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β-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin Jianwei Lv a, b, Xiaolei Sun a, **, Jianxiong Ma a, Xinlong Ma a, b, *, Guosheng Xing a, Ying Wang a, Lei Sun a, Jianbao Wang a, Fengbo Li a, Yanjun Li a a b
Institute of Orthopedics, Tianjin Hospital, No. 122, Munan Road, Heping District, Tianjin TJ 300050, China Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin TJ 300070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2015 Accepted 28 October 2015 Available online xxx
Periostin has an essential role in mechanotransduction in bone. Naringin, a natural flavonoid, has been evidenced for its osteoprotective role in osteoporosis, while its mechanism is far from clear. Here we show that down-regulation of periostin, and up-regulation of its downstream sclerostin and inactivation of Wnt/b-catenin signaling were implicated in neurectomy-induced bone loss. Naringin could upregulate periostin and prevent neurectomy-induced deterioration of BMD, trabecular microstructure and bone mechanical characteristics. In conclusion, naringin could prevent progress of disuse osteoporosis in rats, which may be mediated by increased periostin expression and subsequently inhibition of sclerostin and activation of Wnt/b-catenin signaling pathways. © 2015 Elsevier Inc. All rights reserved.
Keywords: Disuse osteoporosis Naringin Periostin Sclerostin b-catenin
1. Introduction Naringin, the main bioactive flavonoid extracted from citrus fruits [1], has a beneficial role in protecting against osteoporosis caused by post-menopause, glucocorticoid, retinoic acid, orchidectomy and aging [2e6]. In vitro, naringin has been shown to be effective to enhance osteoblast proliferation and differentiation, with increasing expression of osteogenic markers such as osteocalcin (OC), osteopontin (OPN), osteoprotegerin (OPG) and bone morphogenetic protein-2 (BMP) mediated by PI3K-Akt and activator protein-1 (AP-1) signal pathway [3,7,8]. Meantime, naringin could abrogate osteoclastogenesis and osteoclastic differentiation through impeding activation of NF-kB and ERK signaling cascades induced by RANKL, as well as expression of osteoclast gene markers [9]. Although naringin has been exhibited to possess estrogen-like protective effect against postmenopausal osteoporosis [10] and antioxidation in orchidectomized rats [5], the molecular mechanisms implicated in bone anabolic effect of naringin in different
* Corresponding author. Institute of Orthopedics, Tianjin Hospital, No. 122, Munan Road, Heping District, Tianjin TJ 300050, China.. ** Corresponding author. E-mail address: [email protected] (X. Ma).
osteoporotic models are far from clear. The canonical Wnt/b-catenin signaling pathway exerts a pronounced beneficial role in bony homeostasis [11], with critically positive regulation in osteoblastic differentiation [12], and normal activation of which in osteocyte is critical to limit excessive bone resorption of osteoclasts [13]. The antagonist of Wnt/b-catenin pathwayddsclerostin, a secreted protein mainly derived from osteocytes, exerts an essential role in the progress of disuse osteoporosis induced by mechanical unloading by specially suppressing activation of Wnt/b-catenin cascades [14]. Sclerostin is increased by unloading [14] while decreases with mechanical stimulation [15], and mice with gene of which deficiency are resistant to unloadinginduced bone loss [14]. Further studies identify the selectively upstream regulator of sclerostin in response to mechanotransductionddperiostin, is a matricellular protein preferentially expressed by periosteal osteoblasts and osteocytes, level of which displays a reciprocal correlation with that of sclerostin, either with unloading or by mechanical stimuli [16,17]. Mice with gene deficient of periostin show a diminished response to mechanical unloading, accompanied by increased sclerostin [16]. Recently Wnt/b-catenin signaling was found to be involved in the positive effect of Naringin on bone maintenance as naringin promoted nuclear translocation of b-catenin in osteoblast-like UMR-106 cell [18]. While whether naringin displays beneficial
http://dx.doi.org/10.1016/j.bbrc.2015.10.152 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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effects on skeletal properties in disuse osteoporosis through regulation of periostinesclerostineWnt/b-catenin pathway has not been reported. We here evidenced for the first time that naringin could prevent deterioration of skeletal properties induced by immobilization through neurectomy mediated by up-regulation of periostin, inhibition of sclerostin as well as increased nucleus accumulation of bcatenin.
2.4. Bone mineral density(BMD)measurements On the day before sacrifice, BMD in distal femur in vivo was measured by dual energy X-ray absorptiometry (DXA, LunarProdigy; GE). Animals were anesthetized using chloral hydrate (400 mg/kg) by intraperitoneal injection and supine on specimen platform with hind limbs flexed and extorsion. 2.5. Biochemical markers measurements
2. Materials and methods 2.1. Animals All experimental protocols were approved by the Ethics Committee of Tianjin Hospital. Sixty 6-month-old male SpragueeDawley rats (255 ± 10 g) were housed under a 12 h light/dark cycle at 24 ± 0.5 C, with free access to food and water, body weights of which were measured once a week. Rats were allocated into five groups randomly, four of which was unilateral sciatic nerve neurectomized (USN) and one sham-operated. In sciaticneurectomized groups, the right sciatic nerve of each rat was severed, with 1 cm of which excised as previously described [19]. The sham-operated rats only received sciatic nerve exposed with skin and subcutaneous tissues sutured layer by layer then. The four sciatic-neurectomized groups were administered with 30, 100 and 300 mg/kg body weight naringin (NG) or saline by gavage respectively four weeks before and four weeks after neurectomy. The sham-operated group received saline vehicle. Four weeks postoperation, all rats were sacrificed. On the day before sacrifice, urine of overnight fasted rat was collected, while blood was harvested from heart by cardiac puncture on the day of sacrifice and then centrifuged to get serum samples, both of which were frozen at 80 C until analyses of bone turnover markers. Femurs and tibias ipsilateral to neurectomy with soft tissues removed were prepared appropriately for further analysis. 2.2. Micro-computed tomography (Micro-CT) Femurs for Micro-CT analysis were put in 70% ethanol. Distal femoral cancellous bone was scanned by SIEMENS Inveon PET.SPECT.CT (SIEMENS, Berlin, Germany) with energy settings of 500 mA and 80 kV, micro-structure of which was evaluated by bone volume/tissue volume (BS/TV), trabecula number (Tb.N), trabecula thickness (Tb.Th), bone surface/bone volume (BS/BV) and trabecular separation (Tb.Sp). Three-Dimension images were reconstructed by software COBRA Exxim: Licensed to Siemens. 2.3. Biomechanical measurement Femurs subjected to biomechanical testing were preserved within 0.9% saline, biomechanical properties of which were evaluated by three-point bending test and femoral neck-fracture test using Electro-Force 3230 Bose System (BOSE, Minnetonka, MN, USA). In three-point bending tests, femoral shaft were placed two lower bases with a separation of 20 mm. Following a preload of 2 N, a vertical load was applied to midshaft at a displacement speed of 0.01 mm/s until femoral shaft fractured, with load leading to rupture recorded. The proximal part of femur left was collected for femoral neck-fracture test, with its femoral shaft embedded in jig and fixed by methacrylate. Load with the same protocol of threepoint bending test was applied to top of femoral head parallel to long axis of femora shaft until rupture occurred. The forceedisplacement curve was generated, based on which, stiffness, maximal breaking load and energy absorption were obtained.
Cross-linked C-terminal telopeptides of type I collagen (CTX-1, an indicator for osteogenic activity) and amino-terminal propeptide of type 1 procollagen (P1NP, an indicator for osteoclastic activity) in serum, and deoxypyridinoline (Dpd, an indicator for osteoclastic activity) in urine were determined using corresponding ELISA Kits (BlueGene Biotech, Shanghai, China) respectively based on the manufacturer's protocols. And urinary Dpd levels were corrected by urinary creatinine (Cr) levels. 2.6. Quantitative real-time PCR (QRT-PCR) Tibial diaphyses were cut up and flushed with PBS for removing bone marrow. Total RNA was extracted from harvested diaphyses using Trizol reagent (Invitrogen, USA), which was reversetranscribed into cDNA with ReverTra Ace qPCR RT Kit (Toyobo Co. Ltd., Osaka, Japan). Primer sequences of target genes were as following: Sost (gene of sclerostin): F 50 AGC CTT CGT TGC TGT GGA GA 30 and R 50 TGG TGT CAT AAG GAT GGT GG 3'; Postn (gene of periostin): F 50 AGG GTC CTA CAC ATA CTT CG 30 and R 50 GGT CCT TGG TTA GCA TTC TC 30 ; b-catenin: F 50 CAA ACT GCT AAA TGA CGA GG 30 and R 50 GGG AAA GGT TGT GTA GGG TC 30 ; b-Actin: F 50 AGA TCC TGA CCG AGC GTG GC 30 and R 50 CCA GGG AGG AAG AGG ATG CG 30 . The QRT-PCR analysis was performed using SYBR® Green Realtime PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan). Reaction conditions were 94 C for 30 s, followed by 95 C for 5 s and 60 C for 30 s for 40 cycles. The relative changes of transcripts of interest were analyzed according to the 2DDCt method with b-Actin being housekeeper gene [20]. 2.7. Western-blot Tibial diaphyses were grinded up within liquid nitrogen and lysates were prepared within RIPA buffer containing PMSF to extract total protein. Nuclear protein was extracted using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). The isolated protein concentration was determined using BCA Quantitation Kit (Boster, Wuhan, China). 100 mg of protein was separated on SDS-PAGE, electrotransferred to nitrocellulose membranes. Following blocking within 5% skim milk, membranes were incubated with primary antibodies against b-catenin (1:200; Boster, Wuhan, China), Periostin (1:1000; Santa Cruz Biotechnology, CA, USA), Sclerostin (1:1000; Abcam, Cambridge, UK) respectively overnight at 4 C, followed by horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz, CA, USA) for 2 h. Blots were developed using enhanced chemiluminescence (Santa Cruz Biotechnology, CA, USA). 2.8. Immunohistochemistry Following fixed and decalcified, femurs were cut into 10-mmthick sections at midshaft level. Sections were incubated at 60 C for 1 h, incubated in xylene to deparaffinize and subsequently within ethanol to rehydrate. Endogeneous peroxidase quenching was accomplished by pretreating within 3% H2O2. Slices were then incubated in boiling citric acid buffer (10 mM) for antigen retrieval.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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Following pre-treatment in 5% BSA, slices were incubated with primary antibodies against sclerostin (rabbit anti-rat, 1:25; Abcam, Cambridge, UK) at 4 C overnight. Sections were incubated in biotin-coupled goat anti-rabbit secondary antibody, followed by SABC (streptavidinebiotin complex) according to manufacturer's protocols (Boster, Wuhan, China). Detection was performed using 3,3-diaminobenzidine tetrahydrochloride kit (Boster, Wuhan, China), with nuclear counterstaining using hematoxylin. Sclerostin-positive staining was observed by Nikon NiE microscope (Nikon Optical, Tokyo, Japan).
fold (p < 0.001) and 4.81-fold (p < 0.001) respectively compared with the sham-operated rats. In naringin-treating groups, Postn mRNA level raised by 5.14-fold in 300 mg/kg naringin group (p < 0.001) compared to the USN group, whereas Postn expression was not dramatically affected by naringin at 30 or 100 mg/kg. And b-catenin expression increased by 3.59-fold and 5.62-fold respectively in the 100 and 300 mg/kg naringin group compared with the USN group (p < 0.01 and p < 0.001 respectively). However, gene expression of Sost among all groups showed no significant differences.
2.9. Statistical analysis
3.2. Effects of naringin on protein expression of sclerostin and periostin and nucleus b-catenin in each group
We conducted one-way ANOVA to evaluate the significant differences among all groups, followed by Bonferroni multiple comparison test as a post hoc test using GraphPad PrismV6.01 Software (San Diego, CA, USA), with P < 0.05 being as significant difference, which was only indicated when it was considered to be significant. Data are given as mean ± SD (standard deviation). 3. Results 3.1. Neurectomy-induced decreasing of Postn and b-catenin mRNA was attenuated by naringin As illustrated in Fig. 1A and B, following four weeks of neurectomy, Postn and b-catenin mRNA expression decreased by 4.65-
Consistent with change of Postn mRNA expression, periostin protein level was much lower in sciatic-neurectomized rats than the sham-operated rats (p < 0.001), which was dramatically upregulated by naringin at 300 mg/kg (p < 0.001), while administration with naringin at 30 or 100 mg/kg has no significant effect on the decreasing periostin level in neurectomized tibias (both p > 0.05, Fig. 2A and D). Different from change of Sost mRNA level, sclerostin protein expression significantly increased in response to neurectomy compared to Sham group (p < 0.001), which was dramatically decreased by prophylactic application of 300 mg/kg naringin (p < 0.001). However, sclerostin level in 300 mg/kg naringin group was still higher than the Sham group (p < 0.05, Fig. 2B and E).
Fig. 1. Effects of naringin on Sost, Postn and b-catenin mRNA expression in each group analyzed by reverse transcriptase PCR RT-PCR (A) and QRT-PCR (B) respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. USN group. One-way ANOVA, n ¼ 6. Sost: gene of sclerostin; Postn: gene of periostin.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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Fig. 2. Western-blot analysis of periostin, sclerostin and nucleus b-catenin protein in each group (AeC) and the corresponding quantitation histogram (DeF). *p < 0.05, **p < 0.01, *** p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. USN group. One-way ANOVA, n ¼ 6.
Sciatic neurectomy for four weeks significantly down-regulated nucleus b-catenin level compared to that of Sham group (p < 0.01). The b-catenin concentration increased markedly due to administration with naringin at 100 and 300 mg/kg compared to sciaticneurectomized rats (both p < 0.001). And nucleus b-catenin protein level in 300 mg/kg naringin group was even higher than the sham-operated rats (p < 0.01, Fig. 2C and F). 3.3. Effects of naringin on sclerostin protein expression in osteocytes As sclerostin was primarily secreted by osteocytes, we next analyzed sclerostin expression in osteocytes in femoral diaphysis by immunohistochemical staining. Since naringin at 300 mg/kg had a stronger effect on inhibiting sclerostin protein expression than the other two dosages as indicated by western-blot in denervated tibias, we only analyzed effects of 300 mg/kg naringin on sclerostin protein expression in osteocytes. Sciatic neurectomy for four weeks resulted in a stronger intensity of sclerostin staining in osteocytes compared to sham-operated rats, while naringin-treating rats at 300 mg/kg displayed a lower staining intensity than USN group (Fig. 3). However, the staining intensity in naringin-treating group was still higher than the Sham group.
3.4. Effects of naringin on trabecular microstructure Two-dimensional and three-dimensional reconstructed images from micro-CT scanning showed microarchitecture of trabecular bone in distal femoral metaphysis of all rats (Fig. 4). Significant bone loss was observed in the femoral trabecular bone of the USN group compared with the Sham group, while there were denser trabecular bones in higher dosages of naringin-treated rats compared to the USN group. As shown in Table 1, in the USN group, BV/TV ratios, Tb.Th and Tb.N significantly decreased compared with sham-operated rats, while BS/BV and Tb.Sp markedly increased. In the naringin-treating groups, bone quality of trabecular gradually improved, with significantly beneficial effect in 100 and 300 mg/kg naringin group, which showed dramatically increased BV/TV, Tb.Th and Tb.N, and decreased BS/BV and Tb.Sp compared with USN group. 3.5. Effects of naringin on bone turnover markers The level of serum CTX-1 and urinary Dpd, markers of bone resorption, were both dramatically increased in the USN group compared to sham-operated rats, while serum P1NP concentration, a marker of bone formation, was markedly decreased in USN rats,
Fig. 3. Sclerostin-positive osteocytes distributed in femoral diaphysis in each group indicated by immunohistochemical staining. Upper panel magnification 200, Lower panel magnification 400.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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Fig. 4. Representative there-dimensional reconstructed images of trabecular bone located in distal femoral metaphyseal for each group.
Table 1 Analysis of trabecular microarchitecture in distal femur. USN BV/TV(%) Tb.Th(mm) Tb.N(1/mm) Tb.Sp(mm) BS/BV(%)
30 mg/kg NG
17.33 82.02 2.022 180.80 33.07
± ± ± ± ±
2.10c 11.95c 0.165c 12.21c 3.41c
19.44 93.67 2.087 171.50 31.06
± ± ± ± ±
1.74c 11.31c 0.218c 10.04c 1.97c
100 mg/kg NG
300 mg/kg NG
48.48 ± 2.62f 120.20 ± 13.60f 3.665 ± 0.192b,f 122.60 ± 13.96b,f 21.15 ± 2.73a,f
50.73 137.10 4.179 104.80 18.29
± ± ± ± ±
3.45f 9.89f 0.291f 12.34f 2.08f
Sham 53.37 134.30 4.257 93.24 15.90
± ± ± ± ±
4.25 17.55 0.289 12.22 1.63
a p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group. BS/TV, Tb.N, Tb.Th, BS/BV and Tb.Sp indicate bone volume/tissue volume, trabecula number, trabecula thickness, bone surface/bone volume and trabecular separation, respectively.
i.e., USN group showed increased serum CTX-1 of 84.3% (p < 0.001) and urinary Dpd of 39.0% (p < 0.001), with decreased serum P1NP of 33.8% (p < 0.001). However, USN-induced increasing of serum CTX-I and urinary Dpd were dramatically decreased by naringin treatment at higher dosages (100 and 300 mg/kg), while serum P1NP was markedly increased by higher dosages of naringin, the effect of which was most obvious in 300 mg/kg naringin-treating group, i.e., serum CTX-1 decreased by 46.5% (p < 0.001) and urinary Dpd 33% (p < 0.001), and serum P1NP increased by 64.6% (p < 0.001, Table 2).
mechanical properties of distal femoral metaphysis were markedly deteriorated by sciatic neurectomy as indicated by decreasing in maximum energy absorption, stiffness and maximum load compared to the Sham group (all p < 0.001), while all of which were improved by naringin treatment at higher dosages (100 and 300 mg/kg; all p < 0.001 vs. USN group, Table 3). BMD of the denervated distal femur was much lower in USN rats as compared with the Sham group (p < 0.001), while naringin at higher dosages (100 and 300 mg/kg) dramatically suppressed USN-induced BMD decreasing dose dependently (p < 0.01 and 0.001 respectively, Table 3).
3.6. Effects of naringin on femoral biomechanical properties and BMD in distal femur
4. Discussion
No significant differences of the maximal fracture load to femoral diaphysis were found among all groups. However,
Naringin is effective in protection against bone loss caused by ovariectomy [7], Glucocorticoid, retinoic acid [3] and senescence
Table 2 Analysis of bone turnover markers in serum and urine.
Serum P1NP (pg/ml) Serum CTX-1 (ng/ml) Urinary Dpd (nmog/mmol Cr) a
USN
30 mg/kg NG
100 mg/kg NG
300 mg/kg NG
Sham
434.8 ± 41.1c
451.7 ± 47.7c
649.5 ± 56.4f
715.7 ± 42.3f
656.8 ± 42.7
3.87 ± 0.29c
3.91 ± 0.27c
2.49 ± 0.26f
2.07 ± 0.12f
2.10 ± 0.22
63.05 ± 4.54c
61.29 ± 2.95c
42.29 ± 2.91f
42.37 ± 4.64f
45.36 ± 3.00
p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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Table 3 Analysis of BMD in distal femur and femoral biomechanical properties. USN BMD (mg/cm2) Maximum load (FD, N) Maximum load (FN, N) Energy absorption (FN, N) Stiffness (FN, N) a
173.8 123.6 49.71 8.28 395.3
30 mg/kg NG ± ± ± ± ±
7.3c 10.6 4.19c 1.76c 29.17c
174.1 124.6 53.08 8.57 399.0
± ± ± ± ±
12.4c 11.9 5.88c 1.19c 26.15c
100 mg/kg NG 196.4 131.5 97.43 16.17 646.5
± ± ± ± ±
8.4e 11.0 9.77f 1.70f 42.97f
300 mg/kg NG
Sham
9.9f 13.9 9.97f 1.88f 51.24f
203.3 139.6 107.7 16.68 672.6
201.6 138.9 104.6 16.28 668.9
± ± ± ± ±
± ± ± ± ±
12.8 10.9 9.51 1.35 61.75
p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group. FD ¼ Femoral diaphysis, FN ¼ Femoral neck.
[6], however, the molecular mechanisms medicating this osteoprotective effect are far from clear. Thereby the present study was designed to investigate whether periostinesclerostineWnt/b-catenin signaling pathway is involved in effect of naringin on prevention of bone loss induced by immobilization through sciaticneurectomy. Our study showed that naringin exerted a great beneficial role in abrogating disuse-induced bone loss and for the first time we demonstrated that up-regulation of periostin and decreasing of sclerostin by naingin were correlated with the osteoprotective role of naringin through activating Wnt signaling pathway. Sciatic-neurectomy for four weeks caused significant detriment of bone quality as indicated by deterioration of BMD and trabecular microarchitecture in distal femur, as well as bone strength of femoral neck, which was accompanied by a dramatical increment of bone resorption and simultaneously decreased activity of bone formation, as evidenced by bone turnover markers, while treatment with naringin at higher dosages displayed a beneficial effect in preservation of bone quality, together with increasing of osteogenic activity and down-regulation of osteoclastic activity. Wnt proteins by binding with a receptor complex (Frizzled and LRP 5/6) lead to inactivation of cytosolic GSK-3b/Axin2/APC complex, which otherwise leads to phosphorylation and then degradation of b-catenin [21]. Thus b-catenin could accumulate in cytoplasm and then translocate into nucleus, which thereby promotes expression of specific genes, in bone ultimately resulting in differentiation of osteoblast and reduced osteoclastogenesis [12,13]. The results of Western-blot in our study showed that protein levels of nucleus b-catenin in tibias ipsilateral to neurectomy decreased in the USN group compared with the sham-operated rats, which were much higher in higher dosages of naringintreating group. Previous studies reported that mutant mice with increased activation of Wnt/b-catenin signaling showed enhanced bone quality as a result of enhanced sensitivity to mechanical stress compared to wild-type littermates [22], while the increased bone formation due to mechanical stimulation was almost vanished in mutant mice with decreased activation of Wnt/b-catenin signaling [23]. And the activity of Wnt/b-catenin signaling was decreased by mechanical unloading through tail suspension [14]. Additionally, the stimulation of Wnt/b-catenin signaling by naringin was reported to be correlated with improved bone quality by naringin in ovariectomized mice [18]. Thus we propose that inactivation of Wnt/b-catenin signaling pathway contributes to bone loss induced by immobilization through sciatic-neurectomy, and stimulation of Wnt/b-catenin signaling is involved in the beneficial role of naringin in protection of bone quality against disuse. Sclerostin is a predominant inhibitor of canonical Wnt/b-catenin signaling and an antagonistic ligand competing for coreceptors LRP5/6 binding, which is almost exclusively secreted by mature osteocytes [24,25]. Furthermore, increased sclerostin contributed mainly to unloading-induced bone loss [26] and mice with Sost knockout were resistance to unloading-induced bone loss [14]. Thus we wondered whether the change of Wnt signaling activities in our studies was medicated by sclerostin. The results of PCR showed that there were no significant differences of Sost
mRNA levels among all groups, however, as indicated by immunohistochemical staining, the percentage of sclerostin-positive osteocytes in USN group was much higher than the Sham group with stronger staining intensities, which was markedly decreased by the highest dosage of naringin, however. And this result was confirmed by that of western-blot in denervated tibias. These inconsistent results could be explained by the phenomenon that change of protein level is not always linear to that of its corresponding mRNA, which may be due to post-transcriptional and post-translation regulation [27]. And as differential expression of cellular protein determined cellular functional status, the regulation of gene Sost expression by naringin may mainly in the process of post-transcriptional modification. Thereby USN-induced downregulation of Wnt signaling may be mediated by increased sclerostin levels, and administration with the highest dosage of naringin is correlated with the decreasing sclerostin level. However, naringin is still not capable to decrease sclerostin expression to the level of the Sham group. Further studies reported that mechanical unloading-induced up-regulation of Sost was mediated by periostin, a matricellular protein mainly produced by periosteal osteoblasts and osteocytes [28], the expression of which inhibited sclerostin expression and was decreased during tail suspension-induced unloading [16]. And periostin could also trigger Wnt/b-catenin signaling pathway directly [29]. We showed here that the periostin protein level was reduced significantly by sciatic-neurectomy, which was restored to a level higher than that of the Sham group by treatment with naringin at 300 mg/kg. Thereby we propose that decreasing of periostin is also involved in unloading-induced bone loss by sciaticneurectomy and naringin displays an ability to increase periostin expression, which may be implicated in its role in protecting bone integrity against disuse. Besides playing an essential role in mechanotransduction, periostin also mediated the bone anabolic effects of PTH through down-regulation of sclerostin and subsequent stimulation of Wnt signaling [29]. Thereby, periostinesclerostineWnt signaling axis exerts an essential role in regulating bone homeostasis, and through which naringin shows an osteoprotective role in preventing disuse-induced bone loss. Further studies using mice lacking gene Postn or Sost needed to be conducted to verify the above effects of naringin. In conclusion, our studies demonstrated for the first time that periostin-induced down-regulation of sclerostin and subsequent activation of Wnt/b-catenin signaling may be implicated in preventive effect of naringin against sciatic-neurectomy induced bone loss, which makes us better understand the mechanisms mediated the osteoprotective role of naringin and confirms that naringin could be a promising alternative medicine in treatment of disuse osteoporosis.
Acknowledgments This study was funded by the National Natural Science Foundation of China (No. 81501061) and the Traditional Chinese Medicine Administration of Tianjin, China (No. 13123).
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin Jianwei Lv a, b, Xiaolei Sun a, **, Jianxiong Ma a, Xinlong Ma a, b, *, Guosheng Xing a, Ying Wang a, Lei Sun a, Jianbao Wang a, Fengbo Li a, Yanjun Li a a b
Institute of Orthopedics, Tianjin Hospital, No. 122, Munan Road, Heping District, Tianjin TJ 300050, China Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin TJ 300070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2015 Accepted 28 October 2015 Available online xxx
Periostin has an essential role in mechanotransduction in bone. Naringin, a natural flavonoid, has been evidenced for its osteoprotective role in osteoporosis, while its mechanism is far from clear. Here we show that down-regulation of periostin, and up-regulation of its downstream sclerostin and inactivation of Wnt/b-catenin signaling were implicated in neurectomy-induced bone loss. Naringin could upregulate periostin and prevent neurectomy-induced deterioration of BMD, trabecular microstructure and bone mechanical characteristics. In conclusion, naringin could prevent progress of disuse osteoporosis in rats, which may be mediated by increased periostin expression and subsequently inhibition of sclerostin and activation of Wnt/b-catenin signaling pathways. © 2015 Elsevier Inc. All rights reserved.
Keywords: Disuse osteoporosis Naringin Periostin Sclerostin b-catenin
1. Introduction Naringin, the main bioactive flavonoid extracted from citrus fruits [1], has a beneficial role in protecting against osteoporosis caused by post-menopause, glucocorticoid, retinoic acid, orchidectomy and aging [2e6]. In vitro, naringin has been shown to be effective to enhance osteoblast proliferation and differentiation, with increasing expression of osteogenic markers such as osteocalcin (OC), osteopontin (OPN), osteoprotegerin (OPG) and bone morphogenetic protein-2 (BMP) mediated by PI3K-Akt and activator protein-1 (AP-1) signal pathway [3,7,8]. Meantime, naringin could abrogate osteoclastogenesis and osteoclastic differentiation through impeding activation of NF-kB and ERK signaling cascades induced by RANKL, as well as expression of osteoclast gene markers [9]. Although naringin has been exhibited to possess estrogen-like protective effect against postmenopausal osteoporosis [10] and antioxidation in orchidectomized rats [5], the molecular mechanisms implicated in bone anabolic effect of naringin in different
* Corresponding author. Institute of Orthopedics, Tianjin Hospital, No. 122, Munan Road, Heping District, Tianjin TJ 300050, China.. ** Corresponding author. E-mail address: [email protected] (X. Ma).
osteoporotic models are far from clear. The canonical Wnt/b-catenin signaling pathway exerts a pronounced beneficial role in bony homeostasis [11], with critically positive regulation in osteoblastic differentiation [12], and normal activation of which in osteocyte is critical to limit excessive bone resorption of osteoclasts [13]. The antagonist of Wnt/b-catenin pathwayddsclerostin, a secreted protein mainly derived from osteocytes, exerts an essential role in the progress of disuse osteoporosis induced by mechanical unloading by specially suppressing activation of Wnt/b-catenin cascades [14]. Sclerostin is increased by unloading [14] while decreases with mechanical stimulation [15], and mice with gene of which deficiency are resistant to unloadinginduced bone loss [14]. Further studies identify the selectively upstream regulator of sclerostin in response to mechanotransductionddperiostin, is a matricellular protein preferentially expressed by periosteal osteoblasts and osteocytes, level of which displays a reciprocal correlation with that of sclerostin, either with unloading or by mechanical stimuli [16,17]. Mice with gene deficient of periostin show a diminished response to mechanical unloading, accompanied by increased sclerostin [16]. Recently Wnt/b-catenin signaling was found to be involved in the positive effect of Naringin on bone maintenance as naringin promoted nuclear translocation of b-catenin in osteoblast-like UMR-106 cell [18]. While whether naringin displays beneficial
http://dx.doi.org/10.1016/j.bbrc.2015.10.152 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
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effects on skeletal properties in disuse osteoporosis through regulation of periostinesclerostineWnt/b-catenin pathway has not been reported. We here evidenced for the first time that naringin could prevent deterioration of skeletal properties induced by immobilization through neurectomy mediated by up-regulation of periostin, inhibition of sclerostin as well as increased nucleus accumulation of bcatenin.
2.4. Bone mineral density(BMD)measurements On the day before sacrifice, BMD in distal femur in vivo was measured by dual energy X-ray absorptiometry (DXA, LunarProdigy; GE). Animals were anesthetized using chloral hydrate (400 mg/kg) by intraperitoneal injection and supine on specimen platform with hind limbs flexed and extorsion. 2.5. Biochemical markers measurements
2. Materials and methods 2.1. Animals All experimental protocols were approved by the Ethics Committee of Tianjin Hospital. Sixty 6-month-old male SpragueeDawley rats (255 ± 10 g) were housed under a 12 h light/dark cycle at 24 ± 0.5 C, with free access to food and water, body weights of which were measured once a week. Rats were allocated into five groups randomly, four of which was unilateral sciatic nerve neurectomized (USN) and one sham-operated. In sciaticneurectomized groups, the right sciatic nerve of each rat was severed, with 1 cm of which excised as previously described [19]. The sham-operated rats only received sciatic nerve exposed with skin and subcutaneous tissues sutured layer by layer then. The four sciatic-neurectomized groups were administered with 30, 100 and 300 mg/kg body weight naringin (NG) or saline by gavage respectively four weeks before and four weeks after neurectomy. The sham-operated group received saline vehicle. Four weeks postoperation, all rats were sacrificed. On the day before sacrifice, urine of overnight fasted rat was collected, while blood was harvested from heart by cardiac puncture on the day of sacrifice and then centrifuged to get serum samples, both of which were frozen at 80 C until analyses of bone turnover markers. Femurs and tibias ipsilateral to neurectomy with soft tissues removed were prepared appropriately for further analysis. 2.2. Micro-computed tomography (Micro-CT) Femurs for Micro-CT analysis were put in 70% ethanol. Distal femoral cancellous bone was scanned by SIEMENS Inveon PET.SPECT.CT (SIEMENS, Berlin, Germany) with energy settings of 500 mA and 80 kV, micro-structure of which was evaluated by bone volume/tissue volume (BS/TV), trabecula number (Tb.N), trabecula thickness (Tb.Th), bone surface/bone volume (BS/BV) and trabecular separation (Tb.Sp). Three-Dimension images were reconstructed by software COBRA Exxim: Licensed to Siemens. 2.3. Biomechanical measurement Femurs subjected to biomechanical testing were preserved within 0.9% saline, biomechanical properties of which were evaluated by three-point bending test and femoral neck-fracture test using Electro-Force 3230 Bose System (BOSE, Minnetonka, MN, USA). In three-point bending tests, femoral shaft were placed two lower bases with a separation of 20 mm. Following a preload of 2 N, a vertical load was applied to midshaft at a displacement speed of 0.01 mm/s until femoral shaft fractured, with load leading to rupture recorded. The proximal part of femur left was collected for femoral neck-fracture test, with its femoral shaft embedded in jig and fixed by methacrylate. Load with the same protocol of threepoint bending test was applied to top of femoral head parallel to long axis of femora shaft until rupture occurred. The forceedisplacement curve was generated, based on which, stiffness, maximal breaking load and energy absorption were obtained.
Cross-linked C-terminal telopeptides of type I collagen (CTX-1, an indicator for osteogenic activity) and amino-terminal propeptide of type 1 procollagen (P1NP, an indicator for osteoclastic activity) in serum, and deoxypyridinoline (Dpd, an indicator for osteoclastic activity) in urine were determined using corresponding ELISA Kits (BlueGene Biotech, Shanghai, China) respectively based on the manufacturer's protocols. And urinary Dpd levels were corrected by urinary creatinine (Cr) levels. 2.6. Quantitative real-time PCR (QRT-PCR) Tibial diaphyses were cut up and flushed with PBS for removing bone marrow. Total RNA was extracted from harvested diaphyses using Trizol reagent (Invitrogen, USA), which was reversetranscribed into cDNA with ReverTra Ace qPCR RT Kit (Toyobo Co. Ltd., Osaka, Japan). Primer sequences of target genes were as following: Sost (gene of sclerostin): F 50 AGC CTT CGT TGC TGT GGA GA 30 and R 50 TGG TGT CAT AAG GAT GGT GG 3'; Postn (gene of periostin): F 50 AGG GTC CTA CAC ATA CTT CG 30 and R 50 GGT CCT TGG TTA GCA TTC TC 30 ; b-catenin: F 50 CAA ACT GCT AAA TGA CGA GG 30 and R 50 GGG AAA GGT TGT GTA GGG TC 30 ; b-Actin: F 50 AGA TCC TGA CCG AGC GTG GC 30 and R 50 CCA GGG AGG AAG AGG ATG CG 30 . The QRT-PCR analysis was performed using SYBR® Green Realtime PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan). Reaction conditions were 94 C for 30 s, followed by 95 C for 5 s and 60 C for 30 s for 40 cycles. The relative changes of transcripts of interest were analyzed according to the 2DDCt method with b-Actin being housekeeper gene [20]. 2.7. Western-blot Tibial diaphyses were grinded up within liquid nitrogen and lysates were prepared within RIPA buffer containing PMSF to extract total protein. Nuclear protein was extracted using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). The isolated protein concentration was determined using BCA Quantitation Kit (Boster, Wuhan, China). 100 mg of protein was separated on SDS-PAGE, electrotransferred to nitrocellulose membranes. Following blocking within 5% skim milk, membranes were incubated with primary antibodies against b-catenin (1:200; Boster, Wuhan, China), Periostin (1:1000; Santa Cruz Biotechnology, CA, USA), Sclerostin (1:1000; Abcam, Cambridge, UK) respectively overnight at 4 C, followed by horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz, CA, USA) for 2 h. Blots were developed using enhanced chemiluminescence (Santa Cruz Biotechnology, CA, USA). 2.8. Immunohistochemistry Following fixed and decalcified, femurs were cut into 10-mmthick sections at midshaft level. Sections were incubated at 60 C for 1 h, incubated in xylene to deparaffinize and subsequently within ethanol to rehydrate. Endogeneous peroxidase quenching was accomplished by pretreating within 3% H2O2. Slices were then incubated in boiling citric acid buffer (10 mM) for antigen retrieval.
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Following pre-treatment in 5% BSA, slices were incubated with primary antibodies against sclerostin (rabbit anti-rat, 1:25; Abcam, Cambridge, UK) at 4 C overnight. Sections were incubated in biotin-coupled goat anti-rabbit secondary antibody, followed by SABC (streptavidinebiotin complex) according to manufacturer's protocols (Boster, Wuhan, China). Detection was performed using 3,3-diaminobenzidine tetrahydrochloride kit (Boster, Wuhan, China), with nuclear counterstaining using hematoxylin. Sclerostin-positive staining was observed by Nikon NiE microscope (Nikon Optical, Tokyo, Japan).
fold (p < 0.001) and 4.81-fold (p < 0.001) respectively compared with the sham-operated rats. In naringin-treating groups, Postn mRNA level raised by 5.14-fold in 300 mg/kg naringin group (p < 0.001) compared to the USN group, whereas Postn expression was not dramatically affected by naringin at 30 or 100 mg/kg. And b-catenin expression increased by 3.59-fold and 5.62-fold respectively in the 100 and 300 mg/kg naringin group compared with the USN group (p < 0.01 and p < 0.001 respectively). However, gene expression of Sost among all groups showed no significant differences.
2.9. Statistical analysis
3.2. Effects of naringin on protein expression of sclerostin and periostin and nucleus b-catenin in each group
We conducted one-way ANOVA to evaluate the significant differences among all groups, followed by Bonferroni multiple comparison test as a post hoc test using GraphPad PrismV6.01 Software (San Diego, CA, USA), with P < 0.05 being as significant difference, which was only indicated when it was considered to be significant. Data are given as mean ± SD (standard deviation). 3. Results 3.1. Neurectomy-induced decreasing of Postn and b-catenin mRNA was attenuated by naringin As illustrated in Fig. 1A and B, following four weeks of neurectomy, Postn and b-catenin mRNA expression decreased by 4.65-
Consistent with change of Postn mRNA expression, periostin protein level was much lower in sciatic-neurectomized rats than the sham-operated rats (p < 0.001), which was dramatically upregulated by naringin at 300 mg/kg (p < 0.001), while administration with naringin at 30 or 100 mg/kg has no significant effect on the decreasing periostin level in neurectomized tibias (both p > 0.05, Fig. 2A and D). Different from change of Sost mRNA level, sclerostin protein expression significantly increased in response to neurectomy compared to Sham group (p < 0.001), which was dramatically decreased by prophylactic application of 300 mg/kg naringin (p < 0.001). However, sclerostin level in 300 mg/kg naringin group was still higher than the Sham group (p < 0.05, Fig. 2B and E).
Fig. 1. Effects of naringin on Sost, Postn and b-catenin mRNA expression in each group analyzed by reverse transcriptase PCR RT-PCR (A) and QRT-PCR (B) respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. USN group. One-way ANOVA, n ¼ 6. Sost: gene of sclerostin; Postn: gene of periostin.
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Fig. 2. Western-blot analysis of periostin, sclerostin and nucleus b-catenin protein in each group (AeC) and the corresponding quantitation histogram (DeF). *p < 0.05, **p < 0.01, *** p < 0.001 vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. USN group. One-way ANOVA, n ¼ 6.
Sciatic neurectomy for four weeks significantly down-regulated nucleus b-catenin level compared to that of Sham group (p < 0.01). The b-catenin concentration increased markedly due to administration with naringin at 100 and 300 mg/kg compared to sciaticneurectomized rats (both p < 0.001). And nucleus b-catenin protein level in 300 mg/kg naringin group was even higher than the sham-operated rats (p < 0.01, Fig. 2C and F). 3.3. Effects of naringin on sclerostin protein expression in osteocytes As sclerostin was primarily secreted by osteocytes, we next analyzed sclerostin expression in osteocytes in femoral diaphysis by immunohistochemical staining. Since naringin at 300 mg/kg had a stronger effect on inhibiting sclerostin protein expression than the other two dosages as indicated by western-blot in denervated tibias, we only analyzed effects of 300 mg/kg naringin on sclerostin protein expression in osteocytes. Sciatic neurectomy for four weeks resulted in a stronger intensity of sclerostin staining in osteocytes compared to sham-operated rats, while naringin-treating rats at 300 mg/kg displayed a lower staining intensity than USN group (Fig. 3). However, the staining intensity in naringin-treating group was still higher than the Sham group.
3.4. Effects of naringin on trabecular microstructure Two-dimensional and three-dimensional reconstructed images from micro-CT scanning showed microarchitecture of trabecular bone in distal femoral metaphysis of all rats (Fig. 4). Significant bone loss was observed in the femoral trabecular bone of the USN group compared with the Sham group, while there were denser trabecular bones in higher dosages of naringin-treated rats compared to the USN group. As shown in Table 1, in the USN group, BV/TV ratios, Tb.Th and Tb.N significantly decreased compared with sham-operated rats, while BS/BV and Tb.Sp markedly increased. In the naringin-treating groups, bone quality of trabecular gradually improved, with significantly beneficial effect in 100 and 300 mg/kg naringin group, which showed dramatically increased BV/TV, Tb.Th and Tb.N, and decreased BS/BV and Tb.Sp compared with USN group. 3.5. Effects of naringin on bone turnover markers The level of serum CTX-1 and urinary Dpd, markers of bone resorption, were both dramatically increased in the USN group compared to sham-operated rats, while serum P1NP concentration, a marker of bone formation, was markedly decreased in USN rats,
Fig. 3. Sclerostin-positive osteocytes distributed in femoral diaphysis in each group indicated by immunohistochemical staining. Upper panel magnification 200, Lower panel magnification 400.
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Fig. 4. Representative there-dimensional reconstructed images of trabecular bone located in distal femoral metaphyseal for each group.
Table 1 Analysis of trabecular microarchitecture in distal femur. USN BV/TV(%) Tb.Th(mm) Tb.N(1/mm) Tb.Sp(mm) BS/BV(%)
30 mg/kg NG
17.33 82.02 2.022 180.80 33.07
± ± ± ± ±
2.10c 11.95c 0.165c 12.21c 3.41c
19.44 93.67 2.087 171.50 31.06
± ± ± ± ±
1.74c 11.31c 0.218c 10.04c 1.97c
100 mg/kg NG
300 mg/kg NG
48.48 ± 2.62f 120.20 ± 13.60f 3.665 ± 0.192b,f 122.60 ± 13.96b,f 21.15 ± 2.73a,f
50.73 137.10 4.179 104.80 18.29
± ± ± ± ±
3.45f 9.89f 0.291f 12.34f 2.08f
Sham 53.37 134.30 4.257 93.24 15.90
± ± ± ± ±
4.25 17.55 0.289 12.22 1.63
a p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group. BS/TV, Tb.N, Tb.Th, BS/BV and Tb.Sp indicate bone volume/tissue volume, trabecula number, trabecula thickness, bone surface/bone volume and trabecular separation, respectively.
i.e., USN group showed increased serum CTX-1 of 84.3% (p < 0.001) and urinary Dpd of 39.0% (p < 0.001), with decreased serum P1NP of 33.8% (p < 0.001). However, USN-induced increasing of serum CTX-I and urinary Dpd were dramatically decreased by naringin treatment at higher dosages (100 and 300 mg/kg), while serum P1NP was markedly increased by higher dosages of naringin, the effect of which was most obvious in 300 mg/kg naringin-treating group, i.e., serum CTX-1 decreased by 46.5% (p < 0.001) and urinary Dpd 33% (p < 0.001), and serum P1NP increased by 64.6% (p < 0.001, Table 2).
mechanical properties of distal femoral metaphysis were markedly deteriorated by sciatic neurectomy as indicated by decreasing in maximum energy absorption, stiffness and maximum load compared to the Sham group (all p < 0.001), while all of which were improved by naringin treatment at higher dosages (100 and 300 mg/kg; all p < 0.001 vs. USN group, Table 3). BMD of the denervated distal femur was much lower in USN rats as compared with the Sham group (p < 0.001), while naringin at higher dosages (100 and 300 mg/kg) dramatically suppressed USN-induced BMD decreasing dose dependently (p < 0.01 and 0.001 respectively, Table 3).
3.6. Effects of naringin on femoral biomechanical properties and BMD in distal femur
4. Discussion
No significant differences of the maximal fracture load to femoral diaphysis were found among all groups. However,
Naringin is effective in protection against bone loss caused by ovariectomy [7], Glucocorticoid, retinoic acid [3] and senescence
Table 2 Analysis of bone turnover markers in serum and urine.
Serum P1NP (pg/ml) Serum CTX-1 (ng/ml) Urinary Dpd (nmog/mmol Cr) a
USN
30 mg/kg NG
100 mg/kg NG
300 mg/kg NG
Sham
434.8 ± 41.1c
451.7 ± 47.7c
649.5 ± 56.4f
715.7 ± 42.3f
656.8 ± 42.7
3.87 ± 0.29c
3.91 ± 0.27c
2.49 ± 0.26f
2.07 ± 0.12f
2.10 ± 0.22
63.05 ± 4.54c
61.29 ± 2.95c
42.29 ± 2.91f
42.37 ± 4.64f
45.36 ± 3.00
p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group.
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Table 3 Analysis of BMD in distal femur and femoral biomechanical properties. USN BMD (mg/cm2) Maximum load (FD, N) Maximum load (FN, N) Energy absorption (FN, N) Stiffness (FN, N) a
173.8 123.6 49.71 8.28 395.3
30 mg/kg NG ± ± ± ± ±
7.3c 10.6 4.19c 1.76c 29.17c
174.1 124.6 53.08 8.57 399.0
± ± ± ± ±
12.4c 11.9 5.88c 1.19c 26.15c
100 mg/kg NG 196.4 131.5 97.43 16.17 646.5
± ± ± ± ±
8.4e 11.0 9.77f 1.70f 42.97f
300 mg/kg NG
Sham
9.9f 13.9 9.97f 1.88f 51.24f
203.3 139.6 107.7 16.68 672.6
201.6 138.9 104.6 16.28 668.9
± ± ± ± ±
± ± ± ± ±
12.8 10.9 9.51 1.35 61.75
p < 0.05, bp < 0.01, cp < 0.001 vs. Sham group; dp < 0.05, ep < 0.01, fp < 0.001 vs. USN group. One-way ANOVA, n ¼ 6 per group. FD ¼ Femoral diaphysis, FN ¼ Femoral neck.
[6], however, the molecular mechanisms medicating this osteoprotective effect are far from clear. Thereby the present study was designed to investigate whether periostinesclerostineWnt/b-catenin signaling pathway is involved in effect of naringin on prevention of bone loss induced by immobilization through sciaticneurectomy. Our study showed that naringin exerted a great beneficial role in abrogating disuse-induced bone loss and for the first time we demonstrated that up-regulation of periostin and decreasing of sclerostin by naingin were correlated with the osteoprotective role of naringin through activating Wnt signaling pathway. Sciatic-neurectomy for four weeks caused significant detriment of bone quality as indicated by deterioration of BMD and trabecular microarchitecture in distal femur, as well as bone strength of femoral neck, which was accompanied by a dramatical increment of bone resorption and simultaneously decreased activity of bone formation, as evidenced by bone turnover markers, while treatment with naringin at higher dosages displayed a beneficial effect in preservation of bone quality, together with increasing of osteogenic activity and down-regulation of osteoclastic activity. Wnt proteins by binding with a receptor complex (Frizzled and LRP 5/6) lead to inactivation of cytosolic GSK-3b/Axin2/APC complex, which otherwise leads to phosphorylation and then degradation of b-catenin [21]. Thus b-catenin could accumulate in cytoplasm and then translocate into nucleus, which thereby promotes expression of specific genes, in bone ultimately resulting in differentiation of osteoblast and reduced osteoclastogenesis [12,13]. The results of Western-blot in our study showed that protein levels of nucleus b-catenin in tibias ipsilateral to neurectomy decreased in the USN group compared with the sham-operated rats, which were much higher in higher dosages of naringintreating group. Previous studies reported that mutant mice with increased activation of Wnt/b-catenin signaling showed enhanced bone quality as a result of enhanced sensitivity to mechanical stress compared to wild-type littermates [22], while the increased bone formation due to mechanical stimulation was almost vanished in mutant mice with decreased activation of Wnt/b-catenin signaling [23]. And the activity of Wnt/b-catenin signaling was decreased by mechanical unloading through tail suspension [14]. Additionally, the stimulation of Wnt/b-catenin signaling by naringin was reported to be correlated with improved bone quality by naringin in ovariectomized mice [18]. Thus we propose that inactivation of Wnt/b-catenin signaling pathway contributes to bone loss induced by immobilization through sciatic-neurectomy, and stimulation of Wnt/b-catenin signaling is involved in the beneficial role of naringin in protection of bone quality against disuse. Sclerostin is a predominant inhibitor of canonical Wnt/b-catenin signaling and an antagonistic ligand competing for coreceptors LRP5/6 binding, which is almost exclusively secreted by mature osteocytes [24,25]. Furthermore, increased sclerostin contributed mainly to unloading-induced bone loss [26] and mice with Sost knockout were resistance to unloading-induced bone loss [14]. Thus we wondered whether the change of Wnt signaling activities in our studies was medicated by sclerostin. The results of PCR showed that there were no significant differences of Sost
mRNA levels among all groups, however, as indicated by immunohistochemical staining, the percentage of sclerostin-positive osteocytes in USN group was much higher than the Sham group with stronger staining intensities, which was markedly decreased by the highest dosage of naringin, however. And this result was confirmed by that of western-blot in denervated tibias. These inconsistent results could be explained by the phenomenon that change of protein level is not always linear to that of its corresponding mRNA, which may be due to post-transcriptional and post-translation regulation [27]. And as differential expression of cellular protein determined cellular functional status, the regulation of gene Sost expression by naringin may mainly in the process of post-transcriptional modification. Thereby USN-induced downregulation of Wnt signaling may be mediated by increased sclerostin levels, and administration with the highest dosage of naringin is correlated with the decreasing sclerostin level. However, naringin is still not capable to decrease sclerostin expression to the level of the Sham group. Further studies reported that mechanical unloading-induced up-regulation of Sost was mediated by periostin, a matricellular protein mainly produced by periosteal osteoblasts and osteocytes [28], the expression of which inhibited sclerostin expression and was decreased during tail suspension-induced unloading [16]. And periostin could also trigger Wnt/b-catenin signaling pathway directly [29]. We showed here that the periostin protein level was reduced significantly by sciatic-neurectomy, which was restored to a level higher than that of the Sham group by treatment with naringin at 300 mg/kg. Thereby we propose that decreasing of periostin is also involved in unloading-induced bone loss by sciaticneurectomy and naringin displays an ability to increase periostin expression, which may be implicated in its role in protecting bone integrity against disuse. Besides playing an essential role in mechanotransduction, periostin also mediated the bone anabolic effects of PTH through down-regulation of sclerostin and subsequent stimulation of Wnt signaling [29]. Thereby, periostinesclerostineWnt signaling axis exerts an essential role in regulating bone homeostasis, and through which naringin shows an osteoprotective role in preventing disuse-induced bone loss. Further studies using mice lacking gene Postn or Sost needed to be conducted to verify the above effects of naringin. In conclusion, our studies demonstrated for the first time that periostin-induced down-regulation of sclerostin and subsequent activation of Wnt/b-catenin signaling may be implicated in preventive effect of naringin against sciatic-neurectomy induced bone loss, which makes us better understand the mechanisms mediated the osteoprotective role of naringin and confirms that naringin could be a promising alternative medicine in treatment of disuse osteoporosis.
Acknowledgments This study was funded by the National Natural Science Foundation of China (No. 81501061) and the Traditional Chinese Medicine Administration of Tianjin, China (No. 13123).
Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152
J. Lv et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
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Please cite this article in press as: J. Lv, et al., Involvement of periostinesclerostineWnt/b-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/ j.bbrc.2015.10.152