shm group. P< 0.05 n P<0.01, ompre with the moel group. 481 August 15, 2014 Volume 34 ssue 4

6 Xu Y et l. / Experimentl Stuy A B C D E F G H Figure 4 Comprison of effets on honroytes poptosis in weeks 3 n 6 A-H: terminl eoxynuleotie trnsferse-meite UTP niken leling stining ( 200). A-D: the honroytes poptosis of Shm, Moel, EM n EM plus HAM in week 3; E-H: The honroytes poptosis of Shm, Moel, EM n EM plus HAM t the 6 week. : Apoptosis inex of osteorthritis honroytes. Moel group: ACLT + MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.01, ompre with the shm group. P<0.05 n P<0.01, ompre with the moel group. P<0.01, ompre with the EM group. group, in week 3, there were signifintly more positive ells in the EM plus HAM group (P<0.05). However, in the 6th week, there were signifintly more positive ells in oth the formule groups, while the most ells were foun in the EM plus HAM group. There ws signifint ifferene etween the EM n EM plus HAM groups (P<0.05) (Figure 7). The numer of CD34 n SDF-1-positive ells in the untrete group ws signifintly higher in week 3 n lower in week 6 s ompre with tht in the shm-operte group. EM plus HAM group h signifintly greter expressions of CD34 n SDF-1 oth in weeks 3 n 6 s ompre with the untrete group. Only in week 6 ws the CD34 n SDF-1 expression in the EM plus HAM group signifintly higher thn tht in the EM group (P<0.05) (Figure 7). Serum L-1β, TNF-α, NO, inos, BMP, n TGF-β1 Serum levels of L-1β, TNF-α, NO, n inos were signifintly higher, n the levels of BMP-2 n TGF-β1 were signifintly lower in the untrete ACLT + MMx group ompre with the shm group. EM n EM plus HAM groups h signifintly lower levels of L-1β, TNF-α, NO, inos in week 6. Compre with the untrete group, the levels of BMP-2 n TGF-β1 in the EM plus HAM group were signifintly higher (P<0.05) in week 6. However, there were no signifint ifferenes in the ove inexes etween the EM n EM plus HAM groups (Figure 8). DSCUSSON BMSC therpy hs een use in one n rtilge repir for osteorthritis tretment. 16 However, the limite ility of BMSCs to home n repir is the ottlenek for BMSC therpy. 17,18 Therefore, fining gents or methos tht oul promote BMSC homing to lesions is populr in OA tretment reserh. n the present stuy, we evlute the nti-osteorthritis effets of EM n EM plus HAM on the moiliztion of BMSC homing to fol zone using n ACLT+MMx rt moel. We foun signifintly less rtiulr rtilge mge n synovium inflmmtion in the EM n EM plus HAM groups thn in the moel group, n HAM ws le to enhne the urtive effets of EM on OA. Moreover, EM plus the meil guie HAM n promote iretionl homing of BMSCs to pthologilly mge res. Chonroyte poptosis lters the rtilge mtrix (ECM) synthesis, leing to mtrix egenertion n finlly to OA. 19 Crtilge ECM moleules suh s type Ⅱ ollgen n sulfte proteoglyn ply ruil role in regulting honroyte funtions y filitting ell-mtrix intertions. 20 The evelopment n progression of OA is elieve to involve inflmmtion, n L-1β, TNF-α, n NO hve een reporte to inue poptosis in honroytes n use extrellulr mtrix egrtion. 21 MMP-13 n egre type Ⅱ ollgen n its tivities re inhiite y TMP-1. An imlne in the rtio of TMPs to MMPs uses ontinue mtrix estrution in OA. 22 Our results emon- JTCM www. journltm. om 482

7 Xu Y et l. / Experimentl Stuy A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2 A3 B3 E3 F3 C3 D3 G3 H3 J K Figure 5 Comprison of effets on PG, COL, n COL Ⅱ A1-D1: the PG of Shm, Moel, EM n EM plus HAM in week 3 ; E1-H1: the PG of Shm, Moel, EM n EM plus HAM in week 6. A2-D2: the COL of Shm, Moel, EM n EM plus HAM in week 3 ; E2-H2: the COL of Shm, Moel, EM n EM plus HAM in week 6. A3-D3: the COL Ⅱ of Shm, Moel, EM n EM plus HAM in week 3; E3-H3: the COL Ⅱ of Shm, Moel, EM n EM plus HAM in week 6. Dyeing methos of PG, COL, in week 6. A3-D3: the COL Ⅱ of Shm, Moel, EM n EM plus HAM in week 3; E3-H3: the COL Ⅱ of Shm, Moel, EM n EM plusham in week 6. Dyeing methos of PG, COL, n COL Ⅱ were toluiine lue stining, Msson's trihrome stining, n immunohistohemistry, respetively ( 200). -K: Levels of PG, COL, n COL Ⅱ. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). PG: proteoglyn; COL: ollgen; COL Ⅱ: type Ⅱ ollgen. EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.01, ompre with the shm group. P<0.05 n P<0.01, ompre with the moel group. P<0.05, ompre with the EM group. strte tht EM n EM plus HAM tretment h signifintly less rtiulr rtilge mge n synovium JTCM www. journltm. om inflmmtion in OA rts in week 6 thn tht in the moel group. Menwhile, the two formule groups 483 August 15, 2014 Volume 34 ssue 4

8 Xu Y et l. / Experimentl Stuy A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2 J Figure 6 Comprison of expressions of MMP-13 n TMP-1 in the knee joints ( 200) A1-D1: the MMP-13 of Shm, Moel, EM n EM plus HAM in week 3; E1-H1: the MMP-13 of Shm, Moel, EM n EM plus HAM in week 6. A2-D2: the TMP-1 of Shm, Moel, EM n EM plus HAM in week 3; E2-H2: the TMP-1 of Shm, Moel, EM n EM plus HAM in week 6. mmunohistohemistry ws use in ll frmes. n J: OD of MMP-13 n TMP-1. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n=12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). MMP-13: mtrix metlloproteinses-13; TMP-1: tissue inhiitors of metlloproteinses-1; OD: integrte optil ensity; EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.01, ompre with the shm group. P<0.05 n P<0.01, ompre with the moel group. P<0.05, ompre with the EM group. h less honroyte poptosis n more proteoglyn n ollgen ontent, prtiulrly type Ⅱ ollgen expression. Both formule oviously interfere with OA-ugmente expressions of MMPs-13 n EM plus HAM lerly ugmente OA-reue TMPs-1 expression in the knee joints. Moreover, the two formule groups h signifintly less relese of L-1β, TNF-α, NO, n inos in serum n the EM plus HAM group h signifintly higher BMP-2 n TGF-β1 levels. mportntly, the expressions of BrU, CD34, n SDF-1 in rtiulr rtilge were signifintly higher thn those in the moel group. The lrgest hnges in these prmeters were foun in the EM plus HAM group. Artiulr rtilge repir of EM plus HAM is etter thn EM in ACLT+MMx rts. Beuse BMSCs hve honroyte n ECM ifferentition potentil, our results suggest tht the tivity of EM plus HAM my e relte to BMSC repir therpy. JTCM www. journltm. om BrU is n nlogue of thymiine, whih is le to inorporte into the nuleus of ells uring prolifertion or ivision.23 Therefore, it is wiely use to lel stem ells. After n intrperitonel injetion with BrU, n inresing tren of BrU-positive expression ws etete in weeks 3 n 6 in the ACLT + MMx moel. This suggeste tht, fter mge of ompetent ells (suh s osteoytes, honroytes, n one mrrow ells), self-repir mehnism ws initite, n then the tivte stem ells joine in the repiring tivity of the rtiulr rtilge. Although the BrU-positive expression of the moel group ws higher thn tht of the shm-operte group, the homing stem ells were inequte for suffiient repir, n the histopthology feture rtiulr rtilge mge. After tretment with the two formule, there were more BrU positive ells. n week 3, the positive ell ount of the EM plus 484 August 15, 2014 Volume 34 ssue 4

9 Xu Y et l. / Experimentl Stuy A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2 A3 B3 C3 D3 E3 F3 H3 G3 J K Figure 7 Comprison of expressions of Bru, CD34, n SDF-1 in the knee joints ( 200) A1-D1: the Bru of Shm, Moel, EM n EM plus HAM in week 3; E1-H1: the Bru of Shm, Moel, EM n EM plus HAM in week 6. A2-D2: the CD34 of Shm, Moel, EM n EM plus HAM in week 3; E2-H2: the CD34 of Shm, Moel, EM n EM plus HAM in week 6. A3-D3: the SDF-1 of Shm, Moel, EM n EM plus HAM in week 3 ; E3-H3: the SDF-1 of Shm, Moel, EM n EM plus HAM in week 6. mmunohistohemistry ws use in ll frmes ( 200). -K: OD of Bru, CD34, n SDF-1. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT + MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n= 12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). BrU: romoeoxyuriine; CD34: luster of ifferentition 34; SDF-1: stroml ell erive ftor 1; OD: integrte optil ensity; EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. P<0.05 n P<0.01, ompre with the moel group. P<0.05, ompre with the EM group. HAM group exeee tht of the moel group, while the EM n moel groups were not signifintly ifferjtcm www. journltm. om ent. The stem ells oul possily ifferentite into honroytes or extrellulr mtrix, n there ws sig485 August 15, 2014 Volume 34 ssue 4

10 Xu Y et l. / Experimentl Stuy A B β μ μ β α C D E F Figure 8 Comprison of effets on L-1β, TNF-α, NO, inos, BMP-2, n TGF-β1 in serum A-F: the levels of L-1β, TNF-α, NO, inos, BMP-2, n TGF-β 1 in serum in weeks 3 n 6. Moel group: ACLT+MMx without tretment (n=12); shm group: shm operte (n=12); EM group: ACLT+MMx rts trete with EM intrgstrilly t ily ose of 1.6 g/kg (n= 12); EM plus HAM group: ACLT+MMx rts trete with EM (1.6 g/kg) plus HAM (0.3 g/kg) t ily ose of 1.9 g/kg for 3 or 6 weeks (n=12). EM: Ermio Fng; HAM: Xixin (Her Asri Mnshurii); ACLT+MMx: nterior ruite ligment trnsetion n meil menisus resetion. Dt represent the men±sd of smples with 6 rts in eh. P<0.05, P<0.01 ompre with the shm group. P<0.05 n P<0.01, ompre with the moel group. nifintly more repir oserve in the rtiulr rtilge of oth formule groups. n ition, the rtiulr rtilge Mnkin sore of the EM plus HAM group ws mrkely lower thn tht of the EM group. This inite tht HAM oul further promote stem ell homing n the initition of homing ws reltively erlier. The CD34 moleule is trnsmemrne slivry muin, n is onsiere to e positive sign of hemtopoieti stem ells. 24 n this stuy, we foun tht the CD34 expression in week 6 ws lower thn tht in week 3. The reson ws possily euse the ifferentition of tivte stem ells ourre in week 6. EM is le to slightly up-regulte the positive expression of CD34, n this effet ws enhne fter ition of HAM. This ifferene ws lerly oserve in terms of CD34 level in week 6, whih is onsistent with the signifintly higher BrU-positive ell ount t the sme time. These results suggeste tht HAM might promote BMSC homing to the mge re. SDF-1 is the first reporte hemottrtnt protein tht prtiiptes in the homing ourse of stem n pro- JTCM www. journltm. om 486

11 Xu Y et l. / Experimentl Stuy genitor ells. Furthermore, the homing of stem ells is epenent on the SDF-1 onentrtion grient. 25 n this stuy, EM ws le to signifintly up-regulte the positive expression of SDF-1 n therey filitte BM- SC homing tivity. This effet ws signifintly enhne fter ition of HAM in week 6, whih is onsistent with the signifintly higher BrU-positive ell ount t the sme time. Therefore, HAM might promote BMSC homing to mge y inresing SDF-1 levels in pthologi tissues n onsequently oosting the repir tivity. n onlusion, EM n EM plus HAM exert signifint nti-osteorthritis effets in ACLT+MMx rts. mportntly, y stuying the influene of EMR plus HAM ( meiinl guie) on BMSCs homing to mge fous n repir funtion, we primrily expoun the mehnism of HAM in enhning urtive effets of EM for OA my e y promoting the homing of BMSCs of peripherl loo to the mge fous. The filittion of BMSC homing to pthologi res oul e the iologil mnifesttion of HAM guiing other rugs to fol zone. REFERENCES 1 Loeser RF. Aging n osteorthritis: the role of honroyte senesene n ging hnges in the rtilge mtrix. Osteorthritis Crtilge 2009; 17(8): Gupt PK, Ds AK, Chullikn A, Mjumr AS. Mesenhyml stem ells for rtilge repir in osteorthritis. Stem Cell Res Ther 2012; 3(4): Luyten FP. Mesenhyml stem ells in osteorthritis. Curr Opin Rheumtol 2004; 16(5): nrwttn N, Chen G, Tokoro M, et l. Growth ftor omintion for honrogeni inution from humn mesenhyml stem ell. Biohem Biophys Res Commun 2004; 320(3): Mi Z, Ghivizzni SC, Lehmn ER, et l. Aenovirus-meite gene trnsfer of insulin-like growth ftor-1 stimultes proteoglyn synthesis in rit joints. Arthritis Rheum 2000; 43(11): Helmuth L. Stem ells her ll of injure tissue. Siene 2000; 290(5496): Chin Phrmopoei Committee. Chinese phrmopoei. Beijing: Chemil nustry Press, 2010: Liu BY, Hung Y, Liu YL. Moifie Ermio power meiine therpy in treting 31 ses of knee osteorthritis. Hunn Zhong Yi Z Zhi 2012; 28(6): Qu HR, Zhng LY, Su L. Meition hrteristis of Professor Su Li in the tretment of rheumtoi rthritis. Zhong Yi Yo Xue Kn 2002; 20(5): Xu Y, Liu Q, Liu ZL, Li L, Chen WH, Lin N. Tretment with Simiofng, n Anti-rthritis Chinese herl formul, inhiits rtilge mtrix egrtion in osteorthritis rt moel. Rejuvention Res 2013; 16(5): Hymi T, Pikrski M, Wesolowski GA, et l. The role of suhonrl one remoeling in osteorthritis: reution of rtilge egenertion n prevention of osteophyte formtion y lenronte in the rt nterior ruite ligment trnsetion moel. Arthritis Rheum 2004; 50(4): Gruer HE, Mrshll GJ, Nolso LM, Kirhen ME, Rimoin DL. Alkline n i phosphtse emonstrtion in humn one n rtilge: effets of fixtion intervl n methrylte emements. Bioteh Histohem 1988; 63(5): Mnkin HJ, Dorfmn H, Lippiello L, Zrins A. Biohemil n metoli normlities in rtiulr rtilge from osteo-rthriti humn hips Ⅱ. Correltion of morphology with iohemil n metoli t. J Bone Joint Surg Am 1971; 53(3): Yoshimi T, Kikuhi T, Or T, et l. Effets of high-moleulr-weight soium hyluronte on experimentl osteorthrosis inue y the resetion of rit nterior ruite ligment. Clin Orthop Relt Res 1994; (298): Kim KM, Kim JM, Yoo YH, Kim J, Prk YC. Cilostzol inues ellulr senesene n onfers resistne to etoposie-inue poptosis in rtiulr honroytes. nt J Mol Me 2012; 29(4): Brry FP. Biology n linil pplitions of mesenhyml stem ells. Birth Defets Res C Emryo Toy 2003; 69(3): Bentzon JF, Stenerup K, Hnsen FD, et l. Tissue istriution n engrftment of humn mesenhyml stem ells immortlize y humn telomerse reverse trnsriptse gene. Biohem Biophys Res Commun 2005; 330(3): Yn YW, Di QY, Zhng Z, Li WZ, Zhu YQ, Sun BG. The hnges of irulting stem ells n stem ell ftor in ptients with ute myoril infrtion. Zhong Hu Lo Nin Xin No Xue Gun Bing Z Zhi 2007; 9(2): Aigner T, Kim HA. Apoptosis n ellulr vitlity: issues in osteorthriti rtilge egenertion. Arthritis Rheum 2002; 46(8): Eyre D. Collgen of rtiulr rtilge. Arthritis Res 2002; 4(1): Kpoor M, Mrtel-Pelletier J, Ljeunesse D, Pelletier JP, Fhmi H. Role of proinflmmtory ytokines in the pthophysiology of osteorthritis. Nt Rev Rheumtol 2011; 7 (1): Wetzel M, Li L, Hrms KM, et l. Tissue inhiitor of metlloproteinses-3 filittes Fs-meite neuronl ell eth following mil ishemi. Cell Deth Differ 2008; 15 (1): Kong XY, Wng RT, Tin N, Li L, Lin N, Chen WH. Effet of Huogu Ⅱ Formul with meiinl guie Rix Ahyrnthis Bientte on one mrrow stem ells iretionl homing to nerosis re fter osteonerosis of the femorl he in rit. Chin J ntegr Me 2012; 18(10): Kruse DS, Fkler MJ, Civin C, My WS. CD34: struture, iology, n linil utility. Bloo 1996; 87(1): Httori K, Heissig B, Tshiro K, et l. Plsm elevtion of stroml ell-erive ftor-1 inues moiliztion of mture n immture hemtopoieti progenitor n stem ells. Bloo 2001; 97(11): JTCM www. journltm. om 487