Downhill Running Increases Bone Mass Accumulation and Bone Tissues Morphology through the TGF-β/Smad Pathway in the Bone of Growing Mice

doi:10.21203/rs.2.21640/v2

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Downhill Running Increases Bone Mass Accumulation and Bone Tissues Morphology through the TGF-β/Smad Pathway in the Bone of Growing Mice

https://doi.org/10.21203/rs.2.21640/v2

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Background: Substantial evidence has demonstrated that impact exercise can help adolescents to accumulate more bone mass. However, the effect of OB (osteoblast) on promoting bone formation during impact exercise remains to be explored. This study aimed to examine the mechanism for downhill running promoting bone formation ability and bone mineralization ability by activating the TGF-β (transforming growth factor-β)/Smad pathway in growing mice.

Methods: Four-week-old C57BL/6 female mice (n = 28) were randomly divided into exercise and control groups. Mice in the exercise group received a forty-minute downhill training on a descending treadmill (descent 9^o) every day for eight weeks. After eight weeks, BMSCs (bone marrow mesenchymal stem cells) were collected and cultured for assessment of osteoblastic differentiation and the capacity of osteoblastic mineralization. We determined the protein and mRNA expression of TGF-β, Smad2/3/4, and Runx2 (runt-related transcription factor 2) in bone tissues. The BMD (bone mineral density) and histomorphological changes were also examined.

Results: The study showed that the eight-week downhill training significantly increased BMD and ALP (alkaline phosphatase-positive) activity in the tibial epiphysis. Also, downhill running promoted BV/TV (bone volume fraction), Tb.N (trabecular number), and Tb.Th (trabecular thickness), while it decreased Tb.Sp (trabecular separation). Additionally, the numbers of ALP+CFU-f (colony forming units-fibroblastic) cells and the area of mineralized nodule formation were significantly higher in the exercise group compared with that of the control group. Lastly, the mRNA expression of TGF-β, Smad2/3/4, and Runx2 was significantly elevated in the exercise group, and the protein expression of p-Smad2/3/4, and Runx2 was also increased.

Conclusions: The findings indicated that downhill running enhanced bone mass accumulation and bone tissue morphology in growing mice by activating the TGF-β/Smad pathway.

exercise

osteoblast differentiation

bone formation

TGF-β

As the essential part of the human, bones have various functions, such as support, protection, endocrine, etc (1-2). Its growth period is the second peak of bone growth and development, and it is also a sensitive period that affects the peak bone mass. 50% of the peak bone mass will accumulate during growth (3). Exercise-induced mechanical loading plays a vital role in promoting bone formation and improving bone tissue histomorphology and bone mineral density (BMD) during the growth period (4). Several studies have verified the effects of exercise on bone formation, such as treadmill running (5), jumping (6), and swimming (7). Downhill running is an eccentric exercise in which the bone tissues will be subjected to great ground reaction force and muscle tension. Studies found that downhill running produced a better effect on promoting the tibial cross-sectional dimensions formation, tissue-level mechanical properties (8), bone strength, BMD, and bone mineral content (BMC) over the whole body (9).

Exercise improves bone formation and promotes osteoblast (OB) differentiation. OB is mainly differentiated by bone marrow mesenchymal stem cells (BMSCs) and pluripotent differentiated stem cells, as their differentiation and function are the basis of bone formation and growth during the growth period (10). The study of Okuma KF et al. (11) found that climbing exercise promoted OB differentiation, significantly increased the alkaline phosphatase-positive (ALP) positive colony forming units-fibroblastic (ALP+CFU-f) number, and offset the effect of acetaldehyde dehydrogenase 2 (Aldh2) knockout on OB differentiation. Irisin, an essential skeletal muscle actin, enhanced osteoblastogenesis and increased ALP+CFU-f numbers in mice during three weeks of free flight (12). The above studies have shown that exercise promotes OB differentiation, but the molecular mechanism remains unclear.

As a key factor in bone, transforming growth factor-β (TGF-β) enhances OB differentiation and promotes bone development and remodeling (13-14). When the TGF-β is activated, it can transmit signals to the plasma membrane through specific type I and II receptors (15). The type I receptor is activated following the phosphorylation of type II. Type I receptors will phosphorylate Smad 2 and Smad 3 protein (16). Activated Smad 2/3 forms a complex process that combines with Smad 4, and then translocates into nucleus to upregulated runt-related transcription factor 2 (Runx2). Runx2 is key in regulating OB differentiation and osteogenic capacity (17). ALP activity increases when the osteogenic capacity of OB is increased, which promotes the deposition of minerals such as calcium and phosphatase in bones, and improves bone tissue histomorphology and BMD (18). A study reported that TGF-β was more sensitive to the mitotic effect of OB in fetal bone than in other stages. TGF-β promoted the differentiation and proliferation of osteogenic progenitor cells by activating the Smad2/3 pathway in bone (19). Moreover, TGF-β maintains the metabolic balance of OB/osteoclast (OC) in the process of bone remodeling and plays an important part in regulating the bone morphology and mineralization through the synthesis of type Ⅰ collagen, adhesive protein, and osteopontin (20).

The process of exercise promoting OB differentiation and bone formation is regulated by many signaling pathways or key factors, such as mitogen-activated protein kinase (MAPK) (21), Wingless (Wnt)/β-catenin (22), bone morphogenetic proteins (BMPs) and endocrine (23). Although previous studies have found that downhill running promoted bone turnover in rats by improving bone mass, bone histomorphometric indicators, and biomechanical properties (24-25), its effect on OB differentiation and osteogenic capacity remains unknown. However, there is no report that downhill running promotes the differentiation of OB and its osteogenic capacity mediated by TGF-β/Smad pathway in vivo. As mice were considered to be important animal models for exploring significant changes in bone tissue at the cellular and molecular levels (26), this research used the downhill running model to investigate the role of the TGF-β/Smad pathway in promoting OB differentiation and bone formation.

2.1 Experimental Animals and Training Protocol

Twenty-eight C57BL/6 mice (female, 4-week-old, mean ± SD: 15.1 ± 1.9 g) were purchased (B&K Laboratory Animal Company, Shanghai, China). All mice were housed in cages under constant temperature and humidity, fed with standard diet, free water, day/night ratio 12:12. The weight of mice was measured weekly. The study was performed following the guidelines of the Yangzhou University Laboratory Animal Care and Use Committee (license number: YZU-TYXY-31).

After one week of acclimatization, mice were randomly divided into the exercise group and control group, with 14 mice in each group. Downhill running was carried out on a 9^o downhill treadmill (KW-PT, Calvin Biotechnology, Nanjing, China) at a speed of 0.8 Km/h, six days a week. During the first week, mice were trained for 20 min per day for the first two days, and then the training time was increased by 5 min per day until 40 min per day. The exercise intervention lasted for eight weeks.

2.2 BMD

After exercise intervention, we took the left forelimb bones of mice and removed the soft tissues attached to the bone. After 24 h of paraformaldehyde (PFA; V900894, Sigma-Aldrich, Darmstadt, Germany) fixation, the left forelimb was kept in 75% ethyl alcohol at 4 °C until the BMD was measured. BMD at the middle segment of the left radius was measured by Dual-emission X-ray Absorptiometry (DXA; Discovery Wi, Hologic Discovery A, Boston, Massachusetts, USA) (27).

2.3 ALP Activity

After the tibias were fixed with 10% PFA for 12 h, the tibias were treated with 10% ethylenediamine tetraacetic acid (EDTA; E1161, Sigma-Aldrich, Darmstadt, Germany) for 2 weeks of decalcified, followed by alcohol gradient dehydration (70%, 80%, 95%, and 100%), xylene transparent, wax immersion, embedding. Tibia samples were frozen using optical coherence tomography (OCT). Cut a 6-μm section from the frozen samples and choose one for staining from every 4–6 sections. ALP activity in the tibial epiphysis was determined using dye solutions (0.03 g Fast Violet LB salt (F3380, Sigma-Aldrich, Darmstadt, Germany), 0.09 g AS-MX (N4875, Sigma-Aldrich, Darmstadt, Germany)). Dye for one hour. Photograph each section under a microscope (20× magnification).

2.4 Histomorphometry

The right tibias were fixed with 10% PFA, dehydrated with gradient alcohol (75%, 85%, 95%, and 100%), and embedded in methyl methacrylate resin (MMA; M55909, Sigma-Aldrich, Darmstadt, Germany). We cut a 12-μm section from the tibia samples with a sliding microtome (SM2010R, Leica, Mannheim, Baden-Württemberg, Germany). The sections were wiped off and stained using MMA with 1-Acetoxy-2-methoxyethane (AME; 109886, Sigma-Aldrich, Darmstadt, Germany) and did Von kossa/van gieson staining, respectively. Under the electron microscope (DM400, Leica, Mannheim, Baden-Württemberg, Germany), we used the bone measurement software (InSyTe CT, Trifoil Imaging, Chatsworth, California, USA) to detect the bone volume fraction (BV/TV, %), bone number (Tb.N, N/mm), trabecular thickness (Tb.Th, μm) and trabecular separation (Tb.Sp, μm) (28).

2.5 Bone Marrow Stromal Cell Culture

Six mice in each group were prepared for the primary culture of cells. Under sterile conditions, took the femur and tibia of mice. Bone marrow was removed from the bone cavities of the femur and tibia to make single-cell suspension. After counting, according to the concentration standard of 1 × 10⁷ cells/well, the single-cell suspension was put into the 6-well plate and 2 mL α-MEM (α-Minimum Essential Medium) (12561056, GIBCO, Grand Island, State of New York, USA) with 10% fetal calf serum (FBS; 10270-106, GIBCO, Grand Island, State of New York, USA) and 1000 U/mL ciprofloxacin (17850, Sigma-Aldrich, Darmstadt, Germany). The 6-well plates were placed in cell incubator (5% CO₂, 37 ℃). The medium was changed every other day, after seven days of culture, the α-MEM added 100 U/mL glycerophosphoric acid (61688, Sigma-Aldrcih, Darmstadt, Germany) and 1000 U/mL ascorbic acid (A92902, Sigma-Aldrich, Darmstadt, Germany) were used to induce the bone marrow cells differentiate into OB. The medium was changed every 2 days. On day 7, since differentiation began, the ALP+CFU-f was fixed with 10% PFA for 10 min at RT. After washing with phosphate-buffered saline (PBS; P4474, Sigma-Aldrich, Darmstadt, German), cells were stained with 10 mL dye solution (0.03 g Fast Red Violet LB Salt (F3380, Sigma-Aldrich, Darmstadt, Germany), 0.09 g AS-MX (N4875, Sigma-Aldrich, Darmstadt, Germany)) for 20 min in the dark place (29).

2.6 Mineralized Nodule Formation of OB

The method of preparing single-cell suspension is consistent with the ALP staining of OB. The erythrocytes were lysed using an erythrocyte lysis solution and counted using a hematocrit plate. Single-cell suspension were disseminated into 6-well plates according to the standard of 1 × 10⁷cells in 2 mL α-MEM and placed in cell incubator (5% CO₂, 37 ℃), which was the same as supplemented in the CFU-f assay. Change the solution once every two days. On 21-day culture, cells were fixed with 10% PFA for 10 min and washed with deionized water. The cells were then stained with 4% silver nitrate (204390, Sigma-Aldrich, Darmstadt, Germany) and irradiated with ultraviolet for 20 min. A Canon digital camera was used to photograph the surface of each well covered with dark stained parts, and the Image J software (X64, National Institutes of Health, Bethesda, MD, USA) was used to calculate the mineralized nodule area (30-32).

2.7 Western Blot Analysis

50 mg bone tissue was weighed, and protein extract was added for grinding. The bone tissue protein extracted from all mice was placed on ice, and the supernatant was taken after centrifugation. The protein content was determined by the BCA (bicinchoninic acid) method. The protein concentration was adjusted to the same concentration with PBS, the same Loading buffer was added, and denatured at 95 ℃ for 5 min (preparation of separation glue and concentrated glue at the same time). Then mix well and centrifuge. Add protein sample and Marker sample, run glue at 120 V, and transfer film at 100 V. After finishing, the film was placed in 0.2% Lichun red dye solution for 50 s and washed with ddH₂O. PVDF membrane was cut into suitable strips according to Marker strips and cleaned with TBST buffer solution. The strips were then placed in 5% skim milk powder for 1–2 h (33 following incubation with the antibodies. The related antibodies included p-Smad2 (mAb#18338, CST, Danvers, Massachusetts, USA), p-Smad3 (mAb#9520, CST, Danvers, Massachusetts, USA), p-Smad4 (PA5-64712, Thermo Fisher, Waltham, MA, USA), and Runx2 (mAb#12556, CST, Danvers, Massachusetts, USA) in the experiments, both derived from rabbits. All antibodies were diluted according to 1:1000 standard. The Alpha gel imaging system (AlphaImager HP, Protein Simple, silicon Valley, California, USA) was used to test blots. We determined the gray values using Quantity One software (1709600, Bio-Rad, Hercules, California, USA). The expression of the target gene was obtained by calculating the ratio of the gray value of the target gene to the internal reference.

2.8 RT-PCR Analysis

RT-PCR analysis was performed at one step using the One-Step RT-PCR kit (639503, Takara, Beijing, China) to determine the mRNA expression levels of TGF-β, Smad2/3/4, and Runx2. Total RNA was extracted from the treated bone tissues by using Trizol agent (T9424, Sigma-Aldrich, Darmstadt, USA). Its purity and concentration were measured using an ultra-micro spectrophotometer (ND-2000/NanoDrop, Thermo Fisher, Waltham, MA, USA) at 260/280 nm wavelength. cDNA was obtained by using the reverse transcription kit (639506, TakaRa, Beijing, China), amplified through quantitative PCR kit (RR240A, TakaRa, Beijing, China), and the conducted reaction conditions were denaturation at 94°C for 4min, 94°C for 10 min, 55 °C for 30 s, 72 °C for 30 s, 94 °C for 30 s, 55 °C for 30 s, 94 °C for 30 s, for forty-five cycles. The primers were synthesized by Sangon Biotech Co., Ltd. (Primer Premier 5, Canada, Shenggong Biotechnology (Shanghai), Shanghai, China) (TGF-β, Forward: 5′-AGGGCTACCAGCCAACTTC-3′ and Reverse: 5′CCACGTAGTAGACGA TGGGC-3′, Smad2, Forward: 5′-CTGTGACGCATGGAAGGTCT-3′ and Reverse: 5′-CCACGTAGTAGACGATGGG C-3′, Smad3, Forward: 5′-CAGCGAGTTGGGGAGACATT-3′ and Reverse: 5′-TGTAAGTTCCACGGCTGCAT-3′, Smad4, Forward: 5′-CGATGGAATTTTACATACG-3′ and Reverse: 5′-CGCCTAAGTTGCTAGGTGG-3′, Runx2, Forward: 5′-GTCCTATGACCAGTCTTACC-3′ and Reverse: 5′-GATGAAATGCCTGGGAACTG-3′, β-actin: Forward: 5′-ACCCAGAAGACTGTGGATGG-3′ and Reverse: 5′-TTCAGCTCAGGGATGACCTT-3′). Each gene was performed for three repetitions. The 2^−ΔΔCt was used to analyze the specific mRNA amplification of the target gene.

2.9 Statistical Analysis

The way all the data is presented: Mean ± SD. The experimental data was analyzed by SPSS 20.0 (IBM-SPSS Statistics, Chicago, IL, USA), and the independent sample T-test was used to compare the variables' differences. The level of statistical significance was set at p < 0.05.

3.1 Downhill Running Improves ALP Activity and BMD in Growing Mice

The present study designed an animal model to elucidate the effects of exercise on the changes in BMD and the activity of ALP in bone tissues. After an 8-week downhill running exercise, the formation of OB in bone tissues was enhanced; Compared with control group, the ALP activity of epiphyte was obviously increased in exercise group (Fig. 1/A & B). BMD in exercise group was obviously higher than control group (p < 0.001) (Fig. 1/C).

3.2 Downhill Running Improves Bone Morphology in Growing Mice

We further examined the histomorphological changes in bone tissue to prove the promotion effect of downhill running. The results have shown that BV/TV (p < 0.05), Tb. N (p < 0.05) and Tb. Th (p < 0.01) were significantly higher (Fig. 2/A, B & C), while Tb. Sp (p < 0.01) in exercise group was lower than that of the control group (Fig. 2/D), suggesting that downhill running can significantly increase bone morphological structure.

3.3 Downhill Running Promotes OB Differentiation and Mineralization in Growing Mice

We collected bone marrow stromal cells and cultured them in α-MEM. On the 14th day, we stained ALP+CFU-f cells and observed the numbers. Downhill running increased ALP+CFU-f cells (p < 0.01) evidently compared with the control group (Fig. 3/A, B & E). We further examined whether those increased ALP+CFU-f cells have an enhanced capacity for mineralization. Findings demonstrated the mineralized areas were evidently more in exercise group (p < 0.05) compared with control group (Fig. 3/C, D & F). It is shown that downhill running could promote OB formation and mineralization capacity in growing mice.

3.4 Downhill Running Increases Protein Expression and mRNA Expression of the TGF-β/Smad Pathway in Growing Mice

We determined the protein and mRNA expression of TGF-β/Smad pathway related factors in bone tissues to determine the potential mechanism that regulates the impact of exercise on bone formation. Western blot analysis indicated that the expression of p-Smad2 (p < 0.01), p-Smad3 (p < 0.01), p-Smad4 (p < 0.01), and Runx2 (p < 0.01) were higher in the exercise group compared with the control group (Fig. 4). The mRNA levels of TGF-β (p < 0.05), Smad2 (p < 0.05), Smad3 (p < 0.05), Smad4 (p < 0.01) and Runx2 (p < 0.05) were also evidently up-regulated in the exercise group (Fig. 5).

In this study, we found that the 8-week downhill training promoted bone formation in growing mice by activating TGF-β/Smad pathway, significantly improving bone morphological structure indexes and increasing BMD.

The growth period is critical for bone growth, and the accumulation of bone mass during this period determines the amount of peak bone mass, which plays an important role in preventing osteoporosis in the later stage (34). Exercise improves bone metabolism and promotes bone formation in growing mice (34-35). Our study found the indexes of bone histomorphometry in the exercise group were evidently improved. The BMD value at the proximal tibia was also evidently increased. A study found that proper treadmill exercise could significantly promote bone formation and improve the morphology and structure of trabecular bone during growth, as well as the tissue morphology and biomechanical properties of cortical bone (34). Compared to treadmill exercise, downhill running can significantly increase femur bone density in growing rats (25). A recent study found that downhill running promoted bone formation ability and differentiation into chondrocytes of OB in rats and mice (36). In addition, the effect of downhill running on bone formation capacity was more obvious when comparing the impact of uphill running and downhill running on improving bone index (the ratio of bone formation index to bone resorption index) in the previous study (37). To analyze the mechanism based on which downhill running may better promote the increase in BMD, the bone of growing mice was more sensitive to mechanical stimulation during exercise than the bone tissues of the adult and aged mice (38). The mechanical stimulation from downhill running promoted bone formation, which improved bone tissue microstructure and increased BMD.

Exercise promotes bone formation and growth in mice by increasing OB numbers and enhancing osteogenic capacity; however, its molecular regulation mechanism is still unclear. As the key signaling pathway regulating OB differentiation and bone formation, the TGF-β/Smads pathway regulates the differentiation of BMSCs into OB through its downstream target gene Runx2 (39). However, there are studies on the TGF-β/Smad pathway regulating downhill running to promote OB differentiation and bone formation in growing mice. Our study showed that the exercise group's mRNA expression of TGF-β, Smad2/3/4 and Runx2 significantly increased. In addition, the protein expression of p-Smad2/3/4 and Runx2 were also upregulated. These research results showed that downhill running could significantly activate the TGF-β/Smad pathway. After this pathway is activated, TGF-β promoted phosphorylation of downstream Smad2/3/4, which forms a phosphorylation group and rapidly transfers into the nucleus, upregulating the target's expression gene Runx2 and promotes the differentiation of BMSCs into OB (40-41). We found that exercise significantly upregulated the TGF-β expression and activated Runx2 and Osx through the Smads pathway, then increased activity of ALP to promote differentiation of BMSCs into OB, and the effect of downhill running exercise was more evident than other exercises (42-43). An increasing number of OB enhances osteogenic capacity and promotes ALP secretion and activity.

Moreover, we found that the ALP activity in bone was significantly increased, which promoted the deposition of inorganic substances such as calcium and phosphorus in bone and improved bone tissue morphology and BMD (44). In this study, exercise promotes bone formation in growing mice. The ‘puberty’ period is critical for human skeletal growth and development, during which more exercises (such as stairs, jumping, etc.) can significantly promote adolescent growth.

Our study has established that the 8-week downhill running promoted bone formation through up-regulating OB differentiation and mineralization in growing mice. This study also indicated that the TGF-β/Smad pathway partly regulates the exercise-associated benefits on bone.

6. Availability of Data and Materials

The data used to support the finding of this study are available from the corresponding author upon request.

7. Author Contributions

XHC and RQZ—conceptualization; PS and AGC—methodology; WHC, HXP and XLZ—investigation; XHC—resources; XYZ, KY and HLY—data curation; XHC, PS, HXP and HLY—writing original draft preparation; XHC, AGC, HXP and KY—writing review and editing; XHC and RQZ—supervision; KY and HLY—project administration. All authors contributed to editorials changes in the manuscripts. All authors have read and approved the final manuscript.

8. Ethics Approval and Consent to Participate

This study was approved by The Institutional Animal Care and Use Committee of Yangzhou University (YZU-TYXY-31).

9. Acknowledgment

Not applicable.

10. Funding

This study was funded by grants received by Xianghe Chen from The China Postdoctoral Science Foundation (2019M661957), The Special grants from China Postdoctoral Science Foundation (2021T140580), 2020 Yangzhou University “High-end Talent Support Program” and 2021 Yang-zhou University “Qinglan Project”.

11. Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Downhill Running Increases Bone Mass Accumulation and Bone Tissues Morphology through the TGF-β/Smad Pathway in the Bone of Growing Mice

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Abstract

Figures

Introduction

Materials And Methods

2.1 Experimental Animals and Training Protocol

2.2 BMD

2.3 ALP Activity

2.4 Histomorphometry

2.5 Bone Marrow Stromal Cell Culture

2.6 Mineralized Nodule Formation of OB

2.7 Western Blot Analysis

2.8 RT-PCR Analysis

2.9 Statistical Analysis

Results

3.1 Downhill Running Improves ALP Activity and BMD in Growing Mice

3.2 Downhill Running Improves Bone Morphology in Growing Mice

3.3 Downhill Running Promotes OB Differentiation and Mineralization in Growing Mice

3.4 Downhill Running Increases Protein Expression and mRNA Expression of the TGF-β/Smad Pathway in Growing Mice

Discussion

Conclusions

Declarations

References

Supplementary Files

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