The Study Of TGF-β1 Effect On Wallerian Degeneration After Rat Sciatic Nerve Injury

Objective: Wallerian degeneration(WD) remains an important area of research in modern neuroscience. Many protein expressions are regulated by differentially expressed genes of WD, but the precise mechanisms are elusive. Methods: In this study, we profiled the differentially expressed proteins in WD after rat sciatic nerve injury using an antibody array. Results: Functional analysis positively identified cell proliferation, regulation of cell proliferation and immune system process. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis revealed molecular networks relating mainly to cytokine-cytokine receptor interaction, mitogen-activated proteinkinases(MAPK) signaling pathway, apoptosis, toll-like receptor(TLR) signaling pathway and Janus kinase(Jak)- signal transducer and activator of transcription(STAT) signaling pathway. The interactions between these differential proteins were well established and regulated by the key factors transforming growth factor beta 1(TGF-β1), toll-like receptor 4(TLR4), Fas ligand(Fas L) and 5’-AMP-activated protein kinase catalytic subunit alpha-1(PRKAA1). Discussion: These results will provide information relating to the functional analysis of differentially expressed genes in terms of the regulation of nerve degeneration and/or regeneration by WD.Methods: 1. Establishment of rat sciatic nerve injury models; 2. Protein isolation and biotin label-based antibody array scanning; 3. Bioinformatic analysis; 4. Real-time quantitative PCR assay; 5. Western blot analysis; 6. Statistical analysis.Results: 1. Functional analysis positively identified cell proliferation, regulation of cell proliferation and immune system process. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis revealed molecular networks relating mainly to cytokine-cytokine receptor interaction, mitogen-activated proteinkinases(MAPK) signaling pathway, apoptosis, toll-like receptor(TLR) signaling pathway and Janus kinase(Jak)- signal transducer and activator of transcription(STAT) signaling pathway. The interactions between these differential proteins were well established and regulated by the key factors transforming growth factor beta 1(TGF-β1), toll-like receptor 4(TLR4), Fas ligand(Fas L) and 5’-AMP-activated protein kinase catalytic subunit alpha-1(PRKAA1). 2. Real-time PCR was used to verify the differential gene expression, such as CTNNB1, ICAM1, TGFB1, MMP8, FASL, which were consistent with the Microarray results. Western blot was used to verify differential protein expression, such as CTNNB1, ICAM1, TGFB1, MMP8, FASL, which were also consistent with the gene chip results.Conclusions: 1. Many proteins are differential expressed during WD, Functional analysis positively identified cell proliferation, regulation of cellproliferation and immune system process. KEGG analysis revealed molecular networks relating mainly to cytokine-cytokine receptor interaction, MAPK signaling pathway, apoptosis, TLR signaling pathway and Jak-STAT signaling pathway. The interactions between these differential proteins were well established and regulated by the key factors TGF-β1, TLR4, Fas L and PRKAA1. 2. The data of real-time quantitative PCR and Western Blot clearly confirmed the results of Microarray and bioinformatic analysis.Objective By using molecular biology technology and bioinformatic analysis, we investigated the molecular mechanisms of Wallerian degeneration(WD) regulating nerve degeneration and regeneration, and studied the molecular mechanism of TGF-β1 involving in neural restoration and regeneration in this process. The study provided us with a firmer basis on the molecular mechanisms for repairing the nerve injury and functional recovery.Methods 1. Construction of rat sciatic nerve transaction models; 2. Immunohistochemistry(IHC) and Immunocytochemistry(ICC) analysis; 3. Establishment of TGF-β1 recombinant plasmid; 4. RNA interference and overexpression of TGF-β1; 5. Real-time PCR and Western Blot analysis; 6. Ed U proliferation assay, Annexin V apoptosis assay, and Transwell migration assay; 7. Statistical analysis.Results: 1. IHC and ICC analysis the co-location of TGF-β1 and S100B;2. The rate of Schwann cells proliferation and apoptosis decreased after the transfection of si-TGF-β1; 3. The rate of Schwann cells(SCs) proliferation and apoptosis increased after the overexpression of TGF-β1; 4. Real-time PCR analysis of differentially expressed genes associative to nerve injury after the interference and overexpression of TGF-β1; 5. Western Blot analysis the crucial factor in the smad pathway after the interference and overexpression of TGF-β1; 6. Western Blot analysis the signal pathway related to cell proliferation and apoptosis after the interference and overexpression of TGF-β1.Conclusions 1. TGF-β1 promotes SCs proliferation and apoptosis, insignificant change appears in the migration of SCs; 2. The alteration of crucial factor in the smad pathway is consistant with TGF-β1, which means TGF-β1 may regulate SCs by smad pathway; 3. TGF-β1 may interact with AKT signal pathway in the SCs.