Histologic analysis
For histologic analysis, specimens were decalcified in 0.5 M EDTA (G1105, Servecibio, Wuhan, China) for 21 days. Subsequently, the specimens were dehydrated, cleared, paraffin-embedded, and cut into 6 μm sections. Masson staining of femoral sections was performed according to the manufacturer’s instructions using a Masson staining kit (G1006, Servicebio, Wuhan, China). Immunohistochemical analysis of the specimens was conducted using specific antibodies FNDC5 (1:200 dilution, 23,995-1-AP, Proteintech, Wuhan, China). Finally, the sections were photographed using an optical microscope (Leica, Germany).
ELISA assay
Blood samples were collected according to the instructions of ELISA Kits (CUSABIO, Wuhan, China). Briefly, blood samples were centrifuged at 1000 × g for 15 min at 4 °C. Serum samples were aliquoted and stored at − 80 °C. Plasma was then collected and stored at − 80 °C until assayed. Bone formation markers procollagen I N-terminal propeptide (PINP) and osteocalcin (OCN) were measured by PINP ELISA kit and OCN ELISA kit (CSB-E12775m, CSB-E06917m, CUSABIO, Wuhan, China). The absorbance at 450 nm was detected using a microplate reader (Thermo Scientific, USA).
Cell culture and treatment
MC3T3-E1 cells (Procell, Wuhan, China) were cultured in α-MEM (C3060-0500, VivaCell, Shanghai, China) medium supplemented with 10% fetal bovine serum (FBS, FSP500, ExCell Bio). The cells were maintained in a humidified incubator at 37 °C and 5% CO2. To mimic T1DM culture conditions, cells were stimulated with varying concentrations of glucose in the culture medium for 48 h. On this basis, the cells were pretreated with GSK2606414 (GSK, 1 μM) (HY-18072, MedChem Express, Monmouth Junction, New Jersey, USA) at the specified concentration for 2 h, and GSK remained in the media throughout the period of glucose stimulation.
Cell viability assay
Cell viability was evaluated using a CCK8 Assay kit (40,203, Yeasen Biotechnology, Shanghai, China). Cells were treated with different reagents for the indicated times. 10μL CCK-8 reagent was added at indicated time points. Absorbance at 450 nm was measured with a microplate reader (Thermo Scientific, USA).
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNAs were extracted from cells and tissues with the TRIzol reagent (9109, Takara, Shiga, Japan). RNA was reverse transcribed with Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (11,141, Yeasen Biotechnology, Shanghai, China). The qPCR reactions were performed with 1 × Hieff qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China). Specific primers for mRNAs were by Tsingke Biotechnology (Beijing, China). The β-actin forward primer sequence was 5′-AAATCGTGCGTGACATCAAAGA-3′, and the β-actin reverse primer sequence was 5′- GCCATCTCCTGCTCGAAGTC-3′. The FNDC5 forward primer sequence was 5′-ACAGAGCCCAGCCAGTGAGC-3′, and the FNDC5 reverse primer sequence was 5′- GCCCACATGAAGAGGACCACAAC-3′. ACTB (β-actin) was employed as an internal control of mRNAs. The expression levels of genes were calculated via the 2−ΔΔCt method.
Western blot analysis
Total proteins of cells and tissues were extracted using RIPA lysate solution (R0010, Solarbio, Beijing, China). Protein quantification was conducted by a BCA Protein Assay Kit (PC0020, Solarbio, Beijing, China). The SDS-PAGE gel was made with resolving gel and stacking gel. The resolving gel was prepared with 1.5 M Tris base at pH 8.8 (T1010, Solarbio, Beijing, China), 10% sodium dodecylsulfate (SDS, S8010, Solarbio, Beijing, China), and 30% acrylamide (A1010, Solarbio, Beijing, China). The stacking gel contained was prepared with 1.0 M Tris base at pH 6.8 (T1020, Solarbio, Beijing, China), 10% SDS, and 30% acrylamide. The proteins were then subjected to electrophoresis using SDS-PAGE gels and transferred to PVDF membranes (00839B, Pall Corporation, Port Washington, NY, USA). The membranes were blocked with 5% nonfat dry milk and incubated with primary and secondary antibodies. Lastly, the membrane was detected with the super electrochemiluminescence (ECL) kit (S6009M, US Everbright, Suzhou, China).
The primary antibodies used included β-actin (1:3000, P60035, Abmart), alkaline phosphatase (ALP, 1:1000, T55421, Abmart, Shanghai, China), CHOP (1:1000 dilution, T56694, Abmart, Shanghai, China), solute carrier family 7 member 11 (SLC7A11, 1:1000 dilution, ab175186, Abcam, Cambridge, MA, USA), runt-related transcription factor 2 (RUNX2,1:1000 dilution, ab236639, Abcam, Cambridge, MA, USA), ferritin heavy chain (FTH, 1:1000 dilution, 3998, Cell Signaling Technology, Danvers, MA, USA), GPX4 (1:1000 dilution, 52,455, Cell Signaling Technology, Danvers, MA, USA), osteocalcin (OCN, 1:1000, DF12303, Affinity, Changzhou, China), eIF2α (1:1000, AF6087, Affinity, Changzhou, China), phosphorylation-eukaryotic initiation factor 2 alpha (p-eIF2α,1:1000, AF3087, Affinity, Changzhou, China), ATF4 (1:1000, AF5416, Affinity), and FNDC5 (1:2000, 23,995-1-AP, Proteintech, Wuhan, China).
Measurement of ROS levels and lipid peroxidation levels
Intracellular ROS levels were measured by a reactive oxygen species assay kit (S0033, Beyotime Biotechnology, Shanghai, China). Intracellular lipid peroxidation levels were determined by the C11-BODIPY 581/591 fluorescent probe (D3861, Invitrogen, Carlsbad, CA, USA). Firstly, cells were seeded in 24-well plates and treated with specific chemicals for the indicated times. Then the fluorescent probe DCFH-DA or C11-BODIPY581/591 was added to stain the cells. Finally, images were captured with a fluorescence microscope (Olympus, Japan).
Measurement of GSH, MDA, and total iron content
GSH is an important antioxidant, and the depletion of GSH can trigger ferroptosis [
39]. Malondialdehyde (MDA) is an end-product of lipid peroxides, which is used to assess ferroptosis [
40]. The cellular or tissue GSH, MDA content, tissue iron levels and serum iron levels were detected using the reduced glutathione (GSH) assay kit (A006-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the malondialdehyde (MDA) detection kit (S0131, Beyotime Biotechnology, Shanghai, China), the tissue iron assay kit (A039-2-1) and the serum iron assay kit (A039-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. The concentrations of GSH and MDA were determined by measuring the absorbance at 405 nm and 530 nm. The values for the levels of tissue and serum iron were examined at a wavelength of 520 nm.
Transmission Electron Microscope (TEM)
The ultrastructure of mitochondria was observed by TEM. First, cells were collected and fixed in 0.01 MM PBS-buffered 2.5% glutaraldehyde (G1102, Servicebio, Wuhan, China). They were then post-fixed in 1% osmic acid (18,456, Ted Pella, Altadena, CA, USA). Subsequently, samples were dehydrated, embedded in resin, and cut into sections of ∼60 nm. The sections were then dyed with 2% uranyl acetate (02624-AB, SPI, West Chester, PA, USA) and observed with a HITACHI HT7800 electron microscope (Hitachi, Japan).
Overexpression of FNDC5
When the cell density reached 70–80% confluence on a 6-well plate, the cells were transfected with an overexpression plasmid of FNDC5 by Lipo8000 transfection reagent (C0533, Byotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Cells transfected with an empty plasmid vector were used as the negative control (NC).
Alizarin red staining assay and ALP activity test
To induce osteoblast differentiation, MC3T3-E1 cells were treated with osteogenic medium (α-MEM containing 10% FBS, 100 μg/ml ascorbic acid (A8960, Sigma, Sigma-Aldrich, St Louis, MO, USA), 10 mM β-glycerophosphate (G9422, Sigma, Sigma-Aldrich, St Louis, MO, USA), and 10 mM dexamethasone (S4028, Huston, TX, USA) for 14 days. After induction, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Alizarin red staining was performed using 0.1% Alizarin Red S (G8550, Solarbio, Beijing, China) solution for 30 min. The mineralized nodules were observed by a microscope after washing with ddH2O. The alkaline phosphatase (ALP) activity test was performed using the ALP test kit (A059, Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s instructions.
Statistical analysis
Data were expressed as mean ± standard deviation (SD) of at least three independent experiments for the studies. The SPSS 22.0 (SPSS INC, USA) and GraphPad Prism 9.3.1 (GraphPad, USA) were used to analyze experimental data. The Shapiro–Wilk test was performed to evaluate whether the data was normally distributed. Levene’s test was performed to analyze the homogeneity of variances. Statistical analysis was carried out using Student’s t-test between two groups and analysis of variance (ANOVA) among multiple groups in accordance with data normal distribution and homogeneity of variances. The difference was considered statistically significant if P < 0.05.