Fig. 3: Inhibition of acid sphingomyelinase by fluoxetine induces RANKL secretion in bone marrow adipocytes through the COX-2/PGE2 pathway mediated by intracellular S1P.

a Fluoxetine treatment (0, 0.005, 0.01, 0.05, 0.1, 0.5, and 1 µM) resulted in a dose-dependent reduction of intracellular S1P in BMAs. Data were mean ± sem (N = 6/dose). **P < 0.01 compared with no fluoxetine addition group. b RANKL secretion by bone marrow adipocytes (BMAs) that received 24 h of treatment with fluoxetine (5 µM) or the combination of fluoxetine (5 µM) and cisplatin (CDDP, 2.5 µM, a known compound that could upregulate ASM expression). Data were mean ± sem (N = 6/group). **P < 0.01 and ns: not statistically significant. c The dose-dependent RNAKL secretion by BMAs in response to different levels of fluoxetine treatment (0.005, 0.01, 0.05, 0.1, 0.5 and 1 µM). Data were mean ± sem (N = 6/dose). d The increase of RANKL secretion by fluoxetine in the culture of BMAs was normalized with the co-addition of S1P (0.5 µM) and L-serine (1 µM). Data were mean ± sem (N = 6/dose). **P < 0.01 compared with the untreated controls and ns: not statistically significant. (e The significant upregulation of RANKL mRNA expression by fluoxetine addition in BMAs was normalized with the treatment of S1P (0.5 µM) and L-serine (1 µM). f–h The osteoclastogenesis of RAW264.7 cells was stimulated by the supernatant of BMAT from OVX-CUMS rats received 6-week of fluoxetine treatment (OVX-CUMS-Flx), while, the formation of osteoclasts was inhibited by BMAT supernatant from OVX-CUMS-Flx-Ser (OVX-CUMS rats with fluoxetine and L-serine). f The formation of TRAP-positive cells (Osteoclasts) stimulated by BMAT supernatant from the OVX-CUMS-Flx group. g TRAP-positive cells incubated with BMAT supernatant from OVX-CUMS-Flx-Ser. (h The number of TRAP-positive cells was significantly higher in the OVX-CUMS-Flx group than that in the OVX-CUMS-Flx-Ser group. **P < 0.01 (N = 6 wells/group). i S1P negatively regulates COX-2 protein expression in BMAs with the increase of S1P concentrations from 0 to 5 µM. Top: A representative western blot image. Bottom: the quantification of COX-2 expression from 3 replicates. Data were mean ± sem. **P < 0.01 and *P < 0.05 compared with the untreated control. j The addition of S1P led to a significant decrease in PGE2 levels in a dose-dependent manner. Data were mean ± sem (N = 6/dose). **P < 0.01 compared with the untreated controls. k The proposed mechanism for fluoxetine-induced bone loss. The red color indicated an increase (or upregulation) and the blue color indicated a decrease (or downregulation) after fluoxetine treatment. The solid unbroken lines indicated the direct reactions or interactions, and the broken line indicated indirect reactions or interactions (multiple steps) between two sides.