| Due to the extremely high specific surface area and strong interpenetration ability with other substances,nanofibers can effectively imitate the extracellular matrix to provide a good three-dimensional growth space for cells and are conducive to cell adhesion and proliferation.It is easy for most types of cells to adhere and grow on the surface of nanofibers whose diameter is much smaller than the cells’.In addition,nanofibers have excellent degradability and biocompatibility,thus they have been widely employed in applications such as drug controlled release carriers,clinical repairs,and biological tissue engineering scaffolds.The purpose of this paper is to investigate the changes of PC12 cells in cell morphology,cell viability and metabolic level while interacting with poly-L-lactic acid(PLLA)nanofibers.With the help of metabolomics,transcriptomics and proteomics data of our previous studies,the metabolomics data was first analyzed,and then multi-omics approaches of metabolomics,transcriptomics,and proteomics were applied to analyze the effect of PLLA nanofibers on PC12 cells from molecular level.By verifying the changes of important genes and proteins,a path from aligned nanofiber surface → cell adhesion → integrin → gene/protein/metabolite changes→ cell differentiation was established to explore the molecular mechanism of PLLA nanofibers inducing differentiation of PC12 cells.The main content of this paper:1.Preparation of PLLA nanomaterials: PLLA nano-films were prepared by spin coating and PLLA random/aligned nanofibers were prepared by electrospinning technology.These materials were characterized by scanning electron microscopy(SEM),and the results showed that the surface of PLLA nano-films were basically flat and smooth,PLLA nanofibers had uniform thickness without beads and aligned nanofibers have a certain orientation.2.The pre-differentiated PC12 cells were planted on the surface of PLLA nano-film and random/aligned nano-fiber materials respectively.After culturing for 12 h,24h and 36 h,cell morphology observation,cell viability measurement and cell metabolism level detection were performed.Through SEM observation experiment and high content analysis experiment,it is observed that PC12 cells will extend along the direction of nanofibers.The extension direction of PC12 cell protrusions would basically consistent with the orientation of aligned nanofibers.Aligned nanofibers could promote the extension of cell protrusions during PC12 cell differentiation.Cell viability measurement and metabolic level measurement experiments showed that the degree of differentiation of PC12 cells on the surface of nanomaterials increased with the increase of action time,which proved that PLLA aligned nanofibers could promote the differentiation of PC12 cells.3.The online metabolomics analysis software Met PA was employed to analyze differentially expressed metabolite of the metabolomics data and obtain important metabolite pathways related to neural cell differentiation and key metabolic pathways.Under the effect of PLLA random nanofibers,in the key metabolic pathways of phenylalanine,tyrosine and tryptophan biosynthesis and the phenylalanine metabolism pathway of PC12 cells,phenylalanine is mainly converted into phenylpyruvate and 2-phenylacetamide,and the excessive accumulation of phenethylamine,a compound of phenylpyruvate and 2-phenylacetamide,could be detrimental to the growth and differentiation of PC12 cells.Under the effect of PLLA aligned nanofibers,phenylalanine in the key metabolic pathways of phenylalanine,tyrosine and tryptophan biosynthesis and the phenylalanine metabolic pathway could be mainly converted to tyrosine to promote the biosynthesis of dopamine and noradrenaline in PC12 cells,thus provided energy for cell growth.These different differentially expressed metabolites work through corresponding metabolic pathways,making it beneficial for PC12 cells to grow and differentiate on the surface of PLLA aligned nanofiber at the cellular level.4.Bioinformatics method was conducted to perform a comprehensive and systematic multi-omics analysis,including the m RNA expression profiling chip,proteomics,and metabolomics,to obtain metabolic pathways in which the differentially expressed genes,proteins,and metabolites participated together in PC12 cells processed by random nanofiber and aligned nanofibers for 24 h respectively.By analyzing and discussing the upstream and downstream interactions of genes,proteins,and metabolites in important metabolic pathways,the key metabolic pathways for multi-omics analysis of PLLA random nanofibers affecting PC12 cell differentiation were obtained: glutathione metabolism,unsaturated fatty acid biosynthesis and glycerol phospholipid metabolism pathway.The key metabolic pathways for multi-omics analysis of PLLA aligned nanofibers affecting PC12 cell differentiation were obtained: glutathione metabolism,unsaturated fatty acid biosynthesis,and sphingolipid metabolism pathways.From the perspective of systems biology,it was found that the differentially expressed genes,proteins,and metabolites in each pathway could influence each other,and ultimately promote the differentiation of PC12 cells.5.The role of important genes and proteins in PLLA nanofibers,especially aligned nanofibers,in the process of PC12 cell differentiation was detected via q RT-PCR and Western Blot.The expression level of GAP43,an indicator of the degree of differentiation of PC12 cells,was up-regulated.The up-regulated expression levels of integrin-related genes Itga3,Itga5,Itga7,Itgb4 and Tln1,Map2k5,Mapk6 genes proved that the differentiation of PC12 cells induced by PLLA aligned nanofibers was related to the activation of integrin-mediated FAKMEK-ERK pathway.The up-regulated expression levels of genes Pafah1b1,Daam1,Slit2,and Ascl1 verified that they played an important role in the process of PLLA aligned nanofibers promoting the differentiation of PC12 cells.The study proved when PLLA aligned nanofibers affected the differentiation of PC12 cells,the FAK-MEK-ERK pathway mediated by integrin was activated and phosphorylated ERK1/2(p-ERK1/2)entered the nucleus and altered the biochemical reactions of genes such as Pafah1b1,Daam1,Slit2,and Ascl1,which played key roles in important physiological functions such as nervous system development and axon growth. |
