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Osteclast is responsible for bone resorption, osteoclast culture in vitro is an elementary method to study effects of different factors on osteoclasts. Using Chambers’ method and incubation of bone marrow cells, we isolated and cultured osteoclasts. Osteoclasts were identified by observing live cells with inverted light microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits, and then treated by different concentrations of icariin in order to explore the effect of icariin on mature osteoclastic bone resorption and apoptosis, as well as inducing osteoclast formation in vitro. In addition, we investigated the effects of icariin on the expressions ofβ-actin, TRAP , RANK , CK and MMP-9 mRNA in mouse osteoclasts cultured in vitro by using the technique of RT-PCR in order to find the active targets of icariin in osteoclasts and their connections with genic regulation. The experiment included three parts.1. Effects of icariin on osteoclastic bone resorption and apoptosis in vitroUsing, Chambers’ method, osteoclasts were separated from long-limb bones of new born rabbits, cultured with glass slices and bone slices, and treated by 10-7 mol/L, 10-6mol/L, 5×10-6mol/L, 10-5mol/L icariin. Osteoclasts were identified by observing live cells with inverted light microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The number of bone resorption pits were counted and the surface area of bone resorption on bone slice were measured. osteoclasts were stained with acridine orange. The rates of apoptotic osteoclasts were observed under inverted fluorescence microscope. The results were as follows:Comparing with control group, The rates of apoptotic osteoclasts rose obviously and the number and area of bone resorption pits decreased evidently with increasing dose in those groups treated by 10-6 mol/L, 5×10-6mol/L, 10-5mol/L icariin. The differences between control group and those groups treated by 10-6mol/L, 5×10-6mol/L, 10-5mol/L icariin were significant if P <0.05.2. Effects of icariin on inducing osteoclast formation and bone resorption in mouse bone marrow cultureUsing the method of incubation of bone marrow cells, In the process of osteoclast formation from mouse bone marrow induced by M-CSF （25ng/ml）, RANKL（30ng/ml） and 1,25（OH）2VitD3 (10-8mol/L), icariin with different concentration(0mol/L,10-7mol/L, 10-6mol/L,10-5mol/L) was added. Osteoclasts were identified by observing live cells with inverted light microscope, HE staining, TRAP staining and CTR （calcitonin receptors） staining. The number and area of bone resorption pits on bone slices and the number of TRAP positive multinucleate cells on glass slices were counted. The results were as follows:The addition of icariin (10-7mol/L, 10-6mol/L, 10-5mol/L) induced a dose-dependent decrease in the number, area of bone resorption pits and TRAP positive multinucleate cells. Compared with the control group, those groups treated by 10-6mol/L, 10-5mol/L icariin were significant if P <0.05.3. Effects of icariin on the expressions of B-actin, TRAP, RANK, CK and MMP-9 mRNAOsteoclasts were obtained from mouse bone marrow induced by M-CSF（25ng/ml）, RANKL （30ng/ml） and 1,25（OH）2VitD3 (10-8mol/L). After treatment with with different concentrations of icariin (0mol/L, 10-5mol/L ) for 48h, total RNA was isolated from osteoclasts. Expressions ofβ-actin, TRAP , RANK , CK and MMP-9 mRNA were detected by semiquantitative RT-PCR. The results were as followsCompared with the control group, icariin (10-5mol/L ) remarkably decreasedβ-actin, RANK and MMP-9 mRNA levels （P <0.05）, but had no significant effects on the expression of TRAP and CK mRNA （P>0.05） .The conclusions were as follows:1. Icariin can inhibit osteoclastic bone resorption in vitro.2. Icariin can induce osteoclast apoptosis in vitro.3. Icariin could inhibit osteoclast formation from bone marrow induced by M-CSF, RANKL and 1,25（OH）2VitD3 and bone resorption in vitro.4. Icariin can downregulate the expressions ofβ-actin mRNA, which could correlate with inhibiting osteoclast formation and bone resorption.5. Icariin can decrease RANK and MMP-9 mRNA levels, which exhibited icariin could effect on the NF-κB path of signal transduction mediated by RANK to inhibit osteoclastic differentiation and bone resorption.
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