Polygonum multiflorm alleviates glucocorticoid induced osteoporosis and Wnt signaling pathway

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1 970 Polygonum multiflorm llevites glucocorticoid induced osteoporosis nd Wnt signling pthwy MANRU ZHOU 1,2*, JINGKAI WU 1*, YONGJIE YU 1*, YAJUN YANG 1, JIN LI 1, LIAO CUI 3, WEIMIN YAO 4 nd YUYU LIU 1,3 1 Deprtment of Phrmcology, Gungdong Medicl University, Zhnjing, Gungdong ; 2 Deprtment of Phrmcy, Xinhu College of Sun Yt Sen University, Gungzhou, Gungdong ; 3 Gungdong Key Lbortory for Reserch nd Development of Nturl Drugs; 4 Deprtment of Respirtory Medicine, The Affilited Hospitl of Gungdong Medicl University, Zhnjing, Gungdong , P.R. Chin Received December 19, 2016; Accepted October 10, 2017 DOI: /mmr Abstrct. It is known tht long term excessive dministrtion of glucocorticoid (GC) results in osteoporosis. The present study imed to evlute the protective effects of Polygonum multiflorm (PM) on the bone tissue of rts with GC induced osteoporosis (GIO). A totl of 90 6 month old femle Sprgue Dwley rts (weight rnge, g) were rndomly divided into nine groups: Control (norml sline); prednisone (GC; 6 mg kg 1 d 1 ; Model); GC plus PMR30 (the 30% ethnol eluent frction of PM) (H) (400 mg kg 1 d 1 ); GC plus PMR30 (M) (200 mg kg 1 d 1 ); GC plus PMR30 (L) (100 mg kg 1 d 1 ); GC plus PMRF (ft soluble frction of PM) (H) (400 mg kg 1 d 1 ); GC plus PMRF (M) (200 mg kg 1 d 1 ); GC plus PMRF (L) (100 mg kg 1 d 1 ); GC plus clcitriol (CAL; µg kg 1 d 1 ; positive). Rts were dministered intrgstriclly with prednisone nd/or the forementioned extrcts for 120 dys, nd weighed once/week. The serum ws collected for detection of biochemicl mrkers. The left tibi ws used for bone histomorphometry nlysis. The right tibi ws prepred for hemtoxylin nd eosin stining. The left femur ws used to nlyze the protein expression of dickkopf 1 (DKK1), WNT inhibitory fctor 1 (WIF1) nd secreted frizzled relted protein 4 using western blotting. Long term excessive tretment of prednisone inhibited the bone formtion Correspondence to: Professor Yuyu Liu, Deprtment of Phrmcology, Gungdong Medicl University, 2 Wenmingdong Rod, Zhnjing, Gungdong , P.R. Chin E mil: liuyuyu77@163.com Dr Weimin Yo, Deprtment of Respirtory Medicine, The Affilited Hospitl of Gungdong Medicl University, 57 Renminddonn Rod, Zhnjing, Gungdong , P.R. Chin E mil: @qq.com * Contributed eqully Key words: Polygonum multiflorum, glucocorticoid, osteoporosis, Wnt signling pthwy rte ccompnied with decrese in bone mss, growth plte, body weight, nd the level of bone specific lkline phosphtse nd hydroxyl terminl propeptide of type I procollgen in the serum. Furthermore, simultneously increse in the level of trtrte resistnt cid phosphtse 5b nd cross linked crboxy terminl telopeptide of type I collgen in the serum, in ddition to DKK1, nd WIF1 protein expression, ws observed. PMR30 (M nd L) nd PMRF (H) groups were ble to reduce the negtive effects of GC on the bones. PMR30 (M nd L) nd PMRF (H) dose demonstrted protective effect of PM on bone tissue in GIO rts. The mechnism underlying the preventive effect of PM for the tretment of GIO my be ssocited with direct upregultion of the cnonicl Wnt/β ctenin signling pthwy. Introduction Glucocorticoids (GCs) re nti inflmmtory gents used in the tretment of vrious diseses, such s sthm, rheumtoid rthritis, nd systemic lupus erythemtosus (1 4). Although GCs hve been prescribed for mny yers, their potentil side effects (growth retrdtion, osteopeni, drenl insufficiency, etc.), cn prevent their long term use (5,6). Significntly, GC induced osteoporosis (GIO) is thought s the most severe one of these side effects becuse of the incresed frcture risk (7 9). GIO resulting from osteopeni hs been described s the most predictble nd debilitting compliction of long term GCs therpy (5,6). Therefore, the development of medictions tht prevent GIO is of clinicl significnce. Polygonum multiflorum Thunb. (PM, He Shou Wu) is kind of trditionl Chinese medicine (10). PM nd its extrcts cn be used to improve the helth of blood nd blood vessels, blcken hir, strengthen bones, neurosis nd other diseses commonly ssocited with ging (11 16). Bsed on previous evidence in our tem, we found tht PM nd its extrcts exert beneficil effects in the prevention nd tretment of osteoporosis, which hve lredy been pplied for Chin ptents (ZL ) (17). Furthermore, we hve investigted the effects of min components [(emodin nd 2,3,5,4' tetrhydroxystilbene 2 O β D glucoside (TSG)] of PM in vitro. The results showed tht emodin nd TSG cn

2 ZHOU et l: Polygonum multiflorm ALLEVIATES GLUCOCORTICOID INDUCED OSTEOPOROSIS 971 promote Bone Mrrow Mesenchyml Stem Cells (MSCs) to differentite into osteoblsts. Moreover, emodin cn inhibit MSCs differentite into dipocytes (18 21). The underlying mechnism of TSG my be relted to regultion of Wnt signling pthwy (22). However, the exct signling mechnism by which PM rescued impired bone formtion induced by GC hs not yet been investigted. The Wnt/β ctenin signling is n importnt pthwy tht is required by the growth, development nd mintennce of skeletl tissue (9,23). Wnt signls re extrcellulrly regulted by severl secreted ntgonists including secreted frizzled relted protein (sfrp), Wnt inhibitory fctor 1 (WIF1), nd dickkopf 1 (DKK1) (24). Previous evidence showed tht GCs cn promote the expression of DKK1 in cultured humn osteoblsts (25). It is known tht the cell fte of pre osteoblsts is minly determined by the Wnt/β ctenin signl pthwy (26). Therefore, the molecules which induce the ctivtion of Wnt/β ctenin signling re beneficil for the tretment of osteoporosis. PM hs been demonstrted to exert simultory effect on Wnt/β ctenin signling pthwy in vivo nd in vitro (23,27). Whether the extrcts of PM cn increse the bone mss or not in the GIO model chrcteristic of decresed bone formtion? If the extrcts could prevent GIO, nd wht's the mechnism of PM on bone metbolism? Considering the bove questions, this study ims to observe the effect nd the mechnism of PM underlying bone loss in GIO rts. Mterils nd methods Preprtion of PM extrct. The dried roots of PM were purchsed in Yulin Xing Sheng Chinese Herbl Medicine Co., Ltd. (Henn, Chin), nd were uthenticted by Professor Yuyu Liu. A voucher specimen ws deposited t the herbrium of Gungdong Key Lbortory for Reserch nd Development of Nturl Drugs, Gungdong Medicl University (Gungdong, Chin). Air dried roots of PM (56.0 kg) ws extrcted by 75% ethnol t 50~60 C, followed by rinsing with cyclohexne. The orgnic solvent of PMRF ws cquired by evportion under vcuum t 55 C. The PMRF dissolved in wter ws bsorbed by mcroporous resin D 101, nd then eluted with H 2 O, 10, 20, 30, 40, 50, 60, 70, 80 nd 90% ethnol successively, nd PMR30 ws prepred by the collection nd concentrtion of 30% ethnol elution (28). Animl experiments. This study ws crried out in strict ccordnce with the recommendtions in the Guide for the Cre nd Use of Lbortory Animls of Gungdong Lbortory Animl Monitoring Institute, under the Ntionl Lbortory Animl Monitoring Institute of People's Republic of Chin (29). The experiments hve been conducted ccording to protocols pproved for Specific Pthogen-Free niml cre of the Animl Center of Gungdong Medicl University, nd pproved by the Acdemic Committee on the Ethics of Animl Experiments of the Gungdong Medicl University [permit no. SYXK (Gungdong) ; Zhnjing, Chin]. The Sprgue Dwley (SD) femle rts were cclimted to locl vivrium conditions (temperture: C, humidity: 60%) nd under specific pthogen free conditions. Rts were llowed free ccess to wter nd diet. Experimentl protocols. Six month old femle SD rts weighing ( g, n=90) were rndomly divided into ten groups by weight: bsic group, control (norml sline) group, prednisone (GC, 6 mg kg 1 d 1, model) group, GC plus PMR30 (H) (400 mg kg 1 d 1 ) group, GC plus PMR30 (M) (200 mg kg 1 d 1 ) group, GC plus PMR30 (L) (100 mg kg 1 d 1 ) group, GC plus PMRF (H) (400 mg kg 1 d 1 ) group, GC plus PMRF (M) (200 mg kg 1 d 1 ) group, GC plus PMRF (L) (100 mg kg 1 d 1 ) group, GC plus clcitriol (CAL) (0.045 µg kg 1 d 1 ) (positive group). Rts were dministered intrgstriclly with prednisone nd/or the extrcts mentioned bove for 120 dys, nd weighed once per week. Rts were injected subcutneously with clcein on the 3rd, 4, 13, nd 14th dy before killed for the purpose of double lbeling in vivo, respectively (30). Rts were scrified by cridic puncture under sodium pentobrbitl nesthesi t the experimentl endpoint. The serum ws seprted for testing biochemicl mrkers. The left tibi ws used for bone histomorphometry nlysis. The right tibi ws prepred for H&E stining. The left femur ws used to test protein expression of DKK1, WIF1 nd SFRP4 using western blotting ssy (31). Serum mrkers ssy. Blood ws collected in specimen tubes nd kept t 25 C for min in verticl position for completely clotting. And then the serum ws seprted by centrifuging t 1,000 x g for 10 min nd stored t 80 C for biochemicl mrkers ssys. The serum ws seprted for testing biochemicl mrkers, including Bone specific lkline phosphtse (BAP), Hydroxyl terminl propeptide of type I procollgen (PICP), trtrte resistnt cid phosphtse 5b (TRACP 5b), Cross linked Crboxy terminl telopeptide of type I collgen (CTX I), nd DKK1. BAP nd OCN, s serum mrkers of bone formtion, nd OPG, srankl, nd TRAP5b, s the mrkers of bone resorption, were mesured in rts using commercilly vilble ELISAs (Tuoked Bio Tech, Gungzhou, Chin). Bone histomorphometry ssy. For histomorphometric nlysis, the left tibi ws removed, dissected, nd cut. The proximl tibil metphysis (PTM) ws opened to expose the bone mrrow cvity with n Isomet low speed sw (Buehler, Lke Bluff, IL, USA) nd then fixed in 70% ethnol fter they hd been plced in 10% formlin for 24 h, dehydrted in incresing concentrtions of ethnol, deftted in xylene, nd then embedded without declcifiction in methyl methcrylte (32). Frontl sections of the PTM were cut t thicknesses of 5 nd 8 µm. The region of interest in the PTM ws locted between 1 nd 4 mm distl to the growth plte epiphysel junction, not including corticl bone. A semiutomtic digitizing imge nlysis system (OsteoMetrics, Inc., Dectur, GA, USA) ws used for sttic nd dynmic histomorphometry mesurements. For sttic histomorphometry mesurements with Msson Goldner trichrome stining (5 µm sections), the totl tissue re, trbeculr re, trbeculr perimeter, nd osteoclst number (Oc.N) were mesured nd used to clculte the percentge of trbeculr bone volume (BV/TV), trbeculr number (Tb.N), trbeculr seprtion (Tb.Sp), number of osteoclsts (N.Oc), percent osteoclst surfce perimeter (Oc.S.Pm), nd percent osteoblst surfce perimeter (Ob.S.Pm). For dynmic nlyses (8 µm sections),

3 972 Tble I. Serum biochemicl indices of bone mrker in rts (x ± s). Group PICP (µg/l) BAP (µg/l) CTX Ⅰ (nmol/l) TRACP 5b (pg/ml) DKK1 (µg/l) CON 10.59± ± ± ± ±3.11 Pre 9.10± ±2.47 b 45.88± ± ±2.96 CAL 11.47± ±2.46 d 37.40± ± ±1.55 d PMR30(H) 9.94± ±2.61 d 29.84±6.43 d 1959± ±2.62 PMR30(M) 13.58±2.50 d 10.45± ±8.84 d 1960± ±1.68 d PMR30(L) 12.50± ±1.93 d 41.77± ± ±3.07 d PMRF(H) 12.99±2.72 d 13.01±2.31 d 30.03±8.42 d 1649±378 c 10.97±3.16 PMRF(M) 12.01± ±3.06 d 37.28± ± ±4.07 PMRF(L) 11.12± ± ±5.96 d 1820± ±2.08 P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL. single lbeled perimeter, double lbeled perimeter, interlbeled width, percent of lbeled perimeter (%L.Pm), minerl pposition rte (MAR), nd bone formtion rte per unit of bone surfce (BFR/BS), bone formtion rte per unit of bone volume (BFR/BV), nd bone formtion rte per unit of bone tissue re (BFR/TV) were mesured nd clculted. For the tibil shft (40 µm sections), the corticl re (Ct.Ar), percent Ct.Ar (%Ct.Ar), percent mrrow re, percent periostel lbeled perimeter (%P L.Pm), periostel MAR, periostel BFR per unit of bone surfce (P BFR/BS), percent endocorticl lbeled perimeter, endocorticl MAR, nd endocorticl BFR per unit of bone surfce (E BFR/BS) were clculted from the mesured prmeters (33). Western blotting ssy. Left femurs were stored t 80 C before they were used. The whole proteins were extrcted by the method previously described with Totl Protein Extrction kit (Applygen Technologies, Inc., Beijing, Chin) (33). Sixty microgrms of totl protein extrcts ws seprted on sodium dodecyl sulfte polycrylmide gels nd trnsferred to poly (vinylene difluoride) membrnes. The membrnes were blocked with 5% skimmed milk for 2 h t room temperture, nd were incubted overnight t 4 C with rbbit nti DKK1 monoclonl ntibody (b109416), WIF1 ntibody (b155101), nd sfrp4 (b154167) (ll from Abcm, Cmbridge, MA, USA) t dilution of 1:300. This ws followed by incubtion with the corresponding secondry ntibodies nd got nti rbbit IgG ntibodies (Beyotime Institute of Biotechnology, Himen, Chin). Protein expression ws visulized using BeoECL Plus instrument (Beyotime Institute of Biotechnology). GADPH rbbit mab (CST, USA) ws used to normlize the smple loding. The imges of bnds were quntified with Imge Pro Plus 6.0. Sttisticl nlysis. Dt were described s the mens ± stndrd devition (men ± SD) nd nlyzed sttisticlly with SPSS, version One wy ANOVA ws used to detect the differences in chnges between the groups of vrious tretments fter estblishing if the dt were normlly distributed nd equivlency of vrinces. P vlue (the probbilities) <0.05 were considered sttisticlly significnt. Figure 1. Body weight (g) chnges during the experimentl period. Body weight mesurements from vehicle treted controls (CON), prednisone 6 mg. kg 1.d 1 (Pre), clcitriol µg.kg 1.d 1 (CAL), PMR30 (H) 400 mg.kg 1.d 1, PMR30 (M) 200 mg.kg 1.d 1, PMR30 (L) 100 mg.kg 1.d 1, PMRF (H) 400 mg. kg 1.d 1, PMRF (M) 200 mg.kg 1.d 1, nd PMRF (L) 100 mg.kg 1.d 1 treted rts. P<0.05 vs CON, d P<0.05 PMR30 (M) vs. Pre, f P<0.05 PMRF (H) vs. Pre. Results Effects of PM on body weight. The ltertions of body weight of rts were no sttisticlly significnt difference between ech group during the initil stge of experiment, while the weight of rts in prednisone group decresed significntly from the fourth week compred with the CON group (P<0.05) (Fig. 1). Reversely, the chnges of body weight of rts in PMR30 (M) nd PMRF (H) groups incresed compred with the prednisone group (P<0.05). These results indicted tht PMR30 (M) nd PMRF (H) groups cn improve the slow growth of GIO rts. Effects of PM on biomrkers of bone turnover. We confirmed tht GC resulted in the decrese of biomrkers of the bone formtion, including serum bone specific lkline phosphtse (BAP) nd serum Hydroxyl terminl propeptide of type I procollgen (PICP) (Fig. 2 nd Tble I). Contrrily, GC stimulted the increse of the biomrkers relted to bone resorption including serum Trtrte resistnt cid phosphtse 5b (TRAP 5b), DKK1 nd C terminl telopeptides of I collgen (CTX 1) level of bone

4 ZHOU et l: Polygonum multiflorm ALLEVIATES GLUCOCORTICOID INDUCED OSTEOPOROSIS 973 Tble II. Histomorphometric sttic prmeters of proximl tibil of rts (x ± s). Group %Tb.Ar (%) Tb.Th (µm) Tb.N (no./mm) Tb.Sp (µm) CON 29.20± ± ± ±32.59 Pre 18.81±6.60 b 38.21± ± ±49.13 CAL 25.82±5.94 c 50.64± ± ±45.76 PMR30(H) 20.52± ± c,e 4.33± ± PMR30(M) 23.25± ± ± ±49.65 PMR30(L) 22.66± ± ± ±43.53 PMRF(H) 26.76±7.35 c 52.84± ± ±43.48 PMRF(M) 23.62± ± ± ±26.18 PMRF(L) 19.83±3.89 f,g 47.03± ± ±46.87 P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL; g P<0.05, h P<0.01 vs. PMRF(H). Figure 2. Endpoint levels of serum biochemicl mrkers (A) CTX Ⅰ, (B) DKK1, (C) TRACP 5b, (D) BAP, (E) PICP in the rts treted with vehicle (CON), prednisone (Pre), clcitriol (CAL), nd vrious PMR (30, F) dose levels. * P<0.05, ** P<0.01 vs. CON; P<0.05, P<0.01 vs. Pred; # P<0.05, ## P<0.01 vs. CAL. tissue (Fig. 2 nd Tble I). Encourgingly, PMR30 (M, L) nd PMRF (H) groups showed cpcity to reverse the deleterious impcts of bone turnover elicited by GC, s effectively s CAL. Effects of PM on the prmeters of the two dimensionl histomorphometry of the proximl tibil metphysis. The chnges of the two dimensionl histomorphometry of the proximl tibil metphysis: Compred with the control group, %Tb.Ar, percent lbeled perimeter (%L.Pm), minerl pposition (MAR), BFR/TV nd %Ob.S.Pm were decresed (P<0.05), while percentge of osteoclst surfce perimeter (%Oc.S.Pm) nd number of osteoclst per millimeter (Oc.N) were incresed (P<0.05) in prednisone group. Compred with the prednisone group, %Tb.Ar nd BFR/TV were incresed (P<0.05); nd %Oc.S.Pm ws decresed (P<0.05) in CAL group. Compred with the prednisone group, %L.Pm nd new bone formtion rte per unit of bone surfce (BFR/BS) were incresed in PMR30 (H) group (P<0.05). Compred with the prednisone group, number of osteoclst per millimeter (Oc.N) ws decresed (P<0.05) in PMR30 (H, M, L) groups. Compred with the prednisone group, %Tb.Ar nd BFR/TV were incresed (P<0.05) in PMRF (H) group. Compred with the prednisone group, percentge of osteoclst surfce perimeter (%Oc.S.Pm) ws decresed (P<0.05) in PMRF (H, M, L) groups (Tbles II IV nd Fig. 3). These results indicted tht PMR30 (M, L) nd PMRF (H) groups cn improve the bone loss nd decresed ctivity of osteoclst in GIO rts. Effects of PM on the prmeters of two dimensionl histomorphometry of the tibil shft. The chnges of the two dimensionl histomorphometry of the tibil shft: Compred with the control group, Ct.Ar, %Ct.Ar nd P MAR were decresed (P<0.05) while %M.Ar nd E BFR/BS were incresed (P<0.05) in prednisone group. Compred with the

5 974 Tble III. Histomorphometric dynmic prmeters of proximl tibil of rts (x ± s). Group %L.Pm (%) MAR (µm/d) BFR/BS (%/yer) BFR/BV (%/yer) BFR/TV (%/yer) CON 18.26± ± ± ± ±13.99 Pre 12.13± ± ± ± ±13.83 CAL 20.70± ± ± ± ±31.23 d PMR30(H) 40.17±57.19 d,e 1.01± ±21.64 d ± ±22.82 e PMR30(M) 11.67± ± ± ±67.01 f 41.62±18.92 e PMR30(L) 19.05± ± ± ± ±23.95 e PMRF(H) 19.24± ± ± ± ±29.02 c PMRF(M) 16.49± ± ± ±85.37 e 50.16±20.68 e PMRF(L) 18.31± ± ± ± ±20.09 e P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL. Tble IV. Oc nd Ob prmeters of proximl tibil of rts (x ± s). Oc.N %Oc.S. %Ob.S. Group (no./mm) Pm (%) Pm (%) CON 0.10± ± ±0.12 Pre 0.20± ±0.54 b 0.09±0.06 CAL 0.12± ±0.11 d 0.08±0.04 PMR30(H) 0.19± ±0.49 c 0.16±0.28 e PMR30(M) 0.08±0.04 d,g 0.19±0.11 d 0.08±0.04 PMR30(L) 0.10±0.02 c,g 0.18±0.05 c 0.10±0.06 PMRF(H) 0.08±0.03 d 0.12±0.06 c 0.23±0.14 PMRF(M) 0.06±0.05 d,h 0.13± ±0.06 PMRF(L) 0.07±0.03 d 0.12±0.06 c 0.14±0.10 P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL; g P<0.05, h P<0.01 vs. PMR30(H). Tble V. Histomorphometric sttic prmeters of tibil shft in rts (x ± s). Group Ct.Ar (mm 2 ) %Ct.Ar (%) %M.Ar (%) CON 4.59± ± ±1.77 Pre 4.05± ± ±2.72 CAL 4.07± ± ±5.58 PMR30(H) 4.17± ± ±3.56 PMR30(M) 4.21± ± ±4.76 PMR30(L) 4.28± ± ±5.99 PMRF(H) 4.40± ± ±2.18 PMRF(M) 4.49± ± ±2.46 PMRF(L) 4.29± ± ±2.67 P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL. prednisone group, %Ct.Ar nd %P L.Pm were showed trend towrd incresed (P>0.05) while %M.Ar nd E BFR/BS were showed trend towrd decresed (P>0.05) in CAL, PMR30 nd PMRF groups (Fig. 4 nd Tbles V nd VI). These results indicted tht PMR30 nd PMRF groups cn improve the thickness of corticl bone in GIO rts. Effects of PM on the growing epiphysel plte of proximl tibil. The results of growth plte, compred with the control group, the growth plte ws become thinner in prednisone group. Compred with the prednisone group, the growth plte ws become more thicken in CAL, PMRF (M, L) nd PMR30 (M, L) groups (Fig. 4). It is indicted tht PM my be stimulte growth hormone secretion of rt. Effects of PM on mrrow ft tissue deposistion of the proximl tibil metphysis. Compred with the control group, the number of dipocytes ws become more nd dense in prednisone group. Compred with the prednisone group, the number of dipocytes were becme less in CAL, PMR30 (M, L) nd PMRF (H, M) groups (Fig. 5). These results indicted tht PMR30 (M, L) nd PMRF (H, M) groups cn decrese the number of dipocytes in GIO rts. It is prompted tht PM cn inhibit the differentition of bone mrrow stroml cells into dipocytes. Effects of PM on the Wnt signling pthwy. Compred with control group, the expression of DKK1 nd WIF1 were incresed in prednisone group (P<0.05). Compred with prednisone group, the expression of DKK1 were decresed (P<0.05) in CAL, PMR30 (M, L) nd PMRF (H) groups. Compred with prednisone group, the expression of WIF1 ws decresed (P<0.05) significntly in PMR30 (M) nd PMRF (F) groups (Fig. 6). These results indicted tht PMR30 (M) nd PMRF (F) groups cn increse the expression of DKK1 nd WIF1 im to regulte the Wnt signling pthwy in GIO rts. Discussion It is widely known tht GC tretment induces osteoporosis. In our previous study, GC exerts series of deleterious ctions

6 ZHOU et l: Polygonum multiflorm ALLEVIATES GLUCOCORTICOID INDUCED OSTEOPOROSIS 975 Figure 3. Effects of vehicle (CON), prednisone (Pre), clcitriol (CAL), nd vrious PMR (30, F) dose tretments on the proximl tibil metphysis (PTM) bone structure nd minerl bone formtion. Arrows point to the tetrcycline nd clcein lbeling. Quntittive mesurements of histomorphometric prmeters of PTM re showed in Tbles II nd III. (A) Goldner's Trichrome stin, (B) AgNO 3 stin,(c) fluorescence imges of undeclcified sections 1, b 1, c 1:CON; 2, b 2, c 2:Pre (6 mg. 1.d 1 ); 3, b 3, c 3:Pre+CAL (0.045 µg.kg 1.d 1 ); 4, b 4, c 4:Pre+PMR30 (H) (400 mg.kg 1.d 1 ); 5, b 5, c 5:Pre+PMR30 (M) (200 mg. kg 1.d 1 ); 6, b 6, c 6:Pre+PMR30 (L) (100 mg.kg 1.d 1 ); 7, b 7, c 7:Pre+PMRF (H) (400 mg.kg 1.d 1 ); 8, b 8.c 8:Pre+PMRF (M) (200 mg.kg 1.d 1 ); 9, b 9, c 9: Pre+PMRF (L) (100 mg.kg 1.d 1 ). Figure 4. Effects of vehicle (CON) nd vrious prednisone (Pre) dose tretments on corticl bone of the tibil shft nd crtilge growth. Arrows point to interlbeling distnces fter double lbeling with tetrcycline nd clcein. Quntittive mesurements of histomorphometric prmeters of tibil shft re shown in Tbles V nd VI. (A) Fluorescence imges of the tibil shftmiddle nd (B) fluorescence imges of the growth plte on bone tissue in both mle nd femle rts. Furthermore, previous evidence demonstrted tht extrcts of PM exhibits pretective effect on ovriectomized rts (20). In the present study, extrcts of PM (PMR30 nd PMRF) were exhibits pretective effect on bone loss in GIO rts, owing to restortion of bone micro rchitecture nd the serum levels of biomrkers relted to bone formtion. In ddition, Polygonum Multiflorm llevites GIO my be through regultion of Wnt signling pthwy to protect the bone. Physiologiclly, the secretion of endogenous GC ws regulted by hypothlmus hypophysis drenl cortex system (30), thus inhibited the secretion of growth hormone (GH) (34). However, excessive exposure to excessive exogenous cliniclly GC could induce rpidly bone loss resulting to the increse of frcture risk. In the present study, we found tht long term excessive dministrtion of prednisone cused decrese of bone formtion prmeters (MAR nd BFR/TV) in the trbeculr bone re nd decrese of growth of longitudinl bone, nd n increse of bone resorption (Oc.S/BS) (Figs. 1 nd 4). It hs generlly been thought tht GIO results from impired bone formtion s well s exggerted bone resorption. Possible pthologicl mechnisms of GIO re listed in the following: (1) impiring osteoblst or osteoclst function directly, nd (2) secondry hyperprthyroidism, due to the incresed renl excretion nd decresed intestinl bsorption of clcium (35). In this study, PMR30 (M, L) nd PMRF (H) groups were found to be effective t ttenuting GIO in vivo, s evidenced through its restortion of %Tb.Ar, Tb.Th, %L.Pm, BFR/BS, nd BFR/TV (Tbles II IV nd Fig. 3). This indictes tht the compressive strength nd mechnicl properties of the bone tissue nd the ctivity of bone formtion were enhnced. Furthermore, it would promote bone formtion nd increse bone turnover rte. This is benefit to replce bone mtrix between old nd new, nd self repir the bone micro structure, so tht fighting brittle frcture by long term use of prednisone.

7 976 Tble VI. Histomorphometric dynmic prmeters of tibil shft in rts (x ± s). %P L. P MAR P BFR/BS %E L. E MAR E BFR/BS Group Pm (%) (µm/d) (%/yer) Pm (%) (µm/d) (%/yer) CON 27.40± ± ± ± ± ±3.98 Pre 29.18± ±0.05 b 19.27± ± ± ±6.63 CAL 34.49± ±0.13 d 28.72±8.48 c 16.35± ± ±3.30 PMR30(H) 30.67± ± ± ± ±0.25 e 11.38±5.87 PMR30(M) 33.97± ± ±13.47 e 19.08± ±0.24 c,e 6.68±6.04 PMR30(L) 26.64± ± ±6.68 e 14.85± ± ±5.04 PMRF(H) 38.56±6.11 c 0.65± ± ± ± ±5.32 PMRF(M) 29.02± ± ± ± ± ±3.02 PMRF(L) 27.16± ± ±7.39 f 18.26± ± ±3.34 P<0.05, b P<0.01 vs. CON; c P<0.05, d P<0.01 vs. Pred; e P<0.05, f P<0.01 vs. CAL. Figure 5. Effects of vehicle treted controls (CON), prednisone 6 mg.kg 1.d 1 (Pred), clcitriol µg.kg 1.d 1 (CAL), nd vriuos PMR (30, F) dose tretments on dipocyte distribution in bone mrrow of PTM. (A) CON; (B) Pre (6 mg. 1 kg 1.d 1 ); (C) Pre+CAL (0.045 µg.kg 1.d 1 ); (D) Pre+PMR30 (H) (400 mg.kg 1.d 1 ); (E) Pre+PMR30 (M) (200 mg.kg 1.d 1 ); (F) Pre+PMR30 (L) (100 mg.kg 1.d 1 ); (G) Pre+PMRF (H) (400 mg.kg 1.d 1 ); (H) Pre+PMRF (M) (200 mg.kg 1.d 1 ); (I) Pre+PMRF (L) (100 mg.kg 1.d 1 ). PMR30 nd PMRF cn increse the prmeters of the outer membrne nd decrese the inner membrne of tibil shft in different degree (Tbles V VII nd Fig. 4). This shows tht the PM cn promote the periostel bone formtion of the outer membrne nd inhibit endostel bone resorption of the inner membrne, decrese bone turnover, nd increse the bone mss nd thickness of corticl bone, ginst long term use of GC induced %M.Ar increse, then mke corticl become thinner, thereby incresing the qulity nd bone biomechnicl properties to void frcture. Of these results we lso found the rtio of bone conversion re different proximl tibi nd tibi shft. The conversion rte of proximl tibi ws fster thn tibi shft, this my relted to blood vessel is rich in proximl tibil (37). Interestingly, we found tht longitudinl growth plte of proximl tibil were incresed in PMR30 (H, M, L) nd PMRF (H) groups (Fig. 4), compred with prednisone group. This my be relted to PM inducing the growth hormone relese. Lo hs been reported tht n emodin derivtives isolted from PM, this emodin derivtive, tenttively nmed emoghrelin, ws demonstrted to stimulte growth hormone secretion of rt primry nterior pituitry cells, presumbly vi the sme moleculr mechnism of GHSR ctivtion (38). In the present study, the content of combined nthrquinone

8 ZHOU et l: Polygonum multiflorm ALLEVIATES GLUCOCORTICOID INDUCED OSTEOPOROSIS 977 Figure 6. Effects of the extrcts of PM on trget protein of wnt signling pthwys in GIO rts. (A) The scnned imge on X rys ws fter exposure of western blot nlysis. (B) The DKK1 protein of wnt signling pthwys in GIO rts. (C) The SFRP4 protein of wnt signling pthwys in GIO rts. (D) The WIF1 protein of wnt signling pthwys in GIO rts. * P<0.05, ** P<0.01 vs. CON; P<0.05, P<0.01 vs. Pred; # P<0.05, ## P<0.01 vs. CAL. (CAQ) in the smple of PMR30 ws over thn PMRF, tht the longitudinl growth plte of proximl tibil were incresed in PMR30 (H, M, L) over thn PMRF (H) groups. The deficiency is not to determine the longitudinl growth rte (LGR). The disruption of the bone formtion resorption blnce plys key role in osteoporosis (39). Serum BAP is known s mrker of bone formtion, nd TRACP 5b is known s mrker of bone resorption (35). In the present study, we found tht PM recovered the serum BAP nd PICP levels nd significntly reversed the prednisone induced decrese. We hve lso found tht the serum of DKK1 nd TRACP 5b level were incresed in GC group which were consistent with the histomophometric dt (Tbles I VI). TRACP 5b minly exists in bone tissue, which is minly derived from osteoclsts, nd presents the ctivity of osteoclsts nd its function of bone resorption (36). The level of TRACP 5b ws incresed in prednisone group, suggesting tht long term use of GCs incresed osteoclst ctivity. Our study dt indicted tht PM cn promote bone formtion nd inhibit bone resorption. Futhermore, it hs been reported tht high level of DKK1 which suppresses the Wnt signl of bone formtion in osteoblsts, resulted from ctivtion of trnscription through GRE in the DKK1 gene promoter. In the present study, DKK1 ws incresed expression in prednisone group (Fig. 6). The Wnt/β ctenin signling pthwy ws lso found to be re ctivted by PM, which my be relted to the bone protective effects of PM. These results indicte tht PM exerts protective effects ginst GIO. In previous studies, GC treted nimls lso exhibited decresed BMD nd bone minerl content (BMC) (27). In ddition, in the previous experiments, we verified tht TSG, s mjor constituent in PM Thunb, showed nti osteoporosis ctivity in vitro nd in vivo (22). These dt suggest tht the bone protective effects of PM re medited through the regultion of Wnt signling pthwy. Wnt binds with specific cell surfce receptors Frizzled nd LRP5/6, thereby leding to binding with Axin, which in turn medites the proteolysis of β ctenin. DKK1 is lso known to inhibit Wnt signling by binding to LRP5/6 (41). DKK1 re receptor inhibitors which ply key role in the regultion of the Wnt signling pthwy in bone formtion (42). As expected, tretment with PM recovered the ctivity of this signling pthwy. These results suggest tht the Wnt/β ctenin signling pthwy is involved in the bone protective effects of PM ginst prednisone induced osteoporosis. In conclusion, we demonstrted tht PM cn ttenute GIO nd the mechnism of the preventive effect on GIO my be linked to direct up regultion of cnonicl Wnt/β ctenin pthwy. Acknowledgements This reserch ws supported by grnts from Ntionl Nturl Science Foundtion of Chin (no nd ), by the Open Fund Project of Key Lbortory of Gungdong Province (no. 4CX16010G), by the Chrcteristic Innovtion Project (Nturl Science) of Eduction Deprtment of Gungdong Province (no. 2014KTSCX084), by the Science nd Technology Pln of Gungdong Province (no. 2016ZC0178 nd 2016A ), nd by the Open Foundtion of Gungdong Key Lbortory for Reserch nd Development of Nturl Drugs (TRYW201603).

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