The purpose of this study was to investigate the time-dependent alteration in whether concurrent aerobic exercise and bone marrow stromal cell (BMSC) engraftment could regulate myogenic differentiation-related signaling pathway in the soleus up to 35 days after sciatic nerve injury (SNI). The rats were divided as follows: the normal control (CON, n=5), sedentary group (SED, n=20), treadmill exercise group (TEX, n=20), BMSC transplantation group (BMSC, n=20), TEX+BMSC transplantation group (TEX+BMSC, n=20) 7, 14, 21, and 35 days after SNI. SNI was applied into the thigh and treadmill exercise was comprised of walking at a speed of 4 to 8 m/min for 30 min once a day. Harvested BMSC at a density of 5×106 in 50-μL phosphate-buff-ered saline was injected into the injury site. Phosphorylated (p) extracellular signal-regulated kinase 1/2 expression was dramatically upregulated in BMSC and BMSC+EX groups from 21 days after SNI compared to those in the SED group. P-ribosomal s6 kinase (RSK) was sharply increased 14 days later, and then rapidly downregulated from day 21, whereas TEX, BMSC and TEX+ BMSC groups significantly kept up expression levels of p-RSK until 35 days post injury than SED group. TEX+BMSC group significantly increased activation of protein kinase B-mammalian target of rapamycin in the soleus from day 14 and myoblast determination protein 1-myogen-in pathways was activated in TEX+BMSC group from day 21. Present findings provide information that combined intervention of aerobic exercise and BMSC transplantation might be a reliable therapeutic strategy for overcoming the morphological and functional problems in denervated soleus muscle.
The sciatic nerve, which belongs to the peripheral nervous system capable of spontaneous regeneration, is damaged by femoral dislocation and fracture, or by muscle contusion and pressure in sports situations (
Many previous studies on sciatic nerve regeneration have focused on axonal growth through the facilitation of Schwann cell proliferation and differentiation in the distal part of the injured area (
The sciatic nerve indirectly innervates all the skeletal muscles of the lower limbs, and patients with SNI experience limited physical activity due to pain and rapid muscle atrophy (
Among the MRF transcription factors, MyoD and myogenin are muscle-specific proteins involved in muscle hyperplasia and hypertrophy (
With these results presented by previous studies, both signaling pathways are key indicator for verifying muscle atrophy after SNI, and it is thought that exercise can improve muscle metabolism via activating these signaling downstream molecules. However, there is still a lack of studies observing the effects of combined low-volume exercise and BMSC transplantation on biochemical changes from the injured sciatic nerve to the soleus muscle after SNI, and on time-dependent changes in muscle hypertrophy-related proteins for a long time after injury. Therefore, the purpose of this study was to investigate the time-dependent alteration in whether concurrent low-intensity aerobic exercise and BMSC engraftment could regulate myogenic differentiation-related signaling pathway in the soleus up to 35 days after SNI.
Eight-five Sprague-Dawley rats (5 weeks old, male) were used for this experiment and they were divided with randomization method as follows: the normal control (CON, n=5), sedentary group (SED, n=20), low-intensity treadmill exercise group (TEX, n=20), BMSC transplantation group (BMSC, n=20), TEX+ BMSC transplantation group (TEX+BMSC, n=20) 7, 14, 21, and 35 days after SNI. Animals were housed in animal facility under 12/12-hr light-dark regime at 22°C and 60% of humidity, and fed commercial rat chow (Samyang Co., Seoul, Korea) and water ad libitum. The use of rats in this study was approved by Ethical committee of Jeju National University (approval number: 2019–0026). And animal experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals at Jeju National University.
As described in study by
The surgical methods were fundamentally the same as those described in our previous paper (
All rats in this experiment had an adaptation period to treadmill exercise for 2 weeks. Animals in the exercise groups were rested for 2 days after SNI, and they 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 from 7 days to 35 days 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 antiphosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiphosphorylated p90 ribosomal s6 kinase (RSK) rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology, Danvers, MA, USA), antiphosphorylated mTOR rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), antiphosphorylated protein kinase B (Akt) rabbit polyclonal antibody (1:1,000, Cell Signaling Biotechnology), anti-MyoD mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), antimyogenin mouse monoclonal antibody (1:1,000, Santa Cruz 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).
All the data is presented as a mean±standard error. Statistical analysis was performed using one-way analysis of variance followed by Duncan
To confirm the time-dependent effect of combined low-intensity treadmill exercise and BMSC engraftment on expression levels of p-ERK1/2 and p-RSK, proliferative markers of Schwann cell, in the injured sciatic nerve, we performed biochemical experiment using Western blotting techniques. As shown in
To examine the time-dependent metabolic changes of hypertrophy in soleus muscle after SNI, we confirmed activation of Akt-mTOR molecular cascade using Western blotting. As shown in
To identify the time-dependent metabolic changes in myogenic differentiation of reinnervated soleus muscle, we confirmed phosphorylation of MyoD and myogenin proteins using Western blotting. As shown in
Immediately after SNI, Schwann cells undergo demyelination and dedifferentiation along with recruited macrophages and Wallerian degeneration is progressed. Over time, the proliferation of Schwann cells around the injury site becomes active, which is a starting signal for axonal regeneration of the injured sciatic nerve (
Damage of sciatic nerve that control skeletal muscles of the lower limbs is closely related with loss of muscle mass (atrophy) and this muscle atrophy results in behavioral dysfunction due to reduction of strength, endurance and energy metabolism in skeletal muscle (
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.
Combined application of treadmill exercise and bone marrow stromal cell (BMSC) transplantation regulates time-dependent changes of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) and p-ribosomal s6 kinase (p-RSK) in the injured sciatic nerve. (A) Results of Western blot analysis for quantitative analysis of p-ERK and p-RSK in the injured sciatic nerve. (B) The statistical graph on ratio of p-ERK1/2 against actin at 7, 14, 21, and 35 days after sciatic nerve injury (SNI). (C) The statistical graph on ratio of p-RSK against actin at 7, 14, 21, and 35 days after SNI. Actin is an internal loading control. CON, normal control; SED, sedentary group; TEX, low-intensity treadmill exercise group; BMSC, BMSC transplantation group; TEX+BMSC, TEX+BMSC transplantation group. *
Combined application of treadmill exercise and bone marrow stromal cell (BMSC) transplantation controls time-dependent changes of protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling cascade in the denervated soleus muscle. (A) Results of Western blot analysis for quantitative analysis of phosphorylated (p)-AKT and p-mTOR in the denervated soleus muscle. (B) The statistical graph on ratio of p-AKT against actin at 7, 14, 21, and 35 days after sciatic nerve injury (SNI). (C) The statistical graph on ratio of p-mTOR against actin at 7, 14, 21, and 35 days after SNI. Actin is an internal loading control. CON, normal control; SED, sedentary group; TEX, low-intensity treadmill exercise group; BMSC, BMSC transplantation group; TEX+BMSC, TEX+BMSC transplantation group. *
Combined application of treadmill exercise and bone marrow stromal cell (BMSC) transplantation regulates time-dependent changes of myoblast determination protein 1 (MyoD) and myogenin in the denervated soleus muscle. (A) Results of Western blot analysis for quantitative analysis of MyoD and myogenin in the denervated soleus muscle. (B) The statistical graph on ratio of MyoD against actin at 7, 14, 21, and 35 days after sciatic nerve injury (SNI). (C) The statistical graph on ratio of myogenin against actin at 7, 14, 21, and 35 days after SNI. Actin is an internal loading control. CON, normal control; SED, sedentary group; TEX, low-intensity treadmill exercise group; BMSC, BMSC transplantation group; TEX+BMSC, TEX+BMSC transplantation group. *