CC BY
2
STUDY ON THE EFFECT OF CLOBETASOL ON PROTEIN COMPOSITION AND NEUROTROPHIC FACTOR LEVELS IN SOMATIC NERVE INJURY
T.P. Kuzmenko, M.V. Parchaykina*, A.V. Zavarykina, E.V. Popkov, I.D. Molchanov, M.A. Simakova, M.V. Shchankin, V.V. Revin
Department of Biotechnology, Biochemistry and Bioengineering, Faculty of Biotechnology and Biology, Federal State-Financed Academic Institution of Higher Education National Research Ogarev Mordovia State University, 68 Bolshe-vistskaya St., Saransk, Republic of Mordovia, 430005, Russia.
* Corresponding author: [email protected]
Abstract. Previous studies have demonstrated that administration of clobetasol at a concentration of 0.25 mg/kg stimulates the synthesis of neurotrophic growth factors, likely triggering the phosphatidylinositol-3-kinase (PI3K) and mi-togen-activated protein kinase (MAPK) signaling pathways, resulting in increased levels of structural and axonal proteins necessary for the functional recovery of damaged somatic nerves. However, it has been shown that using higher concentrations of the drug (0.5 and 1.0 mg/kg) leads to a reduction in both structural and axonal proteins in both segments of the nerve conduit and exerts an inhibitory effect on the expression of neurotrophic factors responsible for cytoskeletal remodeling and axonal growth regulation.
Keywords: sciatic nerve, injury, regeneration, clobetasol, neurotrophic factors, protein composition.
List of Abbreviations
GAP-43 - Growth Associated Protein 43 MAP - microtubule-associated proteins NF-H - neurofilament-H NF-M - neurofilament-M NGF - nerve growth factor NT-3 - neurotrophin-3 PI3K - phosphatidylinositol-3-kinase signaling pathway
MAPK - mitogen-activated protein kinase signaling pathway
Introduction
The restoration of peripheral nervous system functionality is one of the most challenging and under-researched issues in biology and medicine, driven by a rising incidence of traumatic somatic nerve injuries and the slow pace of functional recovery (Contreras et al., 2022; Ronchi et al., 2023). It is known that proteins of nerve tissue form the basis of the cytoskeleton and play an active role in initiating various signaling cascades necessary for Schwann cell survival, proliferation, and differentiation (Dubovy et al., 2018). Consequently, recent years have seen progress in exploring the neu-roprotective effects of various bioactive compounds and their roles in regulating signaling pathways involved in nerve regeneration
(Revin et al., 2015; Lee et al., 2024). Among the diverse classes of bioactive compounds, glucocorticoids particularly clobetasol - are of special interest (Shi et al., 2019; Zhang et al., 2019). Clobetasol has been shown to stimulate myelination processes in oligodendrocyte precursor cells via glucocorticoid receptor binding, a mechanism reflecting its secondary effects (Morisaki et al., 2010). Additionally, there is evidence of clobetasol's capacity to enhance the expression of major myelin proteins and, in general, to aid in the repair of the axonal myelin sheath (Su et al., 2020). Our previous studies demonstrated that clobetasol administration at a concentration of 0.25 mg/kg increases levels of neurotrophic growth factors and structural proteins, while stabilizing the total protein fraction in damaged somatic nerves, likely through activation of the PI3K and MAPK signaling pathways (Kuzmenko et al., 2023). However, data on its effects at higher concentrations on the molecular and cellular mechanisms that activate regenerative processes in injured somatic nerves remain limited.
Therefore, the objective of this study was to investigate the effects of clobetasol at concentrations of 0.5 and 1 mg/kg on the protein composition and neurotrophic factor levels in injured somatic nerves.
Materials and Methods
The experiments were carried out on mature male Wistar rats weighing 250-350 g, kept in vivarium conditions on a standard diet without restrictions on food and water with a change in the light regime every 12 hours. The object of the study was sciatic nerves. In animals from the first experimental group, the sciatic nerve was transected at mid-thigh level. Animals in the second group, following nerve transection, received daily intramuscular injections of clobetasol at concentrations of 0.5 and 1.0 mg/kg. The proximal and distal ends of the nerves were collected on days 7, 14, and 30, followed by a quantitative assessment of total protein content, individual protein fractions, and neurotrophic factor levels (NGF and NT-3). Intact animals served as the control. All research stages were in compliance with the principles of World Medical Association Declaration of Helsinki (WMA Declaration of Helsinki). The research was also approved by the Local Ethics Committee of Mordovia State University (Protocol № 89 issued in September 12, 2023).
The total protein content was determined using the Lowry method (Lowry et al., 1951). The protein composition of the somatic nerves was analyzed via polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), following the Laemmli method (Laemmli, 1970). Visualization, documentation, and quantitative analysis of the protein gels were conducted using the Gel Doc XR+ system (BioRad, USA), and the results were processed in the ImageLab software. Quantitative determination of growth factors NGF and NT-3 was carried out by enzyme-linked immunosorbent assay (ELISA) using specific commercial kits (Cloud-Clone Corp., China; Puda Scientific, China). Experimental data were statistically analyzed using Microsoft Excel 2016 spreadsheets, and ANOVA was employed for comparison at a 5% significance level.
Results
Literature indicates that Schwann cells contribute to regeneration processes by producing neurotrophic factors, cytokines, and extracellular matrix proteins. Additionally, they provide
supportive effects on developing neurons before their axons reach target organs (Wei et al., 2024). Neurotrophic factors, in turn, can activate signaling pathways involved in the synthesis of cytoskeletal proteins and axonal growth proteins essential for the restoration of function in damaged nerve pathways (Zheng et al., 2024). Based on this, the first stage of our experiment investigated changes in the total protein content and individual protein fractions during degenerative and regenerative processes in somatic nerves following intramuscular administration of clobetasol at concentrations of 0.5 and 1.0 mg/kg.
It was shown that in the proximal nerve segment, protein content decreased by 30.7% compared to the control after 7 days following nerve transection. By day 30, protein levels in this variant increased but remained 10.5% below the control. In the distal segment, a substantial decrease in protein content was observed by day 14, with levels dropping over four-fold relative to control. Extending the postoperative period to 30 days showed that protein content still remained 50% below control levels. Thus, sciatic nerve transection was associated with a reduction in protein content, with the most pronounced degeneration in the distal segment due to the loss of central innervation. With intramuscular administration of clobetasol at 0.5 mg/kg during the first seven days of the experiment, protein content in the proximal nerve segment decreased by 25.3% compared to the control. However, by day 14, this measure had increased by 59.2% compared to the intact nerve. Extending the postoperative period to 30 days showed an increase in protein levels by 44.5% relative to control. In the series with the 0.5 mg/kg concentration, by day 7 post-injury, a significant decrease in protein content (61.2% below control) was observed in the distal nerve segment. Prolonging injury exposure to 30 days led to an increase in total protein, though levels still remained 30.2% below control values (Fig. 1).
Notably, in a series of experiments with clobetasol administration at a concentration of 1.0 mg/kg over a 7-day period, there was no sign-ificant change in protein content observed
in the proximal segment of the nerve. By the 14th day, this parameter decreased by 31.3% relative to the control, and by the 30th day postinjury, protein levels returned to control values. In the distal segment of the nerve pathway,
a significant reduction in total protein content -65.3% compared to the intact nerve - was observed 7 days post-injury with the treatment. By the 30th day, protein levels had increased but remained 59.3% below control values (Fig. 2).
180 160 140 120 100 % 80 60 40 20 0
-
ll
■ №3 n
■ FTTH
■ 1 1 1 1 1 1
I
-rr-«-
I npi I I I I
Proximal Distal
Control ■ 7 days ■ 7 days+Clob
14 days ■ 14 days+Clob ■ 30 days
30 days+Clob
Fig. 1. Changes in total protein content in proximal and distal segments of damaged somatic nerves following treatment with clobetasol (Clob) at a concentration of 0.5 mg/kg (* - p < 0.05)
120 —
100 —
80 —
% 60 —
40 —
20 — 0
I ■
ll L* ±*
*
Proximal
Distal
Control 14 days 30 days+Clob
7 days >7 days+Clob
14 days+Clob ■ 30 days
Fig. 2. Changes in total protein content in proximal and distal segments of damaged somatic nerves following treatment with clobetasol (Clob) at a concentration of 1.0 mg/kg (* - p < 0.05)
It is well established that cytoskeletal proteins play a crucial role in supporting axonal transport and maintaining intracellular skeletal structure (Dubovy et al., 2018). Additionally, during axonal growth and regeneration within the peripheral and central nervous systems, axonal growth proteins, specifically GAP-43, are essential for restoring nerve pathway function (Arabzadeh et al., 2022). Based on these considerations, the next phase of the experiment employed electrophoretic separation on poly-acrylamide gel to assess the qualitative and quantitative composition of individual protein fractions in somatic nerves following injury and clobetasol treatment. In intact nerves, proteins identified included neurofilament-H (190-200 kDa), neurofilament-M (140-160 kDa), tubulin (110 kDa), and GAP-43 (27 kDa) (Fig. 3).
It has been shown that in the proximal segment of the nerve, 14 days post-injury, the quantitative content of neurofilament-H and tubulin fractions decreases by 22.8% and 22.0%, respectively, compared to the control. As the postoperative period extends to 30 days, the
concentration of the studied protein fractions exceeds control values by 9.8% and 34.8%, respectively. Additionally, a decrease in neurofil-ament-M content is observed 7 days post-tran-section by 12.2%, with an increase by day 30 of 13.8% relative to the control. It should be noted that the level of the axonal growth protein, GAP-43, increases both on day 7 and day 30 of the experiment by 1.3-fold and 2.3-fold, respectively, compared to the uninjured nerve (Fig. 4).
In the distal nerve segment, the levels of neurofilaments, neurofilament-M, and tubulin fractions are observed to decrease more than two-fold compared to the control. However, extending the experiment duration to 30 days is accompanied by an increase in their levels: neu-rofilament-H, neurofilament-M, and tubulin concentrations rise by 39.7%, 26.0%, and 14.5%, respectively, compared to the uninjured nerve. The GAP-43 protein level in this series of experiments also decreases by 40.4% by day 14, yet after 30 days post-injury, this indicator increases 1.6-fold compared to the control value (Fig. 4).
Fig. 3. Protein electropherogram (A) and Western blot analysis of GAP-43 levels (B) in damaged somatic nerves collected on Days 7, 14, and 30 Post-Incision
M - marker; a - control; b - proximal segment, day 7; c - distal segment, day 7; d - proximal segment, day 14; e - distal segment, day 14; f - proximal segment, day 30; g - distal segment, day 30; 1 - neurofilament-H, 2 - neurofilament-M, 3 - tubulin, 4 - GAP-43
Fig. 4. Changes in the content of individual protein fractions in the proximal and distal segments of injured somatic nerves (* - p < 0.05)
P - proximal segment; D - distal segment
NF-H NF-M Tubulin GAP-43 Control ■ 7P 7D ■ 14P ■ 14D «30P "30D
Therefore, nerve transection is accompanied by significant alterations in the protein composition, especially in structural proteins such as neurofilaments and tubulin. It is well known that neurofilaments are a major component of the neuronal cytoskeleton, providing structural support for axons (Yuan et al., 2017). Following nerve injury, the level of neurofilaments decreases due to intensive degenerative processes and disruptions in their transport, which compromises nerve conductor functionality. In addition, nerve damage leads to destabilization of microtubules and changes in tubulin levels, affecting transport processes and slowing regeneration (Pero et al., 2023). By extending the experiment period to 30 days, there is a noted trend toward an increase in structural protein content in the proximal segment of the nerve, indicating the activation of regenerative processes in the proximal part of the injured nerve trunk.
Electrophoretic analysis showed that under clobetasol treatment at a concentration of 0.5 mg/kg, the GAP-43 protein band is more intense on days 7 and 14 of observation (Fig. 5).
In the presence of the drug at a concentration of 0.5 mg/kg by day 14 of the experiment, a reduction in neurofilament-H and neurofilament-M levels is observed in the proximal nerve segment by 25.6% and 40.4%, respectively, and by day 30, by 31.9% and 46.1%, respectively, compared to the control. The level of GAP-43 protein significantly increases by day 7 post-injury, exceeding the control value by 57.1%; however, by day 30 of observation, its level decreases by 37.1% relative to the intact nerve (Fig. 6).
In the distal segment of the nerve conduit, clobetasol administration at a concentration of 0.5 mg/kg resulted in a decreased intensity of cytoskeletal protein bands by day 7, specifically neurofilament-H by 34%, neurofilament-M by 41.4%, and tubulin by 46.5% relative to control. By day 30 of the experiment, further degradation of cytoskeletal structural proteins was observed, with neurofilament-H reduced by 53.1%, neurofilament-M by 59.9%, and tubulin by 69.5% compared to control. During the first 7 days, no significant changes in GAP-43 levels were detected relative to control; however, by day 30, GAP-43 content decreased by a factor of 8.6 in comparison to the intact nerve (Fig. 7).
M 110 80
60 40
25
kDa
Fig. 5. Protein electrophoregram (A) and Western blot analysis of GAP-43 levels (B) in injured somatic nerves under clobetasol treatment at a concentration of 0.5 mg/kg
M - marker; a - control; b - proximal segment, 7 days; c - distal segment, 7 days; d - proximal segment, 14 days; e - distal segment, 14 days; f - proximal segment, 30 days; g - distal segment, 30 days; 1 - NF-H, 2 -NF-M, 3 - tubulin, 4 - GAP-43
275 250 225 200 175 150 «/„125 100 75 50 25 0
* T
t
i-
IFÜi b m n ru m 11 ■ MUD
Hill iiraiipimiiii miHin
ni mi
NF-H
Control 14 days 130 days+Clob
NF-M Tubulin GAP-43 7 days 7 days+Clob
114 days+Clob ■ 30 days
Fig. 6. Changes in protein fraction levels in the proximal segment of injured somatic nerves under clobetasol (Clob) treatment at a concentration of 0.5 mg/kg (* - p < 0.05)
Fig. 7. Changes in levels of specific protein fractions in the distal segment of injured somatic nerves 7, 14, and 30 days post-transection following clobetasol (Clob) treatment at a concentration of 0.5 mg/kg (* - p < < 0.05)
%
180 160 140 120 100 80 60 40 20
* * T T *
i • i
T 1 . i i i x 1 —
l ■■ p n P □ □
l l □ A _ A □ 1 III
imn il n9 RnmRMIIIR iiiiimrviiin b
If 111 III IUI II llll II ll li
NF-H
i Control 114 days I 30 days+Clob
NF-M I 7 days
114 days+Clob
Tubulin
GAP-43 7 days+Clob
30 days
Electrophoretic analysis indicated that clobetasol administration at a concentration of 1 mg/kg led to a more pronounced appearance of GAP-43 and tubulin bands on days 14 and 30, respectively (Fig. 8).
It was found that clobetasol administration at a concentration of 1 mg/kg in the proximal segment of the nerve led to a decrease in neurofil-ament-H content on days 7 and 14 by 37.8% and 40.4%, respectively, compared to control. By day 30 of the experiment, no significant changes relative to control were observed. The NF-M level also decreased by 18.1% compared to control, with no significant difference observed on days 14 and 30 relative to control values.
It should be noted that tubulin content significantly increased relative to control by 11.8% and 31.4% on days 7 and 30, respectively. At the same time, GAP-43 levels notably decreased and remained 44.4% below control values by day 30 (Fig. 9).
In the distal segment of the nerve conduit, clobetasol administration at a concentration of 1 mg/kg on day 7 led to a substantial reduction in all examined cytoskeletal proteins, specifically NF-H, NF-M, and tubulin by 73.7%, 81.3%, and 83.9%, respectively, relative to control. On day 14 post-injury, NF-H, NF-M, and
tubulin levels increased but remained below control levels by 28.9%, 25.4%, and 28.4%, respectively. Extending the postoperative period to 30 days resulted in an even greater reduction in NF-H, NF-M, and tubulin levels by 62.3%, 85.6%, and 72.6%, respectively, compared to control values. Notably, GAP-43 band intensity in the distal segment decreased by 70% relative to control, and by day 30 post-injury, it was detected only in trace amounts (Fig. 10).
Therefore, the administration of clobetasol at a concentration of 1 mg/kg leads to destabili-zation of the protein composition in the nerve conductor and inhibits the synthesis of the ax-onal growth-associated protein GAP-43 in both its proximal and distal segments.
It is known that the activation of protein synthesis necessary for axonal regeneration occurs because of triggering various signaling pathways through the binding of neurotrophic factors, particularly nerve growth factor (NGF) and neurotrophin-3 (NT-3), to their respective receptors on the nerve fiber surface (Bruno et al., 2023). Based on this, in the next stage of our experiment, we investigated the levels of NGF and NT-3 in the proximal and distal segments of the sciatic nerve following its transection and intramuscular administration of clobetasol.
The study results demonstrated that, in the proximal segment of the nerve on the 7th and 14th days post-injury, there was a decrease in NGF levels by 31.7% and 26.7%, respectively, compared to the control. By day 30 of the experiment, no significant change in NGF content was observed in this group (Fig. 11A).
In the distal segment of the nerve conductor, NGF levels were also reduced by 24.4% and 22.7% on the 7th and 14th days of observation, respectively, relative to the control. However, as the postoperative period extended to 30 days, the NGF levels increased by 11.8% relative to the control (Fig. 11B).
Fig. 8. Protein electrophoresis (A) and Western blot analysis of GAP-43 levels (B) in injured somatic nerves after clobetasol treatment at a concentration of 1.0 mg/kg
M - marker; a - control; b - proximal segment, day 7; c - distal segment, day 7; d - proximal segment, day 14; e - distal segment, day 14; f - proximal segment, day 30; g - distal segment, day 30; 1 - NF-H, 2 - NF-M, 3 - tubulin, 4 - GAP-43
240 220 200 180 160 140 % 120 /o100 80 60 40 20 0
* * *
Й ill
i i- . i ■ 1 1 1
mm rnii 1 ■ 1 pi ШШЯШШШЯ 11 ■ 11Г11 llllHIIIIIIHIIIII 1 i 1 1 1 ■ 1 i 1 1 1 ■ 1 i 1 1 ■ 1MB ■ 11 m □ ■ 11 R 1 П ■ 111111
NF-H
NF-M Tubulin GAP-43
Control 14 days 130 days+Clob
17 days
114 days+Clob
7 days+Clob 30 days
Fig. 9. Changes in levels of specific protein fractions in the proximal segment of injured somatic nerves 7, 14, and 30 days post-transection following clobetasol (Clob) treatment at a concentration of 1 mg/kg (* - p < 0.05)
Fig. 10. Changes in levels of specific protein fractions in the distal segment of injured somatic nerves 7, 14, and 30 days post-transection following clobetasol (Clob) treatment at a concentration of 1 mg/kg (* - p < 0.05)
Fig.11. Effect of clobetasol (Clob) on changes in NGF ments of injured somatic nerves (* - p < 0.05)
Intramuscular administration of clobetasol at concentrations of 0.5 and 1.0 mg/kg was associated with a reduction in NGF levels by 46.6% and 41.1%, respectively, in the proximal segment of the nerve by the 7th day compared to the control values. After 14 days of the experiment, there was a trend towards a decrease in NGF levels, by 22.7% and 41.4% in the groups receiving 0.5 mg/kg and 1.0 mg/kg of clobetasol, respectively. By day 30 post-injury, NGF levels were shown to increase by 1.7 and 2.2 times, respectively, in the clobetasol groups at
concentration in the proximal (A) and distal (B) seg-
concentrations of 0.5 mg/kg and 1.0 mg/kg, compared to the intact nerve (Fig.11A).
In the distal segment of the nerve, NGF levels decreased by 41.4% and 40.4% within the first seven days of the experiment with clobeta-sol administration at concentrations of 0.5 mg/kg and 1.0 mg/kg, respectively. Notably, the use of clobetasol at 0.5 mg/kg was associated with a substantial increase in NGF content, with levels exceeding the control in the distal segment of the nerve conductor by 3.85 times by day 30 post-injury (Fig. 11B).
Therefore, nerve injury is accompanied by a marked decrease in NGF levels in both segments of the nerve conductor. However, the administration of clobetasol at concentrations of 0.5 and 1.0 mg/kg results in a significant increase in NGF synthesis in the proximal segment of the nerve by day 30 of the experiment, as well as in the distal segment during the same period with the administration of 0.5 mg/kg clobetasol.
In addition to changes in NGF levels in the proximal segment of the nerve, nerve injury also results in an increase in NT-3 concentration by 30.7% and 27.2% on days 7 and 30, respectively, compared to the control. In the distal segment, NT-3 levels exceed the control by 2.0 times on day 7 and by 2.14 times on day 14. By day 30 of observation, NT-3 content decreases by 16.2% compared to the control. With clobet-asol administration at doses of 0.5 and 1.0 mg/kg, NT-3 concentration in the proximal nerve segment decreases by 20.8% and 21.8%,
respectively, by day 7 compared to the control, and by day 30 by 10.4% and 55.0%, respectively, relative to the intact nerve. During the first week of the experiment, clobetasol administration at concentrations of 0.5 and 1.0 mg/kg resulted in decreased NT-3 levels in the distal nerve segment by 25.2% and 18.9%, respectively, relative to the control. With the extension of the postoperative period to 30 days, NT-3 concentration remained below control levels by 40.1% and 25.2% in the clobetasol groups at concentrations of 0.5 mg/kg and 1.0 mg/kg, respectively (Fig. 12).
Based on the results obtained, the administration of clobetasol is associated with an increase in NT-3 synthesis in the proximal end of the nerve on day 14 after administration at doses of 0.5 and 1.0 mg/kg, and on day 30 after intramuscular administration of clobetasol at 0.5 mg/kg. In the distal segment of the nerve, clobetasol in both concentrations inhibits NT-3 synthesis and reduces its level.
Fig. 12. Effect of clobetasol on changes in NT-3 concentration in the proximal (A) and distal (B) segments of injured somatic nerves (* - p < 0.05)
Discussion
Currently, one of the most relevant issues in biomedicine is the search for potential pathways to optimize axonal regeneration of damaged nerve fibers. The use of physiologically active substances appears to be a promising ap-
proach for stimulating post-traumatic nerve conduit regeneration (Revin et al., 2015; Lee et al., 2024). According to various studies, clobet-asol, a glucocorticoid that promotes mye-lination in cerebellar slice cultures and in vivo in mice as well as in experimental focal demy-
elination models, is of particular interest (Mori-saki et al., 2010). However, the molecular mechanisms of its action on somatic nerve regeneration remain unexplored.
Our research demonstrated that intramuscular administration of clobetasol at a concentration of 0.5 mg/kg is associated with an increased synthesis of protein fractions in the proximal segment of the nerve by days 14 and 30 of observation, significantly exceeding control values. A tendency toward protein synthesis was also observed in the distal segment on days 14 and 30. However, the quantitative protein content in this variant of the experiment remained, on average, two times lower than the control values. The use of clobetasol at a concentration of 1 mg/kg stimulated total protein synthesis in the proximal segment of the nerve in the early stages after transection; however, by day 14, its level decreased. Due to profound degenerative processes in the distal segment of the nerve, a reduction in protein fraction content was observed at the higher concentration of the drug. By day 14, there was a slight increase in protein synthesis; however, extending the experiment to 30 days was accompanied by an even more pronounced decrease in protein levels compared to the injury baseline.
According to the literature, various G-pro-teins involved in cytoskeletal rearrangement, as well as MAP family proteins that play an active role in cytoskeletal stabilization, participate in axonal growth (Cai et al., 2020). Additionally, GAP-43, synthesized by the growth cones of regenerating nerves, is one of the markers used to identify regenerative processes in damaged nerve conduits (Chung et al., 2020). Our research demonstrated that clobetasol at concentrations of 0.5 and 1.0 mg/kg reduces the levels of both structural proteins and GAP-43 at the initial stages post-transection. By day 14, there was a trend toward activation of specific protein fractions in the injured nerve conduit. Nevertheless, with an extension of the postoperative period to 30 days, a significant reduction in their levels was observed. The inhibitory effect of the drug was more pronounced at a concentration of 1 mg/kg in the distal nerve segment.
In our previous studies, it was established that clobetasol at a concentration of 0.25 mg/kg enhances recovery processes in injured nerves, resulting in increased levels of neurotrophic growth factors, stimulation of neurofilament-H and neurofilament-M synthesis, tubulin, and GAP-43 protein in both proximal and distal segments of the nerve conduit. We hypothesized that clobetasol participates in activating regenerative processes by binding to glucocorticoid receptors in nerve cells and initiating the synthesis of neurotrophic factors by Schwann cells. Nerve growth factors, in turn, interact with respective receptors to activate several signaling pathways, namely phosphatidylinositol 3-kinase, which is involved in protein synthesis, and mitogen-activated protein kinase (MAPK), which regulates cytoskeletal remodeling and axonal growth (He et al., 2022; Tiong et al., 2020).
However, our findings indicate that higher concentrations of the drug inhibit the synthesis of both structural and axonal growth proteins. These data are consistent with a decrease in the expression of neurotrophic factors, specifically NGF and NT-3, in both segments of the nerve conduit following intramuscular administration of clobetasol at doses of 0.5 and 1.0 mg/kg. Thus, based on our cumulative data, we can suggest that clobetasol, as a glucocorticoid compound, has the potential to stimulate regenerative processes in somatic nerve pathologies by activating the phosphatidylinositol 3-kinase and MAPK signaling pathways, which regulate the recovery of functional activity in damaged nerve conduits. In comparing the effects of different doses, it was shown that intramuscular administration of clobetasol at higher concentrations (0.5 and 1.0 mg/kg) is associated with an inhibitory effect on the expression of neu-rotrophic factors, as well as the synthesis of structural and axonal proteins in regenerating nerve conduits.
Acknowledgements
This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation, grant number FZRS-2024-0005.
References
ARABZADEH E., RAHIMI A., ZARGANI M., SIMORGHI Z.F., EMAMI S., SHEIKHI S. & FEIZO-LAHI F. (2022): Resistance exercise promotes functional test via sciatic nerve regeneration, and muscle atrophy improvement through GAP-43 regulation in animal model of traumatic nerve injuries. Neuroscience Letters 787, 136812.
BRUNO F., ABONDIO P., MONTESANTO A., LUISELLI D., BRUNI AC. & MALETTA R. (2023): The nerve growth factor receptor (NGFR/p75NTR): a major player in Alzheimer's disease. International journal of molecular sciences 24(4), 3200.
CAI Q., WU G., ZHU M., XUE C., CHENG B., XU S. & WU P. (2020): FGF6 enhances muscle regeneration after nerve injury by relying on ERK1/2 mechanism. Life sciences 248, 117465.
CHUNG D., SHUM A. & CARAVEO G. (2020): GAP-43 and BASP1 in axon regeneration: implications for the treatment of neurodegenerative diseases. Frontiers in cell and developmental biology 8, 567537.
CONTRERAS E., BOLÍVAR S., NAVARRO X. & UDINA E. (2022): New insights into peripheral nerve regeneration: The role of secretomes. Experimental Neurology 354, 114069.
DUBOVY P., KLUSÁKOVÁ I., HRADILOVÁ-SVÍZENSKÁ I. & JOUKAL M. (2018): Expression of regeneration-associated proteins in primary sensory neurons and regenerating axons after nerve injury— An overview. The Anatomical Record 301(10), 1618-1627.
HE X., LI Y., DENG B., LIN A., ZHANG G., MA M. & KANG X. (2022): The PI3K/AKT signalling pathway in inflammation, cell death and glial scar formation after traumatic spinal cord injury: Mechanisms and therapeutic opportunities. Cell Proliferation 55(9), e13275.
KUZMENKO T P., PARCHAYKINA M.V., REVINA E.S., GLADYSHEVA M Y. & REVIN V.V. (2023): Influence of neurotrophic factors on protein composition during somatic nerve injury and regeneration. Biophysics 68(2), 259-271.
LAEMMLI U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680-685.
LEE J., YON D.K., CHOI Y. S., LEE J., YEO J.H., KIM S.S. & YEO S.G. (2024): Roles of SMAD and SMAD-Associated Signaling Pathways in Nerve Regeneration Following Peripheral Nerve Injury: A Narrative Literature Review. Current Issues in Molecular Biology 46(7), 7769-7781.
LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J. (1951): Protein measurement with the Folin phenol reagent. Jbiol Chem 193(1), 265-275.
MORISAKI S., NISHI M., FUJIWARA H., ODA R., KAWATA M. & KUBO T. (2010): Endogenous glu-cocorticoids improve myelination via Schwann cells after peripheral nerve injury: An in vivo study using a crush injury model. Glia 58(8), 954-963.
PERO M.E., CHOWDHURY F., & BARTOLINI F. (2023): Role of tubulin post-translational modifications in peripheral neuropathy. Experimental Neurology 360, 114274.
REVIN V.V., ISAKINA M.V., PINYAEV S.I., MOROZOVA A.A. & REVINA N.V. (2015): Influence of potassium hyaluronate on the change of phospholipase activity and state of the membranes of damaged somatic nerves of rats. International Journal of Pharma and Bio Sciences 6(4), 512-520.
RONCHI G., FREGNAN F., MURATORI L., GAMBAROTTA G. & RAIMONDO S. (2023): Morphological methods to evaluate peripheral nerve fiber regeneration: a comprehensive review. International Journal of Molecular Sciences 24(3), 1818.
SHI W., BI S., DAI Y., YANG K., ZHAO Y. & ZHANG Z. (2019): Clobetasol propionate enhances neural stem cell and oligodendrocyte differentiation. Experimental and Therapeutic Medicine 18(2), 12581266.
SU X., YUAN H., BAI Y., CHEN J., SUI M., ZHANG X. & ZHU H. (2020): Clobetasol attenuates white matter injury by promoting oligodendrocyte precursor cell differentiation. Pediatric Neurosurgery 55(4), 188-196.
TIONG Y.L., NG K.Y., KOH R.Y., PONNUDURAI G. & CHYE S.M. (2020): Melatonin promotes Schwann cell dedifferentiation and proliferation through the Ras/Raf/ERK and MAPK pathways, and glial cell-derived neurotrophic factor expression. Experimental and therapeutic medicine 20(5), 1-1.
WEI C., GUO Y., CI Z., LI M., ZHANG Y. & ZHOU Y. (2024): Advances of Schwann cells in peripheral nerve regeneration: From mechanism to cell therapy. Biomedicine & Pharmacotherapy 175, 116645.
YUAN A., RAO M.V. & NIXON R.A. (2017): Neurofilaments and neurofilament proteins in health and disease. Cold Spring Harbor perspectives in biology 9(4), a018309.
ZHANG L., YANG W., XIE H., WANG H., WANG J., SU Q. & WANG Z. (2019): Sericin nerve guidance conduit delivering therapeutically repurposed clobetasol for functional and structural regeneration of transected peripheral nerves. ACS Biomaterials Science & Engineering 5(3), 1426-1439. ZHENG J., ZHANG J., HAN J., ZHAO Z. & LIN K. (2024): The effect of salidroside in promoting endogenous neural regeneration after cerebral ischemia/reperfusion involves notch signaling pathway and neu-rotrophic factors. BMC Complementary Medicine and Therapies 24(1), 293.
