Cistanche Deserticola Extract Increases Bone Formation in Osteoblasts--Part I

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Te-Mao Li^a et a

Abstract
Objectives We investigated the effect of Cistanche deserticola Ma. (CD) on bone formation by cultured osteoblasts. Methods The mineralized nodule formation assay was used to examine the in-vitro effects of CD (Cistanche deserticola)on bone formation. Alkaline phosphatase (ALP), bone morphogenetic proteins (BMP)-2, and osteopontin (OPN) mRNA expression were analyzed by quantitative real-time polymerase chain reaction. The mechanism of action of CD (Cistanche deserticola) extract was investigated using Western blotting. The in-vivo anti-osteoporotic effect of CD (Cistanche deserticola) extract was assessed in ovariectomized mice.
Key findings

CD (Cistanche deserticola) extract had no effect on the proliferation, migration, or wound healing of cultured osteoblasts, but increased ALP, BMP-2, and OPN mRNA and bone mineralization. Mitogen-activated protein kinase (MAPK) or nuclear factor (NF)-kB inhibitors reduced CD (Cistanche deserticola) extract-induced bone formation and ALP, BMP-2, and OPN expression. However, CD (Cistanche deserticola) extract did not affect osteoclastogenesis. In addition, CD (Cistanche deserticola) extract prevented the bone loss induced by ovariectomy in vivo.
Conclusions

CD (Cistanche deserticola) may be a novel bone formation agent for the treatment of osteoporosis.

Keywords bone formation; Cistanche deserticola; Chinese herb; osteoblasts

Cistanche deserticola is good for bone formation

Introduction

Bone is a complex tissue composed of several cell types that undergo a continuous process of renewal and repair termed ‘bone remodeling’. Two major cell types responsible for bone remodeling are osteoclasts, which resorb bone, and osteoblasts, which form new bone. Bone remodeling is regulated by several systemic hormones (e.g. parathyroid hormone, 1, 25-dihydroxy vitamin D3, sex hormones, and calcitonin) and local factors (e.g. nitric oxide, prostaglandins, growth factors, and cytokines).[1] Osteoporosis results when resorption and formation of bone are uncoordinated and excess bone breakdown exceeds bone building.[2] Current treatments for osteoporosis include bisphosphonates, calcitonin, and estrogen, which are bone resorption inhibitors that maintain bone mass by inhibiting osteoclast activity.[3] However, the effect of these drugs in increasing or recovering bone mass is relatively small, certainly no more than 2% per year.[3] There is, therefore, a need for bone-building agents, such as teriparatide, that stimulate new bone formation and correct the change in trabecular microarchitecture that is characteristic of established osteoporosis.[4,5] Because new bone formation is primarily a function of the osteoblast, agents that act by either increasing the proliferation of cells of the osteoblastic lineage or inducing differentiation of the osteoblasts can enhance bone formation.[5,6]
Although the mechanisms of osteoporosis remain unclear, they are likely related to the decreased availability or effects of bone growth factors, such as bone morphogenetic proteins (BMPs).[7] BMPs, which are structurally related to the transforming growth factor-b superfamily, were originally identified by their capacity to induce ectopic bone formation in rodents.[8] BMPs play an important role in bone formation and bone cell differentiation by stimulating alkaline phosphatase (ALP) activity as well as the synthesis of proteoglycan, collagen, and osteopontin (OPN).[9] A recent study found a link between osteoporosis and specific polymorphisms in the BMP-2, ALP, and OPN genes, suggesting they are associated with the development of osteoporosis.[10]

Cistanche deserticola Ma. (Orobanchaceae; abbreviated as CD) (Cistanche deserticola) is a native herb in China and is widely used in traditional medicine as a therapeutic agent due to its sedative, analgesic, and immunostimulatory properties.[11] Modern pharmacological studies have shown that CD (Cistanche deserticola) extracts can stimulate immunity,[12] scavenge free radicals[13], and increase the activity of superoxide dismutase.[14] Anti-inflammatory and antioxidant agents have the potential to treat osteoporosis by increasing bone formation and/or suppressing bone resorption.[15,16] However, the effect of CD (Cistanche deserticola) on bone cell function has not yet been determined. Here, we report that CD (Cistanche deserticola) extract did not affect the proliferation, migration, or wound healing activity of cultured osteoblasts, but did increase ALP, BMP-2, and OPN expression and bone mineralization. In addition, we show that the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kB signaling pathways may be involved in the CD-mediated increase in gene expression and bone mineralization. In contrast, the CD (Cistanche deserticola) extract did not suppress osteoclastogenesis in vitro. Notably, treatment of mice with CD (Cistanche deserticola) extract prevented bone loss induced by ovariectomy in vivo. Our data, therefore, suggest that CD (Cistanche deserticola) may be used to stimulate bone formation for the treatment of osteoporosis.

Cistanche deserticola boost the immunity

Materials and Methods

Cistanche deserticola extract and materials

CD (Cistanche deserticola) extract was purchased from Chuang Song-Zong Pharmaceutical Company (Kaohsiung, Taiwan). Extraction and isolation of CD (Cistanche deserticola) were followed as previously described (on the basis of spectral data they have identified the chemical composition including beta-sitosterol, daucosterol, succinic acid, triacontanol, acteoside, betaine, and polysaccharide).[17] Rabbit polyclonal antibodies specific for p-extracellular signal-regulated kinases (ERK), ERK, p-p38, p38, p-c-Jun N-terminal kinases (JNK), JNK, p-p65, and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). The osteocalcin ELISA kit was purchased from Biosource Technology (Nivelles, Belgium). The C-terminal telopeptides of the type-I collagen ELISA kit were obtained from Cross Laps (Herlev, Denmark). The p38 dominant-negative mutant was provided by Dr. J. Han (South-Western Medical Center, Dallas, USA). The JNK dominant-negative mutant was provided by Dr. M. Karin (University of California, San Diego, USA). The ERK2 dominant-negative mutant was provided by Dr. M. Cobb (South-Western Medical Center). All other reagents were obtained from Sigma-Aldrich (St Louis, USA).

Cell culture

The murine osteoblast cell line MC3T3-E1 was purchased from American Type Culture Collection (ATCC; Rockville, USA). Cells were cultured in 95% air, 5% CO2 with a-MEM that was supplemented with 20 mm HEPES and 10% heat-inactivated fetal calf serum, 2 mm-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Measurement of mineralized nodule formation

Mineralized nodule formation was evaluated as described.[18] In brief, osteoblasts were cultured in a medium containing vitamin C (50 mg/ml) and b-glycerophosphate (10 mm) for two weeks, and the medium was changed every three days. After incubation with CD (Cistanche deserticola) extract for 12 days, 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 compared with a standard curve. One mole of alizarin red-S selectively binds approximately two moles of calcium.

Cistanche deserticola is good for bone formation

Quantitative real-time polymerase chain reaction

Total RNA was extracted from osteoblasts using a TRIzol kit (MDBio Inc., Taipei, Taiwan). Reverse transcription was performed using 2 mg total RNA and an oligo(dT) primer.[19,20] Quantitative real-time polymerase chain reaction (qPCR) was carried out using TaqMan® one-step PCR Master Mix (Applied Biosystems, Carlsbad, USA). Total cDNA was added (100 ng per 25-ml reaction) with sequence-specific primers and Taqman® probes. All target gene primers and probes were purchased commercially, including b-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 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).

Western blot analysis

Cell lysates were prepared as described previously.[21,22] Proteins were resolved by SDS-PAGE and transferred to Immobilon polyvinyl difluoride membranes (Millipore, Billerica, 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 p-65, or p-p65 (1: 1000) for 1 h at room temperature. After three 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, USA).

Ovariectomy-induced osteoporosis

Female ICR mice, four weeks 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 CD (Cistanche deserticola) extract every two days for four weeks. Total body bone mineral density and bone mineral content were determined by a dual-energy X-ray absorptiometer (DEXA; XR-26; Norland, Fort Atkinson, USA) using a mode for small subjects as described previously.[18,23] All protocols complied with institutional guidelines and were approved by the Animal Care Committee of China Medical University.

Statistical analysis

Statistical analysis was performed using Prism 4.01software. (GraphPad Software Inc., San Diego, USA). The values given are mean SEM. Statistical analysis between two samples was performed using the Student’s t-test. Statistical comparisons of more than two groups were performed using a one-way analysis of variance with Bonferroni's posthoc test. In all cases, P < 0.05 was considered significant

Cistanche deserticola is good for bone formation

Results

Cistanche deserticola extract increases bone mineralization by osteoblasts Addition of the CD (Cistanche deserticola) extract for 24 or 48 h did not affect the proliferation of mouse osteoblast MC3T3-E1 cells in an MTT assay (data not shown). Because osteoblast differentiation is a complex process involving cell proliferation and migration, we also tested the migratory ability of MC3T3-E1 cells after treatment with CD (Cistanche deserticola) extract. Using Transwell and wound healing assays, we found that CD (Cistanche deserticola) extract did not affect osteoblast migration (data not shown). The formation of mineralized nodules is one of the markers of osteoblast maturation. Alizarin red S staining showed that mineralized nodules formed when osteoblasts were cultured for two weeks in a medium containing vitamin C (50 μg/ml) and β-glycerophosphate (10 mm), the concentration-dependent manner by the addition of CD (Cistanche deserticola) (Figure 1a). Therefore, CD (Cistanche deserticola) extract induced bone nodule formation by cultured osteoblasts, but not their proliferation or migration. We also examined the role of CD (Cistanche deserticola) extract in RANKL-induced osteoclastogenesis but found no effect (data not shown).

Cistanche deserticola extract increases alkaline phosphatase, bone morphogenetic protein-2, and osteopontin expression in cultured osteoblasts

Differentiated osteoblasts exhibit elevated ALP activity, which correlates with high levels of enzyme expression.[18] We found that treatment with CD (Cistanche deserticola)extract for 72 h significantly increased osteoblast ALP activity (Figure 1b). Given the crucial role of BMP-2, ALP, and OPN in osteoblast differentiation, we tested whether CD (Cistanche deserticola) extract exerts its effect on osteoblast differentiation by regulating BMP-2, ALP, and OPN expression. Treatment of cells with CD (Cistanche deserticola) extracts increased mRNA expression of ALP, BMP-2, and OPN in a concentration-dependent manner (Figure 1c), demonstrating that CD (Cistanche deserticola) extract induced differentiation of osteoblasts by upregulating ALP, BMP-2, and OPN expression.

Cistanche deserticola extract increases bone nodule formation through mitogen-activated protein kinase pathway

It has been reported that MAPK plays an important role in bone formation[24,25] so we next examined whether this signaling pathway is involved in CD (Cistanche deserticola) extract-induced bone mineralization. Pretreatment of cells with ERK inhibitor U0126, p38 inhibitor SB203580, or JNK inhibitor SP600125 reduced CD (Cistanche deserticola) extract-increased bone mineralization (Figures 2a, 3a, and 4a). Furthermore, the inhibitors also blocked CD (Cistanche deserticola) extract-induced ALP, BMP-2, and OPN expression (Figures 2b–4c). Transfection of cells with ERK2, p38, or JNK mutant reduced CD (Cistanche deserticola) extract-increase in ALP, BMP-2, and OPN mRNA expression (Figures 2c, 3c, and 4c). Next, we directly examined the ERK, p38, and JNK activation after CD (Cistanche deserticola) extract treatment. Incubation of cells with CD (Cistanche deserticola) extract induced ERK, p38, and JNK phosphorylation (Figures 2d, 3d, and 4d). Therefore, ERK, p38, and JNK mediate the increased bone formation induced by CD (Cistanche deserticola) treatment of osteoblasts.

Cistanche deserticola extract increases bone nodule formation through the nuclear factor-kB pathway

As previously mentioned, NF-kB activation is necessary for bone formation.[26,27] We next treated osteoblasts with theNF-kB inhibitors pyrrolidine dithiocarbamate (PDTC) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were purchased from Calbiochem (Darmstadt, Germany) to determine whether NF-kB activation is involved in CD (Cistanche deserticola) extract-induced bone mineralization. Figure 5a shows that pretreatment of osteoblasts with PDTC or TPCK inhibited CD (Cistanche deserticola) extract-induced bone nodule formation, and also reduced the increase in ALP, BMP-2, and OPN expression (Figure 5b and 5c). Phosphorylation of the NF-kB p65 subunit is an indicator of NF-kB activation;[28] consistent with this, we found that CD (Cistanche deserticola) extract increased p65 phosphorylation in the osteoblasts (Figure 5d). These results indicated that NF-kB activation is important for CD extract-induced ALP, BMP-2, and OPN expression and bone nodule formation.

Figure 4 Involvement of c-Jun N-terminal kinases (JNK) in Cistanche deserticola (CD) extract-induced bone mineralization in osteoblasts. (a) Cells were plated in 24-well plates and cultured in a medium containing vitamin C (50 mg/ml) and b-glycerophosphate (10 mM) for two weeks. The cells were concomitantly treated with CD (Cistanche deserticola) extract (26 mg/ml) plus SP600125 (3 mM). The mineralized nodule formation was assessed by alizarin red-S staining (n = 5). (b, c) Cells were pretreated with SP600125 for 30 min or transfected with JNK mutant or 24 h followed by stimulation with CD extract, and alkaline phosphatase (ALP) activity and ALP, bone morphogenetic protein (BMP)-2 and osteopontin (OPN) mRNA expression was measured by ALP kit and quantitative real-time polymerase chain reaction. (d) Cells were treated with CD extract for indicated time intervals, and p-JNK expression was examined by Western blotting. *P< 0.05, compared with control group; #P< 0.05, compared with CD (Cistanche deserticola) extract-treated group.

Inhibition of bone loss by Cistanche deserticola extract in ovariectomized mice

To examine the effect of CD (Cistanche deserticola) extract on bone loss, osteoporosis was induced in female mice by ovariectomy. As expected, ovariectomized mice displayed decreased total body bone mineral density and bone mineral content (Figure 6a and 6b). Treatment with CD (Cistanche deserticola) extract for four weeks inhibited the loss of bone mineral density and bone mineral content in a dose-dependent manner (Figure 6a and 6b). Blood concentrations of ALP can reflect the osteoblastic activity.[29] As shown in Figure 6c, CD (Cistanche deserticola) extract inhibited the decrease in serum ALP activity induced by ovariectomy. In addition, CD (Cistanche deserticola) extract also increased the level of osteocalcin, a marker of bone formation, and reduced the level of C-terminal telopeptides of type-I collagen, a marker of bone resorption (Figure 6d and 6e). These findings thus support the potency and efficacy of CD (Cistanche deserticola) extract in preventing bone loss in vivo.

Figure 6 Inhibition of ovariectomy-induced the decrease of bone mineral density and bone mineral content by Cistanche deserticola (CD) extract. Female ICR mice were given a sham operation or were ovariectomized. Ovariectomized mice were treated with the indicated concentrations of CD (Cistanche deserticola) extract by oral feeding. Total body bone mineral density (a), bone mineral content (b), serum alkaline phosphatase (ALP) activity (c), serum osteocalcin (d), and serum C-terminal telopeptides of type I collagen (e), were determined four weeks after surgery. Data are presented as mean SE, n=8–10 mice/group. *P < 0.05, compared with sham; #P < 0.05, compared with ovariectomized group.

DISCUSSION

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From: 'Cistanche deserticola extract increases bone formation in osteoblasts' by Te-Mao Li^a et al

---© 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 897–907