Mechanism Research On Bidirectionally Adjusting Balance Of Bone Metabolism With Extract From Duzhong Leaves

Objective:To study effects of extract from Duzhong leaves on osteoclasts cultured in vitro and effects of extract from Duzhong leaves on the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts. To discuss the mechanism of preventing and treating osteoprosis with extract from Duzhong leaves from both inhibiting bone resorption of osteoclasts and promoting bone formation of osteoblasts.Methods:1. The mature osteoclasts of newborn SD rats were obtained by the mechanical isolated method.2. The extract from Duzhong leaves in different concentration was added into the culture system of osteoclasts and the number of the osteoclasts was detected with tartrate-resistant acid phosphatase (TRAP) staining. The bone resorption lacunae on slices was counted with toluidine blue (TB) staining.3. Bone marrow was obtained from femur and tibia of SD rats. BMSCs were isolated by the method of whole bone marrow.4. The extract from Duzhong leaves in different concentration was added into the culture system of BMSCs and the control group is DMEM medium without extract from Duzhong leaves. Gomori calcium-cobalt staining and alizarin red staining (ARS) were used to observe the expressions of alkaline phosphatase (ALP) and calcific nodules. The activity of ALP in medium was measured in different time.Results:1. Extract from Duzhong leaves in different concentration significantly decreased the number of TRAP-positive multinucleated cells and decreased the number of bone resorption lacunae, as compared with the control group.2. Histochemical examinations showed positive staining of ALP in BMSCs of different groups, in which calcific nodules were formed through durative culture. The results demonstrated that the effects of extract from Duzhong leaves in different concentration were more obvious than the control medium.3. The ALP activity of Duzhong leaves in different concentration (experiment groups) and the control group increased with the extension of time. There was no significant difference between experiment groups and control group on the third day, but the ALP activity of experiment groups were significantly higher than the control group on the sixth day.Conclusions:1. Extract from Duzhong leaves could inhibit the bone resorption of osteoclasts.2. BMSCs can be isolated from bone marrow by the method of whole bone marrow. The obtained BMSCs had stability of growth traits, great ability to proliferate and high survival rate of adherent cells. In primary culture, we can get more BMSCs by changing half medium firstly after 48 hours.3. Extract from Duzhong leaves could promote the differentiation of BMSCs into osteoblasts.4. At present, it is not known which effective components have the effects of preventing and treating osteoprosis. We should ulteriorly study effective components of Duzhong leaves.