Promotion Of Adult Rat Optic Nerve Regeneration By Regulation Of PKC/NF-κB/SOCS3 Signaling Pathway

The traumatic optic neuropathy(TON),often resulting in persistent visual dysfunction,is a common complication of traumatic brain injury(TBI).The TON can be divided into two types,i.e.,direct TON and indirect TON.Direct injury arises from penetrating trauma,especially orbital fractures associated with mid-facial fractures.Several varieties of direct optic nerve injury,such as optic nerve avulsion and transaction,may be recognised ophthalmoscopically or with imaging techniques.There is no effective treatment method for direct TON.However,indirect optic nerve injury is more common in clinic.The incidence of indirect optic nerve injury is 0.5%～5%.The treatment methods of indirect TON include high-dose steroids and surgery to decompress the optic canal.However,the curative effect is not satisfactory.This is related to the physiological characteristics of optic nerve.The optic nerve is composed of a segment of the axon of retinal ganglion cells(RGCs).The RGCs,the only neurons within the retina that are projected to the brain,are located in the inner most layer of the retina,that is the ganglion cell layer.From the perspective of embryology,the retina is derived from the neuroectoderm,belonging to the central nervous system(CNS).Thus,the optic nerve,although nominally belongs to the peripheral nervous system(PNS),but its essence is a part of the CNS,belonging to CNS white matter.Although the adult mammalian CNS,including the optic nerve,has been proved to have certain ability of regeneration and the research of CNS regeneration has made some progress,but the spontaneous regeneration after CNS injury is limited,and it is not enough to restore the injured nerve function.The glial microenvironment plays an important role in the CNS regeneration.Therefore,it has important theoretical and clinical significance to study the molecular mechanism of glial microenvironment on CNS regeneration,which has important theoretical and clinical significance for repairing optic nerve injury or other CNS damage.The Nogo,myelin-associated glycoprotein(MAG)and oligodendrocyte myelin glycoprotein(Omgp)in the adult mammalian CNS myelin and chondroitin sulphate proteoglycans(CSPGs)in glial scar are the major inhibitory molecules of CNS regeneration.The Nogo,MAG and OMgp have a common receptor--the Nogo receptor(NgR).The NgR,a glycosyl phosphatidylinositol anchor chain protein,lacks intracellular domain,so it does not directly transduce the extracellular signal into the cell.Thus,the NgR must be formed with other transmembrane molecules to form a composite receptor to tansduce signal into the cell.The p75,a tumor necrosis factor family member,is an important accessory receptor for NgR.These inhibitory molecules binding to their respective receptors will activate the final common pathway of RhoA/ROCK signaling pathway.The activation of ROCK will further activate LIM kinase to inhibit Cofilin protein,resulting in growth cone atrophy and inhibition of axonal growth.This is the common signaling pathway of the inhibitory molecules in CNS glial microenvironment to inhibit CNS regeneration.Because p75 lacks GDP/GTP exchange factor structure domain,it can not directly activate RhoA,needing for other molecules to mediate the interaction.The protein kinase C(PKC)is an important molecule that mediates the signal transduction of p75 and inhibits the growth of CNS axons in adult mammals.Protein tyrosine phosphatase sigma(PTPσ)is the receptor of inhibitory molecule CSPGs and CSPGs also needs PKC activation.Thus,PKC is an important regulator of Nogo-A,MAG,Omgp,CSPGs and other molecules that inhibit the regeneration of CNS.However,whether PKC is the upstream molecule of RhoA in the process of adult mammalian CNS regeneration is inconsistent in the literatures.It reported that the epriments of rat cerebellar granule cells 7 days after birth cultured in vitro showed that inhibition of PKC could inhibit RhoA activity.In other study of in vitro culture of cerebellar neurons 7 days after birth,it reported that inhibition of PKC did not inflence RhoA activity.This suggests that the downstream signaling molecules acting on PKC may not be the same in different neurons.After optic nerve injury,including trauma,inflammation and hypoxia,the nuclear factor kappa B(NF-κB)and PKC expression significantly increased,and inhibition of PKC or NF-κB significantly decreased the apoptosis of RGCs.The NF-κB is an important transcription factor,which is involved in a series of pathophysiological processes such as inflammation,cancer genesis,neuron degeneration,diabetes and stroke.In normal cells,the NF-κB is present in the cytoplasm of the cell.When the cells were stimulated by extracellular signals,the NF-κB is activated and rapidly shifts to the nucleus,and the transcription of the gene is induced by NF-κB.The activation of NF-κB by PKC has important role in many cellular processes.In addition,activation of NF-κB induces suppressor of cytokine signaling 3(SOCS3)to transfer to the nucleus and bind to DNA to induce gene expression.This process is regulated by PKC.Deletion of SOCS3 gene expression significantly promoted adult mouse optic nerve regeneration.Therefore,we hypothesized that inhibition of PKC,mediating by inhibiting the expression of NF-κB,can down-regulate SOCS3 expression,and then promote the adult rat optic nerve regeneration.We intend to validate this hypothesis by in vivo experiments and in vitro experiments.The obtectives of this study include that the effects of inhibition of PKC/NF-κB/SOCS3 signaling pathway on adult rat optic nerve regeneration in vivo and the role of inhibition of PKC/NF-κB/SOCS3 signaling pathway in Nogo-A inhibits RGC axon regeneration in vitro.Experiment 1:Effect of inhibition of PKC/NF-κB/SOCS3 signaling pathway on adult rat optic nerve regeneration in vivoObjective To investigate the effect of inhibition of PKC/NF-κB/SOCS3 signaling pathway on adult rat optic nerve regeneration in vivo.Methods In this experiment,we used the sciatic nerve autograft transplantation model and the technique of fluorescence retrograde labeling regenerating RGCs.Sixty-one adult SD rats were randomly divided into 6 groups,i.e.,control group(n=17),DMSO group(n=13),Go6976 group(n=13),PDTC group(n=9),and PBS group(n=9).Animal intervention method:① DMSO group,5 μl of DMSO(G66976 solvent)was used by intravetreous injection,once per 5 days,until executing the rats;② Go6976 group,5 μl of PKC inhibitor Go6976 was used by intravetreous injection,once per 5 days,until executing the rats;③ PDTC group,1 ml of NF-κB p65 inhibitor PDTC(concentration at 20 mg/ml)was used by intraperitoneal injection daily until executing the rats;④ PBS group,100 μl of PBS was used by intraperitoneal injection daily until executing the rats;⑤ control group,no reagents intervention was given.Four week after sciatic nerve transplantation,5 rats in each group were killed for analysis of the number of regenerating RGCs,16 rats in DMSO group and Go6976 group were killed for Western blotting analysis of the level of retinal phosphorelated NF-κB p65(4 rats in each group)and preotein expression level of SOCS3(4 rats in each group),and 8 rats in PBS and PDTC groups were killed for Western blotting analysis of the preotein expression level of SOCS3(4 rats in each group.Another 12 normal rats were used for analysis of the normal level of rat retinal PKC activity,phosphorelated NF-κB p65 and protein expression level of SOCS3.The statistical analysis was performed by SPSS 18.0 software.The quantitative data were expressed as mean ± standard deviation.The student-t test,one-way ONOVA and the Turkey test were used.Differences were considered statistically significant when P<0.05.Results The activity of retinal PKC,the leve of phosphorelated NF-κB p65 and the protein expression level of SOCS3 were significantly higher in control group than normal group(P<0.05).The activity of retinal PKC,leve of retinal phosphorelated NF-κB p65 and protein expression level of retinal SOCS3 in Go6976 group were significantly lower than the control group(P<0.05),but DMSO group had no statistical difference with control group(P>0.05).The leve of retinal phosphorelated NF-κB p65 and the protein expression level of retinal SOCS3 in PDTC group were significantly lower than control group(P<0.05),but the activity of retinal PKC had no significant change(P>0.05).The activity of retinal PKC,the leve of retinal phosphorelated NF-κB p65 and the protein expression level of retinal SOCS3 in PBS group had no statistical differences with model group(P>0.05).The number of regenerating RGCs in control group had no statistical differences with DMSO group.The number of regenerating RGCs was significantly higher in Go6976 group than control and DMSO groups(P<0.05).The number of regenerating RGCs in PBS group had no statistical differences with control group(P>0.05).The number of regenerating RGCs was significantly higher in PDTC group than control and PBS groups(P<0.05).Conclusions The activity of retinal PKC,the leve of retinal phosphorelated NF-κB p65 and the protein expression level of retinal SOCS3 significantly increase after optic nerve injury in adult rat.Inhibition PKC can significantly reduce the level of retinal phosphorelated NF-κB and the protein expression level of retinal SOCS3 after optic nerve injury in adult rat.Inhibition NF-κB can significantly reduce the preotein expression level of retinal SOCS3 after optic nerve injury in adult rat.Inhibition PKC and NF-κB can promote axon regeneration in adult rat after optic nerve injury.Our results suggest that inhibition of PKC,mediating by NF-κB,can down-regulate SOCS3 expression,and thus promote regeneration of optic nerve in adult rats.Experiment 2:Role of PKC/NF-κB/SOCS3 signaling pathway in the inhibition of Nogo-A to the axon regeneration of rat RGCs in vitroObjective To investigate the role of PKC/NF-κB/SOCS3 signaling pathway in the inhibition of Nogo-A to the axon regeneration of rat RGCs in vitro.Methods Nogo-A was used to inhibit the axon regeneration of cultured RGCs.Experimental groups:normal group(RGCs were cultured in DMEM with neurothrophic factor),Nogo-A group(culture medium cantaining soluble Nogo-A),Go6976 group(culture medium cantaining soluble Nogo-Aand PKC inhibitor Go6976),PDTC group(culture medium containing soluble Nogo-A and NF-κB inhibitor PDTC)SOCS3-siRNA group(culture medium containing soluble Nogo-A and SOCS3 siRNA to silence SOCS3 gene expression).Each group was duplicated for 4 times.The wesrern blotting analysis was performed to detect the phosphorelated level of NF-κB p65 and the protein expression level of SOCS3.The PKC activity assay was performed using a nonradioactive protein kinase assay kit.The βⅢ-tubulin immunofluorescence staining was performed to measure the nrueite length of cultured RGCs.The statistical analysis was performed by SPSS 18.0 software.The quantitative data were expressed as mean ± standard deviation.The Mann-Whitney U test,one-way ONOVA and the Turkey test were used.Differences were considered statistically significant when P≤0.05.Results The activity of PKC,the level of phosphorelated NF-κB p65 and the protein expression level of SOCS3 in Nogo-A group were significantly higher than normal group(P<0.05).The activity of PKC,the level of phosphorelated NF-κB p65 and the protein expression level of SOCS3 in Go6976 group were significantly lower than Nogo-A group(P<0.05).The level of phosphorelated NF-κB p65 and protein expression level of SOCS3 in PDTC group were significantly lower than Nogo-A group(P<0.01),but the activity of PKC had no significant differences with Nogo-A group(P>0.05).The protein expression level of SOCS3 was significantly lower in SOCS3-siRNA group than Nogo-A group(P<0.05),but the activity of PKC and level of phosphorelated NF-κB p65 in SOCS3-siRNA group had no statistical differences with Nogo-A group(P>0.05).The mean neurite length of cultured RGCs was significantly shorter in Nogo-A group than normal group(P<0.05).The mean neurite length of cultured RGCs was significantly longer in Go6976 group than Nogo-A group(P<0.05).The mean neurite length of cultured RGCs was significantly longer in PDTC group than Nogo-A group(P<0.05).The mean neurite length of cultured RGCs was significantly longer in SOCS3-siRNA group than Nogo-A group(P<0.05).Conclusions Nogo-A can increase the activity of PKC,the level of phosphorelated NF-κB p65 and the protein expression level of SOCS3 in cultured rat RGCs.Inhibition of PKC,mediating by NF-κB,can inhibit SOCS3 expression.Inhibition of PKC,NF-κB and SOCS3 are able to promote axon regeneration of cultured RGCs.Thus,Nogo-A can up-regulate the expression of SOCS3 by activating PKC and NF-κB,thereby inhibiting cultured adult rat RGC axon regeneration.