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ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3β in a mouse sciatic nerve injury model

  • Authors:
    • Shuang Dou
    • Zhijun Li
    • Boyao Zheng
    • Zhenyu Ren
    • Hai Wang
    • Qing Zuo
    • Fang Fang
    • Yuehong Zhuang
  • View Affiliations / Copyright

    Affiliations: Institute of clinical applied anatomy, Fujian Key Laboratory of brain aging and neurodegenerative diseases, School of basic medical sciences, Fujian medical university, Fuzhou, Fujian 350122, P.R. China, Department of pharmacology, Fujian medical university, Fuzhou, Fujian 350122, P.R. China, Orthopedic Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350108, P.R. China
    Copyright: © Dou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 14
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    Published online on: November 5, 2025
       https://doi.org/10.3892/ijmm.2025.5685
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Abstract

The present study investigates the molecular mechanisms of peripheral nerve regeneration by examining the ROCK/PI3K/Akt/GSK3β pathway's role in promoting morphological and functional recovery after peripheral nerve injury (PNI). Using a mouse sciatic nerve crush (SNC) injury model and a dorsal root ganglion (DRG) explant axotomy model, mice and DRG were divided the experimental (treated with DMSO) group, Y27632 group (treated with ROCK inhibitor Y27632), Y + LY group (treated with Y27632 + PI3K inhibitor LY294002), and Y + LY + SB group (treated with Y27632 + LY294002 + GSK3β inhibitor SB216763). Immunofluorescence was used to assess axon density, diameter, myelin thickness and Schwann cell proliferation, while retrograde tracing with cholera toxin subunit B evaluated peripheral‑to‑central reconnection. Behavioral tests measured functional recovery, and in DRG explants, axon regeneration length and growth cone size were quantified. Protein expression analysis of RhoA, ROCK, PI3K, Akt, GSK3β, and their phosphorylated forms was conducted on day 3 post‑axotomy, both in vivo and in vitro. Additionally, RSC96 Schwann cell migration and proliferation were evaluated using scratch assays and EdU staining. Results showed that ROCK inhibition with Y27632 significantly enhanced axonal regeneration, growth cone expansion, retrograde transport, and reinnervation of acetylcholine receptors and Merkel cells, and promoted Schwann cell proliferation and RSC96 migration, leading to thicker myelin sheaths after SNC. These changes mitigated gastrocnemius muscle atrophy, improved muscle strength, gait, and thermal/tactile sensitivity. Co‑treatment with LY294002 blocked these effects, but adding SB216763 restored them. Protein analysis indicated that ROCK inhibition increased phosphorylated PI3K, Akt and GSK3β, whereas PI3K inhibition reduced GSK3β phosphorylation. These findings suggested that ROCK inhibition promotes axon regeneration and remyelination after PNI by enhancing PI3K/Akt phosphorylation and suppressing GSK3β activity, highlighting the therapeutic potential of targeting the ROCK/PI3K/Akt/GSK3β pathway for peripheral nerve repair.
View Figures

Figure 1

Effects of three inhibitors on nerve
regeneration after SNC. (A) Representative gross image of a SN
after SNC injury. The arrowhead indicates the formation of a nerve
defect, with only the epineurium remaining after hemostat clamping.
(B) The asterisk (*) marks a complete absence of NF-200-positive
staining at the clamped site immediately after injury. The arrow
denotes the proximal-to-distal orientation. (C) Representative
longitudinal sections of injured nerves stained with NF-200 at day
14. (D) Axon density is significantly higher in the Y and Y + LY +
SB groups (n=6). (E) Cross-sections stained with NF-200 on day 30
in all groups. a, b, c and d represent the staining from the
experimental, Y27632, Y + LY, and Y + LY + SB groups, respectively.
(F) The diameter of regenerated axons is significantly larger in
the Y27632 and Y + LY + SB groups (n=6). (G) Schematic diagram of
CTB injection into the SN distal to the clamped site for retrograde
labeling of neurons in the ventral horn. (H and I) CTB-labeled
neurons in the ventral horn are significantly more numerous in the
Y27632 and Y + LY + SB groups (n=6). **P<0.01 and
***P<0.001. SN, sciatic nerve; SNC, SN crush; CTB,
cholera toxin subunit B; NS, not significant (P>0.05).

Figure 2

ROCK inhibition promotes
phosphorylation of PI3K/Akt/GSK3β in vivo. (A-E) Increased
expression of RhoA, ROCK1/2, GAP43 and c-Jun in the LE on day 3
after SNC (n=3). (F-K) Y27632 increases the phosphorylation of
PI3K, Akt and GSK3β in the LE on day 3 after SNC (n=3). (L and M)
Co-treatment with LY294002 reduces Y27632-induced phosphorylation
of GSK3β (n=3). (N) Immunofluorescence images of ROCK1/2 and
p-GSK3β in the sciatic nerve and LE. (O-R) Quantification of
ROCK1/2 and P-GSK3β expression (n=6). *P<0.05,
**P<0.01 and ***P<0.001. LE,
lumbosacral enlargement; SNC, sciatic nerve crush; p-,
phosphorylated; NS, not significant (P>0.05).

Figure 3

ROCK inhibition promotes
PI3K/Akt/GSK3β phosphorylation and axon regeneration in
vitro. (A-a), Customized polydimethylsiloxane mold: The
arrowhead and arrow indicate the long and short grooves,
respectively. The red spot indicates the site for placement of the
DRG, ~1.5 mm away from the intersection. (A-b) Representative image
of a DRG placed in the mold. (A-c) Representative image of the DRG
immediately after axon transection. A shallow transection mark is
left, and the distal stumps of the severed axons are washed away.
(A-d) Regenerated axons emerging from the proximal stumps extend
across the transection mark without obvious impediment. (B-F) The
expression of RhoA, ROCK1/2, GAP43 and c-Jun is significantly
elevated after axotomy (n=3). (G-L) Y27632 increases the
phosphorylation of PI3K, Akt and GSK3β in the DRG on day 3 after
axotomy (n=3). (M and N) LY294002 co-treatment reduces
Y27632-induced phosphorylation of GSK3β (n=3). (O and P) The length
of regenerated axons is significantly greater in the Y27632 and Y +
LY + SB groups (n=5). (Q) Comparison of axon outgrowth in
axotomized vs. non-axotomized DRG. ***P<0.001 and
****P<0.0001. DRG, dorsal root ganglion; NS, not
significant (P>0.05).

Figure 4

ROCK/PI3K/Akt/GSK3β pathway regulates
growth cone morphology. (A) Schematic for growth cone size
measurement. (B) Representative images of growth cones stained with
Tuj1 and Phalloidin. Arrowheads indicate splayed microtubules;
arrows mark transition zones. (C) Quantification of growth cone
area (n=6). **P<0.01 and ***P<0.001.
NS, not significant (P>0.05).

Figure 5

ROCK/PI3K/Akt/GSK3β pathway regulates
locomotor and sensory recovery after sciatic nerve crush. (A)
Experimental setup for muscle strength testing. (B) Gastrocnemius
muscle strength ratio ES/NS was significantly greater in the Y27632
and Y + LY + SB groups (n=6). (C) Top row: Representative
appearance of the gastrocnemius muscles on the NS and ES. Middle
row: Representative cross-sections of the gastrocnemius muscles in
ES. Bottom row: Representative neuromuscular junctions of the
extensor hallucis longus on the ES. (D and E) The wet weight ratio
and myofiber size were significantly larger in the Y27632 and Y +
LY + SB groups (n=6). (F) The reinnervation rate of the
acetylcholine receptor was significantly higher in the Y27632 and Y
+ LY + SB groups (n=6). (G) Schematic illustration evaluating the
locomotor function using the sciatic function index. (H-J)
Representative footprints demonstrated significantly improved
recovery of locomotor function in the Y27632 and Y + LY + SB
groups, as indicated by improved toe-spreading ability. (K)
Representative images of reconnection between NF-200-labeled axons
(Yellow) and Keratin 8-labeled Merkel cells (Red). Panels a, b, c
and d represent typical images from the control, Y27632, Y + LY,
and Y + LY + SB groups, respectively. (L) The reinnervation rate of
the Merkel cells in the footpads was significantly higher in the
Y27632 and Y + LY + SB groups (n=6). (M and N) Paw-licking latency
and mechanical pain thresholds significantly improved in the Y27632
and Y + LY + SB groups (n=6). *P<0.05,
**P<0.01 and ***P<0.001. ES,
experimental side; NS, normal side; NS, not significant
(P>0.05).

Figure 6

ROCK/PI3K/Akt/GSK3β Pathway regulates
remyelination after SNC. (A) Representative images of axon and
myelin staining at day 30. (B) The thickness of myelin sheaths was
significantly greater in the Y27632 and Y + LY + SB groups (n=6).
(C) Schwann cell proliferation assessed by S100β and Ki67
co-labeling on day 3 after SNC. (D) The proliferation rate of
Schwann cells was significantly higher in the Y27632 and Y + LY +
SB groups (n=6). (E and F) EdU assay showing increased RSC96 cell
proliferation in the Y27632 and Y + LY + SB groups. (G and H)
Scratch assay showing increased migration of RSC96 cells in the
Y27632 and Y + LY + SB groups. NS, P>0.05;
**P<0.01 and ***P<0.001. SNC, sciatic
nerve crush; NS, not significant (P>0.05).
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Copy and paste a formatted citation
Spandidos Publications style
Dou S, Li Z, Zheng B, Ren Z, Wang H, Zuo Q, Fang F and Zhuang Y: ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model. Int J Mol Med 57: 14, 2026.
APA
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q. ... Zhuang, Y. (2026). ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model. International Journal of Molecular Medicine, 57, 14. https://doi.org/10.3892/ijmm.2025.5685
MLA
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q., Fang, F., Zhuang, Y."ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model". International Journal of Molecular Medicine 57.1 (2026): 14.
Chicago
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q., Fang, F., Zhuang, Y."ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model". International Journal of Molecular Medicine 57, no. 1 (2026): 14. https://doi.org/10.3892/ijmm.2025.5685
Copy and paste a formatted citation
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Spandidos Publications style
Dou S, Li Z, Zheng B, Ren Z, Wang H, Zuo Q, Fang F and Zhuang Y: ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model. Int J Mol Med 57: 14, 2026.
APA
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q. ... Zhuang, Y. (2026). ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model. International Journal of Molecular Medicine, 57, 14. https://doi.org/10.3892/ijmm.2025.5685
MLA
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q., Fang, F., Zhuang, Y."ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model". International Journal of Molecular Medicine 57.1 (2026): 14.
Chicago
Dou, S., Li, Z., Zheng, B., Ren, Z., Wang, H., Zuo, Q., Fang, F., Zhuang, Y."ROCK inhibition promotes axon and myelin regeneration via PI3K/Akt/GSK3&beta; in a mouse sciatic nerve injury model". International Journal of Molecular Medicine 57, no. 1 (2026): 14. https://doi.org/10.3892/ijmm.2025.5685
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