Effects Of Benefiting-bone Capsule Contained Serum On Activity And Apoptosis Of Osteoclast In Osteoblast And Osteoclast Co-culture System

Objectives:To study effects of the Bone-Benefiting Capsule (BBC)-containing serum on activity and apoptosis of osteoclast and the effects on expression of OPG-mRNA of osteoclast, and to study the mechanism of action of BBC in the prevention and treatment of osteoporosis. Methods:1. Establishing the plane osteoblast and osteoclast co-culture system with agarose gel and 0.45 μm aperture microporous membrane.2. Female SD rats aged 10 month were divided into BBC groups (high, middle and low doses) and control group (normal physiological saline) to prepare BBC-containing serum and control serum.3. The MTT method was used to determine the best dosage-efficiency of OB proliferation in osteoblast and osteoclast co-culture system of BBC-containing serum.4. Determine the effect of BBC-containing serum on absorption of lacuna formed by OC in osteoblast and osteoclast co-culture system by toluidine blue staining.5. Determine the effect of BBC-containing serum on activity of TRAP secreted by OC in osteoblast and osteoclast co-culture system by the diazol method.6. Determine the effects of BBC-containing serum on OC survive and apoptosis in osteoblast and osteoclast co-culture system by reverse and fluorescence microscopes.7. Determine the effect of BBC-containing serum on expression of OPG mRNA secreted by OB in osteoblast and osteoclast co-culture system by the FQ-PCR method. Results:1. Protein dispersing experiment: after detection by Coomassie Brilliant blue staining, we demonstrated that the protein in OB culture room dispersed to OC culture room through membrane within 8 hours, which suggested that the establishment of plane osteoblast and osteoclast co-culture system was successful.2. The BBC-containing serum showed a certain quantity-efficiency relationship with OB proliferation i.e., at a density of 25-30%, it improved the proliferation of OBin co-culture system demonstrated by the remarkable differences among different groups at different dosage (PO.01) .3. The number of absorption of lacuna of BBC-containing serum group was obviously less than that of the control group in the osteoblast and osteoclast co-culture system (PO.01) .4. The numbers of TRAP secreted by OC in the osteoblast and osteoclast co-culture system of BBC-containg serum group at 48h> 72h and 96h showed an obvious time-efficiency relationship, which were remarkablaly lower than that of the control group (PO.01) .5. The OC surviving numbers in the osteoblast and osteoclast co-culture system of BBC-containing serum group in 48h > 72h and 96h showed an obvious time-efficiency relationship, which were also remarkablaly lower than that of the control goup CP<0.01) . The OC apoptosis numbers in the osteoblast and osteoclast co-culture system of BBC-containg serum group in 48h> 72h and 96h had an obvious time-efficiency relationship, which were remarkablaly higher than that of the control group (P<0.01) .6. The BBC-containing serum obviously improved the expression of OPGmRNA secreted by OB in the osteoblast and osteoclast co-culture system, which had a remarkable difference compared to that of the control group (P<0.01) .Conclusion:The BBC-containing serum significantly suppressed the activity of OC and improved the withering of OC. Based on the experimental results, we propose that BBC acts on OC directly by suppressing its activity and improving its withering, and/or improving the secretion of OPG by OB through paracrine stimulation to regulate the activity and withering of OC.