key: cord-0687516-kf3b2r13
authors: Tripathi, Ashish Kumar; Rai, Divya; Kothari, Priyanka; Kushwaha, Pragati; Sashidhara, Koneni V.; Trivedi, Ritu
title: Benzofuran pyran hybrid prevents glucocorticoid induced osteoporosis in mice via modulation of canonical Wnt/β-catenin signaling
date: 2022-02-02
journal: Apoptosis
DOI: 10.1007/s10495-021-01702-z
sha: 6c39c62635be6e7456d0f0b959429bc195eeaec5
doc_id: 687516
cord_uid: kf3b2r13

Glucocorticoid induced osteoporosis (GIOP) is the second most leading cause of osteoporosis. We have identified a compound, a benzofuran pyran hybrid compound 4e that has osteogenic potential and we wanted to assess its efficacy in GIOP in male mice. We assessed the effect of dexamethasone and compound 4e on primary osteoblasts using various cell based and immunofluorescence assays. For in vivo studies we administered methylprednisolone and compound 4e as a prophylactic measure in male Balb/c mice for 28 days and then evaluated the effect on bone microarchitecture by microCT, bone formation by histology along with clinically relevant bone markers. Compound 4e preserved osteoblast differentiation as evident by higher ALP positive cells and mineralization in compound treated groups. Compound 4e also increased the expression of osteogenic genes. This compound guarded β-catenin expression both in vitro and in vivo as confirmed by western blot and immunofluorescence assays. This led to the preservation of bone microarchitecture and cortical thickness at 2.5 mg kg(−1) and 5 mg kg(−1) doses. Further compound 4e enhanced bone formation rate and regulated osteocyte death. The osteogenic potential of compound 4e was reflected by an increased level of serum marker osteocalcin and decreased levels of SOST and CTX-I. Overall, Compound 4e is able to overcome the catabolic effect of dexamethasone on bone by targeting the canonical WNT/β-catenin signaling as evidenced by both in vitro and in vivo studies.

Glucocorticoids (GC) are indispensable drugs prescribed by clinicians for various diseases like allergy, rheumatoid arthritis, COPD (chronic obstructive pulmonary disease), SLE (systemic lupus erythematous), inflammatory bowel disease, and other systemic diseases [1] . Approximately 0.5-1% general population is receiving chronic glucocorticoid treatment [2, 3] . Dexamethasone a synthetic glucocorticoid by its anti-inflammatory and immunosuppressive effects has recently shown its benefits and prevented one-third of deaths among Covid-19 patients who were critically ill [4] . However, long-term use of glucocorticoid is associated with fragility of bones and an increased risk of fracture and this is called glucocorticoid-induced osteoporosis (GIOP), the most common form of secondary osteoporosis. The effect of glucocorticoid on bone is time and dose-dependent. Increased risk of fragility fracture may occur even at the lower dose of glucocorticoid in the first month of treatment [5] . About 30-50% of patients receiving long-term glucocorticoid develop glucocorticoid induced osteoporosis [6] . Initial glucocorticoid intake results in a transient increase in bone resorption as glucocorticoids enhance the production of RANKL (Receptor activator of nuclear factor kappa-B ligand) and diminish OPG (Osteoprotegerin) expression [7, 8] . Glucocorticoids suppress bone formation by maneuvering the primary bone formation pathway WNT/β-catenin signaling [9] . Glucocorticoid induces the expression of Sclerostin (SOST) a Wnt pathway inhibitor thus hampers bone formation [9] . Glucocorticoid also inhibits bone formation by inducing BMP-RUNX2 pathway inhibitors [10] . At physiological concentration, glucocorticoid stimulates mesenchymal stem cells to differentiate into osteoblast lineage rather than adipocyte lineage via suppression of sFRP-1 (Secreted frizzled-related protein 1) but at pharmacological doses, glucocorticoid favors adipocyte lineage by increasing the expression of PPAR-γ which might be the physiological basis behind the accumulation of bone marrow fat accompanying bone loss in patients with long-standing glucocorticoid treatment [11] [12] [13] [14] . GC treatment leads to enlarged osteocyte lacunae (osteolysis) and increased autophagy as a defensive mechanism to survive stress whereas overexposure at higher doses leads to apoptosis [15] . Apart from the aforementioned mechanism glucocorticoids also induce osteoblast death by increasing reactive oxygen species (ROS) mediated phosphorylation of JNK and induction of oxidative DNA and anti-oxidative stress defence system damage [16] .

To date, bisphosphonates are used as first-line therapy for GIOP, followed by treatment with PTH and Teriparatide. Bisphosphonates are relatively safe drugs but long term usage carries a warning of osteonecrosis of the jaw and atypical femoral fractures [17, 18] . Another treatment includes Denosumab, a monoclonal antibody against RANKL that increases lumber BMD when compared to Risedronate (bisphosphonate) in a randomized double-blind, double-dummy, active-controlled study [19] . A concern with Denosumab treatment in GIOP is a possible increase in vertebral fracture after stopping the drug [20] . Teriparatide can only be prescribed for a maximum of 24 months of treatment in patients with GIOP; however, due to its higher cost and inconvenience of daily subcutaneous injection, its use in GIOP has been limited. Given these side effects and cost burden, there is a need for a potential candidate with lesser or no side effects.

Compound 4e is a member of the benzofuran-pyran hybrid chemical class. Benzofuran scaffolds are believed to be an essential member of heterocyclic compounds and known for various biological activities like anti-hyperglycemic [21] ), anti-parasitic [22] , antimicrobial [23] , antitumor, and kinase inhibitor activities [24] . The effects of benzofuran compounds as anti-osteoporotic agents have been explored many times. Fontana et.al. had shown bone loss inhibitory activity of benzofuran. Likewise, the derivative of benzofuran is known for its anti-resorptive activity by inhibiting Cathepsin K [25] . Our previous studies have shown the bone forming ability of Benzofuran-dihydropyridine hybrids in the drill hole model of fracture [26] . Studies show that Bergapten, a natural product related to benzofuran ring structure enhances the BMP2 activity by modulating the P38 and ERK dependent pathway in osteoblasts [27] . Pyrans were also extensively studied for their osteogenic activities [28, 29] . Observations by us confirm that compound 4e is known for its osteogenic [30] and anti lipotoxic [31] effects. This led us to explore the potential of Benzofuran-dihydropyridine hybrids in glucocorticoid induced osteoporosis.

Primary osteoblasts were harvested from 2 to 3 days old mice pups. Briefly, calvaria of pups (n = 6) was surgically removed and cleaned. Each calvaria was chopped into small pieces then exposed in enzymatic solution (1 mg/ml of Collagenase I and Dispase II) for 15 min at 37 °C. First digestion was discarded as it contains more brain parts, fibroblast, and other debris. From second digestion onwards supernatant was collected in 10% complete alpha MEM. Cells were plated in T-25 flask and after reaching 70-80% confluency cells were harvested for further use [31, 32] .

Cells were seeded and treated as above mentioned in complete alpha MEM media for 48 h. After the end of the experiment, MTT (5 mg/ml) was added to each well for 2-4 h. After the desired time DMSO was added to dissolve formazan crystal and absorbance was taken at 570 nm [33] .

Osteoblasts were grown in T-25 flask. After achieving 70-80% confluency cells were treated with dexamethasone and compound 4e at 10 nM and 0.1 µM concentration for 48 h. Dead cell apoptosis kit from Invitrogen was used to quantify live and apoptotic cells through flow cytometry. Cells were processed according to the manufacturer's protocol [34] .

Osteoblasts were grown on a coverslip and treated with dexamethasone and compound 4e. Cells were washed with PBS fixed with paraformaldehyde then permeabilized with 0.25% TritonX-100. After the permeabilization, we used Click-iT™ Plus TUNEL Assay (C10617 Invitrogen) to detect apoptotic cells tagged with alexa fluor 488. To detect apoptosis in tissue we dewaxed and rehydrated bone tissue in Ethanol and water. Then processed according to manufacturer's protocol [33] .

Cells were grown in 12 and 96 well plates and treated with dexamethasone, compound 4e at the concentration 10 nM and 0.1 µM for 24 h. Osteoblasts cells were washed twice with PBS and then incubated with 5 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 45 min at 37 °C. Fluorescent images were captured using Evos Fl Fluorescent microscope (Applied Biosystems) at × 10 magnification and absorbance was measured by Spectra max M2 Molecular Devices at 485/535 nm. [35] .

Cells were seeded in 96 well plates at 2 × 10 3 density. After reaching 70% confluency cells were treated with different concentrations, 10 nM, 0.1 µM, 0.5 µM, and 1 µM, of compound 4e with or without dexamethasone (1 µM) for 48 h in osteogenic media (10 mM β-Glycerophosphate and 50 µg/ml Ascorbic acid in Alpha MEM). After 48 h cells were washed with PBS and kept at − 80 °C for overnight. The next day cells were kept at 37 °C, this quick temperature change burst the cell and expose the ALP enzyme. ALP enzymes were quantified by pNPP (para-Nitrophenyl phosphate), which is a substrate for ALP, by a colorimetric ELISA plate reader at 405 nm. For ALP staining cells were treated as above mentioned. At the end of the experiment, cells were washed with PBS and incubated in 1-Step™ NBT/BCIP Substrate Solution for 30 min and then washed with PBS. Imaging was done with Evos Xl (Applied biosystems) at × 10 magnification. ALP positive cells were quantified with IMAGE Pro software [26, 36] .

Calvarial osteoblasts were seeded in 12 well plates and treatment of compound 4e and dexamethasone were given as mentioned in ALP assay for 21 days. Media were changed after every 48 h. After the end of the experiment, cells were washed with PBS and fixed in 4% formaldehyde for 30 min then incubated in Alizarin Red S stain for another 30 min.

Cells were then washed with tap water and PBS. Images of mineralized nodules were captured with Nikon Coolpix B600. For quantification of mineralized nodules, 10% (v/v) acetic acid was added to each well with shaking at room temperature for 30 min. Cells were scraped out and collected in a microcentrifuge tube then centrifuged at 20,000×g for 15 min after that supernatant was collected. 10% (v/v) ammonium hydroxide was added to the supernatant to bring the pH of the solution to 4.5 for color formation. The absorbance of the solution was read at 405 nm [37] .

Primary osteoblasts were treated with dexamethasone and compound 4e as mentioned for ALP assay for 48 h. At the end of the experiment, RNA was isolated by using Quizol (Qiagen) and cDNA was made by High capacity cDNA reverse transcription kit (Thermo Fisher). We performed real-time PCR (Quant Studio 3, Applied Biosystems) for assessment of gene expression by SYBR green (PowerUp SYBR Green master mix Applied Biosystems). We checked gene expression of RUNX2, BMP2, ALP, β-catenin, SOST (Table 1 ) in response to dexamethasone and compound 4e [33] .

Primary osteoblasts cells were treated as above mentioned and lysed in buffer containing 1 × RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor. Protein concentration was quantified using a BCA Protein Assay kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly-vinylidene difluoride membrane (PVDF, Millipore, MA, USA). After blocking with 5% BSA in Tween-20/PBS (PBST) at room temperature for 2 h, membranes were incubated with specific primary antibodies at 4 °C overnight. Thereafter, membranes were probed with HRP-conjugated secondary antibody for 2 h at room temperature and chemiluminescence was detected with an enhanced chemiluminescence system (ECL, MERCK Millipore, MA, USA). Results were normalized to β-actin. [38] .

Cells were grown in 4 well chamber slides. Cells were treated with dexamethasone and compound 4e as above mentioned for 48 h. At the end of the experiment, cells were fixed with 4% formaldehyde and permeabilized with 0.25% Triton × 100. Cells were then blocked with 2% BSA and incubated with RUNX2 antibody (12556 CST, dilution 1:100) overnight at 4 0 C. Further RUNX2 antibody probed with Alexa Fluor 488 (1:200 dilution) and DAPI were used for nuclear stain. β-catenin (SC-7199, dilution 1:100 dilution) was used and Alexa fluor 594 (1:200 dilution) was used as a secondary antibody. Images were taken in Leica confocal microscope at × 40 magnification [31] .

All animal care and experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) [approval no. IAEC/2017/44(234/17)] and performed according to the regulations of the Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India. 10-12 week old male BALB/c mice (weighing 22 ± 2 g each), were obtained from the National Laboratory Animal Centre, CSIR-CDRI, housed in a 12-h/12-h light/ dark cycle with controlled temperature (22-24 °C) and humidity (50-60%). Standard rodent chow diet and water were provided ad libitum. Animals were randomly divided in four groups (n = 10 mice/group), Control, Methylprednisolone (10 mg kg −1 ), Methylprednisolone + Compound 4e 5 mg kg −1 and 10 mg kg −1 for 28 days. Methylprednisolone was given subcutaneously and compound 4e was administered orally. To detect dynamic bone formation each animal was given calcein (20 mg kg −1 ) intraperitoneally 7 days and 1 day before sacrifice.

Micro-computed tomography (μCT) analysis of excised bones was carried out using the SkyScan1276 CT scanner (Aartselaar, Belgium). Mice bone scanning was performed at 9 μM pixel size, using X-ray source 50 kV, 200 mA. Image slices were reconstructed using the cone-beam reconstruction software version 2.6 based on the Feldkamp algorithm (SkyScan). All analyses were executed on CTAn software, SkyScan. For femur trabecular analysis, 100 slices were selected, leaving 30 slices from the start of the growth plate as a reference point. For cortical analysis, 100 slices were selected. For BMD calculation VOI (volume of interest) was used which was made for trabecular region analysis, using hydroxyapatite phantom rods of 4 mm diameter as standards [39] .

For dynamic histomorphometry, undecalcified femora were embedded in acrylic and 50 µm sections were cut by using Isomet-Slow Speed Bone Cutter (Buehler, Lake Bluff, IL) followed by photography using a confocal microscope (LSM 510 Meta, Carl Zeiss, Inc., Thornwood, NY). We enumerated periosteal perimeters, single-labeled surface (sLS), double-labeled surface (dLS) and interlabelled thickness (IrLTh), and these values were employed to calculate mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) [40] .

Femur bones were decalcified in decalcifying solution lite (Sigma-Aldrich, St. Louis, MO, USA) for 1 week and embedded in paraffin, 5 µm sections were cut on RM2265 semi-automatic microtome (Wetzlar, Germany). Sections were blocked with a 5% BSA blocking buffer for 1 h at room temperature. After that sections were incubated with the primary antibody of β-catenin (SC7199 Shantacruz 1:100 dilution) overnight. Sections were then incubated with Alexa Fluor 594 (1:200 [39] .

Decalcified femur bones were fixed in 4% paraformaldehyde and then dehydrate in 70% isopropanol. Distal femora were embedded in paraffin wax and 5 µM sections were cut. Sections were de-deparaffinized in xylene and rehydrated in serial changes of isopropanol and water and stained with hematoxylin and eosin, mounted with DPX (Sigma), and was examined under a microscope. Undecalcified femora were embedded in methylmethacrylate and 5 µM sections were cut with Leica RM2265 semi-automatic microtome (Wetzlar, Germany) equipped with a TC-65, a tungsten carbide cutting knife. Sections were undergone deplastication and stained with Vonkossa and Goldner's Trichrome stain. Bone volume (BV/TV) was measured using Bioquant Osteo Software (Bioquant Image Analysis, Nashville, TN, USA) [38, 41] .

Bone formation marker osteocalcin (OCN), resorption markers Carboxy-terminal collagen crosslinks (CTX-I), and sclerostin (SOST) were determined by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocols from serum. Osteocalcin is exclusively secreted from osteoblasts. A small portion of newly synthesized osteocalcin is released in circulation and can be measured by immunoassays and is a valid marker for bone formation.CTX-1 is a C-terminal cross-linking telopeptide of type I collagen formed after bone resorption [42] .

Data are expressed as mean ± SEM. The data obtained from experiments were subjected to one-way ANOVA followed by Newman Keul's multiple comparison test of significance using GraphPad prism 5.

Dexamethasone (Dex) induced cell death in osteoblasts as assessed by MTT assay. Compound 4e significantly rescued osteoblast from dexamethasone induced cell death at concentrations of 10 nM and 0.1 µM, however, at 0.5 µM and 1 µM concentrations, increase in cell viability was not significant (Fig. 1a) . Further, cell apoptosis was assessed and quantified by Annexin-FITC/PI apoptosis assay and compound 4e at 10 nM restored cell viability as more cells in the lower left quadrant (Fig. 1b and c) . To further confirm cell apoptosis we used TUNEL assay. There are increased TUNEL positive (green fluorescence) cells in the dex panel ( Fig. 1d Dex panel) and compound 4e decreased cell death induced by dex (Fig. 1d Dex + 4e panel). Dexamethasone also increased the intracellular ROS as visible by a greater amount of green fluorescence (H2DCFDA) in dexamethasone treated osteoblasts which were reduced by treatment of compound 4e as visible by lesser green fluorescence at the concentration of 10 nM and 0.1 µM (Fig. 1e and f) . The result was confirmed by fluorescent plate reader analysis of H2DCFDA stained cells. Compound 4e decreased the number of H2DCFDA positive cells at 10 nM and 0.1 µM concentrations (Fig. 1g ).

Treatment of dexamethasone at a concentration of 1 µM impairs the osteogenic potential of osteoblasts in vitro. Treatment for 48 h reduced ALP activity, a crucial osteogenic marker, with a reduced number of alkaline phosphatase (ALP) positive cells. Treatment with compound 4e increased the number of ALP positive cells at the concentrations of 10 nM, 0.1 µM, 0.5 µM, 1 µM (Fig. 2a-c) . P-nitrophenyl phosphate (pNPP) assay was also used to assess the ALP activity. As expected Compound 4e overcame the deleterious effect of dexamethasone and rescued osteoblasts with increased ALP activity at a concentration of 10 nM marginally and at the concentrations of 0.1 µM, 0.5 µM, 1 µM significantly (Fig. 2d) . Dexamethasone attenuated the mineralization ability of osteoblasts which were restored by compound 4e at aforementioned concentrations ( Fig. 2e and f) .

To assess the effect at the molecular level, we observed that dexamethasone downregulated the expression of RUNX2, BMP2, and ALP genes. Treatment with compound 4e reversed the effect of dexamethasone and increased the mRNA expression of RUNX2 by 2.4, 1.6, 1.7, and 2.11 folds at the concentrations of 10 nM, 0.1 µM, 0.5 µM, 1 µM respectively (Fig. 3a) . A similar increase was observed for BMP2 by approximately 2.3, 1.5, 3.1, and 2.9 folds (Fig. 3b ) and for ALP by 1.6, 2.1, at 10 nM and 0.1 µM. (Fig. 3c) . Compound 4e also maintained the RANKL/OPG balance by downregulating RANKL and increasing the mRNA expression of OPG at the concentrations of 10 nM, 0.1 µM, 0.5 µM, 1 µM significantly ( Fig. 3d and e) . The functions of these proteins were also altered by dexamethasone as evidenced in western blots (Fig. 4a) . Dexamethasone showed its negative effect by downregulating RUNX2 (Fig. 4a and d) via inhibiting BMP2 ( Fig. 4a and  b) signaling. Compound 4e restored the BMP2/SMAD1/5/8 ( Fig. 4a and c) signaling pathway and increased RUNX2 expression at 10 nM and 0.1 µM concentrations (Fig. 4d) . Further, we confirmed this result by immunocytochemistry, dexamethasone downregulated RUNX2 expression in the nuclei of the osteoblast cells as visible by less green fluorescence in dexamethasone treated cells, and treatment of compound 4e restored the expression of RUNX2 (Fig. 4e ).

We assessed the possible mechanism by which compound 4e could ameliorate dexamethasone induced inhibition of osteoblast function to strengthen our claim. Wnt/β-catenin is a major pathway by which dexamethasone exerts its effects.

Real-time PCR data shows that dexamethasone increased expression of Wnt inhibitor SOST and downregulated expression of β-catenin. Compound 4e decreased the expression of SOST by 9.3, 6, 3.7, and 4.3 fold at the concentrations of 10 nM, 0.1 µM, 0.5 µM, 1 µM respectively in comparison to dexamethasone treated group (Fig. 5a) . Compound 4e increased an essential mediator of Wnt signaling β-catenin by approximately 2.5, 2.1, 2.2 fold at the concentrations of 10 nM, 0.1 µM, 0.5 µM, respectively (Fig. 5b) . Immunoblotting confirmed the results as dexamethasone increased the expression of SOST (Fig. 5c and d) , and attenuating phosphorylation of GSK3-β (Ser9) (Fig. 5c and  e) . Activation of GSK3-β phosphorylated β-catenin and its degradation. Treatment with compound 4e activated the Ser9 phosphorylation of GSK3-β thus its inactivation and increased the expression of β-catenin ( Fig. 5c and f) . Compound 4e also increased the β-catenin nuclear localization which is an important step for canonical Wnt signaling. We stained β-catenin with Alexa fluor 594 and observed its colocalization with DAPI (Blue) which gives purple color. Dexamethasone inhibits the nuclear localization and compound 4e enhanced this at 10 nM and 0.1 µM (Fig. 5g) .

To assess the effect of compound 4e at the bone microarchitectural level we analyzed the distal femoral metaphysis by microCT after treatment with methylprednisolone (MP), a synthetic glucocorticoid that was used for in vivo administration. Compound 4e protected bone microarchitecture from glucocorticoid induced bone loss as shown in 3D microCT images (Fig. 6a) . Methylprednisolone decreased BMD (Bone mineral density) by approximately 23% (Fig. 6b) , bone volume (BV/TV) by 39.5% (Fig. 6c) , trabecular thickness (Tb.Th) by 22.9% (Fig. 6d) , trabecular number (Tb.N) by 22.8% (Fig. 6e) and increased trabecular separation (Tb. Sp) (Fig. 6f) and SMI (Fig. 6g) 

3D representative images of Cortical bone at mid femoral diaphysis showed that compound 4e abrogated bone loss effect of methylprednisolone (Fig. 7a) . Methylprednisolone reduced the cortical thickness of bone by 14% and increase cortical porosity by 17% thus making the bones fragile. Oral dosing of compound 4e rescued the bones by increasing the cortical thickness at both concentrations of 2.5 and 5 mg kg −1 by 20% and 21% respectively and decreased cortical porosity by 10.8% at 5 mg kg −1 dose (Fig. 7b and c) . Through dynamic histomorphometry studies, we showed the effect of methylprednisolone in reducing bone formation in animals as assessed by a single and discontinuous calcein green fluorescent line (Fig. 7d) . Administration of Compound 4e attenuated the inhibited bone formation at Fig. 1 Effect of compound 4e on dexamethasone induced ROS and apoptosis a Cell viability assessed by MTT. Compound 4e rescues osteoblast at 10 nM, 0.1 µM. b Total dead cell % assessed by c Annexin/Pi apoptosis assay. d TUNEL assay to detect apoptosis in cells. In dexamethasone treated cells there is higher green (alexa fluor 488) positive cells. Compound 4e decreased apoptosis at 10 nM and 0.1 µM. e and f Dexamethasone increased intracellular ROS reflected by higher green fluorescence assessed by fluorescent microscope which was attenuated by treatment of compound 4e at 10 nM, 0.1 µM. g ROS were also measured separately by spectrophotometer and we had the same result as above. Results were obtained from three independent experiments performed in triplicate and are expressed as Mean ± SEM. # p < 0.05, ## p < 0.01 ### p < 0.001 versus control, * p < 0.05, **p < 0.01 ***p < 0.001 versus dexamethasone ◂ 2.5 mg kg −1 and 5 mg kg −1 doses (Fig. 7d) . Methylprednisolone inhibited mineralizing surface/bone surface (MS/BS) (Fig. 7e) and mineral accrual rate (MAR) (Fig. 7f) , and thus bone formation rate (BFR) (Fig. 7g) . Compound 4e dosing for 4 weeks increased the MS/BS, MAR, and BFR.

We observed that administration of glucocorticoid increased SOST mRNA expression in bone by 3.0 folds as compared to animals not receiving glucocorticoid. Dosing of animals with 2.5 mg kg −1 and 5 mg kg −1 compound 4e downregulated SOST by 7.56 folds (Fig. 8a) .

When we estimated circulating levels of SOST in the serum by sandwich ELISA we observed increased levels in the MP group by approximately 381%. Compound 4e at 2.5 mg kg −1 and 5 mg kg −1 dose reduced SOST level by 86% and 81.5% respectively (Fig. 8b) . Immunohistochemistry data unfolds that the expression of the β-catenin protein, a central member of Wnt/β-catenin signaling was found to be abundant in the Control group of mice (red fluorescence alexa fluor 594) co-localized with DAPI (Blue) thus got purple color in merged panel. Expression of β-catenin was decreased in the MP group. The decreased expression was significantly reversed in the co-treatment groups of MP and compound at the doses 2.5 mg kg −1 and 5 mg kg −1 body weight (Fig. 8c) . Thus, making our findings robust and consistent with the previous data exhibiting the osteogenic potential of our compound following the Wnt/β-catenin signaling pathway.

Continuous exposure to Methylprednisolone increased bone marrow adipocytes. We stained bone tissue with hematoxylin and eosin and found the globular spaces in the bone marrow of MP treated animals (Fig. 9a) . These globular spaces (adipocyte ghosts) depict adipocytes in the bone marrow. We performed IHC to show bone marrow adiposity with perilipin antibody, tagged with Alexa fluor 594, which is a surface marker of lipid droplets. In MP treated animals there is an enhanced number of adipocytes thus lipid droplets. Compound 4e abrogated this effect at 2.5 and 5 mg kg −1 doses (Fig. 9b) .

It is well established that glucocorticoid alters osteocyte function by inducing apoptosis. H&E staining also demonstrated increased empty lacunae in the trabecular bone of the MP group suggesting osteocyte death. Treatment of the compound at the above-mentioned doses 2.5 mg kg −1 and 5 mg kg −1 reduced the increased number and size of empty lacunae, representative of osteolysis (Fig. 10a) . We also employed TUNEL to show apoptosis of osteocytes in bone tissue. There are increased TUNEL positive cells in the MP group in comparison to the control. Compound 4e decreased osteocyte apoptosis reflected by lesser TUNEL positive cells in comparison to the MP group (Fig. 10b) .

Von-Kossa and Goldner's trichrome (GT) staining of the femur sections revealed the inadequate number and discontinuous orientation of the trabeculae in the MP treated mice when compared to the Control (without MP) ( Fig. 11a and b  MP panel) . Treatment of the compound at doses 2.5 mg kg −1 and 5 mg kg −1 body weight reversed the deleterious impact caused by MP on the mineralized matrix. Reversal of the disrupted trabecular bone is seen in terms of increased trabecular thickness and number as observed visually in the histological sections ( Fig. 11a and b) . We quantified BV/ TV in GT stained sections by BIOQUANT OSTEO software (Fig. 11c) . Fig. 2 Compound 4e increased ALP and mineralization. a Representative ALP staining (NBT/BCIP) macroscopic images and b at × 10 magnification. c Quantification of ALP staining by Image Pro software. d ALP activity also confirmed separately by pNPP method. Dexamethasone decreased ALP activity and treatment of compound 4e at 10 nM, 0.1 µM, 0.5 µM and 1 µM concentrations increase osteoblast differentiation. e Dexamethasone diminished mineralization capacity of osteoblasts which were restored by compound 4e at 10 nM, 0.1 µM, 0.5 µM and 1 µM concentrations. f Quantification of mineralization nodules. Results were obtained from three independent experiments performed in triplicate and are expressed as Mean ± SEM. # p < 0.05, ## p < 0.01 ### p < 0.001 versus control, *p < 0.05, **p < 0.01 ***p < 0.001 versus dexamethasone ◂ Bone turnover markers in serum were measured in the control (untreated) and treatment groups after 28 days of glucocorticoid administration. Serum CTX-I was robustly increased in the MP group mice than control. The percentage increase in the level of CTX-I is approximately 45% in the MP group in comparison to control. In treatment groups of 2.5 mg kg −1 and 5 mg kg −1 with compound 4e a significant reduction to 23.2% and 29.8% was observed Fig. 11d) . A well-established key osteogenic marker OCN was also evaluated and results indicated a decrease in OCN level in the MP group by 51.9% whereas our treatment group of 2.5 mg kg −1 and 5 mg kg −1 significantly increased the downregulated level by 58.6% and 71.2% respectively (Fig. 11e) . This data indicated an osteoanabolic effect of compound 4e.

Bone loss due to long term use of glucocorticoids (GC) occurs by inhibiting osteoblast survival and function. Excess glucocorticoid acts directly on osteoblast and osteocytes and promotes their death. Studies on transgenic mice show that overexpressing 11β-hydroxysteroid dehydrogenase type-2 converts active glucocorticoid into inactive form preventing osteoblast death [43] . Dexamethasone induces osteoblast apoptosis via GSK3β and Caspases activation [44] . Through this study, we have shown the preventive effect of compound 4e on GC induced bone loss in mice. GIOP is marked by loss of bone mineral density (BMD) and deterioration of cancellous and cortical microstructure [42, 45, 46] . These overall changes in bone volume are due to loss in trabecular thickness (Tb. Th) and trabecular number (Tb. N). Furthermore, erosion of the trabeculae results in increased trabecular separation (Tb. Sp). A higher ratio of rod-shaped trabeculae in comparison to plates in glucocorticoid treated mice indicated poor geometric disposition because rods are mechanically inferior to plates predisposing one to lower bone strength. We observed that compound 4e restored the trabecular microstructure by increasing the trabecular thickness and number and also restored plate like trabaculae. Glucocorticoids initially affect trabecular bone but with long exposure, cortical bone is also affected with reduced bone load bearing capacity. There is evidence that glucocorticoids decreased cortical thickness and increase the porosity of cortical bone [47, 48] . Treatment with compound 4e ameliorated the effect of GC and maintained cortical thickness and cortical porosity. Decreased cortical thickness is a reflection of the lower bone formation rate. There was lower mineralizing surface hence mineral apposition rate in GC treated group due to lower osteoblast activity and higher osteoclast activity as observed by single or discontinuous calcein labeling as compared to control animals. Our data suggest that compound 4e increased mineralizing surface and mineral apposition rate at doses of 2.5 mg kg −1 and 5 mg kg −1 ultimately increasing the bone formation rate.

Wnt signaling is vital for osteoblastogenesis and glucocorticoids use this pathway to stimulate mesenchymal stem cells in favor of osteoblasts at the physiological level and the same pathway is inhibited when glucocorticoid is in excess [13, 14] . Removal of Wntless (Wls), a chaperon required for normal secretion of Wnt ligand from cells, resulted in severe osteoporosis due to less bone formation and high bone resorption [49] . In vitro and in vivo studies have established that glucocorticoid administration suppresses Wnt signaling [16, 50] . In agreement with previous studies, we also observed suppressed Wnt signaling pathways. The inhibitory effect of glucocorticoid on the Wnt pathway is mediated through increased expression of SOST [51] . Compound 4e attenuated the inhibitory effect of glucocorticoid on the Wnt pathway by inhibiting SOST mRNA and protein expression. We also found increased serum levels of SOST in glucocorticoid treated mice which were reduced by compound 4e. BMP2 protein is an important regulator of osteoblast function and bone formation [52] [53] [54] . Several studies have reported that glucocorticoid negatively affects BMP2/SMAD pathway and restored osteogenic property after treatment of recombinant BMP2 protein [55] [56] [57] . In our study treatment of dexamethasone decreased BMP2/ SMAD1/5 pathway. Treatment with compound 4e restored the BMP2 signaling pathway by increasing its expression at mRNA and protein levels.

The above observations were confirmed with the circulating clinical biomarkers, the bone resorption marker CTX-I and bone formation marker Osteocalcin level in serum was perturbed in GIOP [42, 58] . Oral dosing of our compound promoted bone formation by decreasing CTX-I level and increasing OCN level in serum.

Compound 4e belongs to the benzofuran pyran chemical group. Benzofuran is a member of heterocyclic compounds. Heterocyclic compounds provide scaffolds on which pharmacophores can arrange to yield potent and selective drugs [59] . With precise reference to the pharmaceutical industry, Heterocyclic motifs are particularly prevalent in the industry, with over 60 percent of top retail drugs having at least one heterocyclic nucleus as part of the complete topography of the compound [60] . Compounds containing heterocyclic moieties also exhibit improved solubility and can promote salt formation properties, both of which are known to be essential for Fig. 3 Compound 4e ameliorated dexamethasone induced inhibition of osteogenic genes. a Compound 4e increased mRNA expression of RUNX2 b BMP2, c ALP which were down-regulated by dexamethasone. d Compound 4e maintained RANKL/OPG ratio by downregulating RANKL and e increasing OPG expression at 10 nM, 0.1 µM, 0.5 µM and 1 µM concentrations. Results were obtained from three independent experiments performed in triplicate and are expressed as Mean ± SEM. # p < 0.05, ## p < 0.01 ### p < 0.001 versus control, *p < 0.05, **p < 0.01 ***p < 0.001 versus dexamethasone ◂ Fig. 4 Compound 4e maintained RUNX2 and BMP2 at protein level. a and b Compound 4e increased BMP2 protein expression and phosphorylation of a and c SMAD 1/5/8 at 0.1 µM thus increasing, a and d RUNX2 at 10 nM, and 0.1 µM concentration. e Dexamethasone downregulated RUNX2 expression marked by lower green fluorescence and treatment of compound 4e increased RUNX2 expression reflected by higher green fluorescence at 10 nM, 0.1 µM concentration. Results were obtained from three independent experiments performed in triplicate and are expressed as Mean ± SEM. # p < 0.05 versus control, *p < 0.05 versus dexamethasone b Compound 4e increased β-catenin expression at 10 nM, 0.1 µM, 0.5 µM, and 1 µM concentrations which were reduced by dexamethasone. c and d Compound 4e downregulated SOST protein expression and increased inhibition of c and e GSK3-β thus increasing nuclear localization of (c and f) β-catenin. There is a lesser purple signal (alexa fluor 594 + DAPI) (g) in the dex treated group. Compound 4e increased nuclear localization as greater red signal localizes with DAPI. Results were obtained from three independent experiments performed in triplicate and are expressed as Mean ± SEM. # p < 0.05, ## p < 0.01 ### p < 0.001 versus control, *p < 0.05, **p < 0.01 ***p < 0.001 versus dexamethasone Fig. 6 Compound 4e improved micro architecture of femur in MP administered groups. Images obtained from Micro-CT. a Represented 3D/2D Images of femur trabecular of all groups. Femur trabecular parameters were measured. b Volumetric BMD (g HA/cm 3 ), c BV/ TV %, d Tb.Th (mm), e Tb.N (1/mm), f Tb.Sp (mm), and g SMI of animals of study groups were measured. Compound 4e was able to restore microarchitectural parameters in MP treated animals. All values are expressed as Mean ± SEM (n = 8/group); # p < 0.05, ## p < 0.01, ### p < 0.001 compared control group to the MP group.*p < 0.05, **p < 0.01, ***p < 0.001 compared MP to the 4e treated groups Fig. 7 Compound 4e rescue cortical bone from thinning and increased bone formation rate. a Representative 3D images of cortical bone. Oral dosing of compound 4e at 2.5 mg.kg −1 and 5 mg kg −1 doses increased b cortical thickness and c decreased cortical porosity. d Representative images of calcein double labelling. Dexamethasone inhibits bone formation marked by single and discontinuous label. Compound 4e restored the bone formation at 2.5 mg kg −1 and 5 mg kg −1. Compound 4e increased e MS and f MAR thus increased g BFR at 2.5 mg kg −1 and 5 mg kg −1. All values are expressed as Mean ± SEM (n = 8/group); # P < 0.05, ## P < 0.01, ### P < 0.001 compared control group to the MP group.*P < 0.05, **P < 0.01, ***P < 0.001 compared MP to the 4e treated groups Fig. 8 Compound 4e maintained Wnt/β-catenin pathway by reducing SOST in mice. a Glucocorticoid increased SOST mRNA and b serum level which were reduced by compound 4e at 2.5 mg kg −1 and 5 mg kg −1 . c Immunohistochemistry showed markedly reduced β-catenin in MP treated mice reflected by lesser red fluorescence which were abrogated by compound 4e dosing. All values are expressed as Mean ± SEM (n = 3/group); # P < 0.05, ## P < 0.01, ### P < 0.001 compared control group to the MP group.*P < 0.05, **P < 0.01, ***P < 0.001 compared MP to the 4e treated groups Fig. 11 Compound 4e improved histological architecture of trabecular and serum markers in MP administered groups. a Von kossa staining (× 10). b GT stain shows trabecular loss in MP group which were recovered in 4e treated groups. c Trabecular bone were quantified as BV/TV (%) by Bioquant osteo software. In serum of MP only group, collagen degradation marker, d CTX-I increased while bone formation marker, e OCN was decreased. Both markers are restored in 4e treated groups. All values are expressed as Mean ± SEM (n = 3/group); # P < 0.05, ## P < 0.01, ### P < 0.001 compared control group to the MP group.*P < 0.05, **P < 0.01, ***P < 0.001 compared MP to the 4e treated groups oral absorption and bioavailability. In addition, benzofurans are found in a significant number of natural products. Many natural benzofurans have physiological, pharmacological, and toxic effects [61] . Compound 4e is already known for its osteogenic activity [30] . Excess glucocorticoid is associated with increased bone marrow fat [41, 62] . Compound 4e showed its osteoblast rescuing effect against palmitate induced osteoblast death [31] thus suggesting that compound 4e modulates the bone marrow environment in favor of bone formation.

In conclusion, our results provide both in vitro and in vivo proof of the influence of compound 4e on the preservation of bone loss against glucocorticoid induced osteoporosis by modulating Wnt signaling. It can be a promising molecule to treat GIOP by maintaining osteoblast homeostasis.

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Treatment with a soluble bone morphogenetic protein type 1A receptor (BMPR1A) fusion protein increases bone mass and bone formation in mice subjected to hindlimb unloading

Icariin protects against glucocorticoid induced osteoporosis, increases the expression of the bone enhancer DEC1 and modulates the PI3K/Akt/GSK3β/β-catenin integrated signaling pathway

Cortical bone loss caused by glucocorticoid excess requires RANKL production by osteocytes and is associated with reduced OPG expression in mice

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The authors would like to acknowledge Achch-