Effect of diesel exhaust particles on RANK/RANKL expression in in vivo and in vitro models of middle ear inflammation.

Objective: Increasing evidence suggests a link between middle ear inflammation and the development of diesel exhaust particles (DEPs). Chronic middle ear inflammation can lead to bone damage and remodeling. This study aimed to explore the impact of DEPs on the expression of interleukin (IL)-6 and RANKL under conditions of middle ear inflammation.

Methods: DEPs were collected by burning fuel in a diesel engine at the Gwangju Institute of Science and Technology. Human middle ear epithelial cells were cultured to 70-80% confluence in culture plates and then treated with DEPs at concentrations of 0, 5, 10, 20, 40, and 80 μg/mL for 24 h. Cell viability was assessed manually. B6.SJL mice, aged 9 weeks, were exposed to DEPs at a concentration of 200 μg/m for 1 h daily over a period of 28 days. The expression levels of IL-6, tumor necrosis factor α, RANKL, and RANK were evaluated using hematoxylin and eosin staining and western blot analysis of the harvested middle ear samples.

Results: The viability of human middle ear epithelial cells was found to decrease in a dose-dependent manner after 24 h. The mRNA expression level of IL-6 exhibited the most significant increase at the 48-h mark. In contrast, the mRNA expression levels of RANKL and RANK showed a marked increase as early as 6 h post-exposure, with both genes subsequently displaying a time-dependent decrease. Histological analysis revealed that the middle ear mucosa was thicker in the group exposed to DEPs compared to the control group. Additionally, the protein expression levels of IL-6 and RANKL were elevated in the DEP-exposed group relative to the normal control group.

Conclusions: We confirmed the expression of osteoclast-related proteins in the mouse middle ear. These results imply that air pollutants might affect RANKL/RANK signaling, which is associated with bone remodeling.