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Si-Wu-tang extract stimulates bone formation through PI3K/Akt/NF-κB signaling pathways in osteoblasts
© Wu et al.; licensee BioMed Central Ltd. 2013
Received: 18 June 2013
Accepted: 17 October 2013
Published: 24 October 2013
Si-Wu-Tang (SWT), a Traditional Chinese Medicine (TCM) formula, is widely used for the treatment of gynopathies diseases such as menstrual discomfort, climacteric syndrome, dysmenorrhea, and other estrogen-related diseases. Recent studies have shown that SWT can treat primary dysmenorrhea, have anti-pruritic anti-inflammatory effects, and protect against radiation-induced bone marrow damage in an animal model. It has been reported that anti-inflammatory and anti-oxidant agents have the potential to treat osteoporosis by increasing bone formation and/or suppressing bone resorption. However, the effect of SWT on bone cell function has not yet been reported.
Alkaline phosphatase (ALP), bone morphogenetic proteins (BMP)-2, and osteopontin (OPN) mRNA expression was analyzed by qPCR. The mechanism of action of SWT extract was investigated using western blotting. The in vivo anti-osteoporotic effect of SWT extract was assessed in ovariectomized mice.
Here, we report that SWT increases ALP, BMP-2, and OPN expression as well as bone mineralization. In addition, we show that the PI3K, Akt, and NF-κB signaling pathways may be involved in the SWT-mediated increase in gene expression and bone mineralization. Notably, treatment of mice with SWT extract prevented bone loss induced by ovariectomy in vivo.
SWT may be used to stimulate bone formation for the treatment of osteoporosis.
Bone is a mineralized tissue composed of several cell types, which undergoes a continuous renewal and repair process called “bone remodeling”. Bone remodeling is accomplished by bone-forming osteoblasts and bone-resorbing osteoclasts that reside in the bone. The development and differentiation of these 2 cell types are tightly regulated by a number of endogenous substances such as hormones, growth factors, and cytokines . These factors are secreted through the endocrine, paracrine/autocrine, and neurocrine systems, and modulate the balance between bone-forming and bone-resorbing cells in the marrow microenvironment. Osteoporosis results when bone resorption and bone formation are imbalanced and excess bone breakdown exceeds bone building . Bone resorption inhibitors, e.g., bisphosphonates, calcitonin, and estrogen, were designed as therapeutic targets to treat osteoporosis . However, the efficiency of these drugs in improving bone mass is very small, certainly no more than 2% per year . Therefore, teriparatide, an anabolic agent, which stimulates bone formation and corrects characteristic changes in the trabecular microarchitecture in established osteoporosis, is a new approach to treat osteoporosis [4, 5]. Bone remodeling is regulated through a balance of bone-forming and bone-resorbing cell activities that together maintain bone mass and mineral homeostasis. New bone formation is mainly controlled by osteoblasts; therefore, agents that act to either increase proliferation of cells of the osteoblastic lineage or induce differentiation of osteoblasts can enhance bone formation [5–7].
The biological mechanism of osteoporosis is still unclear. However, it is likely related to decreased availability or effects of bone growth factors such as bone morphogenetic proteins (BMPs) . BMPs were first discovered as a result of their capacity to induce ectopic bone formation in rodents, and the protein structure of BMPs are similar to the transforming growth factor-β superfamily . BMPs are secreted proteins, which play crucial roles in bone formation and bone cell differentiation through stimulation of alkaline phosphatase (ALP) activity as well as synthesis of proteoglycan, collagen, and osteopontin (OPN) . A previous study showed linkage of osteoporosis to specific polymorphisms in the BMP-2, ALP, and OPN genes, revealing that they are osteoporosis-associated genes .
Si-Wu-Tang (SWT), a Traditional Chinese Medicine (TCM) formula, is comprised of a combination of 4 herbs; Paeoniae, Angelicae, Chuanxiong, and Rehmanniae, and is widely used for the treatment of women’s diseases such as cutaneous pruritus and chronic inflammation, and other diseases. Modern pharmacological studies have shown that SWT extract has anti-pruritic  and anti-inflammatory effects , and protects against radiation-induced bone marrow damage in an animal model [13, 14]. Previous studies have shown that anti-inflammatory and anti-oxidant agents have the potential to treat osteoporosis by increasing bone formation and/or suppressing bone resorption [15, 16]. However, the effect of SWT on bone cell function has not yet been reported. In the current study, we report that SWT extract increases ALP, BMP-2, and OPN expression and bone mineralization. Furthermore, we show that the phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB signaling pathways are involved in the SWT-mediated increase in gene expression and bone mineralization. Finally, treatment of mice with SWT extract prevented bone loss induced by ovariectomy in vivo. Our data, therefore, suggest that SWT may be used to stimulate bone formation for the treatment of osteoporosis.
SWT extract and materials
SWT extract was kindly provided by Timing Pharmaceutical Company (New Taipei City, Taiwan). The extraction and isolation of SWT were performed as previously described . Rabbit polyclonal antibodies for BMP-2, OPN, p-p85 (Tyr458), p85, p-Akt (Ser473), Akt, p-p65 (Ser536), and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The osteopontin BMP-2 ELISA kit was purchased from Biosource Technology (Nivelles, Belgium). The C-terminal telopeptides of type-I collagen ELISA kit was obtained from Cross Laps (Herlev, Denmark). p85 and Akt siRNAs were purchased from Santa Cruz Biotechnology. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The murine osteoblast cell line MC3T3-E1 was purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). Cells were cultured in 5% CO2 with α-MEM supplemented with 20 mM HEPES and 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL).
Measurement of mineralized nodule formation
Levels of mineralized nodule formation were evaluated as previously described [18, 19]. Briefly, osteoblasts were cultured in medium containing vitamin C (50 μg/mL) and β-glycerophosphate (10 mM) for 2 wks, and the medium was changed every 3 d. After incubation with SWT extract for 12 d, cells were washed twice with 20 mM Tris-buffered saline containing 0.15 M NaCl (pH 7.4), fixed in ice-cold 75% (v/v) ethanol for 30 min, and air-dried. Calcium deposition was determined using alizarin red-S staining. Briefly, ethanol-fixed cells and matrix were stained for 1 h with 40 mM alizarin red-S (pH 4.2) and rinsed extensively with water. The bound stain was eluted with 10% (w/v) cetylpyridinium chloride, and alizarin red-S in the samples was quantified by measuring absorbance at 550 nm and comparing to a standard curve. One mole of alizarin red-S selectively binds approximately 2 moles of calcium.
Quantitative real time PCR
Total RNA was extracted from osteoblasts using a TRIzol kit (MDBio Inc., Taipei, Taiwan). Reverse transcription was performed using 2 μg of total RNA and oligo(dT) primers [20, 21]. Quantitative real-time PCR (qPCR) was carried out using TaqMan® One-Step PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). cDNA (100 ng) was added to a 25-μL reaction containing sequence-specific primers and Taqman® probes. All target gene primers and probes were purchased commercially, including β-actin as an internal control (Applied Biosystems). qPCR assays were carried out in triplicate on a StepOnePlus sequence detection system (Applied Biosystems). The cycling conditions were as follows: 10-min polymerase activation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted CT).
Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. After treatment with SWT extract for 2 days, cultures were washed with PBS. MTT (0.5 mg/ml) was then added to each well and the mixture was incubated for 2 h at 37°C. Culture medium was then replaced with equal volume of DMSO to dissolve formazan crystals. After shaking at room temperature for 10 min, absorbance of each well was determined at 550 nm using a microplate reader (Bio-Tek, Winooski, VT).
Western blot analysis
Cell lysates were prepared as described previously . Proteins were resolved by SDS-PAGE and transferred to Immobilon polyvinyldifluoride membranes (Millipore, Billerica, MA, USA). The blots were blocked with 4% bovine serum albumin for 1 h at room temperature, and then probed with rabbit anti-human antibodies against p85, p-p85, p-Akt, Akt, p65, or p-p65 (1:1000) for 1 h at room temperature. After 3 washes, the blots were incubated with peroxidase-conjugated donkey anti-rabbit secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using X-OMAT LS film (Eastman Kodak, Rochester, NY).
Female ICR mice (4 wks old; 22–28 g) were used for this study. Mice were ovariectomized bilaterally under trichloroacetaldehyde (100 mg/kg) anesthesia and control mice were sham-operated (Sham) for comparison. Bone mineral density and bone mineral content were measured after oral administration of various concentrations of SWT extracts every 2 d for 4 wks. Total body bone mineral density and bone mineral content were determined by a dual-energy X-ray absorptiometer (DEXA; XR-26; Norland, Fort Atkinson, WI) using a mode for small subjects as described previously [19, 23]. All protocols complied with institutional guidelines and were approved by the Animal Care Committee of China Medical University.
Statistical analysis was performed using Prism 4.01 software (GraphPad Software Inc., San Diego, CA, USA). The values given are means ± standard errors of the mean (SEM). Statistical analyses between 2 samples were performed using the Student’s t-test. Statistical comparisons of more than 2 groups were performed using 1-way analysis of variance with Bonferroni’s post-hoc test. In all cases, p < 0.05 was considered significant.
SWT extract increases bone mineralization by osteoblasts
SWT extract increases bone nodule formation through the PI3K/Akt pathway
SWT extract increases bone nodule formation through the NF-κB pathway
Inhibition of bone loss by SWT extract in ovariectomized mice
Si-Wu-Tang, a TCM formula, is widely used in traditional medicine for various therapeutics, including women’s diseases, chronic inflammation, and other diseases because of its anti-pruritic and anti-inflammatory effects . In this study, we showed that SWT extract induced bone mineralization in cultured osteoblasts. In addition, we found that SWT extract increased the expression levels of ALP, BMP-2, and OPN, which requires the activation of PI3K, Akt, and NF-κB signaling pathways. SWT is comprised of a combination of 4 herbs; Paeoniae, Angelicae, Chuanxiong, and Rehmanniae. On the other hand, the major bioactive components in these 4 herbs include phenolics, phthalides, alkaloids, terpene glycosides, and iridoid glycosides. In the current study, we used SWT extract to examine the role SWT in bone formation. However, we did not extract and examine the role of single compound in SWT. Therefore, the next step is to disclose which compound is most important in SWT extract.
Bone is a complex tissue composed of several cell types that are continuously undergoing a process of renewal and repair . Osteoporosis results from an imbalance between bone resorption and bone formation, where bone breakdown overrides bone formation . We took advantage of the ovariectomized mouse model to examine the anti-osteoporotic effects of SWT extract. The results showed that ovariectomized mice had reduced total body bone mineral density and bone mineral content, and this was reversed by treatment with SWT extract. SWT extract also increased serum levels of the osteogenic markers ALP, BMP-2, and OPN. Therefore, SWT is a novel bone formation agent, which prevents bone loss by ovariectomy in vivo.
The molecular mechanisms underlying osteoporosis are not yet entirely clear. However, they are likely correlated with decreased availability or activity of bone growth factors, including ALP, BMP-2, and OPN. These 3 factors play important roles in the process of bone formation and remodeling , and it has been well discussed that stimulation of osteoblast cell differentiation is characterized mainly by increased expression of ALP, BMP-2, and OPN . In this study, we found that SWT extract increased ALP, BMP-2, and OPN expression and enhanced bone mineralization. Therefore, SWT extract mediates bone formation by upregulating the expression of ALP, BMP-2, and OPN.
Previous studies have reported that PI3K and Akt play important roles in bone formation [25, 26]. Phosphorylation of the p85 subunit is required for activation of the p110 catalytic subunit of PI3K . Here, we showed that SWT extract induced PI3K and Akt phosphorylation, and that pretreatment with inhibitors of these signal proteins antagonized the SWT extract-mediated potentiation of bone mineralization, revealing that PI3K and Akt activation play crucial roles in SWT extract-induced bone formation by osteoblasts. Moreover, inhibitors and siRNA of PI3K and Akt reduced SWT extract-dependent enhancement of ALP, BMP-2, and OPN expression. These results suggest that activation of the PI3K and Akt pathways are required for increased ALP, BMP-2, and OPN expression and maturation by SWT extract in osteoblasts. It has been reported that p38 is involved in the regulation of ALP expression during the differentiation of osteoblastic cells ; similarly ERK1/2 is important for the proliferation and differentiation of osteoblasts . JNK is involved in osteoblast formation . However, we did not examine the role of MAPKs (p38, JNK, and ERK) in SWT extract-mediated bone formation in current study. Whether MAPKs are involved in SWT extract-induced bone formation needs further examination.
NF-κB has been shown to control osteoblast function in bone . The results of our study indicate that NF-κB activation contributes to SWT extract-induced bone mineralization and ALP, BMP-2, and OPN expression in cultured osteoblasts, and that inhibitors of the NF-κB signaling pathway, including PDTC or TPCK, inhibited SWT extract-induced bone mineralization and the expression of ALP, BMP-2, and OPN. Phosphorylation at Ser536 of p65 is crucial for p65 transactivation . The results of this study showed that SWT extract increased the phosphorylation of p65. Taken together, these results suggest that NF-κB activation is required for SWT extract-induced bone formation in cultured osteoblasts.
Our present study indicated that SWT extract induces osteoblast differentiation and maturation. SWT extract also increased ALP, BMP-2, and OPN expression, and bone mineralization. SWT extract-mediated bone formation and the expression of ALP, BMP-2, and OPN were mediated through PI3K, Akt, and NF-κB signaling pathways. Furthermore, SWT extract reversed in vivo bone loss induced by ovariectomy. In conclusion, SWT may be beneficial in stimulating bone formation for the treatment of osteoporotic diseases.
This study was supported by grant from the National Science Council of Taiwan (NSC99-2320-B-039-003-MY3; 100-2320-B-039-028-MY3).
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