The Neuroprotective Effect Of Netrin-1 And Its Mechanisms After Spinal Cord Injury In Rats

Objective: Spinal cord injury(SCI)is a serious crippling disease of central nervous system.Due to the non-reproducibility of the neurons,patients often endure different degrees of dysfunction of movement,sensation,sphincter and so on.Spinal cord injury often leads to a significant decline in the qualities of patients' life and high cost of medical care often bring huge financial burden to the families.The pathology of SCI is usually divided into two phases: primary and secondary injury.The primary injury refers to damages caused by direct or indirect external forces on spinal cords and the secondary injury is a complex series of responses including local edema,ischemia,focal hemorrhage,oxidative stress,and inflammatory responses,which occurs on the basis of the primary injury.Because of the controllability of a series of pathophysiological processes happened in the secondary injury,which could affect the prognosis of patients to a large extent.Therefore,the regulation of the pathogenesis in the secondary injury is one of the research focuses in the treatment of spinal cord injury.Autophagy is a lysosomal dependent degradation process,which widely exist in the eukaryotic cells.The cellular components are sequestered by the bilayer membrane to form autophagosomes(Aps)and,subsequently,fused with lysosomes to digest and degrade the contents by different enzymes.Autophagy allows cells to realize metabolisms of itself and renew some organelles,which plays an irreplaceable role in maintaining cellular homeostasis.The studies found that the enhancement of autophagy in injuried region could improve the prognosis in the models of spinal cord injury and traumatic brain injury.For example,Autophagy reduces neuronal damage and promotes locomotor recovery via inhibition of apoptosis after spinal cord injury in rats.Rapamycin could exert neuroprotective effects via improving autophagy in the model of traumatic brain injury in mice.Netrin-1,which belongs to the Netrins family,is initially recognized as a neuronal guidance factor which mediate the migration of neurons and the growth of axons during embryonic development.With the in-depth research,the researchers have found that Netrin-1 were continuously expressed in the nervous systems of vertebrate animals.In addition,Netrin-1 could activate AMPK to affect axon growth of neurons by increasing the phosphorylation of AMPK via interaction with its receptor,Down syndrome cell adhesion molecule(DSCAM).It is well known that the AMPK/m TOR signaling pathway play an important role in the regulation of autophagy,which hint us whether Netrin-1 could play a neuroprotective role through regulating autophagy in neurons from injured spinal cord by activating AMPK/m TOR signaling pathway? the potential mechanisms? Therefore,we have designed the following three parts: Part 1:The experimental study on the changes of autophagic expression in injured spinal cord after spinal cord injury in rats.Part 2: The preliminary experimental study of the neuroprotective role of Netrin-1 and its mechanism in rats after spinal cord injury.Part 3:The further experimental study of the neuroprotective mechanisms of Netrin-1in rats after spinal cord injury.Methods: Part ?: Adult female Sprague-Dawley rats(220-250 g)were randomly divided into two groups,namely sham group and SCI group,then SCI group was divided into five subgroups according to the time points after SCI.After a model of SCI was established by using the modified Allen's method,injured spinal cords were collected at4 h,1d,3d,7d,14 d after SCI.The expressions of LC3 and Beclin-1,two autophagy markers,were detected with Western blots at different time points after SCI.The numbers of double-labeled positive neurons of LC3 or Beclin-1 were detected by immunofluorescence double labeling method.Part ?: Rats were randomly divided into five groups,namely Sham group,SCI group,Netrin-1 group,Netrin-1+Compound C group and Compound C group.After a model of SCI was established by using the modified Allen's method,the injured spinal cords were collected for Western blots or immunofluorescence double labeling at 3d after SCI.The expressions of AMPK,p-AMPK,m TOR,p-m TOR,P70S6 K,and p-P70S6 K,which were associated with AMPK/m TOR signaling pathway,were detected by Western blot at 3d after SCI.In addition,the expressions of autophagy markers LC3 and Beclin-1 as well as apoptosis related protein Cleaved-Caspasse3 were also detected by Western blot.The numbers of double-labeled positive neurons of LC3 or Beclin-1 were detected by immunofluorescence double labeling method at 3d after SCI.The ratios of apoptotic neurons were detected by TUNEL method.The residual areas of transverse section of spinal cords from rats in each group were detected by HE staining at 21 d after SCI.The numbers of surviving neurons in anterior horns of injured spinal cords from rats in each group were counted using Nissl staining at 21 d after SCI.The BBB scores of rats in each group were measured at 0d,1d,3d,7d,14 d,21d after SCI.Part ?: Rats were randomly divided into four groups,namely Sham group,SCI group,Netrin-1 group and Compound C group.After a model of SCI was established by using the modified Allen's method the injured spinal cords were collected for Western blots or immunofluorescence double labeling at 3d after SCI.The expressions of p-AMPK,p-m TOR,p-ACC and p-P70S6 K,which were associated with AMPK/m TOR signaling pathway,were detected by Western blot at 3d after SCI.In addition,the expressions of proteins associated with the synthesis of lysosomes(LAMP1,CTSD and ATP6V1A),apoptosis related protein(Cleaved-Caspasse3,Bax and Bcl-2),autophagy markers(LC3and Beclin-1),autophagic substrate p62 and Transcription factor EB(TFEB)were also detected by Western blot at 3d after SCI.The fluorescence densities of LAMP1 in neurons,the ratios of Nuclear translation of TFEB in neurons and the ratio of LC3,P62 or Cleaved-Caspase3-positive neurons were respectively detected by immunofluorescence double labeling method at 3d after SCI.The numbers of surviving neurons in anterior horns of injured spinal cords from rats in each group were counted using Nissl staining at 28 d after SCI.The BBB scores of rats in each group were measured at 0d,1d,3d,7d,14 d,21d and 28 d after SCI.Result: Part ?: The results of Western blot detection suggested the expressions of Beclin-1 and the ratios of LC3-II/LC3-I in SCI group were both obviously higher than those in Sham group at 4h after SCI.Over time,the expressions Beclin-1 and the ratios of LC3-II/LC3-I gradually increased and peaked at 3d after SCI.Although expressions Beclin-1 and the ratios of LC3-II/LC3-I gradually decreased after that,they were still remarkably higher than those in Sham group at both 7d and 14 d after SCI.With immunofluorescence double labeling method,our results suggested that the numbers of double-labeled positive neurons of LC3 or Beclin-1were both significantly higher than those in Sham group at 3d after SCI.Part ?: The results of Western blot suggested the AMPK/m TOR signaling pathway was activated in injured spinal cords of rats from SCI group compared with that in Sham group,which is characterized by a significant increase in the ratios of p-AMPK/AMPK and a significant decrease in the ratios of p-m TOR/m TOR and p-p70s6k/p70s6 k.In addition,the expressions of Becin-1 and the ratios of LC3-II/LC3-I significantly increased in injured spinal cords of rats with SCI.In comparison to SCI group,Netrin-1treatment further activated the AMPK/m TOR signaling pathway and significantly enhanced the ratios of LC3-II/LC3-I as well as the expressions of Beclin-1 in injured spinal cords of rats with SCI.Compound C,an AMPK inhibitor,could suppress the activation of AMPK/m TOR signaling pathway caused by Netrin-1 and decrease the ratios of LC3-II/LC3-I as well as the expressions of Beclin-1induced by Netrin-1.In comparison with Sham group,the numbers of double-labeled positive neurons of LC3 or Beclin-1 and the ratios of TUNEL-positive neurons in anterior horns of injured spinal cords from rats in SCI group significantly increased.In addition,the residual areas of transverse sections of injured spinal cord and the numbers of surviving neurons in anterior horns of injured spinal cords were obviously reduced.In comparison to SCI group,Netrin-1 treatment not only remarkably increased the numbers of double-labeled positive neurons of LC3 or Beclin-1,the numbers of surviving neurons in anterior horns of injured spinal cords and the residual areas of transverse sections of injured spinal cord,but also significantly decreased the ratios of apoptotic neurons.There is no statistical significance between the BBB scores of rats in Netrin-1 group and those in SCI group before 14 d after SCI.The BBB scores of rats in Netrin-1group were significantly higher than those in SCI group from 14 d after SCI.Part ?: The results of Western blot detection suggested the AMPK/m TOR signaling pathway was activated in injured spinal cords of rats from SCI group compared with that in Sham group,which is characterized by a significant increase in the expressions of p-AMPK and p-ACC together with a significant decrease in the expressions of p-m TOR and p-p70s6 k.In addition,the expressions of LAMP1,CTSD,ATP6V1 A,Cleaved-Caspasse3,p62,TFEB in the nucleus of neurons and the ratios of both LC3-II/LC3-I and Bax/Bcl-2 were remarkably higher than those in Sham group.In comparison to SCI group or Netrin-1+Compound C group,Netrin-1 could further activated the AMPK/m TOR signaling pathway and significantly enhance the expressions of LAMP1,CTSD,ATP6V1 A,TFEB in the nucleus of neurons and increase the ratios of LC3-II/LC3-I in injured spinal cords.In addition,Netrin-1 remarkably reduced the expressions of both Cleaved-Caspasse3 and p62 and significantly decrease the ratios of Bax/Bcl-2 in injured spinal cords.In comparison with Sham group,the ratios of LC3,p62 or Cleaved-Caspase3-positive neurons in the anterior horns of injured spinal cords from SCI group obviously increased.Besides,the fluorescence densities of LAMP1 and the ratios of Nuclear translation of TFEB in neurons from the anterior horns of injured spinal cords in SCI group significantly enhanced.In comparison with SCI group or Netrin-1+Compound C group,Netrin-1 significantly enhanced the fluorescence densities of LAMP1 and the ratios of Nuclear translation of TFEB in neurons from the anterior horns of injured spinal cords.At the same time,Netrin-1 not only obviously enhanced the ratios of LC3-positive neurons but also reduced the ratios of p62 and Cleaved-Caspase3-positive neurons in the anterior horns of injured spinal cords.NISSL staining results indicated the numbers of surviving neurons in anterior horns of injured spinal cords were obviously reduced and Netrin-1 could significantly increase those in Netrin-1 group.However,the combined treatment of Netrin-1and Compound C obviously reduced the numbers of surviving neurons in anterior horns of injured spinal cords.There is no statistical significance between the BBB scores of rats in SCI group and Netrin-1+ Compound C group throughout the whole experiment.There is no statistical significance between the BBB scores of rats in Netrin-1 group and those in SCI group before 14 d after SCI.The BBB scores of rats in Netrin-1 group were significantly higher than those in SCI group or Netrin-1+ Compound C group from 14 d after SCI.Conclusion: Part ?: Autophagy in injured spinal cord tissue from rats with spinal cord injury significantly increased from 4h after SCI and peaked at 3d.Part ?: 1.Netrin-1 could enhance autophagy by AMPK/m TOR signaling pathway in neurons of injured spinal cords following SCI in rats.2.Netrin-1 could exert neuroprotective effects through inhibiting neuronal apoptosis by promoting autophagy in neurons of injured spinal cords following SCI in rats.Part ?: 1.Netrin-1 could enhance lysosomal biogenesis by regulating the Nuclear translation of TFEB via AMPK/m TOR signaling pathway in neurons of injured spinal cords following SCI in rats.2.Netrin-1 could elevate autophagic flux by promoting the degradation of APs via the enhancement of lysosomal biogenesis and upregulating the formation of APs in neurons of injured spinal cords following SCI in rats.3.Netrin-1 could exert neuroprotective effects through inhibiting neuronal apoptosis by promoting autophagic flux in neurons of injured spinal cords following SCI in rats.