Cistanche Deserticola Extract Increases Bone Formation in Osteoblasts
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Te-Mao Lia, Hsin-Chih Huangb, Chen-Ming Suc, Tin-Yun Hoa, Chi-Ming Wua, Wen-Chi Chend, Yi-Chin Fonga,e and Chih-Hsin Tangf,c
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 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 extract was investigated using Western blotting. The in-vivo anti-osteoporotic effect of CD extract was assessed in ovariectomized mice. Key findings CD 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 extract-induced bone formation and ALP, BMP-2, and OPN expression. However, CD extract did not affect osteoclastogenesis. In addition, CD extract prevented the bone loss induced by ovariectomy in vivo. Conclusions CD may be a novel bone formation agent for the treatment of osteoporosis.
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-hydroxyvitamin 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 break-down 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 trans-forming 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) 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 pharmaco- logical studies have shown that CD extracts can stimulate immunity,[12] scavenge free radicals[13], and increase the activity of superoxide dismutase.[14] 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 CD on bone cell function has not yet been determined. Here, we report that CD 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 extract did not suppress osteoclastogenesis in vitro. Notably, treatment of mice with CD extract prevented bone loss induced by ovariectomy in vivo. Our data, therefore, suggest that CD may be used to stimulate bone formation for the treatment of osteoporosis.
Cistanche deserticola
Materials and Methods
Cistanche deserticola extract and materials
CD extract was purchased from Chuang Song-Zong Pharmaceutical Company (Kaohsiung, Taiwan). Extraction and isolation of CD 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 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.
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 Ste- pOnePlus 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 trichloro- acetaldehyde (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 extracts 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 4.01soft- ware. (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 one-way analysis of variance with Bonfer- Roni's posthoc test. In all cases, P < 0.05 was considered significant.
Cistanche deserticola extract
Results
Cistanche deserticola extract increases bone mineralization by osteoblasts
The addition of the CD 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 extract. Using Transwell and wound healing assays, we found that CD 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 mg/ml) and b-glycerophosphate (10 mm), and this was increased in a concentration-dependent manner by the addition of CD (Figure 1a). Therefore, CD extract induced bone nodule formation by cultured osteoblasts, but not their proliferation or migration. We also examined the role of CD 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 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 extract exerts its effect on osteoblast differentiation by regulating BMP-2, ALP, and OPN expression. Treatment of cells with CD extract increased mRNA expression of ALP, BMP-2, and OPN in a concentration-dependent manner (Figure 1c), demonstrating that CD 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 extract-induced bone mineralization. Pretreatment of cells with ERK inhibitor U0126, p38 inhibitor SB203580, or JNK inhibitor SP600125 reduced CD extract-increased bone mineralization (Figures 2a, 3a, and 4a). Furthermore, the inhibitors also blocked CD extract-induced ALP, BMP-2, and OPN expression (Figures 2b–4c). Transfection of cells with ERK2, p38, or JNK mutant reduced CD 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 extract treatment. Incubation of cells with CD 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 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 the NF-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 extract-induced bone mineralization. Figure 5a shows that pretreatment of osteoblasts with PDTC or TPCK inhibited CD 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 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.
Inhibition of bone loss by Cistanche deserticola extract in ovariectomized mice
To examine the effect of CD 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 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 osteoblastic activity.[29] As shown in Figure 6c, CD extract inhibited the decrease in serum ALP activity induced by ovariectomy. In addition, CD 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 extract in preventing bone loss in vivo.
Discussion
Cistanche deserticola Ma, a native herb in China, is widely used in traditional medicine for various therapeutic treatments due to its sedative, analgesic, and immunostimulatory activity.[11–15] Here, we showed that CD extract induced bone mineralization in cultured osteoblasts, but did not affect their cell migration or proliferation. In addition, we found that ALP, BMP-2, and OPN are target proteins of CD extract-induced signaling, which requires activation of ERK, p38, JNK, and NF-kB.
Bone is a complex tissue composed of several cell types which are continuously undergoing a process of renewal and repair.[30] When resorption and formation of bone are imbalanced, bone breakdown overrides bone formation and osteoporosis results.[30] We used ovariectomized mice to examine the anti-osteoporotic effect of CD extract. Ovariectomized mice had reduced total body bone mineral density and bone mineral content, which was alleviated by treatment with CD extract. CD extract also increased serum levels of the osteogenic marker ALP and osteocalcin. Therefore, the CD is a bone formation agent that prevents bone loss brought about by ovariectomy in vivo.
Although the mechanisms of osteoporosis are not entirely clear, they are likely related to the decreased availability, or decreased effects, of bone growth factors, such as ALP, BMP-2 and OPN. These three factors play important roles in the process of bone formation and remodeling,[31] and it has been well documented that stimulation of osteoblast cell differentiation is characterized mainly by increased expression of ALP, BMP-2 and OPN.[32] In this study, we found that CD extract increased ALP, BMP-2 and OPN expression and enhanced bone mineralization. Therefore, CD extract mediates bone formation in part by upregulating the expression of ALP, BMP-2, and OPN.
It has been reported that p38 is involved in the regulation of ALP expression during the differentiation of osteoblastic cells;[33] similarly, ERK1/2 is important for the proliferation and differentiation of osteoblasts.[34,35] JNK is involved in osteoclast formation.[36] We showed here that CD extract induced ERK, p38 and JNK phosphorylation, and inhibitors of these enzymes antagonized the CD extract-mediated potentiation of bone mineralization, suggesting that ERK, p38 and JNK activation play an obligatory role in CD extract-induced bone formation by osteoblasts. In addition, the enzyme inhibitors and dominant-negative mutants of ERK, p38 and JNK reduced CD extract-enhanced ALP, BMP-2 and OPN expression. These data suggest that activation of the ERK, p38 and JNK pathways is required for the increase of ALP, BMP-2 and OPN expression and maturation caused by CD extract in osteoblasts. ERK has been reported to increase osteoblast proliferation and differentiation.[34,35] However, we did not detect an effect of CD extract on osteoblast proliferation. Therefore, other pathways may have counteracted the ERK effect after CD extract stimulation, or be necessary for proliferation. In this regard, we have found that PI3K and Akt inhibitors also reduce CD extract-induced bone mineralization and ALP, BMP-2 or OPN mRNA expression (data not shown). Therefore, these pathways may be required for CD extract-induced bone formation.
NF-kB has been shown to control osteoblast function in bone.[37] The results of this study show that NF-kB activation contributes to CD extract-induced bone mineralization and ALP, BMP-2 and OPN expression in cultured osteoblasts and that inhibitors of the NF-kB-dependent signaling pathway (PDTC and TPCK) inhibited CD extract-induced bone mineralization and ALP, BMP-2 and OPN expression. p65 is phosphorylated at Ser536 by a variety of kinases in several signaling pathways, which enhances the p65 transactivation potential.[38] The results of this study showed that CD extract increased the phosphorylation of p65. Taken together, these results suggest that NF-kB activation is required for CD extract-induced bone formation in cultured osteoblasts. We also found that ERK, p38 and JNK inhibitors reversed CD extract-induced NF-kB luciferase activity (data not shown), consistent with these enzymes being upstream mediators of CD extract-induced NF-kB activation. Therefore, CD extract induces bone formation through the ERK/p38/JNK/ and NF-kB pathways.
Conclusions
This study demonstrated that CD extract induces osteoblast differentiation and maturation but not proliferation or migration. CD extract also increased ALP, BMP-2 and OPN expression and bone mineralization. We showed that the ERK, p38, JNK and NF-kB pathways are involved in CD extract-mediated bone formation and ALP, BMP-2 and OPN expression. Furthermore, CD extract prevented in-vivo bone loss induced by ovariectomy. Therefore, CD may be beneficial in stimulating bone formation in the treatment of osteoporotic diseases.
