Introduction
Osteoporosis is a common postmenopausal disease that markedly affects the quality of life of the patients (1). There is a known association between alveolar bone loss and osteoporosis in women following pausimenia (2). Furthermore, alveolar bone provides essential tooth support through desmodontal fiber anchoring. It was previously reported that alveolar bone mass maybe reduced and alveolar bone structure may be altered in patients suffering from osteoporosis (3). During the early postmenopausal period, alveolar bone loss occurs rapidly, but this process is leveled out in the ~6th postmenopausal year, which likely results from postmenopausal estrogen reduction (4). Alveolar bone loss causes tooth loss or mobile teeth, severely compromising postmenopausal quality of life (5).
Estrogen (6), parathyroid hormone (PTH) (7) and bisphosphonates (8) are currently used for preventing postmenopausal alveolar bone loss. However, it was indicated that long-term use of these agents may be associated with side effects, including higher risk of endometrial and ovarian cancer (9,10), nervous system disorders (11), osteonecrosis of the jaws (12) and venous thromboembolism (13). A substitutive method or drug with verified safety and efficiency is urgently required for treating alveolar bone loss. In recent decades, certain herbal medicines or botanical drugs have been widely recognized as effective remedies for relieving or treating alveolar bone loss (14–16).
Rhizoma Dioscoreae (RD), a Chinese medicinal herb/medicinal food, has long been used to promote bone and tooth strength in China (17). Our previous study demonstrated that RD extract (RDE) exerts a protective effect on maintaining alveolar bone among rats subjected to ovariectomy (OVX) through the regulation of p38 mitogen-activated protein kinase (MAPK) and Wnt signaling pathway (18). However, it has not been fully elucidated whether this effect is associated with other signaling pathways.
Previous studies have demonstrated that the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in osteoclasts and osteoblasts plays a central role in osseous metabolism and remodeling (19,20). Moreover, using bioinformatics methods in our previous study, we predicted that RDE protection against alveolar bone loss may be associated with the IL-6/STAT3 signaling pathway (21). The aim of the present study was to analyze the inhibitory effect of RDE on alveolar bone loss among OVX rats, and investigate the association between this effect and the IL-6/STAT3 signaling pathway.
Materials and methods
Preparation of RDE
RDE was prepared as previously reported (22), and that same batch of extract was used in the present study.
Grouping and treating animals
A total of 48 Wistar female virgin rats (aged 6 months and weighing ~310±20.0 g) were acquired from the Experimental Animal Center in the Academy of Military Medical Sciences [SCXK-(Military) 2014-005, Beijing, China]. The protocol involving animals in the present study was authorized by the Institutional Ethics Committee of China Academy of Chinese Medical Sciences (approval no. 2015-009). Sham surgery (n=12) or bilateral OVX (n=36) using a dorsal incision was conducted on the rats following acclimatization. The rats undergoing OVX were divided in three groups based on the treating agent, namely control (OVX), RDE and 17β-estradiol (E2) groups. Each group contained 12 rats. Subsequently, 17β-estradiol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol and diluted with olive oil. The preparation was used for daily subcutaneous injection in the E2 rats at a dosage of 30 μg/kg body weight. RDE was dissolved with distilled water and force-fed to RDE rats at a dosage of 1.3 g/kg body weight/day, which was calculated using the human recommended dosage (30 g/day) proposed by Chinese Pharmacopeia and the weight ratio of rat and human. OVX and sham rats were force-fed equal quantities of distilled water, and standardized rat food was provided to all subjects throughout the study (Animal Center of the Fourth Military Medical University, Xi'an, China). The rats were treated with agents or distilled water for 7 days postoperatively and the treatment was maintained until the 13th week, with body weight monitoring of each subject once weekly.
Preparation of specimens
On the day after the final treatment, xylazine (12 mg/kg) and ketamine (80 mg/kg) were intraperitoneally injected to anesthetize the animals, which were subsequently sacrificed by exsanguination. The uteri were excised and immediately weighed (23). The abdominal aorta was punctured prior to death to collect blood specimens into heparinized tubes. Subsequently, the blood specimens were separated by centrifugation at 3,000 × g at a temperature of 4°C for 10 min, and then aliquoted and preserved at −80°C until use. The left mandibles were excised and preserved at −80°C for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and microarrays. The right mandibles were excised, immersed into normal saline solution and preserved at −20°C, with the aim of measuring bone mineral density (BMD) and studying the microscopic structure using microscopic computed tomography (micro-CT).
Biomarkers of bone turnover
Enzyme-linked immunosorbent assays (ELISA; Sunbio, Inc., Beijing, China) were conducted to assess plasma concentrations of bone formation and bone absorption biomarkers, such as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), in control, standardized and duplicated experiments. An ELISA reader (Bio-Tek, Colmar, France) was used to read 450 nm absorption values.
Micro-CT analyses
Untreated right mandibles were scanned with high-resolution micro-CT (SkyScan 1172 micro-CT system; SkyScan, Antwerp, Belgium) which applied cone beam reconstruction for the determination of the cone geometric construction of the X-ray source. The desktop SkyScan micro-CT system was operated as previously described (24). The desirable resolution value of 6.8 μm was obtained by placing a specimen on the rotating stage and translating it along the persistently varying magnifying stage. The rotation angle of a specimen was 185°, and an image was generated with the specimen rotating for 0.9°. The repeatability of this protocol was verified by performing repetitive scanning from the start of this experiment. The resulting gray level images were denoised with a low-pass filter, and a constant threshold value was used to determine the trabeculae.
Following image capture (100 keV, 100 μA), a quadrate region of interest (ROI) (1×1 mm) was constructed using CT analyser, which was affiliated to SkyScan on the sagittal planes of the first molar teeth. None of the selected ROIs overlapped with any tooth roots. As a 3-D rebuilding software affiliated to SkyScan, NRecon was applied for establishing a volume of interest (VOI) with a cubical mass (1×1×1 mm) underneath the bottom of the first molar crown, with a vertical distance of 1.5 mm, and the trabeculae in VOI were measured morphologically by the standardized SkyScan software package. Subsequently, degree of anisotropy (DA), structure model index (SMI), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), bone volume fraction (BV/TV) and BMD were assessed for a certain VOI using 3-D analysis (25).
RT-qPCR array assay
Alveolar bone from 6 OVX and 6 RDE rats was used for RT-qPCR. The differential expression profiles of IL-6/STAT3 signaling pathway-related genes were analyzed by the rat IL-6/STAT3 signaling pathway PCR array (PARN-160Z; Qiagen, Valencia, CA, USA) obtained from Kangchen Biotech (Shanghai, China). A total of 84 essential genes that may be involved in the activation of the IL-6/STAT3 signaling pathway and downstream reactions were profiled by this PCR array. RNA was extracted and utilized to synthesize First-Strand cDNA by RT2 First Strand kit (Qiagen, Manchester, UK) on the basis of standardized protocol and the cDNA template was mixed with RT2 SYBR-Green qPCR Master mix (Qiagen, Germantown, MD, USA), which was ready to be used in the specific kit. Subsequently, mixed reagent and template was injected into wells on PCR array plates, which contained cytokines and IL-6/STAT3 signaling-associated genes, at a dosage of 25 μl for 96-well plates, to perform RT-qPCR. An instrument-specific software was utilized to calculate quantification cycles (Cq) of all genes throughout each PCR experiment and the 2−ΔΔCq approach was applied for calculating fold-change in the gene-expressing profiles for comparing between any two means.
Confirmation by RT-qPCR
Alveolar bone from another 6 OVX and 6 RDE subject rats was used for RT-qPCR. The RNeasy mini kit (Qiagen, Valencia, CA, USA) was utilized to purify total extracted RNA and SuperScript First Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) was used to reversely transcribe 4 μg RNA into cDNA. In an ABI-7500 Sequence Detection system (Applied Biosystems, Foster City, CA, USA), cDNA synthesized in the process of RT-qPCR was detected by SYBR-Green based on the manufacturer's instructions. The primers of the RT-qPCR analyses are listed in Table I. The RT-qPCR conditions in the present study were as follows: 10 sec of initiation at 95°C, 5 sec of thermal denaturation at 95°C, and 34 sec of annealing at 60°C for 40 cycles. The expressing quantities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize PCR results and 2-ΔΔCq was applied for data analysis. The purity of the amplified products was assessed by melting curves of each RT-qPCR assay containing negative control without templates.
Statistical analysis
Data are presented as mean ± standardized difference and were statistically analyzed by SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Analysis of variance and the least significant difference (LSD) test were used to determine inter-group differences in the parameters under evaluation. The normality of all data was proven by Kolmogorov-Smirnov tests, and a P-value of <0.05 was set as the threshold of statistical significance.
Discussion
In our previous study, using bioinformatics and PCR or western blotting to confirm the expression of Stat3, it was demonstrated that RDE inhibited osteoporosis in OVX rats, an effect which was associated with the IL-6/oncostatin M (OSM)/STAT3 pathway via miRNA regulation (21). The putative miRNAs targets' gene prediction and pathway analysis are both bioinformatics methods, and the results occasionally do not fully reflect the true effects of RDE; however, the results raise the question whether RDE exerts an anti-osteopenic effect via another IL-6 family cytokine pathway. The IL-6 cytokine family includes 10 members: IL-6, IL-31, IL-27, IL-11, neuropoietin, cardiotrophin-like cytokine, cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), OSM and leukemia inhibitory factor (LIF). All IL-6 family cytokines employ the transducing receptor β-subunit gp130 as part of a multimeric receptor complex (26). The present study aimed to determine the association between the IL-6 family of cytokines pathway and the anti-osteopenic effect of RDE.
Our results demonstrated that treatment with RDE for 12 weeks prevented loss of uterine wet weight and body weight gain resulting from lack of estrogen in rats of the OVX group (Figs. 1 and 2). It was deduced that RDE exerts a mild estrogen-like effect, which may slow down OVX-induced uterine atrophy and weight gain.
Following ovary removal, OVX rats exhibited markedly reduced BMD, resulting from increasing alveolar bone metabolism, compared with the sham rats. By contrast, treatment with RDE or E2 increased the BMD of alveolar bone.
The significant and coincident increases in plasma ALP and TRAP verified mature female OVX rat as an appropriate and reliable animal model for studies on early postmenopausal osteoporosis characterized by high-turnover bone loss. Treating rats with RDE or E2 for 12 weeks inhibited the excessive bone metabolism, as shown by significantly decreased ALP and TRAP levels (Figs. 3 and 4).
From analyses of 3-D bone microarchitecture by micro-CT, it was indicated that the loss of alveolar bone among rats treated with E2 or RDE was less severe compared with that in OVX rats, which was inferred through significant variations in SMI, Tb.Sp, Tb.N and BV/TV. The inhibition of alveolar bone loss with E2 was superior to that of RDE (Table II and Fig. 5).
The results of bone metabolic biomarkers, micro-CT and BMD demonstrated that RDE significantly inhibited alveolar bone loss.
To investigate the association between the IL-6/STAT3 signaling pathway and the anti-osteopenic effect of RDE, the differential expression of genes by was screened using IL-6/STAT3 signaling pathway array. A total of 20 genes (19 downregulated and 1 upregulated) from alveolar bone were successfully identified and validated; their expression profiles differed significantly among RDE rats compared with the OVX group. The IL-6/STAT3 signaling pathway was downregulated following RDE treatment (Fig. 7).
In the IL-6 family cytokine signaling cascade, oligomerization of receptor subunits induced by a specific ligand may activate janus protein-tyrosine kinases (JAKs), which further activate the MAPKs or the STATs (mainly STAT1 and STAT3). Another signaling cascade activated by cytokines of the IL-6 family is the phosphoinositide-3-kinase/AKT (27,28). Receptor complexes that are capable of signaling after being activated by cytokines of the IL-6 family may provide specificity in a given signal transduction pathway. For example, OSM is considered to be a unique gp130-binding cytokine, as it binds first to gp130, and then develops a signaling complex with either the OSM receptor (OSMR) or the LIF receptor (LIFR), which are both capable of intracellular signaling (29,30).
Cytokines of the IL-6 family exert a major effect on osseous remodeling through osteoclasts or osteoblasts, despite seemingly functioning as a double-edged sword. These cytokines maintain bone generation while driving bone absorption induced by a variety of osteolytic factors. This dual effect is apparent in two main signaling pathways (STAT and MAPK signaling) bidirectionally affecting osteocytes (19).
Certain members of the IL-6 family (OSM, CNTF, LIF, IL-11 and IL-6) may promote bone formation. This effect was mainly reflected in three aspects: First, these cytokines promote the differentiation of osteoblast precursors or maturing osteoblasts isolated from the bone marrow or calvaria. During this process, STAT3 must be activated (31,32). Thus, the expression of osteoblastic biomarkers, such as bone sialoprotein, osteocalcin or ALP, was increased, while extracellular matrix mineralization and bone nodule formation were enhanced. Second, the biomarkers inhibit certain osteoblasts from proliferating via activating gp130/STAT3 and increasing the expression of p21WAF1, which is a cell cycle inhibitor and is also required for cytokines of the IL-6 family to induce ALP (33). Finally, these cytokines may protect osteoblasts from apoptosis caused by tumor necrosis factor-α (TNF-α) or serum depletion (34).
As regards bone absorption, cytokines of the IL-6 family promote osteoclast differentiation and bone absorption by accelerating the interactions between osteoclasts and osteoblasts. In co-cultures of osteoclast precursors and stromal cells or osteoblasts, certain IL-6-type cytokines (OSM, CNTF, LIF, IL-11 and IL-6) are able to promote osteoclast differentiation and bone absorption. In fact, these cytokines induce production of various osteoblastic downstream effectors that, in turn, promote osteoclast differentiation or activity, such as IL-1, receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), prostaglandin E2 and PTH-related protein. Recent studies implicated activation of IL-6-type cytokines/STAT3 signaling as the pivotal event in the induction of RANKL in osteoblasts, which leads to pro-resorption action of osteoclasts (35,36).
It was observed that the gene expression of certain signaling molecules (Osm, Osmr, Lifr, Il6st, Jak2, Stat3, Mtor, Nfkb1 and Mapk1) in the IL-6/STAT3 pathway were downregulated following RDE treatment (Fig. 7), which subsequently inhibited IL-6/STAT3 signaling. The RT-qPCR analysis of Osm, Osmr, Lifr, Il6st, Jak2, Stat3, Mtor, Nfkb1 and Mapk1 expression (Fig. 6) proved that RDE effectively reduced excessive alveolar bone formation and bone absorption synchronously caused by attenuated canonical IL-6/STAT3 signaling following OVX.
Further studies are required for RDE to be developed into a novel promising drug for the prevention or treatment of alveolar bone loss in postmenopausal women.
In conclusion, RDE was effective in inhibiting rat alveolar bone loss caused by OVX, by simultaneously inhibiting bone formation as well as bone resorption through regulation of the IL-6/STAT3 signaling pathway. The present study verified that RDE may be used as an oral agent for the treatment of alveolar osteopenia in postmenopausal women.