Limits...
Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts.

Siddiqui S, Arshad M - Daru (2014)

Bottom Line: Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory, Department of Zoology, University of Lucknow, Lucknow, 226007, India. sahabjadabiotech04@gmail.com.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH

Related in: MedlinePlus

Measurement of ALP activity. (A) Photomicrographs stained with fast blue BB salt-ASMX-phosphate complex showing increased formation of ALP in osteoblasts treated with increasing concentrations (10–100 μg/ml) of PG extract at 48 h (B) Quantitative data of ALP level presented in the form of ALP activity relative to control. Values are obtained from three independent experiments and expressed as mean ± SEM. *p < 0.05 and **p < 0.01 as compared with control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4248434&req=5

Fig2: Measurement of ALP activity. (A) Photomicrographs stained with fast blue BB salt-ASMX-phosphate complex showing increased formation of ALP in osteoblasts treated with increasing concentrations (10–100 μg/ml) of PG extract at 48 h (B) Quantitative data of ALP level presented in the form of ALP activity relative to control. Values are obtained from three independent experiments and expressed as mean ± SEM. *p < 0.05 and **p < 0.01 as compared with control.

Mentions: Quantitative estimation of alkaline phosphatase activity is one of the biochemical methods, which described the early cell differentiation of osteoblastic cells [28]. Fast blue BB salt-ASMX-phosphate complex acted on ALP activity which appeared to be blue in color. The qualitative data showed that PG extract stimulated ALP stain by increasing the rate of osteoblast cell differentiation in a dose dependent manner (Figure 2A). As observed from numerical data (Figure 2B), concentrations 10 and 25 μg/ml of extract induced ALP level to 9.66% (p < 0.05) and 22.49% (p < 0.01) significantly as compared to control. Also, 50 and 100 μg/ml of extract induced the significant ALP level to 34.67 and 43.95% (p < 0.01) respectively as compared to control. Exposure of cells to 1 nM of E2 increased ALP activity to 36.66% (p < 0.01) as compared to control. Results of ALP assay were the consistent with MTT assay data which suggested that cell proliferation also correlate with cell differentiation. These results indicate that PG extract induces regeneration of osteoblasts as a function of dose might be due to the presence of estrogenic compounds. PG containing estrogenic compounds daidzein and genistein has been reported to possess stimulatory effects synthesizing alkaline phosphatase by osteoblasts in vitro [29].Figure 2


Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts.

Siddiqui S, Arshad M - Daru (2014)

Measurement of ALP activity. (A) Photomicrographs stained with fast blue BB salt-ASMX-phosphate complex showing increased formation of ALP in osteoblasts treated with increasing concentrations (10–100 μg/ml) of PG extract at 48 h (B) Quantitative data of ALP level presented in the form of ALP activity relative to control. Values are obtained from three independent experiments and expressed as mean ± SEM. *p < 0.05 and **p < 0.01 as compared with control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4248434&req=5

Fig2: Measurement of ALP activity. (A) Photomicrographs stained with fast blue BB salt-ASMX-phosphate complex showing increased formation of ALP in osteoblasts treated with increasing concentrations (10–100 μg/ml) of PG extract at 48 h (B) Quantitative data of ALP level presented in the form of ALP activity relative to control. Values are obtained from three independent experiments and expressed as mean ± SEM. *p < 0.05 and **p < 0.01 as compared with control.
Mentions: Quantitative estimation of alkaline phosphatase activity is one of the biochemical methods, which described the early cell differentiation of osteoblastic cells [28]. Fast blue BB salt-ASMX-phosphate complex acted on ALP activity which appeared to be blue in color. The qualitative data showed that PG extract stimulated ALP stain by increasing the rate of osteoblast cell differentiation in a dose dependent manner (Figure 2A). As observed from numerical data (Figure 2B), concentrations 10 and 25 μg/ml of extract induced ALP level to 9.66% (p < 0.05) and 22.49% (p < 0.01) significantly as compared to control. Also, 50 and 100 μg/ml of extract induced the significant ALP level to 34.67 and 43.95% (p < 0.01) respectively as compared to control. Exposure of cells to 1 nM of E2 increased ALP activity to 36.66% (p < 0.01) as compared to control. Results of ALP assay were the consistent with MTT assay data which suggested that cell proliferation also correlate with cell differentiation. These results indicate that PG extract induces regeneration of osteoblasts as a function of dose might be due to the presence of estrogenic compounds. PG containing estrogenic compounds daidzein and genistein has been reported to possess stimulatory effects synthesizing alkaline phosphatase by osteoblasts in vitro [29].Figure 2

Bottom Line: Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory, Department of Zoology, University of Lucknow, Lucknow, 226007, India. sahabjadabiotech04@gmail.com.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus