Determining the 'in vitro' effect of Padina pavonica on the oestrogen receptor and oestrogen responsive primary cell lines

Abstract

Backgound: Padina pavonica appears to improve the bone mineral density at the lumbar spine and at the hip in post-menopausal women (1). The aims of this project are to compare it to other treatments available on the market, for the treatment of post-menopausal osteoporosis and to shed more light on the mechanism of action of Padina pavonica.

Aims and Objectives: The aim of this research-based experiment is to compare the ability of osteoblasts treated with the extract of Padina pavonica (EPP) to differentiate and fix calcium, with osteoblasts treated with raloxifene and oestradiol. Raloxifene, a selective oestrogen receptor modulator (SERM) and oestradiol in the form of hormone replacement therapy (HRT) are drugs normally used in the management of post-menopausal osteoporosis. A secondary aim is to determine whether the extract exerts its action by modulating the oestrogen receptor. This would imply that the extract of Padina pavonica has a SERM-like activity and is thus potentially prone to the same adverse effects, as other members of this class. Methodology: This is a research-based experiment in which primary osteoblasts were isolated and cultured from human bone explants. Primary cells obtained using this method were used to objectively assess the extract's effect on the differentiation of progenitor bone cells to terminally mature osteoblasts. Results were compared to bone cells treated with raloxifene and oestradiol. Alkaline phosphatase activity was measured as an early marker of osteoblast differentiation using spectrophotometry.

An MTT assay was employed as a measure and marker of cellular viability and proliferation. These tests were performed after seven days of incubation. The alkaline phosphatase: MTT ratio was calculated and used as another end point in order to reflect whether any increases in alkaline phosphatase were due to an increase in cell mass or whether this was due to the formation of more terminally differentiated osteoblasts. Cells were also incubated with the drugs for fifteen days. The Alizarin Red assay was then performed. The latter was employed as measure of calcium fixation and bone matrix mineralization. Oestrogen receptor mono clonal IgG antibodies were used in order to try and assess whether the drugs' activity was oestrogen receptor dependent or independent. The results was analysed usmg multiple-linear regression analysis and the Kruskall-Wallis non-parametric test. Results: Human primary osteoblasts cells can be easily grown in culture and used as a model for the testing of drugs with potential use in the management of postmenopausal osteoporosis. Cells treated with the extract of Padina pavonica expressed alkaline phosphatase activity. This was then found not to be statistically different from oestrogen or raloxifene (p-value: 0.501). A statistically significant difference between drugs was noted in the cell viability assay (MTT) (p-value: 0.002). The highest cell viability was noted in the cells treated with oestradiol. There was no statistically significant difference between cells treated with EPP and cells treated with raloxifene on the cell viability assay (p-value: 0.528). The latter was reflected in the alkaline phosphatase:MTT ratio of the differently treated cultures which revealed a statistically significant difference between groups (p-value 0.034).

Cells treated with the extract of Padina pavonica were only slightly inferior to raloxifene in tenns of osteoblast differentiation as supported by the second highest average estimated marginal means of the alkaline phosphatase to MTT ratio. The different drugs did not show any statistically significant difference in the bone matrix mineralization assay (p-value: 0.548). The oestrogen receptor antibody tests did not reveal any statistically significant results, but suggest a SERM-like activity ofEPP. Conclusions: Our data supports previous studies, which show a potential role for the extract of Padina pavonica in the management of post-menopausal osteoporosis. The mechanism of action of this drug remains to be fully understood. This work indicates a possible direct or indirect modulation (by co-factors) of the oestrogen receptors by EPP and suggests a SERM-like activity of this product. Further Quantitative Polymerase Chain reaction (q-PCR) studies supported and confirmed by Western-blot protein analysis are key steps in understanding the impact of the extract of Padina pavonica on the protein component of bone matrix and to be able to relate differential gene expression signatures to established cell signaling in physiological and pathological pathways.