Lycorine Attenuates Autophagy in Osteoclasts via an Axis of mROS/TRPML1/TFEB to Reduce LPS-Induced Bone Loss

Figure 2

Lycorine inhibits LPS-induced OC differentiation and OC activity in vitro. BMMs (10⁴ cells/well) were prepared and incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further for 48 h (a, b, d) or 24 h (c) with LPS (50 ng/ml)±lycorine (1.6 μM, 3.2 μM) at the indicated concentration in the presence of M-CSF (30 ng/ml). LPS and lycorine were dissolved in PBS as a vehicle. Cells were fixed, and more than 70 TRAP-positive MNCs in each culture were randomly selected. The area, surrounded by a dotted line, and the maximum diameter, indicated by a double arrow of the formed OCs, were measured. The fusion index was presented as the average number of nuclei per TRAP-positive MNC formed in the culture. Representative photos are shown. Scale bar: 200 μm in the representative OC photos (a). Cell viability was measured by MTT assay. No significant difference was found compared with PBS-treated cells (b). RNA from cells stimulated with LPS in the presence or absence of lycorine (1.6 μM) was analyzed by qPCR. The expression level before RANKL pretreatment was set to 1 (c). Mature OCs were incubated further on whole dentine slices with M-CSF and LPS in the presence or absence of lycorine (1.6 μM) for 4 d. After TRAP staining, the cells were removed, and the slices were stained with toluidine blue. Representative photos of TRAP-positive OCs and resorption pits are shown. Scale bar: 50 μm in the representative photos. Total pit area/number of TRAP-positive OCs was calculated (d). , , and compared with PBS-treated pre-OCs. ^#, ^##, and ^### compared with LPS-treated cells. Similar results were obtained from three independent experiments.

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