The purpose of this study was to investigate whether combination of low-intensity exercise with bone marrow stromal cell (BMSC) transplantation could regulate protein kinas B (Akt)-mammalian target of rapamycin (mTOR) and Wnt3a-β-catenin signaling pathways for prevention of soleus muscle atrophy after sciatic nerve injury (SNI). The experimental rats divided into 5 groups (n=10): normal control group, SNI+sedentary group (SED), SNI+low-intensity treadmill exercise group (TEX), SNI+BMSC transplantation group (BMSC), SNI+TEX+BMSC transplantation group (TEX+BMSC). Sciatic nerve crush injury was applied into the middle of thigh twice for 1 min and 30 sec at interval. Low-intensity treadmill exercise was comprised of walking at a speed of 4 to 8 m/min for 30 min once a day. cultured BMSC at a density of 5×106 in 50-μL phosphate-buffered saline was injected into the distal portion of the injured sciatic nerves. TEX+BMSC group dramatically up-regulated expression levels of growth-associated protein-43 in the injured sciatic nerve at 2 weeks postinjury. Also, although Akt and mTOR signaling pathway significantly increased in TEX and BMSC groups than SED group, TEX+BMSC group showed more potent increment on this signaling in soleus muscle after SNI. Lastly, Wnt3a and the nuclear translocation of β-catenin and nuclear factor-kappa B in soleus were increased by SNI, but TEX+BMSC group significantly downregulated activity of this signaling pathway in the nuclear cell lysate of soleus muscle. Present findings provide new information that combination of low-intensity treadmill exercise might be effective therapeutic approach on restriction of soleus muscle atrophy after peripheral nerve injury.
It has been known that peripheral nerve injury, including the sciatic nerve, adversely affects quality of life due to prolonged neuropathic pain and motor dysfunction (
From a molecular biological point of view, SNI-induced muscle atrophy is caused by inhibition of protein kinas B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway and promoting of the Wnt3a-β-catenin cascades (
To accelerate axonal regeneration and functional recovery after SNI, many researchers have widely applied cell transplantation techniques using bone marrow stromal cell (BMSC), neural stem cell, fibroblasts (
With these results presented by previous studies, BMSC transplantation or low-intensity aerobic exercise is a positive effector for improving locomotor functions after SNI. However, experiment studies on combined application of treadmill exercise and BMSC transplantation to examine cellular and molecular mechanisms of muscle atrophy after SNI are still lacking. Therefore, the purpose of this study was to investigate whether combined low-intensity treadmill training with BMSC engraftment could regulate hypertrophy- and atrophy-related signaling pathway in the soleus after SNI.
Male Sprague-Dawley rats (7 weeks old) were purchased for this experiment. The use of rats in this study was approved by Ethical committee of Jeju National University (approval number: 2019-0026). And experimental procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals at Jeju National University. The animals in vivo experiment were divided into five groups with randomization method: the normal control group (CON, n=10), SNI+sedentary group (SED, n=10), SNI+low-intensity treadmill exercise group (TEX, n=10), SNI+ BMSC transplantation group (BMSC, n=10), SNI+TEX+BMSC transplantation group (TEX+BMSC, n=10). Animals were maintained at a constant room temperature of 22°C and 60% of humidity under 12/12-hr light-dark cycle. They were accepted to eat commercial rat chow (Samyang Co., Seoul, Korea) and water ad libitum.
BMSCs were collected from femur and tibia of young rats (4 weeks old), as the extraction technique described by
All experiment animals used anesthetized with using an animal inhalation narcosis control (Jeungdo Bio & Plant, Seoul, Korea). First, the rats were placed into the chamber with 2%–2.5% concentration of isoflurane for anesthesia and then 1.5%–1.8% concentration for maintenance during SNI. Sciatic nerve crush injury was applied into the middle of thigh twice for 1 min and 30 sec at interval (
All rats in this experiment had a treadmill exercise adaptation period for 2 weeks. All animals in exercise groups were rested for 2 days after SNI, and started low-intensity treadmill exercise on third postoperation day. Low-intensity exercise on the treadmill device (Jeungdo Bio & Plant) was comprised of walking at a speed of 4 to 8 m/min for 30 min once a day for 2 weeks postinjury.
Protein lysates were extracted from the dissected sciatic nerve tissues into triton lysis buffer, and the nucleus and cytoplasm were separated by nuclear extraction buffer and cytosol extraction buffer. Denatured proteins were separated on sodium dodecyl sulphate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane on ice at 200 mA for 2 hr. The membranes were blocked with 5% skim milk, 0.1% Tween 20 in tris buffered saline for 30 min at room temperature. Then, the membranes were incubated overnight with primary antibodies at 4°C. Protein (20 μg) was used for Western blot analysis using anti-GAP-43 mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated mTOR rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology, Danvers, MA, USA), anti-phosphorylated Akt rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-Wnt3a rabbit monoclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-β-catenin mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-NF-κB rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-β-actin mouse monoclonal antibody (1:2,000, Santa Cruz Biotechnology), anti-GAPDH rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), and goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1,000, GeneTex Inc., Irvine, CA, USA) were used. The blotting proteins were detected by using Westar ECL substrates (Cyanagen, Bologna, Italy). Analysis of protein density was performed using Chemidoc (Bio-Rad, Hercules, CA, USA).
For detection of sprouting axons in the injured sciatic nerve, tissues were embedded and frozen at 20°C. Cross sections (20 μm) were cut on a cryostat. Sections were fixed with 4% paraformaldehyde and 4% sucrose in PBS at room temperature for 40 min, permeabilized with 0.5% Nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 hr at room temperature. The sections were incubated with anti-neurofilament-200 (NF-200) rabbit polyclonal antibody (1:700) (Sigma-Aldrich, St. Louis, MO, USA). And then, they were incubated with rhodamine-goat anti-rabbit secondary antibody (1:600) (Molecular Probes, Eugene, OR, USA) for 1h at room temperature. The stained samples were viewed with a fluorescence microscope (Nikon model E-600, Nikon, Kawasaki, Japan), and the images were captured with a digital camera, and analyzed using Adobe Photoshop Software (version CS6, San Jose, CA, USA). The number and regenerating axons were evaluated by using i-Solution software (Image and Microscope Technology, Goleta, CA, USA).
All the data is presented as a mean±standard error. Statistical analysis was performed using one-way analysis of variane followed by Duncan
To confirm the effect of combined low-intensity exercise with BMSC transplantation on expression levels of GAP-43, specific regeneration marker, in the sciatic nerve at 2 weeks after SNI, we performed biochemical and histological analysis. As shown in
To examine activation of Akt-mTOR signaling pathway in the soleus at 2 weeks after SNI, we performed Western blot analysis using anti-Akt and mTOR antibodies. As shown in
To examine activation of the nuclear translocation of β-catenin in soleus after SNI, we analyzed induction levels of β-catenin from the whole cell or nuclear cell lysate. As shown in
NF-κB is a key molecule of inflammation during development and tissue regeneration. To confirm translocation of NF-κB to the nucleus in soleus muscle after SNI, we investigated alterations of NF-κB from the whole cell or nuclear cell lysate. As shown in
Various previous studies that applied regular exercise and cell transplantation for sciatic nerve regeneration mainly focused on axonal regrowth in the nerves and pain improvement in dorsal root ganglions, but these results do not provide an appropriate mechanism on exercise-induced skeletal muscle changes for functional recovery after SNI. Therefore, we tried to find the effective molecular mechanisms to suppress atrophy of soleus muscle after SNI.
The injured peripheral nerves undergo Wallerian degeneration, which is morphological and biochemical changes in the injured axons. Regeneration process includes Schwann cell proliferation, migration, and differentiation as well as axonal sprouting and remyelination around the injured areas (
SNI leads to skeletal muscle atrophy and functional dysfunction (
In addition to elucidating the role of canonical Wnt/β-catenin signaling pathway in muscle atrophy, we identified that TEX+ BMSC group sharply downregulated NF-κB levels in soleus at 2 weeks postinjury compared to those in other groups. Skeletal muscle atrophy can result from enhanced protein degradation and downregulated protein synthesis in immobilization (
Given these results obtained in present study, low-intensity treadmill exercise and BMSC transplantation approaches would positively regulate biochemical mechanisms in nerve and soleus muscle during sciatic nerve regeneration. In conclusion, our findings suggest that combination of low-intensity treadmill exercise with BMSC transplantation should provide may be effective therapeutic approach to restrict soleus muscle atrophy after peripheral nerve injury.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1F1A1062392).
No potential conflict of interest relevant to this article was reported.
Combination of low-intensity treadmill exercise with bone marrow stromal cell (BMSC) transplantation regulates expression levels of growth-associated protein-43 (GAP-43) and number of neurofilament-200 (NF-200)-stained axons after sciatic nerve injury (SNI). (A) TEX and TEX+BMSC groups significantly increased GAP-43 induction levels in whole cell lysate of the sciatic nerve 2 weeks after SNI. Upper panel: representative expression of GAP-43 protein. Lower panel: the quantification of the ratio of GAP-43 to actin. (B) To investigate regenerating axons in the injured sciatic nerve, immunofluorescence staining was applied into 10 mm distal region to the injury site 2 weeks after SNI. TEX, BMSC, and TEX+BMSC groups showed a significant increase in the number of NF-200-stained regenerating axons when compared to other groups. Upper panel: immunofluorescence staining of NF-200 antibody. Lower panel: the quantification of the intensity of NF-200-positive axons. wpc, week post crush; CON, normal control group; SED, SNI+sedentary group; TEX, SNI+low-intensity treadmill exercise group; BMSC, SNI+BMSC transplantation group; TEX+BMSC, SNI+TEX+BMSC transplantation group. *
Combination of low-intensity treadmill exercise with bone marrow stromal cell (BMSC) transplantation regulates muscle hypertrophy-related signaling pathway after sciatic nerve injury (SNI). Phosphorylated-protein kinase B (p-Akt) protein was significantly increase in soleus muscle of TEX, BMSC, and TEX+BMSC groups. Phosphorylated-mammalian target of rapamycin (p-mTOR) was further enhanced in BMSC and TEX+BMSC groups than SED group. In particular, both p-Akt and mTOR were dramatically upregulated in TEX+BMSC group. Upper panel: representative expression of p-Akt and mTOR proteins. Lower left panel: the quantification of the ratio of p-Akt to actin. Lower right panel: the quantification of the ratio of p-mTOR to actin. wpc, week post crush; CON, normal control; SED, SNI+sedentary group; TEX, SNI+low-intensity treadmill exercise group; BMSC, SNI+BMSC transplantation group; TEX+BMSC, SNI+TEX+BMSC transplantation group. *
Combination of low-intensity treadmill exercise with bone marrow stromal cell (BMSC) transplantation regulates canonical Wnt3a-β-catenin signaling pathway in soleus muscle after sciatic nerve injury (SNI). In whole cell lysate, Wnt3a was more decreased in soleus of TEX and TEX+BMSC groups than SED group, but applying combination of treadmill exercise and BMSC transplantation sharply downregulated Wnt3a expression levels in soleus compared to TEX group. In nuclear cell lysate, translocation of β-catenin to the nucleus of soleus muscle after SNI was significantly decreased in TEX, BMSC, and TEX+BMSC groups. Upper left panel: representative expression of Wnt3a in whole cell lysate. Upper right panel: representative expression of β-catenin in nuclear cell lysate. Lower left panel: the quantification of the ratio of Wnt3a to actin. Lower right panel: the quantification of the ratio of β-catenin to actin. wpc, week post crush; CON, normal control; SED, SNI+ sedentary group; TEX, SNI+low-intensity treadmill exercise group; BMSC, SNI+BMSC transplantation group; TEX+BMSC, SNI+TEX+BMSC transplantation group. *
Combination of low-intensity treadmill exercise with bone marrow stromal cell (BMSC) transplantation regulates expression of nuclear factor-kappa B (NF-κB), a specific inflammation marker, in soleus muscle after sciatic nerve injury (SNI). In whole cell lysate, NF-κB was more increased in soleus of SED group than CON group, but TEX BMSC and TEX+BMSC groups significantly downregulated NF-κB levels in soleus at 2 weeks postinjury. NF-κB in the nucleus of soleus postinjury was significantly decreased in TEX and BMSC. In particular, TEX+BMSC groups showed the greatest downregulation of NF-κB in both whole and nuclear cell lysates. Upper panel: representative expression of NF-κB protein. Lower left panel: the quantification of the ratio of NF-κB to actin in whole cell lysate. Lower middle panel: the quantification of the ratio of NF-κB to actin in cytoplasm. Lower right panel: the quantification of the ratio of NF-κB to actin in nucleus. wpc, week post crush; CON, normal control; SED, SNI+sedentary group; TEX, SNI+low-intensity treadmill exercise group; BMSC, SNI+BMSC transplantation group; TEX+BMSC, SNI+TEX+ BMSC transplantation group. *