| Objective Nerve damage is a common clinical problem worldwide.Although adult peripheral nervous system(PNS)neurons can spontaneously regenerate after injury,the rate of peripheral nerve regeneration is too limited to ensure that the regenerated nerve reaches the target muscle before muscle atrophy,resulting in poor functional recovery.The general lack of regenerative capacity in the adult central nervous system(CNS)can be attributed,in part,to the lack of intrinsic ability of CNS neurons to activate unique regenerative transcriptional programs after injury.Although studies have reported that transcription factors such as STAT3,c-Jun,CREB,and ATF3 are involved in regulating the activation of the regenerative transcriptional program after peripheral nerve injury,the mechanism of transcription factors in peripheral nerve injury repair is still unclear.Systematic analysis of regulatory networks.This topic systematically analyzes the activity of transcription factors after DRG neuron injury by means of transcription factor activity screening chip,and explores the repair effect and molecular regulation mechanism of transcription factor Hoxd9 on peripheral and optic nerve injury in adult mice.Methods The TF Activation Profiling Plate Array II(Signosis)chip was used to systematically analyze the changes of transcription factor activity in DRG tissues and in vitro cultured DRG neurons at different time points after sciatic nerve injury in mice.Up-regulated;immunohistochemical analysis of Hoxd9 expression changes at different time points during DRG neuron development(P1,P5,P7,P14,P28)and after sciatic nerve injury(0 d,1 d,3 d,7 d);DRG neurons cultured in vitro,infected with Hoxd9 overexpression virus(AAV-Hoxd9),neuron microfluidic chip and in vitro resuspension experiments were used to detect the effect of Hoxd9 overexpression on axonal growth after DRG neuron injury in vitro;mice were intrathecally injected with AAV-Hoxd9 to construct In the sciatic nerve crush model,on the one hand,the pinched sciatic nerve was collected for immunohistochemical analysis of the effect of Hoxd9 overexpression on DRG neuron axon regeneration in vivo.14 d,21 d)Behavioral tests such as thermal withdrawal,mechanical pain,and gait were performed on mice,and finally,electrophysiological testing of regenerative nerves was performed to analyze the effect of Hoxd9 overexpression on sensory and motor function after peripheral nerve injury in mice;RNA-Seq analysis of the effect of overexpression of Hoxd9 on gene transcription in DRG neurons cultured in vitro;Harmonizome and JASPAR Database website to predict the target genes and binding sites of Hoxd9,combined with RNA-Seq results to analyze the molecules that Hoxd9 regulates peripheral nerve axon regeneration Mechanism;immunohistochemical analysis of Hoxd9 expression changes at different time points during optic nerve RGC development(0 d,1 d,5 d,7 d,14 d,28 d)and after injury(0 d,1 d,3 d,7 d);injected into the vitreous of mouse eyes AAV-Hoxd9,an optic nerve pinch model was constructed two weeks later,and the regulatory function of Hoxd9 on optic nerve regeneration was analyzed;finally,AAV-Hoxd9 and PTEN interfering small RNA(sh RNAPTEN)were injected into the vitreous at the same time,and the combined overexpression of Hoxd9 and knockdown of PTEN were analyzed.Effects on regeneration after optic nerve injury.Results Transcription factor activity screening found that Hoxd9 was significantly up-regulated in injured DRG neurons.In the peripheral nervous system,Hoxd9 expression was gradually down-regulated during DRG neuron development,but up-regulated and then down-regulated after sciatic nerve injury;AAV virus-mediated Hoxd9 overexpression promoted primary cultured cells in vitro compared with controls.Axonal growth after DRG neuron injury promotes axonal regeneration after sciatic nerve pinch in vivo;in functional recovery experiments,the results of thermal and mechanical pain show that overexpression of Hoxd9 can promote the recovery of sensory function,and gait results show that Overexpression of Hoxd9 can significantly enhance the recovery of motor function in mice;CMAPs indicators were collected in the electrophysiological experiment of regenerative nerves,and the results showed that compared with the control group,the amplitude of electrical signals in the overexpression Hoxd9 group was significantly improved.The prediction results of RNASeq and Harmonizome and JASPAR Database website showed that Hic2 and Hoxd9 have 3 potential binding sites,and the expression was significantly up-regulated after Hoxd9 overexpression,suggesting that it may be the target gene of Hoxd9.In the central nervous system,Hoxd9 is gradually downregulated during RGC neuron development,but is absent in adult mouse RGCs after optic nerve pinch;overexpression of Hoxd9 significantly improves regeneration after optic nerve pinch compared with controls The length and number of axons did not affect the survival of RGCs;overexpression of Hoxd9 combined with Pten knockdown treatment could significantly enhance the promotion of axon regeneration in optic neurons by a single factor.Conclusion The expression of the transcription factor Hoxd9 is gradually down-regulated during the development of mouse DRG and RGC.Peripheral nerve injury can reactivate Hoxd9 expression,but central nerve injury cannot.Exogenous overexpression of Hoxd9 in adult mouse DRG and RGC can significantly Promotes axonal regeneration after nerve injury.The regulatory effect of Hoxd9 on axon regeneration may depend on its transcriptional regulation of the target gene Hic2. |
