| BackgroundPeripheral nerve injury is a common clinical condition and an important factor leading to serious disability and economic loss.Although peripheral nerves can regenerate after injury,nerve regeneration in the clinicis suboptimal,making it an urgent problem that must be addressed in clinical practice.Following peripheral nerve injury,Wallerian degeneration occurs at the injured distal stump and the axons disintegrate.Myelin and Remak Schwann cells transform into Schwann cells with the repair phenotype.These repair Schwann cells facilitate peripheral nerve regeneration by secreting neurotrophic factors and forming regeneration tracks(called bands of Büngner).However,when distal Schwann cells are in a chronically denervated state,the secretion of neurotrophic factors decreases,and the bands of Büngner become disorganized,significantly diminishing the ability of Schwann cells to promote axonal regeneration.The transcription factor c-Jun plays a crucial role in maintaining the reparative phenotype of Schwann cells,and its sustained overexpression in denervated Schwann cells has been shown to sustain this reparative phenotype,promoting peripheral nerve regeneration after chronic denervation.However,as an inhibitor of myelin sheath formation,the continuous overexpression of c-Jun carries a risk of inhibiting myelin sheath formation.Therefore,identifying a molecular drug that can upregulate c-Jun may serve as a potential therapeutic target for treating peripheral nerve injury after chronic denervation.Previous studies have indicated that c-Jun was upregulated by activating the ERK,p38,and JNK signaling pathways.Neurotrophin-3(NT-3),a neurotrophic factor,activates aforementioned pathways.However,it is unclear whether NT-3 could maintain high expression of c-Jun in the injured distal Schwann cells after chronic denervation and whether it promotes peripheral nerve regeneration after chronic denervation by maintaining high levels of c-Jun expression.Additionally,it is currently unknown whether NT-3 can re-upregulate c-Jun and promote peripheral nerve regeneration when c-Jun expression has already been downregulated in chronically denervated distal nerves.Together,this study aims to explore whether NT-3 can promote peripheral nerve regeneration after chronic denervation and elucidate the underlying molecular mechanisms.Methods1.To elucidate the relationship between the expression of c-Jun in Schwann cells and the time of denervation after peripheral nerve injury,a denervation model of peripheral nerve injury was established in rats.The expression of c-Jun in the injured distal stump at different time intervals was analyzed using Western blot.The denervation process of Schwann cells in the injured distal nerve was mimicked by subculturing Schwann cells in vitro,and the expression of c-Jun in Schwann cells of different passages was detected by western blot.2.To investigate whether NT-3 could upregulate the expression of c-Jun in vitro,primary rat Schwann cells were isolated and subcultured.Eighth passage of Schwann cells were treated with NT-3.Western blot and immunofluorescence were employed to detect the expression of c-Jun in Schwann cells at various time points following NT-3 treatment.3.When the expression of c-Jun was high in the distal tissues of the injured peripheral nerves(5-weekdenervation),NT-3(4 μg in 50 μL PBS)was administered locally at the injured distal site at a frequency of once every other day for a duration of 5 weeks,which aims to investigate whether NT-3 can maintain the high expression of c-Jun in the injured distal nerves.The expression of c-Jun in the injured distal stump was subsequently detected following NT-3 or PBS administration using both western blot and immunofluorescence.4.Adeno-Associated Virus(AAV)vectors carrying shRNA targeting c-Jun mRNA and expressing enhanced green fluorescent protein(EGFP)were constructed.AAV vectors were administered into the distal stump of injured peripheral nerve for selecting AAV vectors with high transfection efficiency and the capability to suppress c-Jun expression using western blot and immunofluorescence.5.Aperipheral nerve degeneration model after chronical denervation was established to investigate whether NT-3 promotes peripheral nerve regeneration after chronic denervation by maintaining the high expression of c-Jun.Four groups were designed as follows:(1)NT-3(5 W)group,NT-3 was administered to the distal stump after 5 weeks of denervation every other day to maintain the high level of c-Jun expression.(2)Control(-)group,administration of PBS only,serving as the negative control.(3)NT3+c-Jun shRNAs group,NT-3 was administered to the distal stump after 5 weeks of denervation.Simultaneously,AAVs carrying shRNA targeting c-Jun were administered into the distal stump to inhibit c-Jun expression.(4)Control(+)group,the peripheral nerves were directly transected and immediately sutured as a positive control.6.To investigate whether NT-3 could re-upregulate and sustain high expression of cJun after chronic denervation of peripheral nerves,two experimental groups were designed.(1)Control(-)group,similar to the aforementioned group,PBS was administered at the distal denervated nerve.(2)NT-3(8 W)group,NT-3 was administered to the distal part of the injured nerve tissue at the 8th week of denervation,once every other day for 2 weeks.Expression levels of c-Jun in the denervated nerve were assessed at the 10th week of denervation using Western blot and immunofluorescence.Concurrently,a peripheral nerve regeneration model after chronical denervation was established to explore the impact of NT-3 on peripheral nerve regeneration.7.In the evaluation of peripheral nerve regeneration,immunofluorescence,transmission electron microscopy,H&E staining,and wet muscle weight were analyzed,which indicated the quantification of axonal regeneration within peripheral nerve units,the degree of myeli nation of regenerating axons,and the reinnervation of target muscles.8.To investigate the downstream pathway,NT-3 was administered to cultured Schwann cells in vitro and denervated distal nerves in vivo.Western blot was employed to measure the expression levels and phosphorylation status of p38,JNK,and ERK.Additionally,inhibitors of the activated pathways were utilized to ascertain whether upregulation of c-Jun occurs through these pathways.9.Immunofluorescence staining was employed to examine the presence of TrkC,a NT-3 receptor,in Schwann cells derived from injured distal nerves and in passaged Schwann cells.The relationship between the expression of TrkC in denervated Schwann cells and the time of denervation was determined by western blot.Additionally,TrkC inhibitor and siRNA were utilized to interfere TrkC expression in passaged Schwann cells to evaluate whether NT-3 could upregulate c-Jun through the TrkC.Results:1.Following peripheral nerve injury,the expression levels of c-Jun in the distal stumps gradually increased from day 3,peaked at week 3,and maintained a high level at week 5.Subsequently,c-Jun began to decrease.In vitro,c-Jun expression decreased progressively with increasing passages(2nd,4th,6th,and 8th generations)by culturing passaged Schwann cells.2.NT-3 upregulated the expression of c-Jun in Schwann cells in vitro.3.The expression of c-Jun was at a high level after 5 weeks of denervation in the distal stumps of the injured peripheral nerves.Following the administration of NT-3 to the injured distal stumps for 5 weeks,NT-3 maintained a high level of c-Jun expression in the distal tissue at 10 weeks of denervation,which was basically consistent with the level of c-Jun expression in tissues at 5 weeks of denervation,and significantly higher than the control(-)group.Addtionally,the upregulated c-Jun mainly existed in the nucleus of Schwann cell in the injured distal nerve.4.AAV vectors containing shRNA that targeted c-Jun could be efficiently transfected into tissues distal to peripheral nerve injury,and most of them were co-labeled with Schwann cells.Among them,c-Jun sh2 significantly inhibited the expression of c-Jun.5.NT-3 maintained a high level of c-Jun expression and promoted peripheral nerve regeneration after chronic denervation.In the distal denervated nerves,the role of NT3 in promoting nerve regeneration was attributed to the maintenance of high expression of c-Jun rather than NT-3 itself.6.When the expression of c-Jun had already decreased in the distal denervated nerve,NT-3 was capable of re-upregulating c-Jun and sustaining its high expression levels,thereby facilitating peripheral nerve regeneration.7.In both in vivo and in vitro,NT-3 induced phosphorylation of ERK in Schwann cells,and ERK inhibitors could abolish the upregulation of c-Jun by NT-3.8.TrkC receptors were consistently expressed in Schwann cells in the distal end of injured peripheral nerve and cultured Schwann cell,and the expression did not change significantly with the time of denervation.Inhibiting TrkC expression using TrkC inhibitors and siRNA results in the suppression of NT-3-induced ERK phosphorylation and c-Jun upregulati on in Schwann cells.Conclusions:1.NT-3 upregulates the expression of c-Jun in Schwann cells in vitro,.2.NT-3 maintains the high level of c-Jun at the distal stump of denervation and promotes the regeneration of chronic denervated peripheral nerve.3.NT-3 promotes peripheral nerve regeneration by maintaining the high expression level of c-Jun rather than NT-3 itself.4.NT-3 re-upregulates the expression of low-level c-Jun after chronic denervation and promotes peripheral nerve regeneration.5.NT-3 upregulates c-Jun mainly through activating the TrkC/ERK pathway.6.TrkC receptors exist in the denervated Schwann cells,which provides a potential and stable therapeutic target for NT-3 in the treatment of peripheral nerve injury after chronic denervation. |
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