Water Extract of Lysimachia christinae Inhibits Trabecular Bone Loss and Fat Accumulation in Ovariectomized Mice

In Asia, extracts of Lysimachia christinae have been used for liver or urinogenital system-related diseases in traditional medicine. In this study, we investigated the effects of the water extract of L. christinae (WELC) on receptor activator of nuclear factor-kappa Β ligand (RANKL)-induced osteoclastic differentiation of bone marrow macrophages, and on osteoporosis and obesity in ovariectomy mice. RANK signaling pathways related to osteoclast differentiation were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Additionally, we performed micro-computed tomography to assess trabecular bone loss, histological analysis for fat accumulation in adipose, liver, and bone tissues, and phytochemical profiling for WELC characterization. WELC significantly inhibited osteoclast differentiation by downregulating RANKL-induced mitogen-activated protein kinase (MAPK)/c-Fos/nuclear factor of activated T-cells (NFAT) signaling in osteoclast precursors and ovariectomy-induced trabecular loss by suppressing osteolcastic bone resorption. WELC markedly decreased ovariectomy-induced body weight gain and fat accumulation in adipose, liver, and bone tissues. Furthermore, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) identified 16 phytochemicals in WELC when compared with the mass fragmentation of standard chemicals. Collectively, these results suggest that WELC might possess beneficial effects on postmenopausal osteoporosis by inhibiting osteoclast differentiation and obesity by suppressing fat accumulation.


Introduction
Menopause and estrogen decline induce significant physiological alterations in women's health, including the development of osteoporosis and obesity. In the bone remodeling unit, estrogen participates in the regulation of bone resorption by osteoclasts and bone formation by osteoblasts [1,2]. Estrogen loss specifically weakens the induction of osteoclast apoptosis or decreases as the expression of osteoprotegerin, a decoy receptor of receptor activator of nuclear factor-kappa B ligand (RANKL) decreasing osteoclast activity, on stromal cells, but increasing the expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6) to stimulate osteoclast resorption [3][4][5]. Thus, estrogen loss increases the bone resorption rate rather than the bone formation rate, resulting in net bone loss and osteoporosis. Furthermore, estrogen is involved in the lipolysis pathway or regulates enzyme expression in glucose and lipid metabolism [6,7]. Estrogen loss significantly increases abdominal fat distribution, lipid accumulation in the adipose tissue, and stimulation of adipocyte differentiation [8,9], possibly related to increased weight gain and metabolic diseases induced by menopausal obesity [10,11]. Estrogen therapy

Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The same quantity of total RNA was reverse-transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was performed using the ABI QuantStudio 6 Flex RT-PCR system with the TaqMan Universal PCR Master Mix (Applied Biosystems). The primers used in this study were c-Fos (Mm00487425_m1), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, Mm00479445_m1), dendrocyte expressed seven transmembrane protein (DC-STAMP, Mm01168058_m1), ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2 (Atp6v0d2, Mm00656638_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm99999915_g1) from the TaqMan Gene Expression Assay (Applied Biosystems). All reactions were run in triplicate. Relative expression of the target genes was calculated using the ∆∆Ct method with GADPH gene as internal reaction control and expressed as fold change relative to the control untreated with RANKL and WELC. Experiments were repeated three times, and results from one representative experiment were shown.

Western Blot
Cells were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were collected by centrifugation at 13,000× g for 30 min at 4 • C. Protein quantification was performed using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal quantities of total protein were separated using Mini-PROTEAN TGX™ Precast Protein Gels and transferred onto polyvinylidene fluoride membranes using Trans-Blot Turbo Transfer System (Bio-rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against c-Fos, NFATc1 (Santa Cruz Biotechnology, Dallas, TX, USA), p-extracellular signal-regulated kinase (ERK), ERK, p-c-Jun N-terminal kinase (JNK), JNK, p-p38, p38, p-IκBα, IκBα, and β-actin (Cell Signaling Technology, Danvers, MA, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies, specific bands were detected with a LAS-4000 image analyzer (Fujifilm, Tokyo, Japan) and Image Gauge software V3.0 (Fujifilm, Tokyo, Japan) using an enhanced chemiluminescence western blotting substrate (Bio-Rad, Hercules, CA, USA).

Animal Study
Animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Knotus (Guri, Korea). Six-week-old female C57BL/6J mice (Japan SLC Inc., Shizuoka, Japan) were housed in a specific pathogen-free environment (22 • C ± 2 • C and 55 ± 5% humidity under a 12 h light/dark cycle). Following one-week acclimatization on a standard chow diet and water ad libitum, the mice were ovariectomized (OVX) or sham-operated. One week after surgery, the mice were randomly divided into four groups of six mice each: (1) Sham, (2) OVX, (3) OVX mice administered WELC 100 mg/kg/day (WELC-L), and (4) OVX administered WELC 300 mg/kg/day (WELC-H). The mice had free access to water and a normal-fat diet containing 10 kcal% fat (D12450B, Research Diets, New Brunswick, NJ, USA). WELC was administered by oral gavage once daily for five weeks. After 7 h of fasting, blood and tissue samples were obtained.

Measurement of Bone Turnover Markers
Serum levels of cross-linked C-telopeptide of type I collagen (CTX-I) and procollagen type I N-terminal propeptide (PINP) were measured using ELISA kits (Immunodiagnostic Systems Ltd., London, UK).

Micro-CT Analysis
Micro-CT analysis (SkySacn 1276, Bruker, Kontich, Belgium) was utilized to scan structural changes in the distal femur of mice. The scanned images were reconstructed using the SkyScan NRecon program and analyzed using SkyScan CTAn software (version 1.17.7.2, Bruker, Kontich, Belgium). The measured volume of the distal femur starting at 80 µm from the lower end of the growth plate and extending across 150 cross-sections (1.2 mm high). Bone morphometric parameters, including trabecular bone mineral density (BMD, g/cm 3 ), trabecular bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm), trabecular separation (Tb.Sp, mm), and trabecular thickness (Tb.Th, mm), were calculated.

Histological Analysis
The tissue samples were fixed using 10% neutral buffered formalin, dehydrated using a series of ethanol, embedded in paraffin, and sectioned to 5-µm thickness. Before dehydration, bone samples were decalcified in RDO Gold (RDO, Aurora, IL, USA). The sections were stained with hematoxylin and eosin, and adipocyte size and lipid accumulation were measured using the ImageJ software.

Statistical Analysis
For the in vitro study, data are presented as mean ± standard deviation, and as mean ± standard error of the mean for the in vivo study. Data were analyzed by a one-way analysis of variance (ANOVA) and Dunnett's post hoc test or a two-way ANOVA and Bonferoni's post hoc test using GraphPad Prism version 8 (GraphPad, CA, USA). p values less than 0.05 were considered statistically significant compared to the control.

WELC Inhibits RANK Signaling during Osteoclast Differentiation
BMMs are precursor cells possessing the potential to differentiate into osteoclasts in the presence of RANKL. RANKL/RANK interaction activates RANK downstream signaling pathways, including mitogen-activated protein kinases (MAPKs) and NF-κB, to stimulate osteoclastogenesis [29]. First, we evaluated Nutrients 2020, 12,1927 5 of 13 the effect of WELC on osteoclast differentiation of BMMs by counting the number of TRAP-stained multinucleated cells. WELC significantly suppressed RANKL-induced osteoclast formation ( Figure 1A) but increased the viability of BMMs ( Figure 1B). As mature osteoclasts demonstrate characteristics distinct from their precursors, we next examined whether WELC affects the bone resorption activity of mature osteoclasts. On the bone mimetic surface, WELC did not affect resorption pit formation by mature osteoclasts (Figure 1C), excluding the inhibitory activity of WELC during the late stage of osteoclast bone resorption. Based on these results, we investigated the effects of WELC on RANKL-induced signaling pathways to initiate osteoclast differentiation. c-Fos and NFATc1 are key transcription factors that are highly increased during RANKL-induced osteoclast differentiation [30,31]. We observed that WELC suppressed RANKL-induced mRNA and protein expression of both transcription factors (Figure 2A). Regarding WELC inhibition of osteoclast formation, as shown in Figure 1A, we further examined the expression of DC-STAMP and ATPv0d2 required for RANKL-induced osteoclast formation [32]. Consistent with WELC inhibition of osteoclast formation, WELC significantly suppressed RANKL-induced DC-STAMP and ATPv0d2 expression ( Figure 2B). Moreover, we investigated WELC activity on RANKL-induced activation of MAPKs and NF-κB, which are early signaling pathways that induce c-Fos and NFATc1 expression [33][34][35][36]. WELC suppressed the activation of ERK, JNK, and p38, but not IκBα phosphorylation and degradation involving the classical NF-κB pathway ( Figure 2C). Therefore, these results suggest that WELC specifically prevents the progress of osteoclast differentiation from precursor cells during the early stages of differentiation, by inhibiting RANKL-induced early signaling components and downstream transcription factors.
Nutrients 2020, 12, 1927 5 of 14 BMMs are precursor cells possessing the potential to differentiate into osteoclasts in the presence of RANKL. RANKL/RANK interaction activates RANK downstream signaling pathways, including mitogen-activated protein kinases (MAPKs) and NF-κB, to stimulate osteoclastogenesis [29]. First, we evaluated the effect of WELC on osteoclast differentiation of BMMs by counting the number of TRAP-stained multinucleated cells. WELC significantly suppressed RANKL-induced osteoclast formation ( Figure 1A) but increased the viability of BMMs ( Figure 1B). As mature osteoclasts demonstrate characteristics distinct from their precursors, we next examined whether WELC affects the bone resorption activity of mature osteoclasts. On the bone mimetic surface, WELC did not affect resorption pit formation by mature osteoclasts (Figure 1C), excluding the inhibitory activity of WELC during the late stage of osteoclast bone resorption. Based on these results, we investigated the effects of WELC on RANKL-induced signaling pathways to initiate osteoclast differentiation. c-Fos and NFATc1 are key transcription factors that are highly increased during RANKL-induced osteoclast differentiation [30,31]. We observed that WELC suppressed RANKL-induced mRNA and protein expression of both transcription factors (Figure 2A). Regarding WELC inhibition of osteoclast formation, as shown in Figure 1A, we further examined the expression of DC-STAMP and ATPv0d2 required for RANKL-induced osteoclast formation [32]. Consistent with WELC inhibition of osteoclast formation, WELC significantly suppressed RANKL-induced DC-STAMP and ATPv0d2 expression ( Figure 2B). Moreover, we investigated WELC activity on RANKL-induced activation of MAPKs and NF-κB, which are early signaling pathways that induce c-Fos and NFATc1 expression [33][34][35][36]. WELC suppressed the activation of ERK, JNK, and p38, but not IκBα phosphorylation and degradation involving the classical NF-κB pathway ( Figure 2C). Therefore, these results suggest that WELC specifically prevents the progress of osteoclast differentiation from precursor cells during the early stages of differentiation, by inhibiting RANKL-induced early signaling components and downstream transcription factors.

WELC Attenuates Bone Loss in OVX Mice
The ovariectomy model is a standard model of postmenopausal osteoporosis exhibiting trabecular bone loss [37]. Ovariectomy increases osteoclast formation from precursors in the bone marrow and the number of osteoclasts per unit of bone surface that lead to increase in bone resorption and trabecular bone loss [38,39] To investigate whether WELC improves bone quality in OVX mice, we analyzed the trabeculae in the femur by micro-CT analysis. Micro-CT images showed that ovariectomy resulted in apparent changes in the trabecular bone structure of the distal femur in OVX mice ( Figure 3A). We observed a compact trabecular structure in the sham, but OVX mice exhibited osteoporotic trabecular bone with less connectivity and thinner rod-like structures. Compared with the sham group, OVX mice exhibited significantly decreased BMD (29%), BV/TV (43%), Tb.N (35%), and Tb.Th (11%), demonstrating an increase in Tb.Sp (65%). A high concentration of WELC (WELC-H, 300 mg/kg) significantly improved OVX-induced bone deterioration by increasing BMD (18%), BV/TV (34%), and Tb.Th (11%), decreasing Tb.Sp (16%). In the animal model, BMD and BV/TV are positively correlated with trabecular bone strength and stiffness, while Tb.Sp is negatively correlated with the same; this predicts the correlation of these parameters with bone strength in the later life of animals [40]. Therefore, it could be suggested that the inhibitory activity of WELC on OVX-induced bone loss could protect against estrogen loss-induced osteoporosis and diminished bone strength observed during menopause.
To explore the mode of action of the bone-protective effect of WELC, we measured serum levels of CTX-I, a maker of bone resorption, and PINP, a maker of bone formation. In line with in vitro anti-osteoclastogenic activity, WELC markedly inhibited OVX-induced increase in CTX levels, while PINP levels were not altered in all groups ( Figure 3B). Therefore, these results suggest that the protective effect of WELC on OVX-induced bone loss is attributed primarily to inhibition of osteoclast-mediated bone resorption.

WELC Inhibits Fat Accumulation in OVX Mice
In OVX rodent models, estrogen deficiency has been indicated as a major factor responsible for fat accumulation and fat distribution toward visceral adipose tissue [41,42]. As expected, OVX mice demonstrated an increase in body weight and gonadal fat, with a decrease in uterine weight, when compared with the sham group ( Figure 4A-C). WELC-treated groups showed significantly reduced body weight and gonadal fat when compared with the OVX group. However, OVX-induced uterine atrophy was not ameliorated following WELC treatment, suggesting that WELC lacks estrogenic effects. Histological staining showed that WELC attenuated OVX-induced increased lipid accumulation in the liver and bone marrow, as well as reduced enlarged adipocytes in fat tissue ( Figure 4D). These findings are consistent with a previous study demonstrating that L. christinae suppresses hepatic lipid accumulation and inflammatory infiltration induced by a high-fat and high-cholesterol diet in mice [21]. Furthermore, it has been reported that L. christinae protects against alcohol-induced liver injury in mice, possibly by inhibiting oxidative stress [22]. Thus, the antioxidant activities of WELC might contribute to its inhibitory action on fat accumulation. However, the underlying mechanisms need to be further investigated. Similar to the results of body weight gain, WELC at the high dose (WELC-H, 300 mg/kg) was more effective than the low dose (WELC-L) to decrease fat accumulation in adipose tissue. However, WELC-H was inefficient to decrease lipid accumulation in non-adipose tissues (liver and bone), whereas WELC-L was highly effective ( Figure 4D). The mechanisms for this differential effect of WELC at the high dose on adipose and non-adipose tissues remain to be further studied.

Phytochemical Profiling of WELC
Next, we investigated the phytochemical profile of WELC to determine the molecular basis of its pharmacological actions. UHPLC-MS/MS analysis identified diverse phytochemical components in WELC, including ten flavonoids (catechin, epigallocatechin, epicatechin, (-)-gallocatechin, kaempferol, myricetin, phlorizin, quercetin, quercitrin, and schaftoside), three polyphenols (chlorogenic acid, neochlorogenic acid, and rosmarinic acid), one cyclitol (quinic acid), one hydroxycinnamic acid (p-coumaric acid), and betaine ( Table 1). The typical UV chromatograms at 254 nm and the extracted ion chromatograms for each component are shown in Figure 5. Reportedly, except for 3 components (quinic acid, schaftoside, and betaine), 13 individual components present inhibitory activities on osteoclast differentiation in vitro and OVX-or glucocorticoid-induced osteoporosis in animal models [43][44][45]. Additionally, some constituents possess anti-adipogenic activity by inhibiting the expression of key adipogenic transcription factors, decreasing the production of pro-inflammatory cytokines in 3T3-L1 cells, or by suppressing fat accumulation in animal models fed a high-fat diet [46,47]. Given that exposure of each WELC component on in vivo target tissue would be lower than the effective dose of individual components, WELC might demonstrate pharmacological efficacy in osteoporosis and obesity through additive and synergistic effects of these components present in WELC. (D) Histological analysis of adipose tissue, liver, and bone was performed using hematoxylin and eosin staining (scale bar, 100 µm). Image analysis of the adipocyte area or lipid droplets in each tissue was performed using the ImageJ program. * p < 0.05, ** p < 0.01 versus OVX group. WELC, water extract of Lysimachia christinae; OVX, ovariectomized.

Phytochemical Profiling of WELC
Next, we investigated the phytochemical profile of WELC to determine the molecular basis of its pharmacological actions. UHPLC-MS/MS analysis identified diverse phytochemical components in WELC, including ten flavonoids (catechin, epigallocatechin, epicatechin, (-)-gallocatechin, kaempferol, myricetin, phlorizin, quercetin, quercitrin, and schaftoside), three polyphenols (chlorogenic acid, neochlorogenic acid, and rosmarinic acid), one cyclitol (quinic acid), one hydroxycinnamic acid (p-coumaric acid), and betaine ( Table 1). The typical UV chromatograms at 254 nm and the extracted ion chromatograms for each component are shown in Figure 5. Reportedly, except for 3 components (quinic acid, schaftoside, and betaine), 13 individual components present inhibitory activities on osteoclast differentiation in vitro and OVX-or glucocorticoid-induced osteoporosis in animal models [43][44][45]. Additionally, some constituents possess anti-adipogenic activity by inhibiting the expression of key adipogenic transcription factors, decreasing the production of pro-inflammatory cytokines in 3T3-L1 cells, or by suppressing fat accumulation in animal models fed a high-fat diet [46,47]. Given that exposure of each WELC component on in vivo target tissue would be lower than the effective dose of individual components, WELC might demonstrate pharmacological efficacy in osteoporosis and obesity through additive and synergistic effects of these components present in WELC.   All data were compared with the retention time (R t ) and MS spectral data of authentic standards. UHPLC-MS/MS, ultra-high-performance liquid chromatography-tandem mass spectrometry; WELC, water extract of Lysimachia christinae.

Conclusions
This study is the first to demonstrate the anti-osteoporotic and anti-osteoclastogenic effects of WELC. WELC inhibited bone loss by suppressing osteoclastic bone resorption in OVX mice and osteoclast differentiation in vitro by suppressing RANK signaling pathways inducing osteoclastogenic transcription factors. In addition, WELC suppressed ovariectomy-induced body weight gain and fat accumulation in adipose and non-adipose tissues. Furthermore, we identified 16 phytochemical components in WELC demonstrating inhibitory potential toward bone loss and fat accumulation. Taken together, these findings suggest that WELC is an attractive, natural therapeutic candidate for the prevention and treatment of postmenopausal osteoporosis and obesity.