Forty four-week-old C57BL/6 mice were provided by B&K Laboratory Animal Company (Shanghai, China). The mice were housed under standard conditions and the room was maintained on a cycle of 12 h light and 12 h darkness, at a temperature of 25 ± 3 °C and with free access to food and water. All animal experiments were approved by the Ethics Committee on Animal Use of Yangzhou University.
Establishment of the T2DM model
All animals were adaptively fed for 1 week and then randomly divided into a normal group (ZC, n = 10) and a T2DM model (n = 30). The T2DM model mice were fed a high-fat diet (31.7% lard, 25.8% casein 30 mesh, 16.3% maltodextrin 10, 9% sucrose, 6.5% cellulose BW200, 3.4% soybean oil, 2.3% potassium citrate, 1% H2O, 1.6% mineral mix S10026, 1.4% dicalcium phosphate, 1.3% vitamin mix V10001, 0.8% calcium carbonate, 0.4% L-cystine, and 0.3% choline bitartrate, purchased from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd.) for 6 weeks, and then STZ (80 mg/kg) was injected 12 h after fasting. The normal group mice were injected with a citric acid-sodium citrate solution. The blood glucose concentration of the mice was detected after 12 h of fasting 2 weeks later. The blood glucose concentration was above 8 mmol/L for the T2DM mice [24, 46], and 27 models were successfully established and randomly divided into the T2DM control group (TC, n = 9), the T2DM swimming group (TS, n = 9) and the T2DM downhill running group (TD, n = 9). Normal mice were fed a normal diet, and T2DM mice continued to have a high-fat diet, with both groups drinking water freely.
TS group and TD group mice were subjected to swimming and downhill running, respectively. For the swimming group, the mice were placed in a (42 cm long × 40 cm wide × 36 cm water depth) container, for 50 min/day, 8 weeks in total. The first week was adaptive training, 30 min a day for the first 2 days, 40 min a day for the 3rd and 4th days, then 50 min a day for the 5th and 6th days, while normal training started in the 2nd week. The conditions for downhill running included: 0.8 km/h, 50 min, slope − 9 degrees, 6 days/week, a total of 8 weeks. The first week was adaptive training, 30 min a day for the first 2 days, 40 min a day for the 3rd and 4th days, then 50 min a day for the 5th and 6th days. Later, normal training started in the 2nd week.
The body weights of each group of mice were weighed using an electronic scale.
Oral glucose tolerance test (OGTT)
After the last exercise intervention in the eighth week, the mice were fasted for 12 h and gavaged 2 g/kg glucose for the OGTT experiment. After gavage at 0, 30, 60, and 120 min, blood glucose was measured through the tail vein, and the area under the curve was calculated. The calculation method of the area under the time of the blood glucose curve was AUC (h mmol L−1) = 1/4 A + 1/2 B + 1/2 C + 1/4 D (A, B, C, and D were the blood glucose values measured after 0, 30, 60 and 120 min of intragastric glucose administration) .
Measurement of bone length and wet weight
A Vernier caliper was used to measure the left tibia lengths of the mice in each group. An electronic balance was used to measure the left tibia wet weights of the mice in each group.
Alizarin red staining
After fixing with 4% PFA for 24 h, the left femurs were decalcified in 4% EDTA for 30 days after washing with PBS. Subsequently, alcohol gradient dehydration was conducted and the left femurs were embedded in paraffin, before being sagittally cut into 6 μm sections. After placing on a baking sheet, the slices were dewaxed with xylene, and rehydrated with an alcohol gradient (from a high to a low concentration). The slices were washed in PBS solution and Alizarin red staining was performed for 5 min followed by washing with PBS. Then, dehydration was performedwith an alcohol gradient (a low to a high concentration), xylene I and II for 5 min each, before sealing with neutral gum. Finally, a Leica microscope was used to take photographs.
Microcomputed tomography (micro-CT) analysis
The left femur of each group was fixed in 4% paraformaldehyde (PFA) for 24 h, washed with PBS and then scanned by a micro-CT scanner (Skyscan, Aarselaar, Belgium) with a resolution of 18 μm per pixel. For 3-dimensional histomorphometric analysis of the trabecular and cortical bone, cross-sectional images of the distal femur were used. The region of interest (ROI) of the distal femur selected for analysis was 5% of the femoral length from 0.05 mm below the growth plate to determine the trabecular and cortical BMD, BV/TV, bone surface area to volume ratio (BS/BV), ratio of bone surface area to tissue volume (BS/TV), trabecular thickness (Tb.Th), Tb.N and trabecular separation (Tb.Sp).
ALP staining of the skull
After washing with PBS, the skull was fixed in 4% PFA at 4 degrees for 24 h. Then, 0.1% Triton-X100 (for permeabilisation) and PBST were used to treat the skull in a 4 degree environment for 24 h. The skull was stained with an ALP dye solution (0.002 g AS-MAX and 0.006 g Fast Red Violet LB Salt dissolved in 5 ml pH 8.3 of Tris–HCl and 5 ml of ddH2O) for 1 h. A camera (Canon, Japan) was used to take photographs and Photoshop software was used to take a screenshot of the seam locations.
ALP and Alizarin red staining of OBs
After euthanising the mice under aseptic conditions, the bilateral femurs and tibias were taken from each mouse and cleaned of all attached soft tissues. The bone marrow cavity was exposed, and the bone marrow was isolated. A single-cell suspension was prepared by repeated aspiration. The total bone marrow cells were counted by haemocytometer and cultured in alpha minimal essential medium (α-MEM; GIBCO, USA) supplemented with 10% foetal calf serum (GIBCO, USA), 1000 U/ml ciprofloxacin (Sigma, USA). The cells were disseminated into 6-well plates (Costar, USA) at a concentration of 1 × 107 cells/well in 2 mL α-MEM, and incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was changed every other day to remove the non-adherent cells. On the 7th day of culture, 100 U/ml glycerophosphoric acid (Sigma) and 1000 U/ml ascorbic acid (Sigma) were added to the α-MEM to induce the bone marrow cells to differentiate into osteoblasts. The medium was changed every other day. On the 7th day after differentiation, the alkaline phosphatase positive colony forming units-fibroblastic (ALP + CFU-f) were fixed with 10% PFA for 10 min at RT. After washing with PBS, the cells were stained with ALP and Alizarin red dye solution. A camera (Canon, Japan) was used to photograph the staining results.
A portion of the right femur (with the bone marrow removed) of the same weight from each mouse was placed in a grinding tube, and pre-soaked steel beads in DEPC water and TRIzol (Takara, Shiga, Japan) were added. Tissue grinding was performed to extract the total RNA. Subsequently, using a reverse transcriptase cDNA kit (Takara, Shiga, Japan), the total RNA was reverse transcribed into cDNA. The mRNA expression of related factors was evaluated with a Real-Time PCR System (Applied Biosystems 7500, Waltham, MA, USA) by using a SYBR Premix Ex Taq kit (Takara, Shiga, Japan). The primers were synthesised by Sangon Biotech Co., Ltd. (Shanghai, China) (Wnt3a, Forward: 5′-AGGACCCATCTGATTCCCCA-3′ and Reverse: 5′-CTTGTGGCAGATGGGCTG TA-3′, β-catenin, Forward: 5′-AGACAGCTCGTTGTACTGCT-3′ and Reverse: 5′ GTGTCGTGATGGCGTAGAAC-3′, Runx2, Forward: 5′-GTCCTATGACCAGTCTTA CC-3′ and Reverse: 5′-GATGAAATGCCTGGGAACTG-3′, Osx, Forward: 5′-GTTCACCTGTCTGCTCTGCTC-3′ and Reverse: 5′-AGCTCCTTAGGG CCACTTGG-3′, β-actin: Forward: 5′-ACCCAGAAGACTGTGGATGG-3′ and Reverse: 5′- TTCAGCTCAGGGATGACCTT-3′). Each gene was analysed in three repetitions. The method of 2−ΔΔCt was used to calculate the mRNA expression of each gene.
Extraction of total bone tissue protein and the procedures and methods for detecting the cytokines protein expression in bone were performed in accordance with a previous study . The antibodies involved in the experiments were directed against Wnt3a (CST, USA, RRID:AB_2215411), β-catenin (CST, USA, RRID:AB_11127855), Runx2 (CST, USA, RRID:AB_10949892) and Osx (abcam, USA, AB_22552), which were derived from rabbits. The dilution ratio of all of the antibodies was 1:1000. Blots were tested by the Alpha gel imaging system (AlphaImager HP, USA) and Quantity One software (Bio-Rad Inc., USA) was used to determine the grey values. The ratio of the grey values of target proteins to the internal reference is defined as the expression of the target proteins.
The experimental results are presented as the mean ± standard deviation (SD). The data were statistically analysed by SPSS 20.0 (The ZC group and the TC group were subjected to independent sample t tests. One-way analysis of variance was used for the TC, TS and TD groups), P < 0.05 and P < 0.01, respectively, indicated that the experimental results had significant differences.