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Effects of Er-Zhi-Wan on Microarchitecture and Regulation of Wnt/β-catenin Signaling Pathway in Alveolar Bone of Ovariectomized Rats*

Wei SUN (孙 为)†, Yuan-qin WANG (王远勤)†, Qi YAN (晏 奇), Rui LU (卢 锐), Bin SHI (施 斌)# State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consist-ing of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariec-tomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchi-tecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phos-phatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/β-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), β-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Fur-thermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and β-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/β-catenin signaling pathway. Key words: osteoporosis; Er-Zhi-Wan; alveolar bone; Wnt signaling pathway

Postmenopausal osteoporosis leads to trabecular bone loss by estrogen-deficiency, which increases the risk of fracture. Some studies showed that as a part of body skeleton, alveolar bone loss bore a positive associa-tion with postmenopausal osteoporosis[1–3]. Ovariectomy (OVX) may increase bone turnover and reduce cancel-lous bone volume in interradicular septum of the first molar in mandible of rats by estrogen-deficiency[4, 5]. To recover the hormone level of postmenopausal osteoporo-sis, hormone replacement therapy (HRT) has been proven to be an effective method in preventing bone loss[6], but it may cause estrogen-like side-effects[7]. Tra-ditional Chinese medicines are an alternative choice to

Wei SUN, E-mail: Suzanne_whu@126.com; Yuan-qin WANG, E-mail: wang2118119@foxmail.com †The authors contributed equally to this work. #Corresponding author, E-mail: shibin_dentist@foxmail.com *This project was supported by grants from the National Natu-ral Science Foundation of China (No. 81170992) and the Fun-damental Research Funds for the Central Universities of Wu-han University (No. 201130402020004).

prevent and treat postmenopausal osteoporosis, which are prepared from plants and have fewer side-effects[8].

Er-Zhi-Wan (EZW), a famous traditional Chinese formula, is mainly composed of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL) in an equal weight ratio. Antiosteoporostic activity of EZW may be carried out via inhibition of osteoclastic bone resorption[9]. It was indicated that EZW had a definite antiosteoporotic effect on osteoporotic femurs in ovariectomized rats, without hyperplastic effect on uterus [8], but the effects of EZW on osteoporotic alveolar bone remain unknown. EZW could also induce the expression of luciferase driven by an estrogen responsive element in a pERE-Luc vector being transfected in MCF-7 cells[10]. In addition, Wnt/β-catenin signaling pathway was reported to play a considerable role in osteoporosis[11], and the inhibition of Wnt/β-catenin signaling pathway could increase bone density, strength and formation in an ovariectomized rat model[12]. It is unknown whether Wnt/β-catenin signaling pathway plays a role in the pathogenesis of osteoporosis in alveolar bone.

Therefore, the present study was aimed to charac-terize the effects of EZW on the microarchitecture and

34(1):114-119,2014J Huazhong Univ Sci Technol [Med Sci]

DOI 10.1007/s11596-014-1241-0

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regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats.

1 MATERIALS AND METHODS

1.1 Plant Components and Preparation of EZW

HE and FLL, two major components of EZW, were purchased from Tong-Ren-Tang Pharmaceutical Group (Wuhan, China), identified as the aerial parts of Eclipta prostrasta L. and the fruit of Ligustrum lucidum Ait. by Prof. Huang (The School of Pharmaceutical Sciences, Wuhan University, Wuhan, China).

EZW was prepared according to the National Phar-macopoeia Commission of P. R. China. Briefly, 1000 g of HE and 1000 g of FLL were broken into coarse pow-der, and then mixed together. The mixture power was immersed in distilled water [weight (kg)/volume (L) ra-tio=1:10] for 1 h, and then boiled in a distillation appa-ratus for 2 h. This process was repeated three times. The extracting solution was filtrated and evaporated using a rotary evaporator in a 70°C water bath and cooled. The concentrated solution was lyophilized with a freeze drier, and stored at 4ºC until use. The yield of dried extract from the starting crude materials was 10%. The extracts (EZW) resolved to 100 mg/mL in distilled water were administered orally to rats at a volume of 1 mL/100 g body weight. The experimental dose for EZW in the present study was equivalent to the corresponding clini-cal prescription dose for a 60 kg human subject. 1.2 Animals and Treatments

Thirty female Spraguee-Dawley rats weighing 189±8.7 g were purchased from the Experimental Ani-mal Center, Wuhan University, China. The rats were randomly divided into three groups: sham operation group (sham, n=10), OVX group (n=10), and OVX with EZW treatment group (EZW group, n=10). Under anes-thesia condition, bilateral ovaries were removed from the rats in OVX group and EZW group, whereas only a small amount of fatty tissue surrounding the bilateral ovaries were removed from the rats in sham group. Be-ginning from one week after the operation, the rats in EZW group received EZW-water solution (100 mg/mL) once daily (1 mL/100 g), whereas the rats in OVX group and sham group were fed with vehicle (distilled water) once daily (1 mL/100 g). The whole feeding process persisted for 12 weeks. During the treatment, body weights of all rats were recorded weekly and dose for EZW was adjusted accordingly. After laparotomy under anesthesia with diethyl ether, blood sample was collected via abdominal aorta puncture, and serum was then pre-pared by centrifugation at 1000 r/min for 15 min. Serum

samples were stored at –80ºC until serum chemical pa-rameters were measured. Uteri were removed and imme-diately weighed. The right mandible from each rat was dissected and stored in 10% phosphate-buffered formalin for 48 h for fixation. The teeth of left mandible from each rat were removed, and a piece of hard tissue around mandibular first molar from each rat was dissected and immediately stored in the liquid nitrogen. All animals were treated according to the Guide for Care and Use of Laboratory Animals with the approval of Institutional Ethics Committee of the School and Hospital of Stoma-tology of Wuhan University on animal experimenting, China. 1.3 Assay for Serum Chemistry

Serum estradiol (E2), bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b) levels were assayed using an enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Elabscience Biotechnology Co., Ltd, Wuhan, China) according to the manufacturer’s instructions. 1.4 Micro-Computed Tomography (CT) Analysis

The right mandible of each animal was scanned on a micro-CT system (70 KV, 114 μA, 300 ms; micro-CT 50 scanner; Scanco Medical, Bassersdorf, Switzerland), with an isotropic voxel size of 15 μm. The following bone morphometric parameters were included: bone volume over total volume (BV/TV), trabecula number (Tb.N), trabecula thickness (Tb.Th), trabecula separation (Tb.Sp), connectivity density (Conn.D). 1.5 Isolation of Total Mandibular mRNA and Real-time RT-PCR

The hard tissue of left mandible from each rat stored in the liquid nitrogen was taken out and then grinded into powder, and then total RNA was extracted from the powder using Trizol-Reagent (Invitrogen, USA) accord-ing to the instructions on the kit. The cDNA was synthe-sized from total RNA of each sample using PrimeScript™ RT Reagent Kit (DDR037S, TAKARA, Japan) and real-time RT-PCR was performed with SYBR® Premix DimerEraser™ Kit (DDR091S, TA-KARA, Japan), following the instructions on the kit. Real-time reverse transcription-polymerase chain reac-tion (RT-PCR) using an ABI-7500 Real-Time PCR ma-chine was performed to evaluate the mRNA expression of target genes of Wnt/β-catenin signaling pathway in mandibles.

The primers are summarized in table 1, including wingless-related MMTV integration site 3a (wnt3a), low density lipoprotein receptor-related protein 5 (LRP5), β-catenin, dickkopf homolog 1 (DKK1) and β-actin. β-actin was used as a housekeeping gene.

Table 2 Effects of EZW on serum levels of E2, BALP and TRAP5b in rats ( ±s)

Groups E2 (ng/L) BALP (U/L) TRAP5b (U/L) Sham 36.80±9.59 70.99±7.77 4.48±0.54 OVX 14.68±5.37** 82.66±9.08** 9.97±0.87** EZW 21.27±5.81# 92.44±8.47# 5.22±0.73##

Data were expressed as ±s. **P<0.01 vs. sham group; #P<0.05, ##P<0.01 vs. OVX group

1.6 Statistical Analysis

Statistical analysis was performed using Prism, ver-sion 5.01. Intergroup differences were analyzed by

Kruskal-wallis test, and the Mann-Whitney test was used to analyze the significance of the differences between the groups. All data were expressed as ±s, and P<0.05

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(two-tailed) was considered statistically significant.

2 RESULTS

2.1 Effects of EZW on Body and Uterus Weights To evaluate the general condition of animals, the

body weights of each group of rats, which had a similar initial mean body weight (189±8.7 g), were observed once a week during the whole process of the study. Body weight was significantly increased in OVX group as

compared with sham group at 3rd week after operation (P<0.05). The OVX-induced weight gain in EZW group was significantly inhibited at 5th week after OVX treat-ment (P<0.01, fig. 1A).

The atrophy of uterus in OVX group was signifi-cantly aggravated as compared with sham group (P<0.01), indicating the successful establishment of OVX, whereas EZW treatment could not alleviate the atrophy of the uterus (fig. 1B).

Fig. 1 Effects of 12-week treatment with EZW on body and uterus weight of ovariectomized rats

A: The body weight of the rats was recorded weekly during the study period; B: The uterus was resected and weighed after sacrificing the rats and uterus index was calculated as uterus weight divided by body weight. Data are expressed as ±s. **P<0.01 vs. sham group

2.2 Effects of EZW on Serum E2, BALP and TRAP5b Levels

Serum levels of E2, BALP and TRAP5b were ana-lyzed to provide an evaluation of the effects of EZW on systemic estrogenic and bone metabolism. The serum biochemical parameters in rats of the three groups are shown in table 2. Serum E2 level was reduced by 60.1% (P<0.01), and serum BALP and TRAP5b levels were

increased by 16.4% and 122.5% respectively (P<0.01 for all) in OVX group as compared with sham group. After 12-week treatment with EZW, serum levels of E2 and BALP were increased by 44.9% (P<0.05) and 11.8% (P<0.05), respectively, and serum level of TRAP5b was decreased by 47.6% (P<0.01), compared with the OVX group.

Table 1 The sequences of the primers

Genes Forward Reverse GenBank accessionWnt3a 5'-TCCATgCCATCgCTTCC-3' 5'-TCACTACAgCCACCCCACTTC-3' XM_220546.5 LRP5 5'-GCTCCTGCAGAACCTGCTGA-3' 5'-CACCAGTGGCACATGCAAAC-3' NM_001106321.2 β-catenin 5'-GTCTGAGGACAAGCCACAGGACTAC-3' 5'-AATGTCCAGTCCGAGATCAGCA-3' NM_053357.2 DKK1 5'-ATGAGGCACGCTATGTGCTG-3' 5'-CTCGAGGTAAATGGCTGTGGTC-3' NM_001106350.1 β-actin 5'-GGAGATTACTGCCCTGGCTCCTA-3' 5'-GACTCATCGTACTCCTGCTTGCTG-3' NM_031144.2

2.3 Effects of EZW on 3-dimensional Trabecular Mi-croarchitecture of Mandibles

The right mandible of each animal was scanned on a micro-CT system (70 KV, 114 μA, 300 ms; micro-CT 50 scanner; Scanco Medical, Bassersdorf, Switzerland) at the time of sacrifice. The 3-dimensional (fig. 2A) and 2-dimensional (fig. 2B) micro-CT images clearly de-picted the differences among groups in trabecular mi-croarchitecture. The area of trabecular bone in both sham and EZW groups was obviously larger than that in the OVX group. Analysis of the mandibular trabecular mi-cro-CT parameters indicated that ovariectomy signifi-cantly decreased mandibular trabecular BV/TV by 39.7% (P<0.01), Tb.N by 27.1% (P<0.01), Tb.Th by 38.7% (P<0.01), Conn.D by 33.9% (P<0.05), while increased Tb.Sp by 35.0% (P<0.01) compared to the sham group. The lowest BV/TV, Tb.Th, Tb.N, Conn.D values and the highest Tb.Sp value in the OVX group were noticed among the three groups (P<0.01, respectively). Com-pared with the OVX group, the EZW group increased the

BV/TV by 45.6% (P<0.01), the Tb.N by 26.7% (P<0.05), the Tb.Th by 29.8% (P<0.01), the Conn.D by 29.4% (P<0.05), and decreased Tb.Sp by 19.5% (P<0.05) (fig. 3). 2.4 mRNA Expression Involved in Wnt/β-catenin Signaling Pathway in the Mandibles

The expression of the mRNAs isolated from the mandibular tissues was estimated by real-time RT-PCR. To determine the expression levels of the Wnt/β-catenin signaling pathway genes, Wnt3a, LRP5, β-catenin, and DKK1 mRNA levels were monitored. In OVX group, the mRNA levels for wnt3a, LRP5, β-catenin and DKK1 were up to 1.42-fold (P<0.05), 1.68-fold (P<0.05), 1.64-fold (P<0.01) and 1.31-fold (P<0.05) of the sham group levels, respectively (fig. 4). The results showed that EZW treatment significantly increased the mRNA expression of Wnt3a to 1.43-fold (P<0.05), LRP5 to 1.95-fold (P<0.01), β-catenin to 2.58-fold (P<0.01), re-spectively, and significantly reduced DKK1 to 0.29-fold (P<0.01), compared with that of the OVX group (fig. 4).

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Fig. 2 The three-dimensional micro-CT images (A) and two-dimensional images (B) of the rat hemi-mandible in the three groups

Scheme of the volume of interest (VOI) for micro-CT assessment was defined as a cuboid extending 180 μm×120 μm×90 μm below mandibular first molar. The images showed significant differences among the three groups in the internal trabecular to-pography.

Fig. 3 Quantitative results of mandibular trabecular microarchitecture within the volume of interest (VOI) by micro-CT assessment

BV/TV: ratio of bone volume to total volume; Tb.N: the mean trabecular number; Tb.Th: the mean trabecular thickness; Tb.Sp: the mean trabecular separation; Coon.D: the mean connectivity density Data are expressed as ±s. *P<0.05, **P<0.01 vs. sham group; #P<0.05, ##P<0.01 vs. OVX group

Fig. 4 The mRNA expression levels of the Wnt signaling proteins (wnt3a, LRP5, β-catenin, DKK1) in the mandible

Data are expressed as ±s. *P<0.05, **P<0.01 vs. sham group; #P<0.05, ##P<0.01 vs. OVX group.

3 DISCUSSION

The ovariectomized rat model has been used as an

animal model for the assessment of new therapies and drugs for postmenopausal osteoporosis[8, 13], also as a

model to evaluate trabecular microarchitecture in jaws[4,

5]. Osteoporosis in alveolar bone was considered as a high risk of alveolar bone resorption and periodontal disease, also a relative contraindication for dental im-plant[14]. Although studies have demonstrated that EZW could restrain osteoclastic bone resorption and prevent

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ovariectomy-induced bone loss in femurs of ovariec-tomized rats[8, 9], the effects of EZW on alveolar bone remain unknown. In the present study, we evaluated the effects of 12-week EZW treatment on trabecular mandi-ble in ovariectomized rats. Our investigation distinctly demonstrated that EZW could restrain the body weight gain with atrophic uterus, increase serum E2 and BALP levels, decrease serum TRAP5b level, prevent degenera-tion of alveolar trabecular bone microarchitecture, and induce the expression of Wnt3a, LRP5, β-catenin, and DKK1, which are involved in Wnt/LRP5/β-catenin sig-naling pathway in ovariectomized rats.

Consistent with other studies[15, 16], the OVX group had a significant increase in body weight with atrophic uterus as compared to the sham group, which was caused by estrogen-deficiency with a direct influence on energy metabolism. Serum E2 was analyzed as a reference com-pound for the influence of estrogenic activity on bone remodeling. In this study, serum E2 level fell off in OVX group significantly, and then EZW treatment increased serum E2 level in OVX rats, indicating that EZW treat-ment enhanced estrogen metabolism in ovariectomized rats. Moreover, serum BALP and TRAP5b were widely analyzed as biochemical markers for bone formation and bone resorption[17, 18]. The results showed the OVX group had a significant increase in BALP and TRAP5b com-pared with the sham group, indicating ovariectomy may enhance both bone formation and bone resorption, but the bone resorption rate was higher. This abnormal bone turnover may cause the bone loss and decrease bone quality, which characterizes postmenopausal osteoporo-sis[19–21]. With 12-week EZW treatment, serum BALP level was significantly increased by 11.8% (P<0.05), meanwhile serum TRAP5b level was significantly de-creased by 47.6% (P<0.01), as compared with the OVX group, indicating EZW treatment could enhance bone formation and inhibit bone resorption. All the results were in accordance with preceding obeservations[18, 22].

The alveolar bone as teeth bearing bone was con-sidered more intricate than long bones. The alveolar bone was observed to have higher rates of bone remodeling compared to the long bones[23]. Micro-CT has been used as a method to evaluate the microarchitecture of trabecu-lar bone in mandible[24]. To investigate the effects of EZW on microarchitecture in trabecular bone of osteo-porotic mandible, the mandibles were scanned in mi-cro-CT analysis. The results showed significant de-creases in trabecular mandible BV/TV, Tb.N, Tb.Th and Coon.D, and obviously increased Tb.Sp in OVX rats (fig. 3), which was consistent with the observations in earlier report[25]. Compared with the OVX group, the EZW group had significantly higher values of BV/TV, Tb.N, Tb.Th and Coon.D, whereas a distinctly lower value of Tb.Sp. EZW with daily oral administration for 12 weeks prevented the deterioration of the trabecular bone in os-teoporotic mandible. The conclusion was in accordance with those mentioned in previous study, which indicated EZW treatment could prevent trabecular bone loss in osteoporotic femurs[8].

Wnt/β-catenin signaling pathway has caught atten-tion as a potential target for osteoporosis treatment, for it was considered to play a important role in bone devel-opment[26]. Wnt/β-catenin signaling is initiated by a Wnt

family member binding to two membrane receptors, Frizzled and LRP5/6, which results in cytoplasmic β-catenin accumulation. Then the accumulated β-catenin translocates to the nucleus, associates with transcrip-tional coactivator, T-cell factor/lymphoid enhancer bind-ing factor (TCF/LEF) and stimulates expression of Wnt target genes involved in skeletal development[27]. Activa-tion of Wnt/β-catenin signaling upgrades differentiation and proliferation of osteoblast precursor cells and stimu-lates osteoblast activity, which promotes new bone for-mation and increases bone mineral density[28]. DKK1 regulates Wnt/β-catenin signaling negatively by binding to the Wnt membrane receptor LRP5/6. To investigate the action mechanism of EZW on alveolar bone at mRNA level, we selected several key genes of Wnt/β-catenin signaling in response to EZW treatment.

In this study, the mRNA expression of wnt3a, LRP5, β-catenin and DKK1 was enhanced in OVX group as compared to sham group. However, with 12-week EZW treatment, the levels of wnt3a, LRP5 and β-catenin ex-pression were upregulated while DKK1 expression was downregulated in EZW group. The results indicated that EZW treatment may activate Wnt/β-catenin signaling by restraining DKK1 expression. The real-time RT-PCR results proved that EZW treatment could prevent bone loss and improve trabecular microarchitecture of mandi-ble in ovariectomized rats, since the significant role of Wnt/β-catenin signaling pathway in osteoblastic differ-entiation was already defined[27, 28].

In conclusion, for the first time the present study il-lustrates the effects of EZW on alveolar bone and eluci-dates the molecular mechanism of EZW treatment for postmenopausal osteoporosis. EZW treatment had posi-tive effects on trabecular bone microarchitecture of al-veolar in ovariectomized rats. Wnt/β-catenin signaling pathway may not be the only signaling pathway up-graded by EZW treatment. Thus, future studies are re-quired to elucidate the role of EZW in Wnt/β-catenin signaling pathway.

Conflict of Interest Statement

The authors declare that there is no conflict of interest with any financial organization or corporation or individual that can inappropriately influence this work.

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(Received May 31, 2013; revised Dec. 20, 2013)