Study On The Effects Of Methotrexate On The Activation And Bone Resorption Function Of Osteoclasts And Its Machanisms

Rheumatoid arthritis(RA), is a kind of chronic, progressing self-immuno disease. The main symptom of RA is chronic inflammation in joints, always accompanied with serious destruction and bone loss around the affected joints. Bone erosion is one of research points in RA. The therapeutic measure is focused on controlling the state of illness in a rapid and effected way, prevent damage of joint structure and function, to inhibit osteoclastic bone destruction and inflammatory bone loss. The numbers and function of osteoclast in affected joints, determine the extent of joint destruction and bone loss. Methotrexate (MTX), a first choice for RA therapy, which is benefit for control of early disease, can affect the abnormal immune function in patients, reduce bone erosion, prevent bone destruction and reduce deformity. But it is still unclear how does low dose MTX affect RA. This study analyzed the effects of MTX on mice osteoclast, attempt to reveal the mechanism of MTX for its inhibitory effects on inflammatory bone destruction.Murine osteoclasts was obtained from co-culture system. 6～8 week male C57BL/6 mice was killed, take thighbone and shankbone in asepsis , bone marrow cells was collected, put the bone marrow cells to culture plate in 1×10⁶ cells·mL^-1 and MC3T3-E1 cells in 2×10⁷ cells·mL^-1. The substrate was half exchanged after two days, and modified to a final concentration of 280 ng·mL^-1 IL-1βand 10 ng·mL^-1 M-CSF. Co-culture was continued for another two days. Then collected cells and transferred to a new dish for purification. We use IL-1βcomparison group and IL-1βwith MTX in different concentration.MTT method was used to determine the effect of MTX on the proliferation of osteoclasts. Flow cytometric analysis and hoechst stain was used to determine the effect of MTX on the apoptosis of osteoclasts. TRAP stain, bone resorption lacuna stain and measurement of lucuna area was executed to determine the effects of MTX on the activity and function of osteoclasts. ELISA method was used to determine the effect of MTX on the MMP-9 secretion from osteoclasts. RT-PCR method was used to determine the effect of MTX on the mRNA expression of RANK and MMP-9 in osteoclasts. Results: The cells growed in co-culture system were large, multinucleated and positive in tartrate-resistant acid phosphase(TRAP) stain, which corresponded with the characters of osteoclasts. When the cells incubated with the bone flap, typical bone resorption lacuna and osteoclast actin ring could be observed.The results of MTT showed that MTX significantly inhibited the proliferation of osteoclasts in a dose dependent manner. The results of flow cytometric analysis showed that IL-1βcould inhibit apoptosis of osteoclast while this inhibition can be antagonised by MTX. That is, MTX could induced the apoptosis of osteoclasts. High concentration of MTX has stronger effect than low dose. Hoechst stain assay has similar results.The results of bone resorption test showed that IL-1βelevated the bone resorption function of osteoclasts, which was inhibited by MTX in a does dependent manner.The results of MMP-9 secretion of osteoclasts showed that MTX could inhibit MMP-9 secretion only in high dose, low dose showed no influence or adverse effect, and not to a statistically significant degree.MTX also decreased the mRNA expression of RANK and MMP-9 but showed a weaker effect on MMP-9 mRNA expression.In conclusion, These resultes indicated that the inhibitory effects of MTX on the proliferation and function of osteoclasts were related with its inhibition on inflammatory bone destruction.