Method: Monocytes obtained from murinic bone marrow (BMC) were submitted to different concentrations of recombinant AaCDT in the presence and absence of RANKL. After 6 days, viability was determined (MTT) and TRAP + cells presenting > 3 nuclei were counted. After 2-6 days, RT-qPCR was performed to evaluate expression of genes associated with osteoclastogenesis (rank, ctpk, nfatc1 and irf-8) and gapdhwas used as control. The data were analyzed by ANOVA followed by Tukey or Bonferroni tests (p < 0.05).
Result: At the tested concentrations, CDT did not affect cells viability, but the number of TRAP+ multinucleated cells increased with increasing AaCDT concentrations, in the presence or absence of RANKL. The expression of rank was down regulated after 4 days of incubation in AaCDT-treated cells in the absence of RANKL. This effect was also seen in BMC added with RANKL after 2-6 days. Furthermore, after 4 and 6 days, AaCDT down regulated the expression of transcription factor nfatc1 and ctpK (encoding cathepsin K). Thus, although an increase in TRAP+ cells was observed in AaCDT treated pre-osteoclastic cells, genes involved in osteoclastogenesis were repressed by the toxin, even in presence of RANKL.
Conclusion: AaCDT may affect osteoclastogenesis by inhibiting signaling in pre-osteoclastic cells, and by down regulating the expression of genes associated with mature osteoclasts.