Mice (clean level) were bought from Shanghai Slyke Experimental Animal Co., Ltd., China, and housed in specific pathogen-free conditions with ad libitum access to food and water at the Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy. Forty 7-week-old male ICR mice were divided into four groups randomly (n = 10 per group): control group; model group (diabetic osteoporosis group); low-dose group (diabetic osteoporosis group with 30 mg/kg catalpol); and high dose group (diabetic osteoporosis group with 90 mg/kg catalpol). The type 2 diabetes model was induced through a high-fat diet and intraperitoneal injection of low-dose streptozotocin. The mice (except the normal group) were fed with 55% high-fat diet fed (4 weeks) and then intraperitoneally injected with streptozotocin (0.5% of STZ solution was prepared in 0.1 mol citrate buffer, using 1 ml syringe) at a dose of 40 mg/kg for five consecutive days to build type 2 diabetes osteoporosis model (blood glucose level of >16.0 mmol/L; FBG more than 13 mmol/L) (Xie et al., 2018). The health status of all the mice was observed during the modeling. The success rate of modeling was 100%, and the injured mice did not exhibit death following the impact of STZ. After 12 weeks of intragastric administration of vehicle control or catalpol, serum and femurs were immediately harvested for a variety of analyses.

MC3T3-E1 cells were purchased from the typical Culture Collections Committee cell library of the Chinese Academy of Sciences (Shanghai, China). After resuscitation, MC3T3-E1 cells were maintained in minimum essential medium-α (MEM-α) (HyClone, Logan, UT, United States) containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO₂ in air at 37°C. The mineralizing medium was used to induce osteogenic differentiation containing 10% fetal bovine serum, 50 mg/ml ascorbic acid, 10 mM ß-glycerophosphate phosphate, and 10⁻⁸ M dexamethasone (Dong et al., 2017). In a high glucose experiment, 25.5 mM glucose was added to a-MEM to mimic high glucose concentration while 25.5 mM mannitol was used as an osmotic control for high glucose conditions (Zhou et al., 2019). MC3T3-E1 cells (reached approximately 80%) were maintained in mineralizing medium and divided into the following four groups: normal control group, high-glucose group, and catalpol groups (1 and 10 μM). To test the osteogenesis, Western blot analysis of the osteoblastic markers, ALP activity assay, Alizarin Red staining, and other assays were performed (Ho-Shui-Ling et al., 2018).

MC3T3-E1cells were suspended in a-MEM containing 10% FBS and plated in 12-well culture plates at a density of 5 × 10⁴ cells per well. When the cells reached approximately 80% of the well, the cells were maintained in mineralizing medium and cultured with catalpol (1 and 10 μM). After 2 h, the cells were treated with high glucose (25.5 mM glucose in the culture medium) to mimic the diabetic state for 7 days (Dong et al., 2017). The medium was changed every 3 days. ALP activity was determined in the cell culture supernatants according to the manufacturer’s instructions of ALP activity assay kit (Jiancheng, Nanjing, China). The test samples and buffers were prewarmed to room temperature. 50 µl/well of p-nitrophenol was added to a 96-well plate, and 100 µl of samples was mixed well with the substrate, then incubated at 37°C for 10 min, and added 100 µl stop solution. The color change of p-NPP to p-nitrophenol was measured at 405 nm using a microplate reader (Li et al., 2012).

MC3T3-E1 cells were cultured suspended in a-MEM containing 10% FBS and plated in 24-well culture plates at a density of 5 × 10⁴ cells per well. The cells were incubated with osteoblasts differentiation medium containing 50 mg/ml of ascorbic acid, 10 mM of ß-glycerophosphate, and 10 nM of dexamethasone. Cultured for 21 days, cells were washed with ice-cold PBS and fixed with 4% formalin in PBS for 15 min at 25°C. Then, the cells were washed twice with dH₂O and stained with 0.1% AR-S-Tris-HCl (pH 4.2) for 30 min at room temperature. The mineralized nodules were observed under an inverted light microscope, and the mineralized nodules were dissolved with 10% acetyl chloride in 10 mM sodium phosphate, followed by the absorbance measurement at 562 nm using a multiplate reader (Zhang et al., 2019).

MC3T3-E1 cells were plated in 6-well plates at a density of 1 × 10⁵ cells per well. After overnight incubation, the cells were maintained mineralizing medium and cultured with catalpol (1 and 10 μM). After 2 h, the cells were subjected to high glucose (25.5 mM) for 5 days. The cells were collected using a rubber scraper and incubated for 30 min at 4°C with shaking and centrifuged (10,000 rpm, 15 min, 4°C), and the supernatants were collected. The protein concentration was determined by a BCA protein assay kit (Jiancheng, Nanjing, China). The proteins separated through electrophoresis were transferred onto polyvinylidene difluoride (PVDF) membranes. Afterward, membranes were incubated with blocking buffer (5% (v/v) skimmed milk in 0.05% (v/v) PBS/Tween) for 1 h at RT and, respectively, incubated overnight at 4°C with Runx2, type I collagen, osteocalcin, OPG, RANKL, and mouse GAPDH antibodies. The primary antibodies were detected using alkaline phosphatase-conjugated immunoglobulin G (IgG; Bioworld Technology, St. Louis, United States) at room temperature for 1 h. Membranes were developed colorimetrically by BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, Nantong, China). The band density was quantified using ImageJ soft (LI-COR Biosciences) (Lu et al., 2018).

For migration assay, the wound-healing assay was done. MC3T3-E1 cells were plated in 12-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 h. The monolayer was then scratched with pipette tips and washed with PBS. Subsequently, the cells were then cultured with catalpol (1 and 10 μM). After 2 h, the cells were subjected to high glucose (25.5 mM) for 24 h according to references and results of pretests. Slides were observed and photographed with a Leica DM LB microscope (Leica Mikrosysteme Vertrieb, Wetzlar, Germany) (Schwarting et al., 2015). The transwell cell migration assay was performed by using a Boyden chamber (polycarbonate membrane with 8-µm pores in 12-well plates). MC3T3-E1 cells were plated onto the upper chamber in 12-well plates at a density of 1 × 10⁵ cells per well in a-MEM containing 0.3% FBS, and the lower chambers were filled with 10% FBS medium. The cells were then cultured with catalpol (1 and 10 μM), added to the upper chamber. After 2 h, the cells were subjected to high glucose (25.5 mM) for 24 h. After 24 h of incubation at 37°C, the cells on the upper surface of the membrane were mechanically removed, and the cells adherent to the underside of the transwell membrane were fixed with 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole solution (Kawabata et al., 2018).

After mice were sacrificed, serum was immediately harvested. The concentrations of TRACP and ALP were measured via ELISA (Jiancheng Bioengineering Institute, Nanjing, China) kits according to the manufactures’ guidelines and read at 450 nm using a microplate reader (Bio-Rad).

For analysis of bone microstructure and mineralization, the left femurs were subjected to scanning using micro-CT (GE Healthcare, United States). After removing the soft tissues, the left femurs were placed with gauze in the sample holder and were scanned using micro-CT using a resolution of 6 μm, 80 kV, 80 μA, 400 number of views to obtain 3-dimensional images. The eXplore Reconstruction Utility software was used for three-dimensional reconstruction. Trabecular morphometry was characterized by measuring the volumetric parameters of bone mineral density (BMD), bone volume fraction (BVF), BS/BV, trabecular thickness (Tb.Th), trabecular number (Tb.N), and decreases in trabecular spacing (Tb.Sp) (Lontos et al., 2018).

The sections of the femur, thoracic vertebra, and lumbar vertebra were stained histochemically for ALP and TRACP activity as an OB and OC marker. The femurs were shortly washed with cold PBS, fixed in 4% paraformaldehyde overnight, and decalcified in 19% EDTA for 7 days. Then, the femur, thoracic vertebra, and lumbar vertebra were dehydrated in an alcohol series and embedded in paraffin and, respectively, cut into serial sections of 10 μm (Li et al., 2017). Respectively, the ALP and TRACP staining were, respectively, performed in accordance with the kit instructions (D001-2-1, D023-1-1, Nanjing Jiancheng Bio, Nanjing, China).

Data are expressed as the mean ± SD of at least three independent experiments. One-way analysis of variance followed by Dunnett’s t-test was used for the statistical analysis (SPSS 13.0 software; SPSS, Chicago, United States). A p value of < 0.05 was conventionally considered to be statistically significant. Graphs were drawn using GraphPad Prism (version 6.0 for Windows).

We established a diabetic osteoporosis mice model by 55% high-fat diet fed and injecting streptozotocin. Bone mass and bone formation were obviously decreased in osteoporotic femur, compared to normal group mice. The microarchitecture of the distal femur was analyzed by micro-CT and the corresponding bone morphometric parameters shown in Figure 1. The observed degeneration in trabecular bone structure in the model group compared with the normal group and the degeneration of femurs were clearly inhibited by the treatment with catalpol. Histograms represent the parameters of three-dimensional trabecular structural of the distal femur: BMD, BVF, BS/BV, Tb.Th, Tb.N, and Tb. Sp. Compared with the normal group, a high-fat diet and streptozotocin treatment deteriorated the trabecular microarchitecture of the femur, which was demonstrated by decreased BMD (p < 0.05), BVF (p < 0.01), Tb.Th (p < 0.05), and Tb.N (p < 0.01) as well as increased Tb. Sp (p < 0.01) and BS/BV (p < 0.01). Administration of catalpol (90 mg) significantly improved the deteriorated trabecular of the femur. Compared with the model group, the parameters of BV/TV, Tb.N, Tb.Th, and trabecular BMD were significantly enhanced by catalpol, while Tb. Sp, and BS/BV were significantly decreased by catalpol (p < 0.05).

To assess the role of catalpol on bone formation and bone resorption of the bone of diabetic osteoporosis mice, the serum levels of bone metabolism markers, and the levels of ALP (Figure 2) and TRACP (Figure 3) in the femur, thoracic vertebra, and lumbar vertebra were assayed by ELISA and ALP/TRACP staining. The levels of ALP in femur, thoracic vertebra, and lumbar vertebra of diabetic mice were reduced significantly compared to the control group. Red color shows the ALP in the femur, thoracic vertebra, and lumbar vertebra with significantly elevated levels in catalpol treated groups. This provides further evidence that catalpol stimulates bone formation in different bone tissues of diabetic osteoporosis mice. In addition, serum ALP and osteocalcin levels of diabetic mice in the model group were decreased significantly (p < 0.05), and administration of catalpol (90 mg) significantly improved the ALP and osteocalcin levels. These results illustrated that catalpol promoted bone osteoblast differentiation and bone formation in vitro and in vivo. However, the TRACP levels in the femur, thoracic vertebra, and lumbar vertebra, as well as in serum of diabetic mice, had no significant differences among groups. Catalpol did not show inhibitory effects on bone resorption.

In the process of bone regeneration, osteoblast precursors and their progeny are required to migrate within the three-dimensional bone space (Xie et al., 2018). Therefore, inducing the migration of osteoblast might play a role in osteoporosis. To investigate the effects of catalpol on the migration capacity of osteoblasts, a transwell assay was performed. As shown in Figure 7, MC3T3-E1 cells treated with high glucose have lower migration behavior compared with the normal group. Catalpol (1 and 10 μM) significantly enhanced the migration behavior of MC3T3-E1 cells treated by high glucose after 24 h of culture. In addition, cell migration activity was also assessed using the wound-healing assay. In Figure 8, compared with the MC3T3-E1 cells in normal situations, the environment of high glucose had an obvious inhibiting effect on cell migration activity. Catalpol enhanced cell motility and scattering following gap formation in MC3T3-E1 cells treated by high glucose. Collectively, these findings from the transwell cell migration assay and the wound-healing assay demonstrate that catalpol promotes migration of MC3T3-E1 cells treated by high glucose.