Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts
© Siddiqui and Arshad; licensee BioMed Central Ltd. 2014
Received: 3 August 2014
Accepted: 1 November 2014
Published: 20 November 2014
Osteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal rats.
The ethanolic extract of PG was subjected to evaluate cell proliferation, regeneration, mineralization and formation of collagen matrix using MTT, alkaline phosphatase, Alizarin Red-S staining and Sirius Red dye, respectively. Cell cycle progression and osteogenic gene Runx2 expression were carried out by flow cytometry and real time PCR, respectively.
Exposure of different concentrations (10-100 μg/ml) of the extract on osteoblastic cells showed characteristic morphological changes and increment in cell number. A significant growth in cell proliferation, ALP activity, collagen contents and matrix mineralization of osteoblasts in a dose dependent manner (p < 0.05), suggested that PG has a stimulatory effect on osteoblastic bone formation or potential activity against osteoporosis. In addition, PG extract also enhanced DNA content in S phase of cell cycle and Runx2 gene expression level in osteoblasts.
The data clearly indicated that PG promoting bone cell proliferation and differentiation in primary osteoblasts might be due to elevating the osteogenic gene Runx2 expression. The present study provides an evidence for PG could be a promising herbal medicinal candidate that able to develop drugs for osteoporosis.
KeywordsCell cycle Osteoblast Osteogenesis Punica granatum Runx2
Osteoporosis is a metabolic bone disorders that afflicts about 200 million people worldwide. It is mainly prevalent in women (approximately 80%) and also older men . The bone remodeling process is the alternative of this severe concern, and it's dependable for repair of damage or formation and resorption of bones to maintain the integrity of the skeleton. However, any abnormalities in remodeling that lead to fracture in the bones and osteoporosis.
Recently, several conventional and antiresorptive drugs are used to reduce fracture risk in osteoporosis including hormone replacement therapy (HRT), bisphosphonates, selective estrogen receptor modulators (SERMs) and calcitonin . However, these drugs and therapy have multiple side effects, which causes heart related issues, headache, dizziness, anorexia, cramping of legs and gastrointestinal related problem, particularly pain in stomach and heartburn . The research still continues for the enhancement of such benefit to lower risk involved in human beings.
Anabolic or osteogenic therapies are preferred for pharmacological development to treat osteoporosis . Parathyroid hormone (PTH 1-34) only anabolic agent for the treatment of postmenopausal osteoporosis that is also recommended by the FDA (Food and Drug Administration) which regulates the formation of bones by enhancing the cell proliferation of the osteoblastic lineage or inducing differentiation of osteoblast progenitor cells. Whilst, this therapy is also related to an increased risk of cancer, such as osteosarcoma ,.
In the recent time, several medicinal plants are used for health care treatments and management especially bone related diseases. Although compared with other drug treatments, herbal products create no/little side effects. Punica granatum (Pomegranate, PG) is one of the most potent fruit-bearing medicinal herbs widely distributed throughout the Mediterranean region of southern Europe, northern Africa and tropical Africa, Indian subcontinent, Central Asia and the drier parts of South-East Asia. PG seeds contain punicic acid, ellagic acid, steroidal estrogen and non-steroidal phytoestrogens, including comesten, coumoestrol and isoflavones genistein, daidzein and ascorbic acids. PG also contains estrogenic compound such as luteolin, quercetin, kaempferol, estrone and estradiol, which are responsible for bone formation and also inhibit the resorption process -. A non-isoflavone phytoestrogenic compounds such as quercetin, rutin, apigenin and coumestrol has also been reported in various legumes . A crude PG extract and its seed oil enhance bone healing properties and prevent loss of bones because of the proliferation of osteoblast, inhibition of osteoclast cell and also decrease the inflammation. ,. Recently, PG has been also utilized to inhibit acetyl cholinesterase activity as a new source for management of Alzheimer's disease .
Osteoblast differentiation and proliferation mediated by different growth factors such as bone morphogenetic proteins (BMPs), transforming growth factor beta (TGFβ) and core-binding factor alpha1 (Cbfα1) are known to be targeted the osteogenic Runt-related transcription factor2 (Runx2) gene . Runx2 is the key transcription factor initiating and regulating the early osteogenesis and late mineralization of bone. Furthermore, Runx2 triggers the expression of major bone matrix genes during the early stages of osteoblast differentiation .
There is no evidence regarding the investigation of the PG seed extract on bone cell proliferation, differentiation, and collagen matrix formation in primary culture of osteoblasts. The present study also described the matrix mineralization activity along with osteogenic gene Runx2 expression by real time PCR and DNA content analysis in the S phase of the cell cycle in osteoblastic cells. The findings suggested that PG may be a potent osteogenic herbal drug that induces bone cells proliferation and regeneration following increased DNA contents and Runx2 gene expression which provide future prospects in the development of anti-osteoporotic drugs and therapy.
Materials and methods
Reagents and chemicals
Alpha modified minimum essential medium (α-MEM), fetal bovine serum (FBS), MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) dye, p-nitrophenyl phosphate (pNPP), naphthol ASMX phosphate, fast blue BB salt, ascorbic acid and Sirius Red dye were purchased from Himedia, India. β-glycerophosphate, Ribonuclease (RNase) A and propidium iodide (PI) were purchased from Sigma-Aldrich, USA. RNAiso Plus reagent was procured from Takara, India. cDNA synthesis kit was purchased from Thermo Scientific, USA and SYBER green kit from Roche, USA. All the reagents used were of high purity grade.
Plant materials and extraction
The fresh cultivated PG plant was collected from nearby University of Lucknow, Lucknow, India. Plant materials were identified and authenticated from Department of Pharmacognosy, Faculty of Pharmacy, Integral University, Lucknow. A reference specimen (voucher No. IU/PHAR/HRB/14/08) has been deposited in the herbarium of Faculty of Pharmacy, Integral University, Lucknow. Seed parts of collecting plant were air dried in the shade and crushed to a powder in a mechanical grinder. The 95% ethanolic extract of PG was prepared with the help of Soxhlet apparatus (Borosil Glass Works Limited, India) at 60°C and Whatman No. 1 filter paper was used to obtain filtrate of extract. The filtrate was concentrated in vacuum at 40°C using a rotary evaporator (BUCHI Rotavapor R-205, Switzerland).
Primary culture of osteoblasts
Osteoblastic cells were isolated from neonatal rat pups calvaria using sequential digestion with slight modification . Briefly, calvaria were dissected from four to five neonatal (1-2 days old) rat pups. After removal of sutures and adherent mesenchymal tissues, calvaria were subjected to five sequential (10-15 min) digestions at 37°C in shaking water bath at 120 rpm containing each of 0.1% dispase and collagenase type II enzymes. Supernatants were pooled from the second to fifth digestions in a tube containing 800 μl FBS. Cells were re-suspended in α-MEM containing 10% FBS with 1% penicillin/streptomycin solution and transfer in T-25 cm2 culture flasks. The flasks were incubated at 37°C with 5% CO2 in CO2 incubator (Excella ECO-170, New Brunswick). The study was approved by the Institutional Animal Ethics Committee of Azad Institute of Pharmacy and Research, Lucknow (Ref. No.: AIPR/2013-14/1398).
The cellular morphology was also observed in other sets of PG treatment under trinocular inverted phase contrast microscopy (Nikon ECLIPSE Ti-S, Japan).
Alkaline phosphatase (ALP) activity
ALP assay is based on the hydrolysis of pNPP by ALP into a yellow colored product at alkaline pH. ALP activity of osteoblasts was determined with a slight modification . A 100 μl of cell suspension containing 2 × 103cells /well was seeded in 96-well plates using α-MEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 1% penicillin/streptomycin (osteoblast differentiation medium) and treated with different concentrations (10-100 μg/ml) of the extract for 48 h. E2 at the concentration of 1 nM was used as a positive control. After completion of incubation period, osteoblast cultures were fixed in 4% paraformaldehyde and stained with a solution containing 0.1 mg/ml naphthol ASMX phosphate, 0.5% N, N- dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml of fast blue BB salt in 0.1 mM Tris hCl (pH 8.5) for 20 min. The formation of color was examined and images were taken under an inverted phase contrast microscope. For the quantitative estimation of ALP, the plate was fixed and kept in -70°C for 20 min, and then brought to 37°C for freeze fracture. Next, 50 μl of chilled p-nitrophenyl phosphate (pNPP) substrate was added to each wells and incubated at 37°C for 30 min for color development. The absorbance was measured at 405 nM using an ELISA reader.
Assessment of collagen deposition
Sirius Red is an anionic dye that binds strongly to collagen molecules. Collagen deposition was quantified using Sirius Red dye following slightly modification . Treated cells were washed with PBS and dried at 37°C in 96-wells plate for overnight incubation and then stained with 20 μl of Sirius Red dye (0.1% in saturated picric acid) for 1 h with mild shaking. Sirius Red dye solution (pH 3.5) was prepared in saturated aqueous picric acid (1.3% in H2O) at a concentration of 0.1 mg/ml. The stained cell layers were extensively washed with 0.01 N HCl to remove all non-bounded dye. After rinsing, photographs were taken under inverted phase contrast microscope. For quantitative analysis, the stained cells were dissolved in 0.2 ml 0.1 N NaOH at shaker for 30 min. Next, absorbance was measured colorimetrically at 550 nm against 0.1 N NaOH serve as a blank.
Alizarin Red S, an anthraquinone derivative, was used to identify calcium content in osteoblasts according to a method reported previously . Approximately, 2 × 104 cells/well were seeded in 12-wells culture plate in osteoblast differentiation medium with 10-7 M dexamethasone. Cells were treated with PG extract at various concentrations (10-100 μg/ml) for 21 days and the medium was changed every alternate day. At the end of the experiment, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. The fixed cells were stained with 40 mM Alizarin Red-S (pH 4.5) for 30 min followed by washing with distilled water. Calcified nodules appearing as bright red color were photographed under inverted phase contrast microscopy. For quantification of staining, 100 mM cetylpyridinium chloride solution was added for 1 h in each well to solubilise and to release calcium-bound alizarin red into solution. A 100 μl of the supernatant from each well were transferred in 96 well plate in triplicate and the absorbance was recorded at 570 nm by a microplate reader.
Cellular DNA content
Cell cycle phase distribution with cellular DNA content was analyzed using flow cytometry with some modification . Osteoblasts were planted in 6-wells plate at a density 1 × 106 cells/well and treated with different concentrations (10-100 μg/ml) of extract for 48 h. E2 at the concentration of 10 nM was used as a positive control. Cultured cells were washed with cold PBS and fixed in 70% ethanol at -20°C for 2 h. Fixed cells were treated with RNase A (10 mg/ml) and stained with PI dye in the dark for 30 min at room temperature. The PI dye fluorescence of individual nuclei was measured by using a flow cytometer (BD FACS Calibur, Becton Dickinson, USA) and data were analyzed using Cell Quest Pro V 3.2.1 software (Becton Dickinson, USA).
Quantitative real-time PCR (qPCR)
The total RNA was isolated from cultured osteoblasts treated with PG extract using RNAiso Plus reagent. Aliquots of 2.0 μg of total RNA in a 10 μl reaction volume were subjected to PCR using a cDNA synthesis kit. Quantitative real-time PCR was performed in light cycler PCR system (LightCycler 480, Roche, USA) using SYBER green kit following manufacturer's instruction. Runx2 gene expression in calvarial osteoblasts was determined by qPCR using an optimized protocol . Sequence of primer pairs of the genes used in the present study were; runx2- CCACAGAGCTATTAAAGTGACAGTG (F), AACAAACTAGGTTTAGAGTCATCAAGC (R) and GAPDH (housekeeping gene) - CAGCAAGGATACTGAGAGCAAGAG (F), GGATGGAATTGTGAGGGAGATG (R). All the data were normalized to GAPDH expression to study the relative expression of the targeted gene.
All results were represented as the means ± SEM of results from all replicates and statistically significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. Probability values of p < 0.05 were considered to be statistically significant. All analysis was conducted using the Graph Pad Prism (Ver. 5.1) software.
Results and discussion
Microscopic observation of osteoblastic cells
Effect of PG on the cell proliferation of osteoblasts
The effect of different doses (10-100 μg/ml) of PG extract on osteoblastic cell proliferation was tested at 48 h (Figure 1B). As compared to control group (cells without extract treatment), PG significantly increased the cell proliferation to 13.03 (p < 0.05) and 24.28% (p < 0.001) at 10 and 25 μg/ml, respectively. Moreover, 50 and 100 μg/ml of PG extract induced cell proliferation to 39.64 and 62.59% (p < 0.001) respectively. The results revealed that PG extract induced the significant cell proliferation in a dose dependent manner. Exposure of cells to 1 nm of 17β-estradiol as a positive control, increased the cell proliferation to 41.72% (p < 0.001) as compared to control. The proliferative effects of PG extract might be due to their estrogenic nature of its contents, including quercetin, kaempferol, estrone and estradiol, which promote bone cell proliferation . A study has shown that PG promoted osteoblast MC3T3-E1 cell proliferation up to approximately 2-fold at 250 μg/ml of plant extracts . Both osteoblast and MCF-7 (human breast adenocarcinoma) cells are an estrogen receptor positive (ER+) cells. A similar study has also revealed that MCF-7 cells exposed to legume extracts containing quercetin, daidzein, genistein and kaempferol glycosides at various concentrations (1-1000 μg/ml) showed a maximum cell proliferation at 100 μg/ml of the extracts .
Effect of PG on ALP activity of osteoblasts
Effect of PG on collagen deposition by osteoblasts
Effect of PG on osteoblast mineralization
Effect of PG on cellular DNA content and cell cycle distribution of osteoblasts
Effect of PG on osteogenic gene runxexpression
The present study showed that osteogenic potential of PG extract in primary calvaria osteoblasts is based on two salient features. (1) Cytochemical studies including cell proliferation, ALP stain, collagenation, matrix mineralization and DNA content in the S phase of the cell cycle in osteoblasts are the key parameters, which favor the osteogenic potential of PG. (2) A molecular marker Runx2 is a highly conserved osteogenic transcription factor involved in the regulation of bone cells proliferation and differentiation, which is also favors the osteogenic nature of PG.
Even though E2 used as a positive control shows the slightly higher effect on the proliferation, differentiation, collagenation, mineralization, DNA content and Runx2 gene expression of osteoblasts than lower doses, however, the side effects of the long-term use of estrogen, such as a higher incidence of endometrial cancer, cardiovascular disease and breast carcinoma, could not be ignored.
Thus, the results from this study suggest that PG promotes the function of osteoblasts and plays an important role in remodeling of the bone, which indicated that it may be one of anti-osteoporotic herbal candidate free from side effects. Accordingly, PG might be useful for alternative pharmacological agent of osteoporosis and skeletal tissues may benefit from the consumption of PG.
SS and MA participated in the design of the study. SS performed the experimental work and data interpretation. SS and MA involved in review of the paper. Both authors read and approved the final version of the manuscript.
Sahabjada Siddiqui, M.Sc. in Biotechnology; Md Arshad, Ph.D. in Endocrinology, CDRI, Lucknow and Assistant Professor at University of Lucknow.
Alpha modified minimum essential medium
Bone morphogenetic proteins
Core-binding factor alpha-1
Enzyme linked immunosorbent assay
Fetal bovine serum
Food and drug administration
Hormone replacement therapy
Phosphate buffered saline
Quantitative real-time PCR
Runt-related transcription factor 2
Standard error mean
Selective estrogen receptor modulators
Transforming growth factors
Authors are thankful to UGC Major Research Project File No. 37-436/2009 (SR), New Delhi, India for financial support. Author Sahabjada Siddiqui is thankful to ICMR, New Delhi, India for the award of Senior Research Fellowship (No. 45/26/2013/BMS/TRM). Authors are thankful to the Director, Azad Institute of Pharmacy and Research, Lucknow for providing research facilities for animal experiment.
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