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

Figure 4

Lycorine reduces TFEB nuclear translocation by attenuating oxidation of TRPML1 via decreased mitochondrial ROS upon LPS stimulation in OC. BMMs were prepared, incubated with RANKL (40 ng/ml) in the presence of M-CSF (30 ng/ml) for 40 h, washed, and then incubated further with the indicated conditions (DPI, 5 nM; Mito-TEMPO, 100 nM; lycorine, 1.6 μM) in the presence of LPS (50 ng/ml) and M-CSF (30 ng/ml). Mitochondrial ROS and cytosolic ROS were determined by flow cytometry using MitoSOX Red after 16 h and using H₂DCF-DA after 24 h, respectively (a, b). After 48 h, cell lysates were prepared (c) or fixed (d). Cell lysates were subjected to Western blot to determine p62 and LC3II with the addition of bafilomycin A1 (25 nM) for 4 h. The quantified levels of p62 and LC3II are shown normalized to β-actin (c). Cell lysates were labeled with N-(biotinoyl)-N-(iodoacetyl) ethylenediamine, and TRPML1 was immunoprecipitated (IP) from each sample. HRP-streptavidin immunoblotting was performed to evaluate the reduced form of TRPML1 (e). Cells were transfected with 50 nM of scRNA or siTRPML1 and incubated further for 48 h with LPS and M-CSF. siRNA-mediated silencing of TRPML1 was confirmed by RT-PCR and qPCR (f). After fixation, more than 70 TRAP-positive MNCs in each culture were randomly selected to determine the area and fusion activity of the formed OCs (d, f). Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with anti-TFEB Ab. Abs for β-actin and lamin B1 were used for the normalization of cytoplasmic and nuclear extracts, respectively. Quantification of TFEB normalized to β-actin or lamin B1 was plotted (g). , , and compared with RANKL-pretreated cells treated with PBS. ^#, ^##, and ^### compared with LPS-treated cells. ^† and ^††† compared with scRNA-treated cells. Similar results were obtained in three independent experiments.

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