Materials and reagents

All reagents used for cell culture, including low-glucose Dulbecco’s Modified Eagle’s Media (DMEM; Gibco, 11054, 020), fetal bovine serum (FBS; Gibco, 10099, 141C), non-essential amino acids (Gibco, 11140, 050), antibiotics (P/S; Gibco, 15070, 063) and trypsin (Gibco, 15400, 054) were purchased from Thermo Fisher Scientific Incorporation. Recombinant basic fibroblasts growth factor (bFGF; 100-18B), epidermal growth factor (EGF; AF-100-15) were purchased from PeproTech Inc.. PBS (P1020), Chloroquine (CQ; IC4440), rapamycin (R8140) and 3-Methyladenine (3-MA; IM0190) were purchased from Beijing Solarbio Science & Technology Co., Ltd (Solarbio Inc.). Hydrogen peroxide solution (H2O2; Sigma, 88597) was from Merck group. All materials and regents for western blotting were purchased from Beyotime Institute of Biotechnology (Beyotime), including polyvinylidene difluoride membranes (FFP19), Bovine Serum Albumin (BSA; ST023), TBST buffer (ST673).

All drugs used for in vitro differentiation of hBMSCs, including dexamethasone (Sigma; D4902), β-glycerophosphate (Sigma; G9422), L-ascorbic acid (Sigma; A4544), indomethacin (Sigma; I7378), isobutyl methylxanthine (Sigma; IBMX, I7018) were purchased from Merck. Insulin (I8830) was from Solarbio Inc. Verteporfin was from MedChemExpress LLC (MCE; CL318952). CXM102 was synthesized in our lab as described in a patent (CN23NN14118I).

Primary and secondary antibodies used in this study were shown in Table S1. Cytochemical staining dyes, including Alizarin red S (G8550) and Oil red O (O8010) were from Solarbio Inc. The working solution was prepared based on the manufacture’s instruction.

Animal approval

All animal experiments were performed in accordance with the protocol approved by Guangxi University of Chinese Medicine Institutional Welfare and Ethical Committee (Approval No. DW2022). C57/BL6 mice were reared in a pathogen-free facility at a controlled temperature (23–25 °C), under a 12 h light and dark cycle with food and water provided ad libitum. The cage and bedding were changed once a week. 3-month-old and 14-month-old mice were used in this study.

Cell culture

Young, old and immortal hBMSCs, normal synoviocytes, FEK4 and RA-FLS were kindly gift from Dr. Li Yang (Chongqing University) and maintained as described before.50,71 Human normal liver cells (L02) and cancer cell lines, including Hep3B, HCCLM3, HCT116, DU145, A549 and THP-1 were from Dr. Erwei Hao (Guangxi University of Chinese Medicine). Briefly, 5 × 105 cells/mL were plated in a 100-mm diameter cell culture dish containing low-glucose DMEM supplemented with 10% FBS, 100 U/mL P/S, 0.1 mmol/L non-essential amino acids, 20 ng/mL bFGF and 20 ng/mL EGF. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. When 70% to 80% confluent, adherent cells were trypsinized with 0.05% trypsin-1mmol/L EDTA at 37 °C for 2 min, harvested, and expanded for future use.

Effects of CXM102 on cell growth

To study the effects of CXM102 on cell growth, a density of 2000 cells per well of indicated cell types were seeded into a 96-well plate as described before.71 cells were treated with different concentrations of CXM102 (0, 5, 10, 20, 30, 50 μmol/L) for different time points (12, 24 or 36 h). Cell activities were determined by Cell Counting Kit-8 kit (Beyotime, C0038) following the manufacturer’s instruction.

Effects of CXM102 on hBMSCs senescence

To study the effects of CXM102 on cellular senescence, hBMSCs were pretreated with different concentrations of CXM102 (0, 5, 10, 20 μmol/L) 2 h before 200 μmol/L H2O2 addition into culture medium for 2 h at 37 °C, as described by others,72 followed by fresh medium change containing 20 μmol/L H2O2 for 4 h. Then hBMSCs were fixed and analyzed the levels of senescence β-Galactosidase activities (SA-β-Gal; Beyotime, C0602), reactive oxygen species (ROS; Beyotime, S0033S), mitochondrial membrane potential (MMP; JC-1, Beyotime, C2006) according to the manufacturer’s instruction. Molecules levels of p16INK4a and γH2AX, osteogenic and adipogenic differentiation were also detected as described below.

Effects of CXM102 on autophagy

To investigate the roles of autophagy and transcription factor EB (TFEB) in hBMSCs senescence, 2.5 mmol/L 3-MA or 50 μmol/L CQ was added into the culture medium for 4 h before CXM102 treatment. Then cells were fixed and detected by immunofluorescence (IF) or western blotting for the analysis of autophagic influx, autophagosomes, autolysosomes and TFEB activities as described below.

Immunofluorescent (IF) /Immunohistological (IHC) analysis

For immunofluorescent/immunohistochemical staining, cells or deparaffined sections of femurs were fixed with 4% paraformaldehyde (Beyotime, P0099), permeatilized with 0.2% Triton X-100 (Solarbio, T8200), and blocked with 1% bovine serum albumin (Solarbio, SW3015). Then samples were incubated with primary antibodes (Table S1) or non-immune antiserum as a negative control at 4°C overnight. After washing with PBS, sections were incubated with HRP-conjugated or fluorescent secondary antibodies (Table S1) for 1 h, and counterstained with DAPI for 10 min at room temperature. Images were obtained by upright microscope (BX53, Olympus), or fluorescence microscopy (DMI8, Leica). For quantitative analysis, at least four independent samples of each group were analyzed by Image-Pro Plus 6.0 software.

RNA interference and transfection

To study the role of TFEB in CXM102-mediated autophagy, the following sequence of siRNA was used for TFEB downregulation. siRNA scramble sense, 5’-UAGCGACUAAACACAUCAAU‑3’ and antisense, 5’‑UUGAUGUGUUAGUCGCUAU‑3’; siTFEB sense, 5’-GGAUCAAGGAGCUGGGAAUUU‑3’ and antisense, 5’- AUUCCCAGCUCCUUGAUCCUU-3’ (GenePharma, Suzhou, China). The transfection of siRNA was conducted as we did before.73 Briefly, hBMSCs (2 × 105 cells per well) were seeded into a 24-well plate, then 50 nmol/L siRNA scramble or siTFEB was transfected into hBMSCs with Lipofectamine 2000 (Invitrogen, 11668, 019) according to the manufacturer’s instructions. Finally, the protein level was assessed by IF staining or western blotting.

Quantitative real-time PCR

Total cellular RNAs were isolated using RNA Extraction Kits (Solarbio, R1200) according to the manufacturer’s instructions and quantified by Nanodrop One C (Thermo Fisher Scientific, Madison, USA). 1 μg RNA was analyzed by using a TaqMan One Step RT-qPCR Kit (Solarbio, T2210) on LightCycle 96 Instrument (Roche, Mannhein, Germany) according to the manufacturer’s instructions. The primers used in this study are shown in Table S2. The relative expression of target genes was normalized to Gapdh and calculated using the 2−∆∆CT method.

Western blotting

Total protein was extracted using a protein extraction kit (Solarbio, BC3710) and the protein concentration was determined by BCA (Beyotime, P0012) assay according to the manufacturer’s instructions. Next, 25 μg of protein sample was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes. Then the membranes were blocked with 3% BSA in TBST buffer for 2 h at room temperature, followed by incubation with the indicated primary antibodies (Table S1) at 4 °C overnight and horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Targeted proteins were visualized with enhanced chemiluminescence solution (Beyotime, P0018S) and detected using a ChemiDoc Touch Imaging system (Bio-Rad, CA, USA).

Effects of CXM102 on differentiation induction

Osteogenic, adipogenic, and bivalent differentiation and detection of BMSCs were strictly conducted as we described in a recent method article.50

Animal experiments

To study the effects of CXM102 on aged-related bone metabolism, 14-month-old mice were divided into four groups, followed by a period of 4-month-long treatment with DMSO (Indicated as “Old”), CXM102 alone (OC; 20 mg/kg body weight), verteporfin alone (OV; 10 mg/kg) and CXM102 combined with verteporfin (OVC). In OVC group, verteporfin was pretreated 2 weeks before CXM102 injection. All drugs were dissolved into 50 μL DMSO and injected subcutaneously twice a week. Mice were sacrificed 1 month after the final injection, serum and femurs were collected for analysis.

BMD measurement

To assess the effects of CXM102 on BMD changes in vivo, the BMD values of 14-month-old mice were measured as the initial time and then followed by a period of 4-month injection with DMSO (Marked as “Old”), CXM102 (OC), verteporfin (OV) alone or both (OVC). In addition, 3-month-old untreated mice (Marked as “Young”) were used as positive control in this experiment. BMD was measured once a month by dual energy x-ray absorptiometry (DXA) as described by others.74 Briefly, mice were anesthetized with isoflurane (Yuyan Corporation, Shanghai, China) in a chamber, then were placed on the Lunar PIXImus densitometer platform (GE Medical-Lunar, Madison, WI, USA). Half of the distal femur was selected as area of interest and perform a scout scan. BMD value was recorded by the PIXImus software (Lunar Piximus 2.0).

ELISA assay

Mouse blood serum was diluted 1:1 using PBS and the diluted serum levels of P1NP (Sangon Biotech, D721053), CTX-1; (Sangon Biotech, D721204), TNF-α (Sangon Biotech, D721217), IL-1β (Sangon Biotech, D721017) and IL-6 (Sangon Biotech, D721022) were measured by ELISA kits according to the manufacture’s instruction.

MicroCT analysis

MicroCT analysis was performed using the settings according to the manufacture’s instruction. Generally, Mouse femurs were dissected, fixed with 4% paraformaldehyde (Beyotime, P0099) for 48 h and stored in 70% ethanol, loaded into 10-mm diameter scanning tubes, and scanned with a peak tube voltage of 50 kV and current of 0.145 mA at 18 μm resolution, (Latheta LCT200, Hitachi Aloka, Japan). The 2D and 3D model visualization software (BeeViewer v3.4.1) and data analysis software (VGStudioMAX v2.2) were applied to analyze the distal femoral metaphyseal trabecular bone and the parameters of the diaphyseal cortical bone. We defined a region of interest (ROI) as the area between 2%–6% proximal to the growth plate in the distal femora. Cross-sectional images of the femora were established for 3D histomorphometry analysis of the trabecular bone. The percentage of bone volume (BV/TV), the number (Tb. N), thickness (Tb. Th) and space (Tb. Sp) of trabecular bone were analyzed.

Histological analysis

Histological analysis of femurs was performed as we did before.71 Briefly, the dissected distal femurs were fixed in 4% paraformaldehyde (Beyotime, P0099) for 48 h, decalcified with 15% EDTA (pH7.2-7.4) and were paraffin embedded. 5mm-thickness sections were cut and subjected to haematoxylin and eosin (H&E; Solarbio, G1120) or Masson’s trichrome staining (Solarbio, G1340). Images were taken by a microscope (BX53, Olympus) and analyzed by Image-Pro Plus 6.0 (Media Cybernetics, MD, USA) software.

Biomechanical testing

To investigate mechanical strength of femurs, an electronic universal material testing machine (AGS-10kNJ, Shimadzu, Japan) was used for the three-point bending test as described in other’s experiment.31 Before the test, the long and short diameters of the femoral shaft were measured with a Vernier caliper, then femurs were placed between two support abutments. The load was applied to middle of the femoral shaft through a touching probe. During this test, the posterior surface was in tension, and the anterior surface was in compression. The strength variables (bending modulus of elasticity, bending energy, maximum bending stress, and bending rigidity coefficient) were calculated with a standard engineering formula.

Lifespan analysis

To compared the effects of CXM102 and rapamycin on lifespan, middle-aged mice were divided into three groups, which were administered with rapamycin (8 mg/kg weight), CXM102 (8 mg/kg weight) or DMSO twice a week for a period of 6-month-long term. 1 month after the final injection, three mice from each group were sacrificed for assessment of histological changes of liver, kidney and heart. The other mice were freely fed until natural death. Survival curves were calculated by Origin 8.0 (OriginLab, Guangzhou, China).

Statistical analysis

Results are represented as means ± standard deviations. Statistical analysis was performed using Student’s t-test as well as one-way analysis of variance (ANOVA) followed by the Tukey HSD test for post hoc comparison (Origin 8.0, OriginLab). Difference was considered significant when P < 0.05 indicated as *, while more significant when P < 0.01 indicated as **.