Synergistic effects of magnesium ions and simvastatin on attenuation of high-fat diet-induced bone loss.

INTRODUCTION: Magnesium (Mg) has a prophylactic potential against the onset of hyperlipidemia. Similar to statin, Mg is recommended as lipid-lowering medication for hypercholesterolemia and concomitantly exhibits an association with increased bone mass. The combination of statin with Mg ions (Mg2+) may be able to alleviate the high-fat diet (HFD)-induced bone loss and reduce the side-effects of statin. This study aimed to explore the feasibility of combined Mg2+ with simvastatin (SIM) for treating HFD-induced bone loss in mice and the involving mechanisms.
MATERIALS AND METHODS: C57BL/6 male mice were fed with a HFD or a normal-fat diet (NFD). Mice were intraperitoneally injected SIM and/or orally received water with additional Mg2+ until sacrificed. Enzyme-linked immunosorbent assay was performed to measure cytokines and cholesterol in serum and liver lysates. Bone mineral density (BMD) and microarchitecture were assessed by micro-computed tomography (μCT) in different groups. The adipogenesis in palmitate pre-treated HepG2 cells was performed under various treatments.
RESULTS: μCT analysis showed that the trabecular bone mass was significantly lower in the HFD-fed group than that in NFD-fed group since week 8. The cortical thickness in HFD-fed group had a significant decrease at week 24, as compared with NFD-fed group. The combination of Mg2+ and SIM significantly attenuated the trabecular bone loss in HFD-fed mice via arresting the osteoclast formation and bone resorption. Besides, such combination also reduced the hepatocytic synthesis of cholesterol and inhibited matrix metallopeptidase 13 (Mmp13) mRNA expression in pre-osteoclasts.
CONCLUSIONS: The combination of Mg2+ and SIM shows a synergistic effect on attenuating the HFD-induced bone loss. Our current formulation may be a cost-effective alternative treatment to be indicated for obesity-related bone loss.

Introduction

Obesity is associated with increased risk of metabolic syndrome, including insulin resistance, abdominal obesity, hypertension, and dyslipidemia [[1], [2], [3]]. Epidemiological and pathophysiological evidences also show the link between osteoporosis and dyslipidemia, where the low bone mineral density (BMD) appears to be an indicator of cardiovascular mortality in obese individuals [[4], [5], [6]].

A high-cholesterol diet in rats shows a progressive osteopenic bone in line with increased serum osteocalcin and carboxyterminal collagen crosslinks [7]. Whereas, another in vivo study demonstrates that cholesterol potentiates osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) [8]. The explanation for this paradox is that the type of cholesterol (free cholesterol or cholesterol ester) is the major cause of the biological diversity on osteogenesis [8]. The elevation of free cholesterol concurrent low BMD conveys the negative effect of hypercholesterolemia on the process of osteoporosis [8]. Thus, lowering the free cholesterol level in circulatory system may rescue the hypercholesterolemia induced bone metabolic disorders.

Statins are commonly recommended as lipid-lowering medications for hypercholesterolemia [9,10], and are also able to increase BMD [11,12]. However, high dose of statins may lead to asymptomatic hepatotoxicity and serious muscle injury [[13], [14], [15]]. The risk of statin-induced newly diagnosed diabetes mellitus elevates about 0.2% per year [16]. Statins have an effect on risk reduction of atherothrombotic stroke and total stroke, but they possibly increase the risk of hemorrhagic stroke in patients with cerebrovascular diseases [17]. Statins competitively inhibit HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway, inactivation of which requires the presence of magnesium ions (Mg2+) [18,19]. In addition, Mg2+ has a prophylactic potential against the onset of hyperlipidemia and cardiovascular diseases by reducing lipid indices [20]. In contrast, obesity-induced hyperglycemia could cause the hypomagnesemia in non-diabetic individuals [[21], [22], [23]]. Therefore, we formulated the notion that the combined supplementation of Mg2+ with simvastatin (SIM) at a lower dose might synergistically reduce cholesterol synthesis, replenish the Mg2+ scarcity in obese mice, and bypass the side-effects from statins treatment alone.

Bone remodeling requires the differentiation of BMSCs-derived osteoblasts along with subsequent collagen synthesis and the recruitment of hematopoietic-derived osteoclasts for bone resorption [24]. Still up for debate is the question on the explicit roles of Mg2+ on bioactivities of the bone resident cells. Knockdown of Mg2+ transporter SCL41A1 mitigates the inhibition of Mg2+ for the osteogenic differentiation in BMSCs and promotes bone mineralization [25]. However, Mg-based implant exhibits biodegradability and osteo-inductive properties in our previous study [26]. Mg2+ scarcity results in an increased formation of osteoclasts, but has no discernible effect on osteoclasts viability [27]. Whereas, the high levels of degradative products from Mg metal arrest the proliferation and mature differentiation of osteoclasts progenitors [28]. With regards to statins, the major mechanism on bone metabolism refers to promote osteogenesis by increasing viability and differentiation of osteoblasts [29], while inhibits osteoclastogenesis by impairing the prenylation of GTP-binding proteins [30]. No matter the direct regulation of bone resident cells activities or shrinkage the effects of hypercholesterolemia on these cells activities, Mg2+ and SIM offer the possibility to alleviate the bone loss in high-fat diet (HFD)-fed mice.

Therefore, our overall hypothesis is that the combined supplementation of Mg2+ and SIM could improve the lipid profiles in circulatory system while alleviates the bone loss in HFD-fed mice. Our findings confirmed that the combined therapy of Mg2+ and SIM attenuated the HFD-induced bone loss via synergistically impaired osteoclast formation and activities, as well as limited the cholesterol synthesis.

Materials and methods

Animals

C57BL/6 mice (Twelve-week-old, male) were fed with a HFD (60% of total calories from fat, 20% from carbohydrate and 20% from protein, cat# D12492, Research Diets Inc.) [31]. The control group received a normal-fat diet (NFD; 10% of total calories from fat, 70% from carbohydrate and 20% from protein, cat# D12450J, Research Diets Inc.). Body weight was measured weekly. The mice in SIM group were intraperitoneally injected SIM 20 mg/kg/day until sacrificed [32,33]. The mice in Mg group were orally received water supplemented with Mg2+ (magnesium chloride, 100 mg/kg/day) until sacrificed. The mice in the combination of Mg2+ and SIM group were intraperitoneally injected SIM (20 mg/kg/day) and orally received Mg2+ (100 mg/kg/day). The dosage was calculated based on the clinical trials on Mg supplementation (about 500 mg/day) in diseases with Mg deficiency [[34], [35], [36]]. If animals showed signs of suffering during the observation periods, they were humanely euthanized. All animals were housed under a 12-h light/dark cycle at the Experimental Animal Center at the Prince of Wales Hospital and received NFD/HFD and water ad libitum. The specified experimental protocols were approved by the Animal Experiment Ethics Committee of the Chinese University of Hong Kong (17-184-MIS-5-C and 19-036-MIS-5-C).

Histological and histomorphometric analysis

After fixation in 4% paraformaldehyde (PFA) for 24 h, bone tissues were decalcified in 10% ethylene diamine tetraacetic acid (EDTA, pH 7.4, Sigma, cat# 798681) for 14 days, embedded in paraffin to prepare sections (5 μm thick) for routine staining [37].

Calcein green (10 mg/kg, Sigma, cat# C0875) and xylenol orange (90 mg/kg, Sigma, cat# 398187) were subcutaneously injected into the abdomen at day 10 and day 3 before sacrifice, respectively. After euthanasia, dehydrated tibiae were embedded in the methyl methacrylate (MMA, Sigma, cat# M55909) to prepare sections with 5 μm in thickness (Leica RM2255) along the sagittal plane. The mineral apposition rate (MAR, μm/day) and bone formation rate (BFR/BS, μm3/μm2/day) were calculated based on the fluorescence labeling of the proximal tibia under the growth plate [38]. Histomorphometric analysis was carried out using OsteoMeasure System (Osteometrics, Atlanta, GA, USA) according to a standard histomorphometry protocol [39].

Micro-computed tomography (μCT) imaging and analysis

Trabecular bone microstructure under the growth plate near the proximal tibia was reconstructed and analyzed, including about 1.5 mm length of bone mineral density (BMD), bone volume over total volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) [40]. The microstructure of cortical bone at the femoral midshaft was reconstructed and analyzed, including 0.8 mm length of average cortical thickness (Ct.Th) and cortical porosity (Ct.Po) [40].

Isolation of bone marrow cells for mRNA analysis

Fresh bone marrow from proximal tibia was flushed out with phosphate-buffered saline (PBS) containing 1% fatty acid-free bovine serum albumin (BSA) and 1% RNase and DNase-free water using a 25-gauge needle [41]. Red blood cells were lysed using erythrocyte lysis buffer for 3 min at room temperature. After centrifugation at 1500 rpm for 5 min, cells were washed with PBS one time and added into Trizol reagent.

Isolation of bone marrow mesenchymal stem cells (BMSCs)

For BMSCs isolation, bone marrow cells from C57BL/6 mice (6-week-old, male) were flushed out with minimum essential medium (MEM, Gibco, cat# 61100061) [42]. The erythrocyte-deleted BMSCs were cultured in MEM supplemented with 15% fetal bovine serum (FBS, Gibco, cat# 16140071) and 1% Penicillin-Streptomycin-Neomycin (PSN, Gibco, cat# 15640055) and incubated at 37 °C with 5% CO2. The medium was changed every 2 days. Cells were passaged until the 80% confluent. Immunofluorescent staining for stem cell surface markers (CD105 and CD44) in primary BMSCs and tri-lineage differentiations assays (adipogenesis, osteogenesis, and chondrogenesis) were performed.

Cell viability assay

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen, cat# M6494) assay was performed to evaluate the effect of Mg2+ (Sigma, cat# M8266) or SIM (Abcam, cat# ab120505) on the viability of BMSCs and bone marrow macrophages, respectively. For MTT test, the cells were seeded into 96-well plates at a density of 3 × 103 cells/well. After overnight incubation, the cells were treated with different concentrations of Mg2+ or SIM for 0, 24, 48, and 72 h, respectively. At each time point, the cells were incubated with 50 μl (1 mg/ml) MTT solution at 37 °C for 4 h in dark. After discarding the MTT solution, 50 μl DMSO was added into each well, and the absorbance at 570 nm was detected.

Tri-lineage differentiation of BMSCs

BMSCs at passage 2 were exposed to Mg2+ (0.8, 1.8, and 5 mM) or SIM (0, 0.1, 0.2, 0.5 and 1 μM) in osteogenic induction medium (low glucose DMEM medium containing 10% FBS, ascorbic acid (50 μg/ml, Sigma, cat# 795437), dexamethasone (10 nM, Sigma, cat# D1756), and β-glycerolphosphate (5 mM, Sigma, cat# 50020)). Medium was changed every 2 days. Cell lysates were homogenized for mRNA level analysis after induction for 3 days. Alkaline phosphatase (ALP, Sigma, cat# 86R-1 KT) staining was performed after induction for 6 days. Alizarin Red S (Sigma, cat# 130-22-3) staining was performed after induction for 10 days.

Besides, BMSCs at passage 2 were exposed to Mg2+ (0.8, 1.8, and 5 mM) or SIM (0, 0.1, 0.2, 0.5 and 1 μM) under adipogenic induction medium (high glucose DMEM medium containing 10% FBS, 3-isobutyl-1-methylxanthine (0.5 mM, IBMX, Sigma, cat# I7018), insulin (5 μg/ml, Sigma, cat# I2643), indomethacin (50 μM, Sigma, cat# I7378), and dexamethasone (500 nM)). The mRNA expression level was analyzed after induction for 3 days. Oil red O (Sigma, cat# O0625) staining was performed for lipid droplet formation after induction for 7 days.

BMSCs at passage 2 (5 × 105 cells) were centrifuged to a pellet at 1200 rpm for 5 min using centrifuge tubes and then cultured in chondrogenic induction medium (high glucose DMEM medium containing 10% FBS, 1% PSN, TGF-β3 (10 ng/ml), BMP-2 (500 ng/ml), dexamethasone (100 nM), and ascorbate-2-phosphate (50 μg/ml)) at 37 °C incubator with 5% CO2. The medium was carefully replaced with fresh induction medium every 2 days. Fast green and safranin O staining was performed to evaluate the chondrogenesis after induction for 21 days [43].

Bone marrow-derived macrophages (BMDMs) and osteoclastogenic differentiation

For isolation of BMDMs, the bone marrow from C57BL/6 mouse (6-week-old, male) was flushed out with MEM and plated in MEM containing 10% FBS and 1% PSN. Non-adherent cells were collected after 12 h, and the erythrocyte-deleted cell pellet was resuspended in DMEM/F12 (Gibco, cat# 10565018) medium containing 10% FBS, 1% PSN, and macrophage colony-stimulating factor (M-CSF, 30 ng/ml, Gibco, cat# PHG6054).

BMDMs at passage 1 were cultured in DMEM/F12 medium containing 10% FBS, 1% PSN, M-CSF (30 ng/ml), and RANK ligand recombinant human protein (RANKL, 30 ng/ml, Gibco, cat# PHP0034) for 2 days to generate pre-osteoclasts and above 5 days to generate multinucleated mature osteoclasts [44].

Fatty acid treatment

HepG2 cells, a human hepatoma cell line (American Type Culture Collection, Rockville, MD, USA), were grown in high-glucose DMEM at 37 °C containing 10% FBS, 1% PSN. Palmitic acid powder (Sigma, cat# P9767) was added to a 10% solution of fatty acid free BSA and dissolved by shaking gently overnight at 37 °C to yield the stock solution (8 mM). The final molar ratio of free fatty acid to BSA was 5.2:1 [45]. Sub-confluent monolayer HepG2 cells were pre-treated with or without Mg, SIM, or Mg + SIM for 24 h, and then stimulated with or without palmitic acid (0.4 mM) for another 24 h. BSA (0.5%, w/v) was added in the control group. Cells were stained with BODIPY (Invitrogen, cat# D3922) to examine the amount of neutral lipid accumulation in HepG2 cells.

Serum measurements

Blood samples were collected before mice euthanasia. After clotting and centrifuging, serum was collected and stored at −80 °C until assayed. Cholesterol (Abcam, cat# ab65390), procollagen type I N-terminal propeptide (P1NP, Abclonal, cat# RK03119), C-terminal telopeptide of type I collagen (CTX1, Abclonal, cat# EMC0850), high-density lipoprotein (HDL, Abcam, cat# ab65390), low-density lipoprotein (LDL, Abcam, cat# ab65390) and very low-density lipoprotein (VLDL, Abcam, cat# ab65390) in serum were measured as manufacturer's instructions. The concentrations of Mg2+ and Ca ion (Ca2+) in serum were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES, Shimadzu Europa Gmbh, Germany) at 280 nm and 318 nm absorbances, respectively [46].

Liver lysates for cholesterol measurements

For analysis of liver lipid content, frozen liver tissue (20 mg) was homogenized in cholesterol assay buffer (100 μl, Abcam, cat# ab65390) on ice. Collection of the supernatant was regarded as total cholesterol after centrifugation for 8 min at 4 °C and 13,000 rpm. After incubation of mixture of sample (100 μl, above supernatant) with 2X Precipitation Buffer (100 μl) for 10 min at room temperature, the supernatant was collected as HDL fraction after centrifugation for 10 min at 2000 g. The resuspended precipitation in PBS (200 μl) was regarded as LDL/VLDL fraction.

Cholesterol solution

To yield the stock solution (12.5 mg/ml) of cholesterol (Sigma, cat# C8667), we added cholesterol (25 mg) in absolute ethyl alcohol (2 ml) and incubated at 75 °C with gentle vibration until dissolved.

Mechanical testing

Three-point bending strength was performed using a mechanical testing machine (H25KS Hounsfield Test Equipment, Redhill, Surrey, UK). The femur (with the anterior surface upwards) was positioned horizontally on the two holding pivots setting with a constant span length of 10 mm. The pressing force was vertically directed to the femoral midshaft. Each bone was compressed with a constant speed of 5 mm/min until failure. Breaking force was defined as failure load. Stiffness was calculated by the slope of the linear (elastic) part of the load-displacement curve [47].

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using Trizol reagent. RNA was quantified spectrophotometrically based on the A260 using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). mRNA was reversely transcribed to cDNA using the cDNA kit (Takara). cDNA was amplified in a mixture reaction system containing SYBR Green qPCR SuperMix-UDG (Takara) and specific primer sequences (Table S1) [48]. qRT-PCR analysis was performed using ABI 7900HT platform (Applied Biosystems, Waltham, MA, USA).

Statistical analysis

All the mice were randomly assigned to the indicated groups. The performances such as the distributions, treatments, and assessments were not blinded, but the results were confirmed by three independent investigators. Statistical analyses were performed using one-way ANOVA with Tukey's post hoc test for comparison of control/normal and other treatment groups or two-way ANOVA with Sidak's post hoc test for among-group comparisons (with the purpose to determine if there is synergistic effect). Numerical values for each measurement were displayed as mean ± standard deviation (SD). P < 0.05 was defined as statistical significance. GraphPad Prism (version 8.2.1) was used for all statistical analysis.

Results

HFD-fed group has a susceptibility to lower trabecular bone mass measured at proximal tibia as compared with that of NFD-fed group

The body weights began to diverge at week 2 of the HFD-fed group showing a significantly greater body weight than that of NFD-fed group (Fig. 1A). μCT and histological analysis of the trabecular bone showed that BV/TV in the HFD-fed group was significantly lower than that in the NFD-fed group after week 8 (Fig. 1B–D). Linear regression analysis showed that the body weight was negatively correlated with BV/TV under HFD-fed group (Fig. 1E). μCT analysis of the cortical bone at the femoral midshaft showed a significantly lower value of cortical thickness and a significantly higher cortical porosity in the HFD-fed group than that in the NFD-fed group at the week 24 (Fig. 1F and G). Linear regression analysis showed that body weight was negatively correlated with cortical thickness in mice with HFD-fed (Fig. 1H). These results demonstrate that HFD-fed group has a susceptibility to lower trabecular bone mass measured at proximal tibia as compared with that of NFD-fed group.

Fig. 1

HFD-fed group has a susceptibility to lower trabecular bone mass measured at proximal tibia as compared with that of NFD-fed group.

(A) Comparison of the body weights between NFD- and HFD-fed mice (n = 5). (B) Representative H&E staining of proximal tibiae from NFD- and HFD-fed mice at the indicated time points (Scale bar: 500 μm). (C, D) Representative μCT images and quantification (n = 5) of trabecular bone from standard region of interest (ROI) in proximal tibiae (Scale bar: 400 μm). (E) Linear regression analysis of the BV/TV of trabecular bone and body weight in HFD-fed mice within 24 weeks (n = 20). (F, G) Representative μCT images and quantification (n = 5) of cortical bone in the midshaft of femora (Scale bar: 400 μm). Ct.Th: cortical thickness; Ct.Po: cortical porosity. (H) Linear regression analysis of the Ct.Th of femur and body weight in HFD-fed mice within 24 weeks (n = 20). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by two-way ANOVA with Sidak's post hoc test (A, D, and G).

Combination of Mg2+ and SIM attenuates the trabecular bone loss in HFD-fed mice

The concentration of serum Mg2+ in the HFD-fed group showed a significantly lower level than that of the NFD-fed group since week 16 (Fig. 2A). Rather, the concentration of serum Ca2+ in HFD-fed group was similar to that in NFD-fed group (Fig. 2B). In line with what we mentioned previously, obesity-induced hyperglycemia was the cause of hypomagnesemia [23]. The concentration of serum cholesterol was higher in the HFD-fed group than that in the NFD-fed group since week 16 as well, indicating a potential relationship between serum Mg2+ and cholesterol (Fig. 2C). Nitrendipine, the Mg2+ channel antagonist, did not affect the gene expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in HepG2 cells (a commonly used human liver cancer cell line) in vitro (Fig. 2D). While BAY K8644, the Mg2+ channel agonist, significantly inhibited the gene expression of HMGCR in HepG2 cells (Fig. 2E). These results suggest that the HMG-CoA reductase synthesis could be affected by the concentration of Mg2+ in hepatocyte where the cholesterol is produced.

Fig. 2

The alterations of Mg2+, Ca2+, and cholesterol levels in serum.

(A) Comparison of the concentrations of serum Mg2+ between NFD- and HFD-fed mice (n = 6). (B) Comparison of the concentrations of serum Ca2+ between NFD- and HFD-fed mice (n = 6). (C) Comparison of the concentrations of serum cholesterol between NFD- and HFD-fed mice (n = 5). (D) Relative expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in HepG2 cells treated with different concentrations of nitrendipine (an inhibitor of Mg2+ channel) for 2 days (n = 3). (E) Relative expression of HMGCR in HepG2 cells treated with different concentrations of BAY K8644 (an agonist of Mg2+ channel) for 2 days (n = 3). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by two-way ANOVA with Sidak's post hoc test (A, B, and C), by one-way ANOVA with Tukey's post hoc test (D and E).

Based on the individual variation of trabecular bone mass, we defined two stages of HFD-induced bone loss: early stage ranged from week 8 to week 12, and late stage started from week 12 to week 16. In order to perform the early-stage therapy, at week 8 post HFD-feeding, the mice were treated with Mg2+ and/or SIM for another 4 weeks (Fig. 3A). The body weight of combination of Mg2+ and SIM group (combined group) was significantly lower than that of the HFD-fed group (Fig. 3B). Both BMD and BV/TV of proximal tibia in the combined group were significantly higher than that in the HFD-fed group (Fig. 3C and D). Although the concentration of serum Mg2+ in combined group was slightly higher than that in the HFD-fed group, combined treatment could not normalize the concentration of serum Mg2+ in early-stage therapy (Fig. 3E). In order to perform the late-stage therapy, at week 12 post HFD-feeding, the mice were treated with Mg2+ and/or SIM for another 4 weeks (Fig. 3F). The body weight of the combined group was significantly lower than that of the HFD-fed group (Fig. 3G). Both BMD and BV/TV of the combined group were substantially higher than that of the HFD-fed group (Fig. 3H and I). The concentration of serum Mg2+ in combined group was substantially greater than that in HFD-fed group (Fig. 3J).

Fig. 3

Combination of Mg2+ and SIM attenuates the trabecular bone loss in HFD-fed mice.

(A) Schematic diagram illustrating the early-stage therapy. (B) Comparison of the body weights between NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups at the early-stage therapy (at week 8 post HFD-feeding, the mice were received treatment for another 4 weeks) (n = 5). (C) Representative μCT images and H&E staining of proximal tibiae at the early-stage therapy (Scale bar: 400 μm). (D) Quantification of trabecular bone from standard ROI in proximal tibiae at the early-stage therapy (n = 5). (E) Comparison of the concentrations of serum Mg2+ at the early-stage therapy (n = 5). (F) Schematic diagram illustrating the late-stage therapy. (G) Comparison of the body weights between NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups at the late-stage therapy (at week 12 post HFD-feeding, the mice were received treatment for another 4 weeks) (n = 5). (H) Representative μCT images and H&E staining of proximal tibiae at the late-stage therapy (Scale bar, 400 μm). (I) Quantification of trabecular bone from standard ROI in proximal tibiae at the late-stage therapy (n = 5). (J) Comparison of the concentrations of serum Mg2+ at the late-stage therapy (n = 5). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by one-way ANOVA with Tukey's post hoc test (B and G), by two-way ANOVA with Sidak's post hoc test (D, E, I, and J).

To prevent cortical bone loss, at week 16 post HFD-feeding, the mice were treated with Mg2+ and/or SIM for another 8 weeks (Fig. 4A). The body weight of the combined group was significantly lower than that of the HFD-fed group (Fig. 4B). The concentration of serum Mg2+ in the combined group was substantially greater than that in HFD-fed group (Fig. 4C). However, the cortical thickness and porosity of the combined group were similar to the HFD-fed group, indicating that the combination of Mg2+ and SIM supplement had no synergistic effect on cortical bone mass in HFD-fed mice (Fig. 4D and E). The three-point bending biomechanical test also confirmed such finding with no significant difference found in the maximum load and stiffness between the combined treatment group and HFD-fed group (Fig. 4F). These results demonstrate that the combination of Mg2+ and SIM leads to a synergistic effect on prevention of trabecular bone loss but fails to rescue the cortical bone loss in our designed experiments.

Fig. 4

Combination of Mg2+ and SIM fails to attenuate the cortical bone loss in HFD-fed mice.

(A) Schematic diagram illustrating the combination of Mg2+ and SIM to prevent the cortical bone mass loss. (B) Comparison of the body weights among NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups (at week 16 post HFD-feeding, the mice were received treatment for another 8 weeks, n = 5). (C) Comparison of the concentrations of serum Mg2+ in the NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups (at week 16 post HFD-feeding, the mice were received treatment for another 8 weeks, n = 5). (D, E) Representative μCT images and quantification (n = 5) of cortical bone mass at the femoral midshaft (Scale bar: 400 μm). (F) Maximum load and stiffness at the femoral midshaft (n = 5). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by one-way ANOVA with Tukey's post hoc test (B and F), by two-way ANOVA with Sidak's post hoc test (C and E).

The combination of Mg2+ and SIM arrests the osteoclast formation and bone resorption when starting treatment at the late stage of bone loss

To avoid the skeletal adaption to the changes of body weight, we thus explored the mechanism of combined therapy of bone loss based on the steady body weight at the late-stage therapy. Due to large individual variation of trabecular mass between NFD- and HFD-fed mice, the levels of bone resorption marker, CTX1, and bone formation marker, P1NP, could not clearly reflect the activities or numbers of osteoblasts and osteoclasts at the late stage (Fig. 5A). An apposition rate represents the activity of a group of osteoblasts, and a formation rate is determined by the remodeling activation and the number of osteoblast [39]. The double labeling of calcein green and xylenol orange suggested the similar new-bone formation in the different treatment groups as compared with the HFD-fed group (Fig. 5B and C). The histomorphometric analysis showed similar osteoblast number and significantly decreased osteoclast number in proximal tibiae of combined group as compared with the HFD group (Fig. 5D and E). The expression level of bone formation marker Runt-related transcription factor 2 (RUNX2) was similar between the combined group and the HFD-fed group (Fig. 5F). However, the expression level of bone resorption marker Cathepsin K (CTSK) in the combined group was significantly lower than that in the HFD group (Fig. 5G). These results suggest that combination of Mg2+ and SIM may attenuate the HFD-induced bone loss through inhibiting the osteoclast formation and bone resorption.

Fig. 5

Combination of Mg2+ and SIM decreases the osteoclasts number and activity in HFD-fed mice at the late-stage therapy.

(A) Bone formation marker of procollagen type I N-terminal propeptide (P1NP) and bone resorption marker of C-terminal telopeptide of type I collagen (CTX1) in serum of the NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups at the late-stage therapy (n = 5). (B, C) Representative calcein green and xylenol orange images and quantification of bone remodeling in proximal tibiae of the NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups at the late-stage therapy (n = 3, Scale bar: 100 μm). (D, E) Representative Goldner's trichrome staining and quantification of osteoblast/osteoclast number on the surface (n = 4) of trabecular bone in proximal tibiae of the NFD, HFD, HFD + Mg, HFD + SIM, and HFD + Mg + SIM groups at the late-stage therapy (Scale bar: 25 μm; osteoclasts, black arrows; osteoblasts, yellow arrows). (F) Representative images and quantification of immunohistochemical stained RUNX2-positive cells in proximal tibiae at the late-stage therapy (n = 5, Scale bar: 50 μm). (G) Representative images and quantification of immunohistochemical stained CTSK-positive cells in proximal tibiae at the late-stage therapy (n = 5, Scale bar: 50 μm). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by two-way ANOVA with Sidak's post hoc test (A, C, E, F, and G).

Mg2+ and SIM play roles in the bone marrow cells differentiation

We isolated the mouse BMSCs that positively expressed stem cell markers and possessed tri-lineage differentiation capacities (adipogenesis, osteogenesis, and chondrogenesis) (Figs. S1A–D). MTT assay showed that high dose of Mg2+ (higher than 5 mM) or SIM (higher than 1 μM) exerted cytotoxicity (decreased the cellular metabolic activity by the value of half maximal inhibitory concentration) to BMSCs, as well as BMDMs (Figs. S1E–H).

ALP activity, mineralized nodule formation, and osteogenic mRNA expression in the groups of Mg2+ supplementation (1.8 mM and 5.0 mM) were similar to the group without Mg2+ treatment (0.8 mM) under osteogenic induction (Fig. 6A and B). Besides, Mg2+ supplementation at tested concentrations did not show effect on adipogenesis and osteoclastogenesis as compared with the group without Mg2+ treatment (0.8 mM) (Fig. 6A and 6C-E).

Fig. 6

Combination of Mg2+ and SIM arrests the osteoclast formation and activities in vitro.

(A) Representative images of ALP staining (top, BMSCs after osteogenic induction for 6 days (Scale bar: 250 μm). The top right corner is the representative images of Alizarin Red S staining.), Oil red O staining (middle, BMSCs after adipogenic induction for 7 days, Scale bar: 100 μm), and TRAP staining (bottom, BMDMs after osteoclastogenic induction for 7 days, Scale bar: 50 μm) under different concentrations of Mg2+. (B) Relative expression of Sp7 and Runx2 in BMSCs after osteogenic induction for 3 days under different concentrations of Mg2+ (n = 3). (C) Relative expression of Pparg and Cebpa in BMSCs after adipogenic induction for 3 days under different concentrations of Mg2+ (n = 3). (D) Quantification (n = 5) of Trap + osteoclast number in BMDMs after osteoclastogenic induction for 7 days under different concentrations of Mg2+. (E) Relative expression of Ctsk, Nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), matrix metallopeptidase 2 (Mmp2), Mmp9, and Mmp13 in BMDMs after osteoclastogenic induction for 3 days under different concentrations of Mg2+ (n = 3). (F) Representative images of ALP staining (top, BMSCs after osteogenic induction for 6 days (Scale bar: 250 μm). The top right corner is the representative images of Alizarin Red S staining.), Oil red O staining (middle, BMSCs after adipogenic induction for 7 days (Scale bar: 100 μm), and TRAP staining (bottom, BMDMs after osteoclastogenic induction for 7 days (Scale bar: 50 μm) under different concentrations of SIM. (G) Relative expression of Sp7 and Runx2 in BMSCs after osteogenic induction for 3 days under different concentrations of SIM (n = 3). (H) Relative expression of Pparg and Cebpa in BMSCs after adipogenic induction for 3 days under different concentrations of SIM (n = 3). (I) Quantification of Trap + osteoclast number in BMDMs after osteoclastogenic induction for 7 days under different concentrations of SIM (n = 5). (J) Relative expression of Ctsk, Nfatc1, Mmp2, Mmp9, and Mmp13 in BMDMs after osteoclastogenic induction for 3 days under different concentrations of SIM (n = 3). (K, L) Representative images of TRAP staining and quantification (n = 5) of Trap + osteoclast number in BMDMs after osteoclastogenic induction for 7 days (Scale bar: 50 μm). (M) Relative expression of Ctsk, Nfatc1, Mmp2, Mmp9, and Mmp13 in BMDMs after osteoclastogenic induction for 3 days with indicated treatments (n = 3). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by one-way ANOVA with Tukey's post hoc test (B, C, D, E, G, H, I, J, L, and M).

Although SIM did not diverge the process of osteogenesis or adipogenesis of BMSCs (Fig. 6F–H), it could significantly arrest the process of osteoclastogenesis and the synthesis of bone matrix degrading enzyme, except for the significantly increased matrix metallopeptidase 13 (Mmp13) with 1 μM SIM intervention (Fig. 6F and 6I-J). We subsequently combined a low dose of SIM (0.5 μM) with different concentrations of Mg2+ (0.8, 1.8, and 5 mM) in vitro. The combined application had an inhibitory effect on the mRNA expression of Mmp2 and Mmp13 in pre-osteoclasts (Fig. 6K-M). These findings suggest that SIM substantially inhibits the process of osteoclastogensis and the synthesis of bone matrix degrading enzyme (Ctsk and Mmp9), while the combination of Mg2+ with a low dose of SIM further dampens the bone matrix degrading enzyme (Mmp2 and Mmp13) in vitro.

Cholesterol activates the Mmp13 expression in pre-osteoclasts

Histomorphometric analysis revealed a significant increased number of osteoclasts in the HFD-fed group. Then we incubated pre-osteoclasts with different concentrations of cholesterol under osteoclastogenic induction condition. The high concentration of cholesterol (50 μg/ml) activated the expression of Mmp13 without affecting other genes expression, such as Ctsk, Nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), Mmp2, and Mmp9 (Figs. 7A and S2A). However, the combination of Mg2+ and SIM could not directly arrest the gene expression of Mmp13 in cholesterol-treated pre-osteoclasts in vitro (Figs. 7B and S2B).

The combination of Mg2+ and SIM reduces the synthesis of cholesterol in hepatocytes of HFD-fed mice

The combined utilization of Mg2+ and SIM might produce synergistic effects on lowering cholesterol, while replenished the Mg2+ for HFD-fed mice that mostly occurred the hypomagnesemia. We found that the cholesterol level in serum of combined group was significantly lower than that of HFD-fed group for the late-stage therapy (Fig. 7C). The high-density lipoprotein (HDL) transports cholesterol mostly to the liver or steroidogenic organs such as adrenals and ovary [49]. Low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) released from the liver delivers carried fat molecules to cells (adipose, cardiac muscle, and skeletal muscle) [50]. We found that the HDL and LDL/VLDL levels were similar between combined group and HFD-fed group in the late-stage therapy (Fig. 7D and E).

Fig. 7

Cholesterol activates the Mmp13 expression in pre-osteoclasts, but the combined therapy compromises the cholesterol synthesis in hepatocytes.

(A) Relative expression of Ctsk, Nfatc1, Mmp2, Mmp9, and Mmp13 in BMDMs after osteoclastogenic induction for 4 days and following the treatment of cholesterol (CHL) for 12 h (n = 3). (B) Relative expression of Ctsk, Nfatc1, Mmp2, Mmp9, and Mmp13 in BMDMs after osteoclastogenic induction for 4 days and following the treatment of mixed cholesterol (50 μg/ml) with Mg (5 mM), SIM (0.5 μM), or 5 mM Mg + 0.5 μM SIM for 12 h (n = 3). (C–E) The levels of serum cholesterol (C), HDL (D), and LDL/VLDL (E) at the late-stage therapy (n = 4). (F, G) Representative H&E staining and quantification (n = 4) of lipid area in liver at the late-stage therapy (The top right corner is a magnification, Scale bar: 250 μm). (H) Cholesterol level of liver lysates at the late-stage therapy (n = 5). (I) Representative BODIPY staining of neutral lipids in HepG2 cells pre-treated by Mg, SIM, or Mg + SIM for 1 day and following palmitic acid (0.4 mM) treatment for another 1 day (The top right corner is a magnification; Scale bar: 100 μm). (J) Relative expression of PPARG, GPAM, SCD, FASN, ACACA, SREBF1, and SREBF2 in HepG2 cells pre-treated by Mg, SIM, or Mg + SIM for 1 day and following palmitic acid (0.4 mM) treatment for another 1 day (n = 3). All data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; by one-way ANOVA with Tukey's post hoc test (A and J); by two-way ANOVA with Sidak's post hoc test (B, C, D, E, G, and H).

Therefore, we speculated that the decreased level of cholesterol in serum was correlated with the compromised cholesterol synthesis in liver instead of the alteration of lipid transportation. Quantification of the lipid-containing area in liver sections demonstrated that the combination of Mg2+ and SIM substantially reduced the accumulation of lipid in liver from HFD-fed mice at both early and late stage (Fig. 7F–G and S3A-B). Meanwhile, the cholesterol level of liver lysate in the combined group was significantly less than that in HFD-fed group (Fig. 7H). Moreover, the adipogenesis was substantially inhibited by the combination of Mg2+ and SIM in palmitate pre-treated HepG2 cells, which imitated the micro-milieu of cholesterol synthesis in vitro (Fig. 7I and J). Therefore, the combination of Mg2+ and SIM reduces the hepatocytic synthesis of cholesterol in vivo and in vitro.

Discussion

SIM is widely used for preventing cardiovascular diseases. SIM also exerts positive biologic effects on bone formation partially via competitively binding HMG-CoA reductase, whose inactivation requires the participation of Mg2+ [51]. However, the possibility of the combination of Mg2+ and SIM in preventing bone loss remains elusive. As summarized in Fig. 8, our results corroborate for the first time that the combination of Mg2+ and SIM leads to synergistic effects on the attenuating HFD-induced bone loss via impairing the osteoclast maturation and bone resorption. The combination could directly limit the hepatocytic synthesis of cholesterol and inhibits Mmp13 mRNA expression in pre-osteoclasts.

Fig. 8

Illustration of the underlying mechanism of combined therapy of Mg2+ and SIM that leads to a synergistic effect on attenuating the HFD-induced bone loss via impairing osteoclast maturation and bone resorption in bone marrow. Meanwhile, the combination can directly limit the hepatocytic synthesis of cholesterol.

Recent clinical studies demonstrate that body weight per se negatively correlates with bone mass when the effect of gravity is eliminated [[52], [53], [54]]. Growing epidemiological evidences, as well as animal studies, offer an explanation that the metabolic syndrome in abdominal obesity is responsible for the bone loss [[55], [56], [57]]. Indeed, a mouse strain (C57BL/6) with a susceptibility to atherosclerosis is prone to bone loss in response to a HFD than atherosclerosis-resistant strains [58].

All statins possess a structural component that is extremely similar to the HMG portion of HMG-CoA, and statins sterically shield substrate binding by integrating to the active site of HMG-CoA reductase. In fact, Mg2+ is also another regulator for the deactivated HMG-CoA reductase [18,59], which is the fourth abundant element in the body and the second abundant in the intracellular compartment [35]. Mg2+ is a cofactor in more than 300 enzymatic reactions and critically stabilizes enzymes, including many adenosine triphosphate (ATP)-generating reactions. Moreover, Mg2+ is universally required for the ATP-consumption reactions of glucose utilization, synthesis of fat, proteins, nucleic acids and coenzymes, muscle contraction, methyl group transfer and many other processes [60,61]. At least two Mg2+-dependent reactions are required to deactivate HMG-CoA Reductase [18,36]. The effect of the systemic administration of statins is limited due to its metabolism in the liver and clearance from first-pass elimination. In addition, high-dose administration of SIM is reported to cause severe adverse sides [62]. Previous study suggests that Mg2+ supplementation could increase the SIM passive diffusion in hepatocytes and its pharmacological actions on cholesterol biosynthesis [63]. Low dietary Mg intake and hypomagnesemia are strongly associated with increased risk of developing lipid metabolic disorders [64,65]. In addition, because obesity is usually related with hypomagnesemia, it has been suggested that obesity deteriorates the Mg status [66]. However, the regression analysis shows no correlation between body weight status and the hypomagnesemia, indicating that hypomagnesemia is an epiphenomenon related with obesity rather than the causal relationship between body weight.

Low-density lipoprotein (LDL) is the major source of transferred lipids in the extracellular fluid, making the fats available to cells for the endocytosis, whereas HDL is negatively correlated with the high loading condition of cholesterol [67]. The classical LDL particle function facilitates cholesterol release from liver and transports it to the circulatory system [67]. The HDL particle definitely functions as the efflux capacity of carrying free cholesterol from cells and delivering cholesteryl esters to various tissues, especially the liver where cholesterol is processed for excretion through the bile [67]. However, we do not find pharmaceutical effects on the adjustment of LDL/VLDL and HDL, indicating the negligible influence on the comprehensive transportation and utilization of cholesterol in the body. Both in vivo and in vitro experimental results demonstrate the blocked synthesis of cholesterol in hepatocytes under the combination of Mg2+ and SIM.

Given the numerous biomolecules regulated by the mevalonate pathway, it comes with a little surprise for the usage of statins experiencing a beneficial effect on prevention or treatment of osteoporosis and bone fracture, in addition to its known effect on limiting hyper-cholesterolemia [8]. Statins can particularly intervene in the process of bone turnover and remodeling via producing effects on bone resident cells, such as BMSCs and osteoclasts [68]. We fail to find the osteogenic feature of SIM in vitro. However, our results corroborate that SIM substantially inhibits the osteoclast formation and activities, and further arrests the synthesis of matrix degrading enzyme in osteoclast under the combined treatments. SIM exerts the inhibitory effect on the MMP release via a post-transcriptional mechanism, and this effect is reversed by mevalonate and geranylgeranyl-pyrophosphate [69]. We find that the BMDMs treated with high dosage of SIM (1 μM) increases the Mmp13 expression under the osteoclastogenic induction in vitro. This result partly explains that SIM increases the risk of trigger finger and shoulder tendinopathy, possibly correlated with the increased MMP13 release [70]. We thus choose the low dosage of SIM (0.5 μM) to limit the osteoclasts activity by inhibiting the Ctsk and Mmp2/9 expression and avoiding the enhanced Mmp13 expression in vitro. The dosage in vivo of SIM (20 mg/kg/day) is also lower as compared with other investigation [71].

In our study, we acquire a desirable result with a physiologically safe concentration of Mg2+, which is substantially lower than that in other investigation [36]. Supplementary Mg2+ could directly reach bone resident cells and hepatocytes from circulation. Namely, studies should inevitably focus on the effects of Mg2+ on these types of cells. Actually, the circulatory system is less sensitive to reflect the scarcity of Mg2+ due to the reservoir of Mg2+ in bone tissue [72]. About 60% of total Mg2+ is stored in the bone. One third of skeletal Mg2+ resides on cortical bone either on the surface of hydroxyapatite or in the hydration shell around the crystal [61]. Then the level of Mg2+ in serum may be kept constant despite of bone loss at trabecular dominant skeletal sites. Therefore, the time point of supplementary Mg2+ for the early-stage therapy (at week 8 of HFD) is earlier than the initial time point of decreased concentration of Mg2+ (at week 16 of HFD). We select saline as the matched group to avoid the interference of Cl− from MgCl2. Moreover, the Mg2+-evoked currents are not affected with deletion of external sodium by substitution with choline or replacement of chloride with nitrate [73]. Niflumic acid, a Cl− channel antagonist, does not alter Mg2+ currents, indicating that extracellular Cl− has no influence on the transport of Mg2+ [73]. Besides, both in vitro and in vivo results demonstrate that the high mineralization is realized with a concentration of 0.8 mM Mg2+, which is consistent with previous study [37].

Due to lesser density in trabecular bone as compared with cortical bone, trabecular bone provides only about 20% of the bone mass, but it is predominantly responsible for the dynamic bone turnover (34). We find that the bone loss mainly occurs at trabecular bone in the stage (within 16 weeks of HFD), but the cortical bone becomes more and more porous in following stage (later than 16 weeks post HFD). These results are in line with the fact that the higher expression of Wnt/β-catenin signaling is mainly distributed in trabecular bone and therefore more easily regulated by this signaling than cortical bone under a HFD-fed condition [74,75]. Most studies confirm the independent correlation between HFD and cortical bone mass [76,77]. In the current study, we find the significantly increased cortical porosity and decreased cortical thickness in HFD-fed mice at week 24. Our follow-up time point is longer than other studies, suggesting that the duration of HFD may be crucial for investigating the alteration of cortical bone mass.

Nevertheless, some uncertainties do still exist in our study. Firstly, we have not fully explored the mechanisms of combined therapy on the regulations of hepatocytic metabolism and the synthesis of matrix degrading enzyme in osteoclasts. Secondly, it is now well established that combined therapy could improve the bone mass through two ways, however, their exact contributions remain unclear. Thirdly, our study is not able to eliminate the effect of free cholesterol on osteogenesis. Notwithstanding these limitations, this study does suggest the synergistic effects on lowering cholesterol and improving bone mass in HFD-fed mice.

Conclusion

Our study demonstrates that combination of Mg2+ and SIM leads to a synergistic effect on attenuating the HFD-induced bone loss via impairing osteoclast formation and activities. This study provides a new avenue for clinical translation of statin combined with Mg2+ to ameliorate obesity-related bone loss.

CRediT authorship contribution statement

Bingyang Dai: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Software, Validation. Xu Li: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft, Software, Validation. Jiankun Xu: Conceptualization, Methodology, Investigation, Funding acquisition, Writing - original draft, Software, Validation. Yuwei Zhu: Methodology, Formal analysis, Data curation. Le Huang: Methodology, Formal analysis, Data curation. Wenxue Tong: Methodology, Formal analysis, Data curation. Hao Yao: Methodology, Formal analysis, Data curation. Dick Ho-kiu Chow: Methodology, Formal analysis, Data curation. Ling Qin: Supervision, Writing - review & editing, Data curation, Project administration, Funding acquisition.