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

Lycorine, a plant alkaloid, exhibits anti-inflammatory activity by acting in macrophages that share precursor cells with osteoclasts (OCs). We hypothesized that lycorine might decrease bone loss by acting in OCs after lipopolysaccharide (LPS) stimulation, since OCs play a main role in LPS-induced bone loss. Microcomputerized tomography (μCT) analysis revealed that lycorine attenuated LPS-induced bone loss in mice. In vivo tartrate-resistant acid phosphatase (TRAP) staining showed that increased surface area and number of OCs in LPS-treated mice were also decreased by lycorine treatment, suggesting that OCs are responsible for the bone-sparing effect of lycorine. In vitro, the increased number and activity of OCs induced by LPS were reduced by lycorine. Lycorine also decreased LPS-induced autophagy in OCs by evaluation of decreased lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3) (LC3II) and increased sequestosome 1 (p62). Lycorine attenuated oxidized transient receptor potential cation channel, mucolipin subfamily (TRPML1) by reducing mitochondrial reactive oxygen species (mROS) and decreased transcription factor EB (TFEB) nuclear translocation. Lycorine reduced the number and activity of OCs by decreasing autophagy in OCs via an axis of mROS/TRPML1/TFEB. Collectively, lycorine protected against LPS-induced bone loss by acting in OCs. Our data highlight the therapeutic potential of lycorine for protection against inflammatory bone loss.


Introduction
Inflammatory osteolysis is caused by the pathogenesis of infectious and inflammatory disease, resulting in irreversible bone erosion. This bone loss has been reported to be due to inflammation that stimulates osteoclasts (OCs) either directly or indirectly by activating osteoblasts/stromal cells [1]. Injection of lipopolysaccharide (LPS) leads to increased eroded surface area by increasing the number of OCs in rat femurs [2]. LPS has been shown to increase the number and activity of OCs, leading to bone loss [3,4]. Increased OC number by LPS has been reported to be due to the induction of differentiation via reactive oxygen species (ROS) [5] and enhancing OC survival [6,7]. LPS-induced autophagy has been demonstrated to be responsible for increased OC formation and OC activity [4,5,8].
We investigated whether lycorine can attenuate LPSinduced bone loss in mice. The present studies assessed the detailed molecular mechanisms of lycorine for inhibitory activity on LPS-induced autophagy in OCs.

Animals and Study
Design. C57BL/6J female mice (10 weeks old) were kept in the pathogen-free animal facility of IRC. All mice were treated in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Immunomodulation Research Center (IRC), University of Ulsan, following the approval by the IACUC of IRC. The approval ID for this study is #HSC-15-011. Animals were randomly divided into the following 4 groups: vehicle control (n = 5), vehicle+lycorine (n = 5), LPS (n = 5), and LPS+lycorine (n = 6). Mice were injected with LPS (5 mg/kg, i.p.) in 200 μl phosphate-buffered saline (PBS) once per week for 3 weeks [19]. Lycorine was solubilized with 1 N HCl in PBS and neutralized with sodium hydroxide to reach pH 7.2; mice were treated with lycorine once every two days intraperitoneally in 200 μl PBS (or with PBS as a vehicle) at a dose of 6 mg/kg for 3 weeks. Mice were sacrificed by CO 2 . To analyze bone mineral density (BMD) and microarchitecture, the right femur was scanned in a high-resolution micro-CT (μCT) SkyScan 1176 System (Bruker Micro-CT, Kontich, Belgium) following the methods of Park et al. [4]. Serum collagen-type I fragment (CTX-1) level was measured with commercial RatLaps EIA assay kits. Osteocalcin and alkaline phosphatase (ALP) of serum were assessed using an osteocalcin EIA kit and a colorimetric kinetic determination kit, respectively. Serum MCP-1 was evaluated by sandwich ELISA using the recommended Abs according to the manufacturer's instructions.
2.3. OC Formation. At 4-5 weeks of age, C57BL/6J bone marrow cells were isolated from the tibiae and femurs according to a previously described method [20]. Further steps were performed following the methods of Park et al. [4]. After incubation for the indicated times, the cells were fixed in 10% formalin for 10 min and stained for tartrate-resistant acid phosphatase (TRAP). The number of TRAP-positive multinucleated cells (MNCs) (three or more nuclei), area, and maximal diameter of the formed OCs were measured, and the fusion index was presented as the average number of nuclei per TRAP-positive MNC [21].

Bone Resorption.
To evaluate the ability of OCs to form pits, mature OCs were cultured on dentine slices [22]. To generate mature OCs, preosteoclast was cultured with M-CSF and RANKL in 60 mm dishes. After 3-4 days, detached mature OCs and seeded equal numbers of cells on top of dentine slices. The cells were incubated with M-CSF (30 ng/ml) and LPS (50 ng/ml) in the presence or absence of lycorine (1.6 μM) for 4 days. Further steps followed the methods of Park et al. [4].

Detection of Oxidized TRPML1 by Carboxymethylation.
OCs were generated from pre-OCs by treatment with M-CSF (30 ng/ml) and LPS (50 ng/ml) in the presence or absence of lycorine (1.6 μM) for 2 d. The medium was removed, and the cells were frozen rapidly in liquid nitrogen. The frozen cells were transferred to 100 μM N-(biotinoyl)-N′-(iodoacetyl) ethylenediamine (BIAM)-containing lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10 μg/ml aprotinin, and 10 μg/ml leupeptin; the buffer was rendered free of oxygen by bubbling nitrogen gas at a low flow rate for 20 min). After sonication in a bath sonicator for three periods of 1 min each, the lysate was then clarified by centrifugation and subjected to immunoprecipitation with 1 μg of TRPML1-specific Ab. Immunocomplexes labeled with BIAM were detected with HRP-conjugated streptavidin and developed with an enhanced chemiluminescence kit.

Transfection of siRNA.
After treatment with M-CSF and RANKL for 40 h, BMMs were transfected with small interfering RNA (siRNA) against TRPML1 or with scrambled siRNA (scRNA) using Lipofectamine 3000. Further steps followed the method of Park et al. [4].
2.11. Statistical Analysis. All experiments were repeated at least three times. The data are expressed as mean ± standard deviation. Statistical analysis was performed by Student's t-test when two groups were compared or by one-way ANOVA followed by Bonferroni posttests when multiple groups were compared. Two-way ANOVA was used when two variables were analyzed. p value less than 0.05 was considered statistically significant.

Lycorine Protects against LPS-Induced Bone Loss in Mice.
Lycorine has been shown to be anti-inflammatory [10], with ROS scavenging activity [11] as well as inhibition of autophagy [17]. Our previous results suggested that LPS induces bone loss by increasing autophagy in vivo [4] and LPS induced autophagy to enhance differentiation and activity of OCs by upregulating ROS [5]. This prompted us to hypothesize that lycorine attenuates LPS-induced bone loss in mice. To investigate the effect of lycorine on LPSinduced inflammatory bone loss, μCT scans of femurs from mice treated with lycorine or vehicle after administration of LPS or PBS were analyzed. A 6 mg/kg dose of lycorine exhibited maximum protection compared to doses of 2.5 mg/kg and 4 mg/kg, respectively (Figure 1(a)). Body size or shape showed no change among the 4 groups when the mice were 13 weeks old. LPS induced significant bone loss with decreased bone mineral density (BMD), bone volume (BV/TV), and trabecular thickness (Tb.Th.) and increased trabecular space (Tb.Sp.) compared with PBS alone (Figure 1(b) and Table 1). The serum concentration of MCP-1 was also elevated upon LPS treatment (Table 1), indicating that LPS induced systemic inflammation. However, administration of lycorine with LPS attenuated the LPS-induced bone loss (Figure 1(b), Table 1). Treatment of lycorine with LPS increased BMD, BV/TV, and Tb.Th. compared with LPS alone, in addition to decreasing the   (Table 1), but lycorine alone did not exhibit any significant difference compared with vehicle (Figure 1(b), Table 1). As shown in Figure 1(c), OC.N/BS (the ratio of OC number to total bone surface area) and OC.S/BS (the ratio of OC surface area to total bone surface area) as assessed by in vivo TRAP staining were also significantly reduced when lycorine was injected in LPS-treated mice, indicating that lycorine reduced both number and size of OCs in LPS-treated mice. Consistent with these findings, serum CTX-1, a marker of bone resorption in vivo that was elevated by LPS treatment, was decreased when LPS-injected mice were treated with lycorine. However, cotreatment with lycorine did not significantly affect the levels of serum ALP and osteocalcin, which act as markers of bone formation in vivo, compared with LPS alone (Table 1). LPS-induced serum MCP-1 was also reduced by lycorine (  the number of nuclei per OC (Figure 2(a)). Lycorine did not change the viability of BMMs under the assayed conditions (Figure 2(b)). Consistent with this result, the mRNA levels of TRAP, calcitonin receptor, cathepsin K, DC-STAMP, and ATP6v0d2 were markedly reduced in lycorine-treated cells (Figure 2(c)). Next, we examined whether lycorine attenuated in vitro bone resorption induced by LPS. Mature OCs generated from cells treated with lycorine in the presence of LPS showed significantly reduced total pit area/number of OCs compared to cells stimulated with LPS only (Figure 2(d)), indicating that lycorine inhibits OC activity.

Lycorine Decreases LPS-Induced Autophagy in OCs.
Excess autophagy has been reported to be responsible for inflammatory bone loss conditions such as rheumatic arthritis [23]. Since LPS induced autophagy to affect differentiation and activity of OC in our previous results [4,5,8], we hypothesized that lycorine inhibits autophagy to attenuate differentiation and activity in LPS-induced OCs. Therefore, we assessed whether lycorine decreased autophagy induced by LPS in OCs. Formation of autophagosomes was determined by immunoblotting cell lysates with an antibody against microtubule-associated protein light chain 3 (LC3) as a marker for activation of autophagic stimulus [24]. LPS increased the lipidated form of LC3 (LC3II), and addition of bafilomycin A1 led to accumulation of LC3II, whereas lycorine treatment significantly attenuated this accumulation ( Figure 3). Degradation of p62/STSQM1 was evaluated as a surrogate marker for autophagic flux [25]. The expression level of p62 that was decreased by LPS stimulation was recovered by lycorine treatment in the presence of LPS (Figure 3).

Lycorine Reduces TFEB Nuclear Translocation by Attenuating Oxidation of TRPML1 via Decreased
Mitochondrial ROS in OC. Mitochondrial ROS have been reported to contribute to autophagy by oxidizing TRPML1, a lysosomal Ca channel [26], suggesting a critical role of ROS in inducing autophagy. Our previous results showed that LPS induces autophagy in OCs by stimulating cytoplasmic ROS (cROS) production [5]. That prompted us to hypothesize that lycorine decreased ROS to reduce LPSinduced autophagy in OCs. As we expected, lycorine treatment dramatically decreased mitochondrial ROS (mROS) and cROS after 16 h and 24 h LPS stimulation, respectively (Figure 4(a)). DPI, an inhibitor of NADPH oxidase, and Mito-TEMPO, an mROS scavenger, decreased mitochondrial ROS induced by LPS, and no further decrease was found when lycorine was added to DPI or Mito-TEMPO (Figure 4(b)). DPI and Mito-TEMPO decreased autophagic flux stimulated by LPS by decreasing LC3 II level and increasing p62 level (Figure 4(c)), supporting a role of ROS in LPS-induced autophagy. Lycorine did not further decrease the effect of DPI or Mito-TEMPO on area or number of OCs upon LPS stimulation (Figure 4(d)), indicating that the inhibitory activity of lycorine in OCs was mainly mediated by decreasing ROS levels induced by LPS. Since mROS has been reported to be responsible for transcription factor EB (TFEB) nuclear localization by oxidation of transient receptor potential cation channel, mucolipin subfamily (TRPML1) [26], we determined whether lycorine increased the reduced form of TRPML1. As shown in Figure 4(e), LPS decreased the level of reduced TRPML1, whereas lycorine treatment recovered the reduced form, as Mito-TEMPO did in the presence of LPS. To confirm the role of TRPML1  to exhibit the effect of lycorine on OCs, knockdown of TRPML1 was performed. TRPML1 silencing significantly reduced OC number, OC area, and fusion index that were increased by LPS (Figure 4(f)). No further decrease was found with lycorine treatment when TRPML1 was downregulated (Figure 4(f)), suggesting that TRPML1 is responsible for the inhibitory effect of lycorine in OCs. We determined whether lycorine delayed TFEB nuclear translocation stimulated by LPS as a next stage. As shown in Figure 4

Discussion
We demonstrated that lycorine, a plant alkaloid purified from Lycoris radiata [9], protects mice from inflammatory bone loss induced by LPS. LPS injection led to a significant decrease in bone density with increased number and surface area of OCs with no change in bone formation, suggesting a 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). * p < 0:05, * * p < 0:01, and * * * p < 0:001 compared with RANKL-pretreated cells treated with PBS. # p < 0:05, ## p < 0:01, and ### p < 0:001 compared with LPS-treated cells. † p < 0:05 and † † † p < 0:001 compared with scRNA-treated cells. Similar results were obtained in three independent experiments. critical role of OCs in LPS-induced bone loss. When lycorine was administered to LPS-injected mice, OC.N/BS, OC.S/BS, and serum CTX-1 levels were significantly reduced, whereas no changes were observed with in vivo bone formation markers, serum ALP and osteocalcin, implying that lycorine decreases bone loss induced by LPS in mice through OCs. In vitro, lycorine decreased TRAP-positive MNCs as well as the area and maximum diameter of OCs and fusion index that were increased by LPS. Bone resorption assessed in dentine slices was also decreased by lycorine. These results indicated that lycorine affected not only differentiation but also activity of OCs. Lycorine decreased LPS-induced autophagy when evaluated by decreased LC3II and increased p62. Our previous studies showed that LPS increased number and activity of OCs by inducing autophagy [4,5]. These findings suggested that lycorine decreased number and activity of OCs by decreasing autophagy, finally leading to protection against bone loss.
We showed that lycorine decreased mROS, which act as a trigger signal for an axis of mROS/TRPML1/TFEB to induce autophagy. Lycorine also decreased cytoplasmic ROS induced by LPS. Our previous data exhibited that LPS increased the fusion of autophagosomes and autolysosomes [4] as well as autophagy induction by increasing cROS [5]. Blockade cROS by inhibition of NADPH oxidase induced by LPS decreased mROS, indicating that cROS also affected mitochondrial ROS. Many studies have demonstrated the association of ROS with autophagy, but the detailed mechanisms by which ROS induce autophagy are not completely explained. ATG4, which participates in the autophagy process, acts as a direct target of ROS [27], and ROS induce activation of NRF2 and FOXO3 as transcription factors to induce autophagy [28]. Increasing mitochondrial ROS has been reported to activate lysosomal TRPML1 channels, leading to nuclear translocation of TFEB and subsequent stimulation of autophagy [26], implying an important role of TRPML1 oxidation in TFEB nuclear translocation to induce autophagy. TRPML1, a lysosomal membrane protein, has been reported to play a role in autophagosome and lysosome fusion [29,30] as well as acting as an ROS sensor [26]. Lysosome function and autophagy are modulated by an interaction between TRPML1 and TFEB [29][30][31], although there are other pathways for the activation of TFEB [30] such as mTOR inhibition. Our present study also shows that LPS induces mitochondrial ROS and subsequently oxidizes TRPML1 to induce TFEB nuclear translocation, suggesting that the effect of LPS on autophagy induction in OCs is regulated by an interaction loop of TRPML1 and TFEB. Removal of mROS by Mito-TEMPO reduced oxidized TRPML1 induced by LPS, suggesting that mROS participates in TRPML1 oxidation. Lycorine also decreased oxidized TRPML1 and TFEB nuclear translocation as well as mROS levels stimulated by LPS. Knockdown of TRPML1 decreased the effect of LPS and the inhibitory effect of lycorine on number of OC, OC area, and fusion index, suggesting that TRPML1 is required for the effects of LPS and lycorine on number and activity of OCs. Taken together, these results suggest that lycorine inhibits inflammatory bone loss by decreasing mROS that lead to decreased TFEB nuclear translocation by decreasing oxidation of TRPML1 in OCs.
Lycorine has been reported to have diverse pharmacological effects on various diseases with anti-inflammatory and antioxidant activity as well as downregulating autophagy with low toxicity and mild side effects [10,11,13,14,16,17]. Our data demonstrated the protective effects of lycorine on inflammatory bone loss by attenuation of LPS-induced autophagy via an axis of mROS/TRPML1/TFEB, implying that decreasing oxidative stress in OCs is a potential therapeutic strategy for inflammatory bone loss.

Data Availability
All data used to support the findings of this study are included within the article.