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Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis.

Penolazzi L, Lampronti I, Borgatti M, Khan MT, Zennaro M, Piva R, Gambari R - BMC Complement Altern Med (2008)

Bottom Line: Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments.This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene.Accordingly, we suggest the application of Emblica officinalis extracts as an alternative tool for therapy applied to bone diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioPharmaNet, ER-GenTech, Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy. pnlmlt@unife.it

ABSTRACT

Background: Osteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of Emblica officinalis fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts.

Methods: The effects of extracts from Emblica officinalis on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of Emblica officinalis extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments.

Results: Extracts of Emblica officinalis were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. Emblica officinalis increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that Emblica officinalis extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that Emblica officinalis extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene.

Conclusion: Induction of apoptosis of osteoclasts could be an important strategy both in interfering with rheumatoid arthritis complications of the bone skeleton leading to joint destruction, and preventing and reducing osteoporosis. Accordingly, we suggest the application of Emblica officinalis extracts as an alternative tool for therapy applied to bone diseases.

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TRAP staining analysis of human primary osteoclasts obtained after 14 days of culture in presence of 0.5, 5, and 50 μg/ml of Emblica officinalis extracts, as indicated (A); the same percentage of multinucleated TRAP-positive cells was obtained when mature osteoclasts were grown for 60 hours with the same amount of Emblica officinalis extracts (B). Cells were photographed at the 20 × magnification. In the lower part of the panel data from five determinations are presented (average ± SD).
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Figure 1: TRAP staining analysis of human primary osteoclasts obtained after 14 days of culture in presence of 0.5, 5, and 50 μg/ml of Emblica officinalis extracts, as indicated (A); the same percentage of multinucleated TRAP-positive cells was obtained when mature osteoclasts were grown for 60 hours with the same amount of Emblica officinalis extracts (B). Cells were photographed at the 20 × magnification. In the lower part of the panel data from five determinations are presented (average ± SD).

Mentions: Human primary osteoclasts were obtained from peripheral blood and cultured in complete D-MEM plus MCSF, PTH and RANKL for 14 days. OCs differentiation was tested by tartrate-acid resistant acid phosphatase (TRAP)-positivity (Fig. 1) and metalloproteinase-9 (MMP-9) expression (data not shown). In order to test the effect of E. officinalis extracts on osteoclast differentiation, mature OCs (Fig. 1A) or monocytes during the two weeks of induction (Fig. 1B) were exposed to 0.5, 5, 50 μg/ml of plant extracts. The conditions used for these experiments correspond to the concentrations of E. officinalis extracts leading to 50% of inhibition (IC50 value) of cell growth, previously analyzed in different cell lines [55]. As reported in Figure 1, the presence of comparable levels of TRAP-positive cells cultured both in presence and in absence of E. officinalis extracts did not affect the process of osteoclastogenesis, at the concentrations employed. Quantitative data from three independent experiments are presented in the lower sides of Figure 1, demonstrating that treatment of the cultures with E. officinalis extracts does not have inhibitory effects on the development of TRAP-positive OCs. Cytotoxic effects of E. officinalis extracts were then analyzed. Human primary OCs were treated with increasing amount of E. officinalis extracts (0.5–500 μg/ml) for 72 hours and the viability of the cells was examined by the colorimetric MTT assay [56]. As shown in Figure 2, 0.5, 5 and 50 μg/ml of E. officinalis extracts did not cause any cytotoxic effect on the total cell population (1–5% of which is constituted by OCs). Only E. officinalis extracts used at 500 μg/ml were found to induce a slight but not significant decrease of viability.


Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis.

Penolazzi L, Lampronti I, Borgatti M, Khan MT, Zennaro M, Piva R, Gambari R - BMC Complement Altern Med (2008)

TRAP staining analysis of human primary osteoclasts obtained after 14 days of culture in presence of 0.5, 5, and 50 μg/ml of Emblica officinalis extracts, as indicated (A); the same percentage of multinucleated TRAP-positive cells was obtained when mature osteoclasts were grown for 60 hours with the same amount of Emblica officinalis extracts (B). Cells were photographed at the 20 × magnification. In the lower part of the panel data from five determinations are presented (average ± SD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2587459&req=5

Figure 1: TRAP staining analysis of human primary osteoclasts obtained after 14 days of culture in presence of 0.5, 5, and 50 μg/ml of Emblica officinalis extracts, as indicated (A); the same percentage of multinucleated TRAP-positive cells was obtained when mature osteoclasts were grown for 60 hours with the same amount of Emblica officinalis extracts (B). Cells were photographed at the 20 × magnification. In the lower part of the panel data from five determinations are presented (average ± SD).
Mentions: Human primary osteoclasts were obtained from peripheral blood and cultured in complete D-MEM plus MCSF, PTH and RANKL for 14 days. OCs differentiation was tested by tartrate-acid resistant acid phosphatase (TRAP)-positivity (Fig. 1) and metalloproteinase-9 (MMP-9) expression (data not shown). In order to test the effect of E. officinalis extracts on osteoclast differentiation, mature OCs (Fig. 1A) or monocytes during the two weeks of induction (Fig. 1B) were exposed to 0.5, 5, 50 μg/ml of plant extracts. The conditions used for these experiments correspond to the concentrations of E. officinalis extracts leading to 50% of inhibition (IC50 value) of cell growth, previously analyzed in different cell lines [55]. As reported in Figure 1, the presence of comparable levels of TRAP-positive cells cultured both in presence and in absence of E. officinalis extracts did not affect the process of osteoclastogenesis, at the concentrations employed. Quantitative data from three independent experiments are presented in the lower sides of Figure 1, demonstrating that treatment of the cultures with E. officinalis extracts does not have inhibitory effects on the development of TRAP-positive OCs. Cytotoxic effects of E. officinalis extracts were then analyzed. Human primary OCs were treated with increasing amount of E. officinalis extracts (0.5–500 μg/ml) for 72 hours and the viability of the cells was examined by the colorimetric MTT assay [56]. As shown in Figure 2, 0.5, 5 and 50 μg/ml of E. officinalis extracts did not cause any cytotoxic effect on the total cell population (1–5% of which is constituted by OCs). Only E. officinalis extracts used at 500 μg/ml were found to induce a slight but not significant decrease of viability.

Bottom Line: Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments.This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene.Accordingly, we suggest the application of Emblica officinalis extracts as an alternative tool for therapy applied to bone diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioPharmaNet, ER-GenTech, Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy. pnlmlt@unife.it

ABSTRACT

Background: Osteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of Emblica officinalis fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts.

Methods: The effects of extracts from Emblica officinalis on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of Emblica officinalis extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments.

Results: Extracts of Emblica officinalis were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. Emblica officinalis increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that Emblica officinalis extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that Emblica officinalis extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene.

Conclusion: Induction of apoptosis of osteoclasts could be an important strategy both in interfering with rheumatoid arthritis complications of the bone skeleton leading to joint destruction, and preventing and reducing osteoporosis. Accordingly, we suggest the application of Emblica officinalis extracts as an alternative tool for therapy applied to bone diseases.

Show MeSH
Related in: MedlinePlus