| PurposeAxon regeneration after peripheral nerve injury not only plays an important role,but is also currently a difficult problem in the clinical.Peripheral nerve injury is mainly manifested by the disruption of axon continuity,which leads to the loss of neuroinflammatory response and axonal signal transmission,which ultimately leads to the loss of sensory and motor functions in patients.It is well known that inflammatory response is one of the important factors regulating the regeneration of axons.Nuclear factor kappa-B(NF-?B),as a classic inflammatory factor,participates in many inflammatory reactions and plays an important regulatory role in it.However,the role of NF-?B in axon regeneration in adult mammals is unknown.Here,we find that after peripheral nerve injury,both NF-?B and STAT3 are activated in adult sensory neurons.In addition,using PTDC to inhibit NF-?B can inhibit the axon growth of peripheral sensory neurons in vitro and in vivo.On the contrary,the addition of VP 16 activates its activity,and axon regeneration in vitro has not shown a significant promotion effect.Our results also show that the use of S3I-201 to inhibit signal transduction of transcription factor(STAT3)can block the axon growth of peripheral sensory neurons in vitro and in vivo.Conversely,using CLN to activate its activity,in vitro Axon regeneration can be promoted to some extent.In addition,we found that activation of STAT3 significantly promotes the regeneration of the optic nerve in vivo.More importantly,when NF-?B is inhibited by PTDC,phosphorylated STAT3 is also inhibited to a certain extent.In summary,our results show that NF-?B/STAT3 has an intrinsic cascade effect and has the ability to regulate the axon growth of adult sensory neurons.Methods1.In this study,a peripheral nerve injury model was used.Dorsal root ganglion(DRG)was taken 1,3 and 7 days after injury to perform Western blotting on the expression of NF-?B and STAT3 in sensory neurons Western Blot)quantitative detection.2.DRG sensory neuron culture model was applied in vitro to inhibit the expression of NF-?B and STAT3 through specific chemical drugs and small interfering RNA(siRNA).After 3 days of in vitro culture,specific neuron markers were used.The antibodies were immune fluorescently stained,labeled neurons regenerating axons,and statistically measuring their length to determine the functional effects of NF-?B and STAT3 on axon regeneration.3.Culture of DRG sensory neurons in vitro,activate the expression of NF-?B and STAT3 by specific chemical activators.After 2 days of in vitro culture,use specific neuron-labeled antibodies to label regenerating axons by immunofluorescence staining and statistically measure their length in order to detect the functional effects of NF-?B and STAT3 on axon regeneration.4.Using in vivo DRG electroporation,transfection of specific siRNA and green fluorescent protein(GFP)mixed solution to lumbar spine 4,5(Lumbar 4?5,L4?5)to inhibit NF-?B and STAT3 activity.Two days later,the sciatic nerve was crushed after 5 days,the crushed sciatic nerve was removed for compression,and the neuron labeled with GFP transfection was used to measure the regeneration of axons.5.Using vitreous injection and optic nerve injury model,inject 20%ethanol or STAT3-specific activator into the vitreous and perform optic nerve injury at the same time.Alexa-488-labeled cholera toxin B(Alexa-488-CTB)was re-injected into the intraocular vitreous body 12 days after surgery to label the regenerating axons.Fourteen days after surgery,the optic nerve was fixed with 4%paraformaldehyde and dehydrated with a 10%,20%,and 30%sucrose gradient.After frozen sections,the survival rate of optic neurons and axonal regeneration were measured.6.Using the optic nerve injury model,take out the retina at 14 days after injury,and freeze the sections,then use the specific neuron immunofluorescence staining to count the survival rate of retinal ganglion cells to determine that activating STAT3 promotes ganglion neurons to some extent survival.Results1.NF-?B regulates axon regeneration in sensory neurons.1)After peripheral nerve injury,the expression of phosphorylated p65 protein in DRG neurons gradually increased,and it was most obvious on days 3 and 7.2)The PDTC inhibits the activity of NF-?B,or transfects p65 small interfering RNA to knock down its protein,which hinders the growth of DRG sensory neuron axon cultured in vitro.3)The p65 small interfering RNA was electro-transfected into L4/L5 DRG in vivo.After knocking down the NF-?B protein,sciatic nerve axon regeneration was inhibited.4)On the contrary,although the drug VP 16 promoted the increased expression of phosphorylated p65,the growth of sensory axons in vitro did not show a significant increase.2.STAT3 regulates axon regeneration in sensory neurons.1)After peripheral nerve injury,the expression of phosphorylated STAT3 in DRG neurons reached a peak on the first day,and then gradually decreased.2)The drug of S3I-201 inhibits the activity of STAT3,or transfection of siRNA knocks down its protein,and the regeneration of DRG sensory neurons axon cultured in vitro is hindered.3)The specific small interfering RNA of STAT3 was transfected into L4/L5 DRG sensory neurons,and the growth of sciatic axons was inhibited.4)The drug of CLN not only promotes the marked increase of phosphorylated STAT3 expression,but also significantly promotes axonal regeneration of adult sensory neurons.5)Intravitreal injection of CLN activates the activity of STAT3,which not only promotes the growth of optic nerve axons,but also improves the survival rate of retinal ganglion cells.3.NF-?B regulates axon regeneration of sensory neurons through STAT3.1)Inhibition of NF-?B activity by PDTC can not only reduce phosphorylated p65,but also reduce phosphorylated STAT3 expression.2)Using the dug of CLN to activate the STAT3,this can reverse the effect of NF-?B inhibitors on the axon regeneration of sensory neurons to a certain extent.ConclusionThe process of axon regeneration after peripheral nerve injury is more complicated,and a variety of genes are strictly regulated.In general,nerve injury is often accompanied by inflammatory response and activation of related transcription factors to regulate essential substances required for axon regeneration after injury.The results of this study indicate that both NF-?B and STAT3 can regulate sensory nerve axon regeneration,and revealed that NF-?B can regulate axon regeneration by STAT3.More importantly,activating STAT3 can not only promote axon regeneration after optic nerve damage,but also improve the survival rate of retinal ganglion cells. |
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