IL-37 suppresses the osteogenic response of AVICs by inhibiting IRAK-M degradation. (A, B) rIL-37 inhibited LPS-induced inflammatory and osteogenic responses in AVICs (n = 6 samples per group, Bar = 50 μm, two-way ANOVA and Student’s t-test). (C, D) rIL-37 treatment reduced LPS-induced calcium deposition and ALP activity in AVICs (n = 5 samples per group, Bar = 100 μm, Student’s t-test). (E, F) rIL-37 inhibited LPS-induced phosphorylation and nuclear translocation of p65 (n = 6 samples per group, Bar = 75 μm, Student’s t-test). (G) The rIL-37-mediated suppression of inflammatory and osteogenic responses was abolished after IRAK-M knockdown (n = 6 samples per group, two-way ANOVA). (H) Transvalvular peak jet velocity was measured in ApoE^−/− mice and rIL-37 treatment groups, showing a decrease in the rIL-37 treatment mice (n = 7 male mice per group, Student’s t-test). (I) H&E staining revealed thinned aortic valve leaflets in the rIL-37 treatment mice compared with the ApoE^−/− mice (n = 7 male mice per group, Bar = 100 μm, Student’s t-test). (J) Detection of calcium nodules using Von Kossa staining revealed less calcium deposition in the rIL-37 treatment mice (n = 7 male mice per group, Bar = 100 μm, Student’s t-test). Areas of calcium deposition are indicated by arrows. (K) The expression of IRAK-M, ICAM-1, and BMP-2 in ApoE^−/− mice and rIL-37 treatment groups was determined using immunofluorescence staining (n = 7 male mice per group, Bar = 25 μm, Student’s t-test). (L) rIL-37 inhibited the expression of YTHDF2 but not METTL3 (n = 6 samples per group, two-way ANOVA). (M) The m⁶A level of IRAK-M was determined by MeRIP-qPCR after treatment of AVICs with rIL-37 (n = 4 samples per group, two-way ANOVA). (N) The binding of IRAK-M mRNA and YTHDF2 was reduced after rIL-37 treatment, with HPRT1 serving as a negative control (n = 4 samples per group, two-way ANOVA).