Bioactivity effects of Vitex agnus-castus L. (chaste berry) on the modulation of in vitro osteogenesis and on the protection against mandibular and femoral bone loss in a experimental model of rat Osteoporosis

Abstract

Osteoporosis is a disease of high prevalence worldwide, associated with oxidative stress and leading to an imbalance in the process of bone homeostasis, with high resorption and low deposition of mineralized matrix. There are several current treatments for Osteoporosis, which are effective but presenting a high incidence of undesirable side effects, increasing in the search for effective alternative therapies without damaging the organism. Evidence shows that phytoestrogens, found in abundance in vegetables, can be used as alternatives treatment for Osteoporosis because of their 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 (ROS) produced by oxidative stress. Therefore, the purpose of this study is to evaluate the effect of phytoestrogen Vitex Agnus-Castus (VAC) in vivo and in vitro, compared to ß-estradiol (EST), on the osteogenic function of osteoblastic cells in the presence and absence of oxidative stress as well as bone loss in rats submitted to an experimental model of Osteoporosis. For this purpose, cells of MC3T3-E1 osteoblastic cell line will be cultured and divided into control and in vitro oxidative stress induction groups. These two groups, will be further subdivided into 1) Control; 2) Group with 10-8 mol.L-1 of ß-estradiol and 3) Group with the addition of Vitex agnus-castus (concentration to be defined). The 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 quantitative expression of genes related to osteogenesis and oxidative stress. Mesenchymal stem cells from bone marrow will also be utilized for mineralization tests. 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 three groups: 1) Water administration; 2) Administration of 20 µg/mL of ß-estradiol; 3) Administration of 200 mg/mL of Vitex agnus-castus. 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 estereological microarchitecture analysis. The data obtained will be analyzed statistically by the Graph Pad Prism 5.0 software with a significance level of 5%. (AU)