The Effects Of GPNMB In Promoting The Repair Of Peripheral Nerve Injury And Its Mechanism

Peripheral nerve injury(PNI)is a common clinical injury,and there are many factors involved in its repair and regeneration,in which schwann cells(SCs)play an important role.After PNI,SCs up-regulate the expression of various neurotrophic factors(NTFs)and adhesion molecules,and recruit macrophages locally in the lesion,phagocytose the cleaved axons and myelin together with macrophages,and improve the regeneration microenvironment.SCs also form Büngner's band in the basement membrane and guide axon regeneration.Furthermore,SCs surround regenerated axons to form myelin sheath and promote nerve functional recovery after axonal regeneration.Long-term denervation of the SCs in the injured distal segments leads to a decrease in the ability of peripheral nerve regeneration and affects nerve repair.Therefore,improvement of the regeneration microenvironment,which is improved by activation of denervated SCs,promotion of SCs proliferation,differentiation,migration,expression and secretion of NTFs or supplementing exogenous trophic factors,small molecules and so on,are very important strategies for the treatment of PNI.Glycoprotein Non-Metastatic Melanoma Protein B(GPNMB),also known as osteoactivin(OA),or heparin integrin ligand dendritic cells(DC-HIL),or hematopoietic growth factors induce neurokinin-? type(HGFIN),is a type ? transmembrane protein that was first found in melanoma cells.GPNMB is distributed in the central nervous system widely and plays vital roles including improving memory,anti-inflammation,reducing neuronal death and neuroprotection.GPNMB is also expressed in the peripheral nervous system,especially in SCs.However,the role of GPNMB in the activation or protection of SCs and peripheral nervous systems,as well as the role in peripheral nerve regeneration and repair,remains unclear.Based on the above considerations,in this study we will investigate the expression trend of GPNMB after PNI by microarray analysis of distal segments of sciatic nerve injury(SNI)model,and explore the effects of GPNMB on peripheral nerve regeneration and repair and SCs,as well as the underlying mechanisms,in order to provide theoretical and experimental basis for the clinical application of GPNMB.Part ? Expression change of GPNMB after SNIObjective: Using microarray analysis of the distal segments from SNI model to explore the expression changes of genes at the distal stumps after PNI,especially the expression change of GPNMB.Methods: Microarray analysis was performed on the distal stumps at 0,1,3,7,14,21 and 28 d after sciatic nerve transection.STEM analysis was used to select gene expression profiles with significant tendency,and then bioinformatics prediction and cluster analysis were performed.The gene which was the most obviously changed was selected to validation by qRT-PCR,WB,and IHC.Results: It was showed that there were 12 gene expression profiles with significant tendency at 0,1,3,7,14,21,and 28 d after sciatic nerve transection.And the expression tendency of profile 41 was increased,peaked,and then decreased which was consistent with the proliferation of acute denervated SCs in the distal segments.GO and KEGG analysis predicted that the genes in profile 41 might be involved in cell proliferation.Cluster analysis showed that the expression of GPNMB which was the most significantly changed gene in profile 41 rose from 1 d,increased rapidly from 3 d,peaked at 7 d which is about 48 times of that at 0 d,then decreased from 14 d,and dropped to about 15 times of that at 0 d until 28 d.Furthermore,results of qRT-PCR,WB,and IHC showed that GPNMB had the same tendency as the results of microarray analysis.Conclusion: The expression tendency of GPNMB which was the most obviously changed in profile 41 was increased,peaked,and then decreased at 0,1,3,7,14,21,and 28 d after SNI.And GPNMB might be involved in the process of self-regeneration and repair after SNI,especially in the process of cell proliferation.Part ? GPNMB promotes the regeneration and repair of SNIObjective: To explore the effects of GPNMB on regeneration and repair of SNI.Methods: Subepineurium injection of recombinant human GPNMB(rhGPNMB)or PBS into multisite from the distal to proximal stump of sciatic nerve was performed after three weeks when sciatic nerve severe crush model was established.And rats were randomly divided into three groups: PBS group(phosphate-buffered saline(PBS)injection),100 ng rhGPNMB group(100ng rhGPNMB injection),and 500 ng rhGPNMB group(500ng rhGPNMB injection).Then four weeks later,the rats were sacrificed for further detection.Immunofluorescence was performed to detect the proliferation of SCs and the regeneration of axons and myelin sheath.The ultrastructure of myelin sheath was observed by transmission electron microscope(TEM)and G-Ratio was calculated.The nerve excitation conduction velocity(NCV)and action potential amplitude(APA)of these injured nerves was also measured in vitro.The walking footprints of rats were observed weekly after the injection of rhGPNMB,and then sciatic nerve functional index(SFI)was calculated to investigate the functional recovery of injured nerves.The gross morphology of gastrocnemius was first observed,and then gastrocnemius muscle was weighed and stained by HE.Results: The number of SCs,regenerated axons and myelin,and the expression of proliferating cell nuclear antigen(PCNA)in the rhGPNMB groups were significantly higher than those in the PBS group where the differences were statistically significant.The number of SCs which had more regular shape and arrangement in the 100 ng rhGPNMB and 500 ng rhGPNMB groups was 1.55±0.21 and 2.75±0.28 times of that in the PBS group,respectively.In addition,the number of regenerated axons which possessed more uniform distribution,larger diameter,and more complete continuity in the 100 ng rhGPNMB and 500 ng rhGPNMB groups was 1.38±0.19 and 1.78±0.18 times of that in the PBS group,respectively.Furthermore,the number of regenerated myelin sheath which had better integrity and more uniform distribution in the 100 ng rhGPNMB and 500 ng rhGPNMB groups was 1.18±0.18 and 1.67±0.08 times of that in the PBS group,respectively.The results of TEM showed that the number of regenerated myelin sheath which was with more complete shape,thicker,and denser myelin lamina,and more regular arrangement of microtubules,filaments and other ultrastructures in the rhGPNMB groups was larger than that in the PBS group.Then we calculated the G-Ratio value and found that the G-Ratio value in the 500 ng rhGPNMB group was lowest among these three groups where the differences were statistically significant,indicating that the thickness of the regenerated myelin sheath in this group was the thickest.It was showed that APA of injured sciatic nerve from the 100 ng rhGPNMB and 500 ng rhGPNMB groups was 1.10±0.22 and 1.44±0.30 times of that from the PBS group,respectively.And the NCV from the 100 ng rhGPNMB and 500 ng rhGPNMB groups was 1.32±0.08 and 2.15±0.17 times of that from the PBS group,respectively.And the differences among these three groups were statistically significant.It was found that SFI of these three groups which was calculated based on the walking footprints was in decline.And four weeks after injection,SFI of the PBS,100 ng rhGPNMB,and 500 ng rhGPNMB groups was-72.83±5.80,-60.06±4.77,and-40.60±5.00,respectively,and the differences among these three groups were statistically significant,meaning that there was functional recovery,to some extent,in all these three groups during sciatic nerve regenerative process and rhGPNMB promoted functional recovery of sciatic nerve after injury.According to the gross morphology of gastrocnemius from these three groups,it was found that the gastrocnemius atrophy of the rhGPNMB groups was less than that of the PBS group,especially the 500 ng rhGPNMB group.And the weight ratio of gastrocnemius in the PBS,100 ng rhGPNMB,and 500 ng rhGPNMB groups was(44.68±4.08)%,(54.55±3.05)%,and(66.85±4.10)%,respectively,and the differences among these three groups were statistically significant.Furthermore,the results of HE staining revealed that the diameter and cross-sectional area(CSA)of the muscle fibers in the rhGPNMB groups were larger than those in the PBS group where the differences among these three groups were statistically significant,especially the 500 ng rhGPNMB group.Conclusion: GPNMB promoted the proliferation of SCs and regeneration of axons,and accelerated the remyelination of injured nerves.Furthermore,GPNMB improved the electrophysiological properties,promoted functional recovery of injured nerves,and alleviated the gastrocnemius atrophy.In general,GPNMB promoted the regeneration and repair of SNI.Part ? Effects of GPNMB on SCs and the underlying mechanismsObjective: To investigate the effects of GPNMB on SCs and the underlying mechanisms.Methods: The proliferation of RSC96 was detected by CCK8 and WB when RSC96 was treated with 12.5ng/ml or 25ng/ml rhGPNMB in vitro.And the apoptosis and migration of RSC96 were detected by TUNEL and cell scratch assay,respectively.Then qRT-PCR,WB,and ELISA were used to measure the expression and secretion of neurotrophic factors(NTFs)and adhesion molecules.The phosphorylation of Erk1/2 and Akt was also detected by WB to explore the underlying mechanisms.Co-immunoprecipitation(Co-IP)and double-labeling immunofluorescence were performed to confirm the protein which rhGPNMB bound on the membrane of RSC96.Co-cultured inhibitor of this confirmed protein or lentivirus which carried siRNA of this confirmed protein and rhGPNMB with RSC96,and then the proliferation and migration of RSC96 were detected by CCK8 and cell scratch,respectively.And then,the expression and secretion of NTFs and adhesion molecules was also tested by WB and ELISA.The phosphorylation of Erk1/2 and Akt was detected by WB.Results: There was no significant difference in proliferation when RSC96 were treated with rhGPNMB at 1 d,however,there were significant differences in proliferation among these three groups from 2 d.And the expression of PCNA in RSC96 of the rhGPNMB groups were significantly higher than that of the control group at 3 d,especially the 25 ng/ml rhGPNMB group.The 25 ng/ml rhGPNMB group proliferated significantly faster than the 12.5 ng/ml GPNMB and control groups and the differences among them were statistically significant.rhGPNMB had no significant effect on apoptosis of RSC96.The wound healing rate of the 25ng/ml rhGPNMB group was faster than the other two groups.At 48 h,wound healing reached(70.35±0.20)% in the 25ng/ml rhGPNMB group,while they were(59.74±1.76)% and(41.23±0.82)% in the 12.5ng/ml rhGPNMB group and the control group,respectively,and the differences among them were statistically significant.rhGPNMB promotes the expression and secretion of NGF,BDNF,NT-3 and N-cadherin at transcriptional and protein level.Meanwhile,the p-Erk/total-Erk and p-Akt/total-Akt of the 25 ng/ml rhGPNMB group was 4.20±0.21 and 2.58±0.52 times of that of the control group,which was higher than the other two groups,and the differences among them were statistically significant.The results of Co-IP and double-labelling immunofluorescence showed that rhGPNMB could co-precipitate and co-localized with NKA ?1 of RSC96,indicating that rhGPNMB bind with NKA ?1 on the membrane of RSC96.It was found that the proliferation rate,wound healing rate,expression and secretion of NTFs and N-cadherin,p-Erk/total-Erk,and p-Akt/total-Akt of Ouabain(NKA antagonist)+rhGPNMB group were lower than rhGPNMB group and the difference between them was statistically significant when Ouabain and rhGPNMB co-treated with RSC96.However,there were no significant differences among the Ouabain+rhGPNMB,control,and Ouabain groups.The proliferation rate,wound healing rate,expression and secretion of NTFs and N-cadherin,p-Erk/total-Erk,and p-Akt/total-Akt in the NKA ?1 siRNA+rhGPNMB group was lower than the rhGPNMB,NC siRNA+rhGPNMB groups,but higher than the other three non-rhGPNMB treated groups when NKA ?1 siRNA and rhGPNMB co-treated with RSC96.And the differences among these groups were statistically significant.Conclusion: GPNMB might bind with NKA ?1 on the cell membrane of SCs and promoted the proliferation,migration,expression and secretion of NTFs and N-cadherin in SCs via promotion of the phosphorylation of Erk1/2 and Akt.