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Phytoestrogens X hormonal replacement therapy with beta-estradiol: potential of Vitex agnus-castus L. and extract of red-purple pitaya in the protection of mandibular and femoral bone loss: in vitro and in vivo analyses in ovariectomized rats


Osteoporosis is a multifactorial disease of high prevalence in the population, and its pathogenesis may result from several factors such as the use of drugs, alcohol consumption, genetic factors, aging, and hormone deficiency, leading to an imbalance in bone remodeling, with high resorption and low deposition of mineralized matrix. The association of this imbalance with estrogen deficiency that occurs in women at the post-menopause phase is under investigation, with the hypothesis that this deficiency may promote oxidative stress, influencing the functional activity of bone cells. There are several current treatments for osteoporosis, but they are characterized by the high incidence of undesirable side effects, which leads to an increase in the search for effective alternative therapies without damaging the organism. Evidence shows that phytoestrogens, found in abundance in vegetables, can be used as treatment alternatives for osteoporosis because it has a similar effect to estrogen, which acts by increasing the rate of bone deposition and reducing the rate of bone resorption, besides having an antioxidant effect which might protect cells against the effects caused by reactive oxygen species (ROSs) produced by oxidative stress. Therefore, the purpose of this study is to evaluate the effect of two phytoestrogens, Vitex agnus-castus (VAC) and pitaya extract (EPY) with in vivo and in vitro parameters, compared to b-estradiol (E2), on the osteogenic function of osteoblastic cells in the presence and absence of oxidative stress and on bone loss in rats submitted to an experimental model of osteoporosis. For this purpose, MC3T3-E1 osteoblastic cell line and mesenchymal cells from femoral bone marrow will be cultured and divided into control and groups with in vitro oxidative stress induction. These two groups, will be subdivided into: i) Control; ii) group with 10-8 mol.L-1 b-estradiol; iii) Group with the addition of Vitex agnus-castus (concentration to be defined) and iv) Group with the addition of pitaya extract (concentration to be defined). Such cell groups will be submitted to biochemical and molecular biology assays, such as analysis of cell proliferation (MTT), detection and quantification of the enzyme alkaline phosphatase and mineralized nodules, in addition to the intracellular detection of ROS and analysis of the expression of genes related to osteogenesis and oxidative stress. Mesenchymal cells from femoral bone marrow will be cultured for confirmation of mineralization. For in vivo analysis, Sprague-Dawley rats will be divided into a control group (SHAM) and a group with experimental osteoporosis induction. These two groups will be subdivided into four groups: i) water administration; ii) administration of 20 ug / mL of b-estradiol; iii) administration of 200 mg/mL of Vitex agnus-castus and iv) administration of 200 mg/mL of pitaya extract. The administration will take place for 12 weeks immediately after ovariectomy followed by euthanasia and collection of samples for qualitative and qualitative histological analysis as well as microtomographic and dynamics histometric analysis through fluorochromes, for microarchitecture evaluation. The data obtained will be analyzed statistically by the Graph Pad Prism 5.0 software with a significance level of 5%. (AU)

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