Ps-58: The Antioxidative Effects of Soybean on Embryonic Chondrogenesis


Khodabandeh Jahromi Z *, Vojdani Z , Talaee T ,

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Objective: Free radicals such as reactive oxygen species are formed during a variety of biochemical reactions and cellular functions. Oxidative stress is responsible for a variety of degenerative processes in some human diseases. In aerobic cells, free radicals are constantly produced mostly as reactive oxygen species. Once produced, free radicals are removed by antioxidant defenses including enzyme catalase, glutathione peroxidase, and superoxide dismutase. Oxidative stress has been implicated in the pathogenesis of various diseases affecting chondrogenesis or the function of articular cartilage. DNA damage caused by oxidative stress may trigger the activation of the nuclear enzyme, poly (ADP-ribose) polymerase-1 (PARP-1) which may contribute to tissue injury. It has been demonstrate that increasing ROS levels induce chondrocyte hypertrophy. Reactive oxygen species (ROS) have been implicated in cellular damage; however, the physiological role of ROS in chondrogenesis is not well characterized. Diet can ameliorate oxidative stress. Diet has been known for years to play a key role as a risk factor for chronic diseases. Food contains many chemicals that have various effects on biological phenomenon. Some phytochemicals may contain anti-oxidative agents. It has been demonstrated that the incidence of the different type of cancer is less in ascians than the other nations and it can be related to using soybean in their diet. Soybean contains phytoestrogen, oxygenase-1 and tocopherol. It has been demonstrated that they play a protective role for mammalian cells against oxidative stress. The soy extract and its fractions displayed a strong free radical scavenger activity. It has been demonstrated that soybean extract impact on embryo chondrogenesis and ossification in vivo (JAAR). Many researches revealed that soybean has beneficial effects on cartilage. There is evidence that flavonoids and in particular isoflavones, may exert positive effects on cartilage metabolism and skeletal health. Flavenoid and isoflavenoid are the phytoestrogen and soybean is rich source of these components. There are many investigations that focus on the mechanism of soybean effects. Some of them focus on the its anti-inflammatory effects and the other mentioned its antiestrogenic or estrogenic properties With regards to these considerations, the Objective of this project will be to find the impact of soybean extract on reduction of detoriation effects of oxidative stress in embryonic chondrogenesis in vitro. Materials and Methods: 12-13 days old mice embryos used to extract mesenchymal cells. For this purpose, the limb bud of embryos removed and after rinsing in PBS containing antibiotic, incubated in dispase for 90 minutes in order to separate ectoderm from mesenchyme. After separation, the limb bud rinse and incubate in the trypsin for 20 minutes. The limb bud became dissociation by adding culture media (DMEM/F12 contain 10% FAS, 2 mM glutamine and antibiotic) and pipetting. The cell was count by hemocytometer and the number of the cells adjusts to 1-2 × 105. Superconfluent micromass culture of limb bud mesenchyme established by spotting 10 μl of cell suspention in 96 well tissue culture plate. After incubation for 1.5-2 hours at 37 ºC and 5% CO2 to permit cell attachment, the plate will be flooded with culture media. The cells allowed to confluent for 4 days. The culture media changed daily. Some wells stained with alcian blue to improve that the cartilage was differentiated. At day 5 first, the sample divided to 10 groups and different concentration of H2O2 and soybean separately and mixed with each other added to fresh media and media without any soybean extract as negative control and incubated for 24 hours in order to induce oxidative stress. The day after that, the medium changed by culture media (DMEM/F12 contain 10% FAS, 2 mM glutamine and antibiotic) without any soybean extract as negative control added to the wells. Each group will be done in triplicate. Each well stained with alcian blue and the size and the number of the nodules calculated. Results: The viability rates of each group were separately compared to control and the results showed that there were significant different between each experimental group as compared to control (p ≤ 0.05). The best result was for the group that the soybean was mixed with the 1000 μm concentration of H2O2. We also detected that the high concentration of soybean reduced the viability. So between the different concentrations of soybean the lowest concentration (10 μm) was the best. Conclusion: It has been demonstrated that soybean extract impact on embryo chondrogenesis.Flavenoid and isoflavenoid were the phytoestrogen and soybean was rich source of these components.we found the impact of soybean extract on reduction of detoriation effects of oxidative stress in embryonic chondrogenesis in vitro.