Abstract
The effects of water extract of Cajanus cajan (Linn.) Millsp. (Leguminosae) leaves (WECML) on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipocytic trans-differentiation of mouse primary osteoblasts (OBs) were studied. The results indicated that WECML promoted the proliferation of BMSCs and OBs at most concentrations. WECML promoted the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentrations of 0.1, 1, and 10 μg/mL, but inhibited the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentration of 0.01 μg/mL. WECML inhibited the adipogenic differentiation of BMSCs and adipocytic trans-differentiation of OBs at concentrations of 0.001, 0.1, 1, 10, and 100 μg/mL, but had no effects at concentration of 0.01 μg/mL. The results suggest that WECML has protective effects on bone and these protective effects may be mediated by decreasing adipocytic cell formation from BMSCs, which may promote the proliferation, differentiation, and mineralization function of OBs. The defined active ingredients in the WECML and the active mechanism need to be further studied.
Introduction
As the general population is aging, osteoporosis is becoming more prevalent. In the western world, the prevalence of osteoporosis and osteopenia in those aged 55 to 64 years is 20% and 37%, respectively. After the age of 80 years, the prevalence of osteoporosis reaches almost 70%. Therefore, the incidence of hip fractures is expected to increase to three-fold the current value by 2050 (CitationFok et al., 2008).
There are two primary types of drug therapy for osteoporosis: antiresorptive agents and anabolic agents. Antiresorptive drugs reduce bone loss, while anabolic agents stimulate new bone formation. Antiresorptive agents include bisphosphonates, estrogen, selective estrogen receptor modulators, and calcitonin. Anabolic agents include fluoride and anabolic steroids. Among them, estrogen replacement therapy (ERT) used to be a popular regime for prevention and treatment of postmenopausal osteoporosis. However, recent evidence suggests that ERT is associated with increased risk of development of breast, ovarian, and endometrial cancer (CitationDavison & Davis, 2003). Thus, alternative treatment or prevention regimes for osteoporosis are urgently needed (CitationSakamoto et al., 2000).
Traditional Chinese medicine has been widely used in orthopedic clinical practice for thousands of years for the treatment of fractures and joint diseases. Their therapeutic actions are believed to be mediated through multiple signal pathways and cellular targets. Many herbs that are known to possess “kidney-toning” activities or “bone and gonad nourishing” effects demonstrated to be effective in reducing bone loss and promoting fracture healing (CitationWu et al., 2003). Cajanus cajan (Linn.) Millsp. (Leguminosae) is an important drug in traditional Chinese medicine for reducing swelling, alleviating pain, toning kidney, and strengthening bone, and can treat a wide range of diseases including osteoporosis (CitationSun et al., 2001; CitationDornstauder et al., 2001). A drug made of (WECML) has been approved for clinical use by the State Food and Drug Administration (SFDA) in China, and has a remarkable preventive effect on glucocorticoid-induced avascular necrosis of the femoral head. Some experimental results on animals indicate that this drug could increase the number of osteoblasts (OBs) and decrease the number of osteoclasts (OCs) (CitationYuan et al., 2005). So WECML may prevent osteoporosis. But effects of WECML on the osteogenic and adipogenic differentiation of mouse primary BMSCs and the adipogenic trans-differentiation of mouse primary OBs have not been reported so far. In this paper the effects of WECML on the osteogenic and adipogenic differentiation of mouse primary BMSCs and the adipogenic trans-differentiation of mouse primary OBs were studied in order to further elucidate the bone metabolism mechanism of WECML.
Materials and methods
Materials
Kun Ming (KM) mice were purchased from Guangming Weiwu Biological Product Factory (Shenzhen, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Carlsbad, CA). Benzylpenicillin, streptomycin, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), β-glycerophosphate, dexamethasone, ascorbic acid, insulin, alizarin red S (ARS), collagenase II, and oil red O were obtained from Sigma (St Louis, MO). Demethyl sulfoxide (DMSO) was purchased from Sangon (Shanghai, China). An alkaline phosphatase (ALP) activity kit was obtained from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China), micro-protein assay kit was purchased from Beyotime Biotechnology (Haimen, China). The leaves of Cajanus cajan were purchased in Hainan province in China in August 2005 and were identified by Zhu Guo-Yuan (Shenzhen Research Institute of City University of Hong Kong). A voucher specimen was deposited in Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China.
Preparation of WECML
Leaves of Cajanus cajan (5 kg) were extracted with water (50 L) three times, 1.5 h each time. Three extracts were combined, filtered and concentrated to reach a relative density of 1.05~1.10 (60°C), and then subjected to spray drying to yield a syrup residue (0.96 kg). The total flavonoids were measured according to the method previously reported (CitationHuang et al., 2006). The dried substance (50 mg) was dissolved in 10 mL water and subjected to column chromatography over Amerlite XAD 7HP and eluted with distilled water and ethanol respectively. The eluant was collected and evaporated to dryness, the residue was dissolved in ethanol and AlCl3 was added. The content of vitexin was determined by spectrophotometry at a wavelength of 274 nm. The vitexin was calculated according to standard curve and is no less than 0.8 mg in the WECML (0.5 g). Polysaccharide was measured according to the method previously reported (CitationZhao & Cheng, 2006). The dried substance (50 mg) was dissolved in water (5 mL) and ethanol (15 mL) was added. The solution was centrifuged 4,000 rpm/min and the supernatant was removed. The sediment was dissolved in water (5 mL), and ethanol (20 mL) was added again. The solution was centrifuged 4,000 rpm/ min and the supernatant was removed again. The sediment was dissolved in water again and 0.6% phenol solution was added. The content of glucose was measured by spectrophotometry at a wavelength of 490 nm. Glucose was calculated according to standard curve. The total flavonoids calculated as vitexin are no less than 50 mg in the WECML (0.5 g), polysaccharide calculated as glucose is no less than 50 mg in the WECML (0.5g).
Isolation and culture of primary BMSCs
The mouse BMSCs were obtained from adult KM mice (4-6 weeks old) using a modification of the method previously reported (CitationVerma et al., 2002). Briefly, mice were sacrificed by decapitation. Femora and tibiae were aseptically harvested, and the whole bone marrow was flushed using DMEM in a 1 mL syringe and a 25-gauge needle. The cells were collected and cultured in a culture flask. After 3 days incubation in a 37°C, 5% CO2 humidified incubator, the non-adherent cells were removed by gentle aspiration and the medium was replaced with fresh DMEM. Then the medium was changed every 3 days in all the experiments.
Isolation and culture primary of OBs
The mouse OBs were isolated mechanically from newborn mouse skull using a modification of the method previously reported (Carpenter et al., Citation1998). Briefly, skull was dissected from KM mice, endosteum and periosteum were stripped off, cut into approximately 1-2 mm2 pieces, digested with trypsin (2.5 g/L) for 30 min and the digestion was discarded. Then the bone was digested with collagenase II (2 g/L) twice for 1 h each time, and the cells were collected and cultured in a culture flask. After overnight at 37°C in a 5% CO2 humidified incubator, the DMEM was removed. The medium was changed every 3 days in all the experiments.
Cell proliferation assay
The protocol described by CitationMosmann (1983) was followed with some modifications. Briefly, BMSCs (3 × 106 cells per well) or OBs (3 × 104 cells per well) were plated in 96-well culture plates overnight at 37°C in a 5% CO2 humidified incubator. WECML was added to the wells at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/ mL. Control wells were prepared by adding DMEM. Wells containing DMEM without cells were used as blanks. The plates were incubated at 37°C in a 5% CO2 incubator for 44 h. Upon completion of the incubation, MTT dye solution (20 μL, 5 mg/mL) was added. After 4 h incubation, the supernatant was removed and DMSO (100 μL) was added to solubilize the MTT. The optical density (OD) was measured on a microplate spectrophotometer (BioRad Model 3550, BioRad, Ca, USA) at a wavelength of 570 nm. The proliferation rate (%) was calculated according to the formula: (ODtreated/ODcontrol−1) × 100%.
Measurement of ALP activity
The BMSCs were isolated as above. BMSCs (3 × 106 cells per well) were plated in 48-well culture plates, after being induced by osteogenic supplement (10−7 M dexamethasone, 5 mM β-glycerophosphate, 50 μg/mL ascorbic acid) and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL for 2 days. The plates were washed thrice with ice-cold PBS and lysed by two cycles of freezing and thawing. Aliquots of supernatants were subjected to ALP activity and protein measurement by an ALP kit and a micro-protein assay kit, respectively. The osteogenic differentiation promotion rate (%) was calculated according to the formula: (ALP activitytreated/ALP activity control−1) × 100% (CitationLi et al., 2005).
Assay for mineralized matrix formation
The BMSCs were isolated as above. BMSCs (2 × 105 cells per well) were plated in 48-well culture plates overnight at 37°C in a 5% CO2 humidified incubator. After being induced by mineralization supplement containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid and treated with WECML at final concentrations 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL for 20 days. The formation of mineralized matrix nodules was determined by ARS staining. Briefly, the cells were fixed in 70% ethanol for 1 h at room temperature. The fixed cells were washed with PBS and stained with 40 mM ARS, pH 4.2, for 30 min at room temperature. Quantitation of ARS stain was performed by elution with 10% (w/v) cetylpyridium chloride for 10 min at room temperature and measuring the absorbance at 570 nm (CitationGori et al., 2001). Results were expressed as moles of ARS per mg total cellular protein.
Oil red O stain and measurement
The BMSCs or OBs were isolated as above. BMSCs (3 × 106 cells per well) or OBs (1 × 104 cells per well) were plated in 48-well culture plates, after being induced by adipogenic supplement (10 μg/mL insulin, 10−7 M dexthamethone) and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL, the adipocytes from BMSCs and trans-differentiated adipocytes from OBs were measured using the oil red O staining method described by CitationSekiya et al. (2002) with some modifications. Briefly, cell monolayers were fixed in 4% formaldehyde, washed with water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 15 min at room temperature. For quantification, cell monolayers were washed extensively with water to remove unbound dye, and recorded by inverted phase contrast microscopy (Olympus IX 51), and isopropyl alcohol (1 mL) was added. After 5 min, the absorbance was measured by a spectrophotometer at 510 nm. The adipogenic differentiation inhibition rate (%) and adipocytic trans-differentiation inhibition rate (%) was calculated according to the formula: (1-ODtreated/ODcontrol) × 100%.
Statistical analysis
Data were collected from at least three separate experiments. The results were expressed as means ± standard deviation (SD). The statistical differences were analyzed using the SPSS t-test. P values less than 0.05 were considered to indicate statistical differences.
Results
Effect of WECML on the BMSC proliferation
As shown in , WECML promoted the proliferation of BMSCs at all concentrations; moreover, the proliferation rate reached maximal value at a concentration of 100 μg/mL.
Figure 1. Effect of WECML on the proliferation of BMSCs (*P <0.05, **P <0.01 versus control, n = 6). BMSCs were cultured at a density of 3 × 106 per well and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL. MTT dye solution was added when BMSCs were cultured for 44 h. After 4 h incubation, culture medium was removed; DMSO was added and the OD was measured at a wavelength of 570 nm. The proliferation rate (%) was calculated according to the formula: (ODtreated/ODcontrol−1) × 100%.
Effect of WECML on the OB proliferation
As shown in , WECML (0.001~10 μg/mL) promoted the proliferation of OBs, moreover, the proliferation rate reached maximal value at a concentration of 1 μg/mL. WECML inhibited the proliferation of OBs and the inhibition rate was about 12.12% at the higher concentration of 100 μg/mL.
Figure 2. Effect of WECML on the proliferation of OBs (*P <0.05, **P <0.01 versus control, n = 6). OBs were cultured at the density of 3 × 104 per well and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL. MTT dye solution was added when OBs were cultured for 44 h. After 4 h incubation, culture medium was removed, DMSO was added and OD was measured at a wavelength of 570 nm. The proliferation rate (%) was calculated according to the formula: (ODtreated/ODcontrol−1) × 100%.
Effect of WECML on the osteogenic differentiation of BMSCs
As shown in , WECML had no effect on the osteogenic differentiation of BMSCs at concentrations of 0.001 and 100 μg/mL, promoted osteogenic differentiation of BMSCs at concentrations of 0.1, 1, and 10 μg/mL and the proliferation rate reached maximal value at a concentration of 0.1 μg/mL, but inhibited osteogenic differentiation of BMSCs at a concentration of 0.01 μg/mL.
Figure 3. Effect of WECML on the osteogenic differentiation of BMSCs (*P <0.05, **P <0.01 versus control, n = 6). The BMSCs were cultured in a culture medium, after being induced by osteogenic supplement (10−7 M dexamethasone, 5 mM β-glycerophosphate, 50 μg/mL ascorbic acid) and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL for 2 days. The medium was removed; ALP activity and protein were measured by an ALP kit and a micro-protein assay kit respectively. The osteogenic differentiation promotion rate was calculated according to the formula: (ALP activity treated/ALP activity control−1) × 100%.
Effect of WECML on the formation of mineralized matrix nodules
As shown in , WECML had no effect on the formation of mineralized matrix nodules of BMSCs at concentrations of 0.001 and 100 μg/mL, promoted the formation of mineralized matrix nodules of BMSCs at concentrations of 0.1, 1, and 10 μg/mL and the proliferation rate reached maximal value at concentration of 0.1 μg/mL, but inhibited the formation of mineralized matrix nodules of BMSCs at a concentration of 0.01 μg/mL.
Figure 4. Effect of WECML on the mineralized nodule formation of BMSCs (*P <0.05, versus control, n = 6). The BMSCs were cultured in a culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL for 20 days. The mineralized matrix nodules were determined by ARS staining. Quantitation of ARS staining was performed by elution with 10% (w/v) cetylpyridium chloride for 10 min at room temperature and measuring the absorbance at 570 nm. Results were expressed as moles of ARS per mg total cellular protein.
Effect of WECML on the adipogenic differentiation of BMSCs
As shown in , WECML inhibited adipogenic differentiation of BMSCs, except for 0.01 μg/mL, and the inhibition rate reached maximal value at concentration of 100 μg/mL. The morphological observation was accorded with the experimental results ().
Figure 5. Effect of WECML on the adipogenic differentiation of BMSCs (*P <0.05, **P <0.01 versus control, n = 6). The BMSCs were plated in 48-well culture plates after being induced by adipogenic supplement (10 μg/mL insulin, 10−7 M dexthamethone) and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL. The adipocytes from BMSCs were fixed in 4% formaldehyde, washed with water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 15 min at room temperature. Then cell monolayers were washed extensively with water to remove unbound dye, isopropyl alcohol (1 mL) was added. After 5 min, the absorbance was measured by a spectrophotometer at 510 nm. The adipogenic differentiation inhibition rate (%) was calculated according to the formula: (1-ODtreated/ODcontrol) × 100%.
Figure 6. Effect of WECML on the adipogenic differentiation of BMSCs. The BMSCs were plated in 48-well culture plates, after being induced by adipogenic supplement (10 μg/mL insulin, 10−7 M dexthamethone) and treated with WECML at final concentrations of 0.1 and 100 μg/mL. The adipocytes from BMSCs were fixed in 4% formaldehyde, washed with water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 15 min at room temperature. Then cell monolayers were washed extensively with water to remove unbound dye, and recorded by inverted phase contrast microscopy (Olympus IX 51). (A) adipogenic supplement, (B) adipogenic supplement + 0.1 μg/mL WECML, (C) adipogenic supplement + 100 μg/mL WECML.
Effect of WECML on adipocytic trans-differentiation of OBs
As shown in , WECML inhibited adipocytic trans-differentiation of OBs except for 0.01 μg/mL.
Figure 7. Effect of WECML on the adipocytic trans-differentiation of OBs (*P <0.05, **P <0.01 versus control, n = 6). The OBs were plated in 48-well culture plates after being induced by adipogenic supplement (10 μg/mL insulin, 10−7 M dexthamethone) and treated with WECML at final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μg/mL. The adipocytes from OBs were fixed in 4% formaldehyde, washed with water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 15 min at room temperature. Then cell monolayers were washed extensively with water to remove unbound dye, isopropyl alcohol (1 mL) was added. After 5 min, the absorbance was measured by a spectrophotometer at 510 nm. The adipocytic trans-differentiation inhibition rate (%) was calculated according to the formula: (1-ODtreated/ODcontrol) × 100%.
Discussion
BMSCs are pluripotent cells which have the capacity to differentiate into OBs, adipocytes, chondrocytes, myoblasts, or fibroblasts (CitationDominici et al., 2001). It is now hypothesized that an increase in the number of adipocytes occurs at the expense of OBs in osteopenic disorders. Furthermore, there is more and more evidence that suggests a large degree of plasticity exists between OBs and adipocytes, and their trans-differentiation is reciprocal (CitationBeresford et al., 1992). The precise role of adipocytes in bone marrow is unknown. It has been suggested that they act a purely passive role to fill marrow cavities and play a role in lympho-hematopoiesis. They may also serve an active role in the energy metabolism. Recent data suggest that medullary adipocytes are secretory cells that may affect hematopoiesis and osteogenesis (CitationAilhaud et al., 1992). It was reported that preadipocytes isolated from mouse marrow may regulate the activity and final differentiation of OBs. The condition medium harvested from mouse stromal preadipocytes decreased the ALP activity of a mouse stromal osteoblastic cell line (CitationBenayahu et al., 1993). Some investigators suggest that adipocytes might be involved in hematopoietic and osteogenic process by supplying the necessary soluble cell surface factors for OC differentiation and function in vitro (CitationKelly et al., 1998). CitationSakaguchi et al. (2000) demonstrated that adipocyte-enriched stromal cells support OC formation. CitationBenayahu et al. (1994) reported that preadipocytes also have the potential to stimulate OC differentiation. Adipocytes synthesized and released a variety of peptide, non-peptide compounds or secreted cytokines such as tumor necrosis factor-α (TNF–α) and interleukin (IL-6), and the main effect of these cytokines is a stimulation of bone resorption (CitationAilhaud et al., 1992). It was reported that there was a therapeutic opportunity to either prevent or treat osteopenic disorders by inhibiting marrow adipogenesis (CitationNuttall & Gimble, 2000). So a reversal of adipogenesis will provide an important therapeutic approach to prevent aged-related and steroid-induced osteoporosis (CitationSong, 2002).
In this study we have examined the effects of WECML on the osteogenic and adipogenic differentiation of BMSCs and the adipogenic trans-differentiation of OBs in vitro by employing isolated mouse primary BMSCs and OBs. Our results showed that: 1) WECML promoted the proliferation of BMSCs and OBs at most concentrations; 2) WECML promoted the osteogenic differentiation of BMSCs except individual concentrations; 3) WECML promoted the formation of mineralized matrix nodules of BMSCs at concentrations of 0.1, 1, and 10 μg/mL; 4) WECML inhibited the adipogenic differentiation of BMSCs and the adipocytic trans-differentiation of OBs except for 0.01 μg/mL. These results indicated that WECML may have a protective effect on bone and these protective effects may be mediated by decreasing adipocytic cell formation from BMSCs, which may promote the proliferation, differentiation and mineralization function of OBs.
Cajanus cajan leaves have been reported to contain vitexin, isovitexin, apigenin, luteolin, naringenin-dimethylether, longistyline A, longistyline C, pinostorbin, salicylic acid, etc. (CitationBhanumati et al., 1979; CitationCooksey et al., 1982; CitationDuker-Eshun et al., 2004). The defined active ingredients in the WECML and the mechanism of the effects of them on the osteogenic and adipogenic differentiation of BMSCs and the adipogenic trans-differentiation of OBs remains to be further studied.
Declaration of interest: This work was supported by the Foundation for Key Program of the Ministry of Education of China (No.208018), the Natural Science Foundation of Hebei University and Returned Scholars of Hebei Province (No.207041). The authors alone are responsible for the content and writing of the paper.
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