Fig. 4

Exosomal miR-140-3p reduces osteoclastogenesis by inhibiting PDLC pyroptosis and reversing M1/M2 ratio. (A) The schedule of administration for miR-140-3p inhibitors and exosomes. (B) Micro-CT demonstrates Force-Exos with miR-140-3p inhibitors significantly increased the RR volume, which was reduced by Force-Exos. Scale bar: 1 mm. n = 4. (C, D) TRAP staining of osteoclasts reveals the. Compared with Force + Force-Exo + NC group, TRAP⁺ osteoclasts were increased by Force-Exos with miR-140-3p inhibitors. Scale bar: 50 μm. n = 6. (E) TUNEL staining indicates apoptosis or pyroptosis (green) of PDLCs in the root tip and mesial sides. The dashed line delineates the boundary of the PDL. The count of TUNEL-positive PDLCs was upregulated following the inhibition of miR-140-3p, while Force-Exos without miR-140-3p inhibitors downregulated apoptosis or pyroptosis of PDLCs. Scale bar: 200 μm. n = 5. (F, G) Immunofluorescence staining for TUNEL/NLRP3 (F) and TUNEL/caspase-1 (G) co-localization shows the PDLC pyroptosis. The dashed line delineates the boundary of the PDL. Force-Exos with miR-140-3p inhibition significantly increased the PDLC pyroptosis compared with Force + Force-Exo + NC group. Scale bar: 50 μm. n = 5. (H, I) The level of IL-1β (H) and IL-18 (I) on the compression side of the mesial roots was increased in Force + Force-Exos + anti-miR-140-3p group, which was decreased in Force + Force-Exo + NC group. Scale bar: 50 μm. n = 6. (J, K) Immunofluorescence staining of CD68/iNOS (CD68-green and iNOS-red) (J) and CD68/CD163 (CD68-green and CD163-red) (K) demonstrates the co-localization of M1 and M2 macrophages. The dashed line delineates the boundary of the PDL. The inhibition of miR-140-3p resulted in more M1 macrophages and fewer M2 macrophages and the effect was reversed by Force-Exos. Scale bar: 50 μm. (L) The quantities of M1 and M2 macrophages were subjected to statistical analysis, as well as the M1/M2 ratio. Force-Exos with miR-140-3p inhibition upregulated the M1/M2 ratio significantly in comparison to Force + Force-Exo + NC group. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001