The Identification Of Dynamic Proteomics, Bioinformatics Analysis Of NRCF And Its Preliminary Function Experimental Study

Peripheral nerve injuries (PNI) are a common sequalae of trauma with a high incidence of disability. PNIs are often caused by accidental injury, illness or from accidental collateral damage from surgical procedures. Improper handling of PNIs may result in partial or total loss of function in the involved segments of the body; affecting the patient’s quality of life as well as greater socioeconomic costs.Clinically, the most common method of treatment for peripheral nerve injury is direct conventional suturing and autologous nerve graft transplantation. But because of problems such as neurologic beam dislocation and anastomotic scar tissue formation, it is difficult to further improve functional outcome. Further, secondary damage to the graft harvest site may occur. Therefore, peripheral nerve regeneration has become an important area of research to simulate biological processes. Current research shows that peripheral nerve regeneration relies on local microenvironment, which has the ability to to make self-regulate and self-improve. Currently, biological treatment has achieved a breakthrough and become a hotspot for nerve regeneration research. Biological treatment is based on the renovation of nerve regeneration chambers (NRC), integrated application of functional cells implantation, gene therapy and a variety of other measures.1980s, Lundborg et al successfully repaired NRC rat peripheral nerve transection defect by using a cylindrical model, which becoming a standard animal model in peripheral nerve injury. Since then, researchers developed a greater understanding about duct material, luminal contents, soluble regulation elements, functioning cells and other components of Lundborg NRC model. The closed microenvironment, provided by the NRC model, is an important window for study of peripheral nerve regeneration. Nerve Regeneration Conditioned Fluid (NRCF) is a form of functional liquid secreted by the nerve stump in the NRC model. NRCF is mainly composed of proteins and derives from the composition cells from the peripheral nerve. NRCF diffuses through the axon towards the injury site. In different periods of peripheral nerve regeneration, its expression has dynamic changes in protein types and quantities. Studies have shown that NRCF provides an important role in supporting nerve regeneration, nutrition, orientation and so on.Repair of peripheral nerve injury is a complex and sophisticated physiological process. The functional regeneration of peripheral nerve depends on specific interactions between neurons and their local microenvironment. Nerve regeneration chamber (NRC) provides a closed microenvironment, which is an important research window for peripheral nerve regeneration. Nerve regeneration conditions liquid (NRCF) is the functional component in NRC. It is an important channel to reveal the mechanism of peripheral nerve regeneration. Until now, researchers have made significant advances on microenvironment factors of peripheral nerve regeneration, such as the extracellular matrix, neurotrophic factors and cytokines. But current literature is random, scattered and speculative. Therefore, the depiction of protein components spectrum in NRCF and direct evidence for the role of microenvironment in the study of peripheral nerve regeneration is still lacking. The development of proteomics technology offers a new method to understand further the contents of NRCF. Relative and absolute quantification of isobaric tags for relative and absolute quantitation (iTRAQ) technology is an advanced proteomics technique. It can make a direct comparison of relative content or absolute content of proteins in 4 or 8 different kinds of samples, which is an effective way to study the dynamic nature of protein expression in NRCF.Given the important role of the microenvironment in peripheral nerve injury repair, conducting research regarding its functional components (NRCF) will be an important way to understand the mechanism of peripheral nerve regeneration. This paper produces for the first time a panoramic and dynamic presentation, with analyses for four key time points (post nerve transection days 3,7,14 and 25) of NRCF protein ingredients in peripheral nerve regeneration using iTRAQ technology. By comparing proteomic databases, we conducted bioinformatics analyses of co-expression proteins and shown the relationship between certain key proteins and peripheral nerve regeneration. Through experiments we preliminarily discuss the impact of NRCF on the proliferation of fibroblast and Schwann cells. Combined with previous research results, we discuss the dynamic changes and significant of different complement components in NRCF involved in peripheral nerve regeneration process.Part 1 The establishment of NRC model, extraction of NRCF and its impact on the proliferation of Schwann cells and fibroblast cellsPurposeThe typical Nerve Regeneration Chamber (NRC) model is an enclosed space formed by cylindrical silicone or other material that connect the two nerve stumps. The closed microenvironment that it provides is an important window for study of peripheral nerve regeneration. Nerve Regeneration Conditioned Fluid (NRCF) is a secretion from nerve stumps in the NRC model, which can provide support, nutrition and biological guidance for nerve regeneration. Peripheral nerve regeneration is mainly mediated by Schwann Cells (Sc) and fibroblast cells (Fb). In this part of the study, we used sciatic nerve of the Lewis rat to initially create the NRC model, then extracted NRCF, and used CCK-8 method and flow cytometry to investigate the impact of NRCF on the proliferation of Schwann cells and fibroblasts.MethodsSciatic nerve of the Lewis rat was detached and cut with a 10mm defect. The NRC model was set using a specifically sized silicone tube with the NRCF extracted 7 days later. Concentration of the NCRF was measured using the BCA method with the Schwann cells and fibroblast cells sub-cultured. Subsequently, the impact of NRCF on the proliferation of Schwann cells and fibroblast cells was evaluated using CCK-8 and flow cytometry.ResultsNRCF is a clear, pale yellow and mildly viscous fluid that is similar in appearance to serum. The physiological concentration is 15.01μg/μl. The cell bodies of Schwann cells are oval or spindle-shaped, with some having two or more dendritic processes. The bodies of fibroblasts cells are larger, have a prison or irregular triangle shape, and fuses in a braided pattern. NRCF can significantly promote the proliferation of Schwann and fibroblast cells in a time and concentration-dependent manner. Our results show that optimal proliferation of the two cell types occur at 10000ng/ml of NRCF.Conclusion1. Using sciatic nerve of Lewis rat to establish the NRC model is a stable and reliable method. NRCF extracted from this model were of a high quality; which satisfies experimental requirements. The Lewis Rat Sciatic Nerve-NRC model is an effective way to study peripheral nerve regeneration.2. Using CCK-8 method and flow cytometry techniques, preliminary results show that nerve regeneration conditions fluid (NRCF) has biological properties to promote the proliferation of Schwann cells and fibroblast cells.Part 2 Proteomics study and bioinformatics analysis of NRCF-iTRAQPurposeNerve regeneration conditions fluid (NRCF) is a proteinous secretion produced by nerves stumps in NRC that contains functional ingredients for nerve restoration. Investigation of the protein component in NRCF will provide a platform to understand the mechanisms of peripheral nerve regeneration. Using iTRAQ proteomics technology combined with mass-spectrometry technology, we extracted NRCF at the four key time points of peripheral nerve regeneration (post sciatic nerve transection days 3,7,14 and 25) and analyzed the protein expression in NRCF at each set time point, all time point and time among time points. Finally, we used the samples obtained and compared it to proteomic databases to gather a greater amount of understanding into what other proteins are also co-expressed in this process.MethodsA NRC model was established with NRCF materials extracted at days 3,7,14 and 25. The samples were analyzed using SDS-PAGE electrophoresis, iTRAQ proteomics, mass spectrometry, GO analysis, KEGG pathway analysis, hierarchical cluster analyses, co-trend analyses and genetic analyses of expression timing. These systematic investigations provided an excellent basis for qualitative and quantitative analysis of NRCF protein expression.ResultsThe protein bands in 4 groups samples of NRCF are uniform, and with good parallelism.876 proteins were identified in total. The number of protein identified in NRCF in days 3,7,14 and 25 were 536,645,376 and 548 respectively, the number of unique proteins is 95,137,18 and 58 respectively; the number of identical proteins in 4 groups was 264.Bioinformatics analysis of co-expression protein showed:TOP 10 of KEGG Pathway are 1) complement and coagulation cascades 2) staphylococcus aureus infection 3) systemic lupus erythematosus 4) pertussis 5) focal adhesion 6) prion diseases 7) amoebiasis 8) regulation of actin cytoskeleton 9) glycolysis/ gluconeogenesis 10) ECM-receptor interaction. TOP 7 proteins of co-trend analysis in Group 2, Group 3 and group 4 are 1) PROFILIN-1 2) DESTRIN-LIKE 3) ACTIN-RELATED PROTEIN 2/3 COMPLEX SUBUNIT 2 4) ANNEXIN A1 5) CORONIN-1A 6) PROTEIN S100-A8 7) PROTEIN S100-A9. CLUSTERIN, EPIDERMAL GROWTH FACTOR RECEPTOR, RETINOL-BINDING PROTEIN 4, COLLAGEN ALPHA-2(I) CHAIN, HIGH MOBILITY GROUP PROTEIN B2-LIKE, LIVER CARBOXYLESTERASE 1 and COMPLEMENT FACTOR I; EUKARYOTIC TRANSLATION ELONGATION FACTOR 1 ALPHA 2, HEMOGLOBIN SUBUNIT ALPHA-1/2, HEMOGLOBIN ALPHA 2 CHAIN, SUPEROXIDE DISMUTASE [CU-ZN], GALECTIN-5, ISOFORM 2 OF GROWTH FACTOR RECEPTOR-BOUND PROTEIN 2 and CARBONIC ANHYDRASE 2 separately. Proteins with significant differences and high connectivity information are GRB2, ACTB, ALB, VIM, VCP, A2M, CALR, THBS1, TF and PEBP1 and so on.Conclusion1. A comprehensive database of NRCF protein expression was identified using iTRAQ proteomics technology combined with mass spectrometry with samples obtained at different stages of the nerve injury repair process. Certain proteins were expressed ubiquitously throughout the experiment whilst there were also time specific proteins expressed at each stage of sample extraction. These changes reflect different stages of peripheral nerve regeneration.2. Analysis of NRCF samples showed a plethora of proteins produced in the nerve regeneration process, suggesting that they may have a significant and currently unknown role in the nerve regeneration process. Unique protein expression at different stages of the regeneration process suggests an underlying mechanical process. These data not only enrich the understanding of peripheral nerve regeneration, but also provide numerous gene and protein research targets for future nerve regeneration studies.3. The panoramic analysis of NRCF proteins and related literature review partially validate previous research understanding of nerve regeneration and open new avenues for further study of nerve regeneration.Part 3 Complement components in NRCF and its relationship with peripheral nerve regenerationPurposeNRCF protein spectra suggested the presence of a large number of the complement system components with significantly dynamic changes at each nerve regeneration period. Biological analysis (KEGG pathway analysis) showed that the complement pathways belong to’Top 10’enriched pathways. The complement system is currently understood as mediators of the immunity process by orchestrating the inflammatory response. Little is currently known about their role in nerve regeneration. From what was observed, the author believes the role of the complement cascade is worth exploring further. A large number of complement components found through NRCF-iTRAQ proteomics and its dynamic changes are described, analyzed and summarized. Western blot technique is used to verify some complement components of NRCF and possible mechanisms of action for nerve regeneration are discussed.MethodsNRC model was established and NRCF extracted at 7 days. iTRAQ proteomics analysis was carried in the same way as Section 2. Western blot technique was used to detect Clq, C3, C7 and CFD expressed in NRCF.ResultsUsing NRCF-iTRAQ proteomic analysis,10 types of complement components were obtained:CFI, Clq-A, Clq-B, C2, C3, C4, C5, C7, C8β, CFD. Western blot confirmed the expression of Clq, C3, C7 and CFD in NRCF.ConclusionIn NRCF, there exists more than 10 kinds of complement components which have qualitative and quantitative information. Quantitative analysis of complement C7 in different periods of regeneration have significant changed. Complement Clq, C3, C4 and C5 etc are closely related to nerve regeneration.