| Objectives The study is aimed to examine the effect of zoledronate acid on cellular Ca2+oscillation and gene expression of Ca2+signal molecules, Ca MKII, Ca MKIV and NFATc1 during differentiation of the osteoclasts. The results of study will broaden our knowledge on molecular mechanism of zoledronate for its inhibiting function on osteoclastogenesis and bone resorption function, and benefit development of new drugs targeted on osteoclasts to treat excessive bone resorption diseases.Methods 1 Mice leukemia mononuclear macrophage RAW264.7 cell line was used in this study and the cells were divided to seven groups, group A to group G. Group A was the control and received no treament, while group B to G were treated with zoledronate(ZOL) for 5 days at concentration of 1×10-7mol/L, 1×10-6mol/L, 5×10-6mol/L, 1×10-5mol/L, 5×10-5mol/L and 1×10-4mol/L, respectively. Cellular morphology was observed under microscope and MTT test was performed to explore the effect of zoledronate on osteoclast proliferation. 2 RAW264.7 cells underwent osteoclast induction with 100ng/ml receptor activator for nuclear factor-κ B ligand(RANKL) for 5 days and the cells were divided into two groups, the control and ZOL group. The latter was treated with 1×10-6mol/L ZOL for 48 h from 2nd day of RANKL induction. Five days later, the cells were harvested for following examination.(1) Tartrate-resistant acid phosphotase(TRAP)staining and dentin resoreption lacunae analysis were performed to evaluate the difference of osteoclastogenesis and bone reorption between the two groups;(2) Ca2+oscillation examination. The cells were harvested at 0h, 1h, 3h, 6h, 12 h, 24 h, 48 h, 72h(24h after withdraw of ZOL) and 96h(24h after withdraw of ZOL)after ZOL treatment and Fluo-4, Fluo-red were used for labeling of intracellular Ca2+. Then the change of Ca2+concentration was detected under laser scanning confocal microscope to explore the effect of ZOL on Ca2+ oscillation in osteolcasts;(3) Real-time PCR, immunofluorescent cytochemistry and Western-blot were also performed to evaluate m RNA and protein levels of Ca MKII, Ca MKIV and NFATc1, three important signal molecules downstream of Ca2+, in control and ZOLgroup.Results 1 Zoledronate at low level from 1×10-7mol/L to 5×10-6mol/L exert no significant effect on cellular morphology and proliferation of RAW264.7 cells(p>0.05),while 1×10-5mol/L zoledronate slightly decreased the proliferation of RAW264.7 cells(P<0.01), although cellular morphology was not affected. Obvoius influence of zoledronate was observed at concentration of 5×10-5mol/L and 1×10-4mol/L, which lead to significant inhibition of cellular proliferation(P<0.01) and decreasement of cellular size, or even disruption and death of cells. 2 After RANKL induction and ZOL treatment,cells in control and ZOL group underwent different examinations and the results were demonatrsted as follows.(1) TRAP staining and scanning electron microscope observation showed that TRAP+multinuclear osteoclasts and dentin absorption lacunae were formed in both groups, whereas number of osteoclasts, number and size of dentin resorptiom lacunae were 33.0±1.0,46.0±3.5and 4125.9±674.8μm2 respectively in ZOL group, which were significantly lower than those(66.6±3.2, 86.0±9.2 and 9418.3±1260.8μm2, respectively) in control group(P<0.01).(2) The effect of zoledronate on intracellular Ca2+ oscillation was time-dependent. Zoledronate exert no significant effect on Ca2+oscillation at the early stage of treatment(0~3h), while with the increasement of trament period(6h~48h), Ca2+ oscillation was gradually decreased and it was almost disappeared at 48 h. After removing of ZOL, Ca2+ oscillation in osteoclasts gradually returned to control level at 72 h and 96 h.(3) Real-time PCR showed that m RNA level of Ca MKII, Ca MKIV and NFATc1 decreased about 39.3%, 51.7% and 54.7% respectively,when compared with their controls(P<0.01).(4) Western-blot also showed significant decrease of protein levels of the genes in ZOL group when compared with control and the decreases were 58.9%, 46.8% and 39.8%(P<0.05)respectively for Ca MKII, Ca MKIV and NFATc1, respectively. The changes of protein level were also verified by immunofluorescent cytochemistry, in which the fluorescent intensity of the three genes were also obviously decreased(P<0.05).Conclusions Our study demonstrates that zoledronic could significantly inhibit Ca2+oscillation and the gene expression of Ca MKII, Ca MKIV and NFATc1, which may play a certain role in zoledronate-induced inhibition of osteoclastogenesis. |
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