| Objective:In the previous study of the research group,it was found that the extracts of the tail-amputated Eisenia foetida for three days(DL3)contained active substances that promote wound healing.On the basis of the previous studies,this project aims to screen more active protein components from the DL3 that promote wound healing,and the shotgun proteomics strategy was used to analyze and identify the active protein components,which revealed the material basis for the tail-amputated earthworm on wound healing.Methods:(1)The extracts of the tail-amputated Eisenia foetida for three days was prepared and salting out was performed in stages to obtain different salting out components.More active components was screened out by studying salting out components on the proliferation of mouse embryonic fibroblasts(NIH3T3),then the active components were further separated by the anion exchange chromatography and the different components obtained by the exchange chromatography were applied to NIH3T3 cells,and the best active components were screened by comparing the cell proliferation rate.(2)The active protein bands were analyzed by SDS-PAGE,and the active proteins were digested followed with the peptides were extracted,then LC-MS/MS was used for protein analysis combined with the shotgun proteomics strategy.(3)The difference of active proteins between extracts of Eisenia foetida(CL)with DL3 and the difference of active proteins between the tail-amputated Pheretima guillelmi(HL3)with DL3 were analysed and compared.The both extracts of Eisenia foetida(CL)and the tail-amputated Pheretima guillelmi(HL3)for three days was prepared to compared with DL3 by SDS-PAGE,and then LC-MS/MS was used to identify the salting out components of CL and HL3.(4)Combined with the results of protein identification,an initial research of signaling pathway of DL3 on NIH3T3 proliferation was performed by studying MEK1 inhibitor(PD98059)and MEK1/2 inhibitor(U0126)on the proliferation of mouse embryonic fibroblasts.Results:(1)DL3 was separated into four fractions(S1,S2,S3,S4)by salting-out.Each drug group showed the effect of promoting the proliferation on NIH3T3,which the S3 group had the strongest effect on proliferation of fibroblasts with more broader and more stable coverage.Therefore,S3 was selected for anion exchange chromatography and five eluents were obtained:I1,I2,I3,I4,and 15.Each drug group showed the effect of promoting the proliferation on NIH3T3 too,which the 13 group had the strongest effect on proliferation of fibroblasts with more broader and more stable coverage.(2)According to the SDS-PAGE results,it was found that the molecular weight of the eluent 13 was mainly distributed around 11KD,17KD,25KD,35KD and 48KD.The eluent 13 was identified by LC-MS/MS,then the NCBI Uniprot Eisenia fetida database and Uniprot Sprot database were searched by MASCOT,and 24 proteins were identified.Among then,six proteins identified by the Uniprot Eisenia fetida database were Lombridine kinase,Heat shock protein 70,Cytochrome c oxidase subunit 1,Fibrinolytic protease P-Ⅲ-1,Lysozyme and 1,3-beta-glucanse.。(3)Compared with the CL,the protein bands of the DL3 samples were significantly increased,and the color was deeper.And Compared with the DL3 and HL3,those were slightly different in the distribution of active proteins.The protein distribution of DL3 was more extensive and there was a large amount of protein distribution between 11KD～17KD and 25KD～48KD,and there were significant protein bands at molecular weights of 11KD and 17KD,while at the molecular weight of 11KD the HL3 samples has a significant band and the protein content is relatively small.Eighteen proteins in CL were identified by searching the NCBI Uniprot Eisenia fetida database.Compared with DL3,Cytochrome c oxidase subunit 1,Fibrinolytic protease P-Ⅲ-1 and Lysozyme were only identified in DL3.Six species of proteins in HL3 were identified by searching for NCBI Uniprot Pheretima guillelmi database.The Cytochrome c oxidase subunit 1 protein was only identified in DL3 compared with the CL and DL3 identification results.(4)After adding MEK1 inhibitor and MEK1/2 inhibitor,the effect of active protein on NIH3T3 cell proliferation was significantly inhibited,and the two inhibitors had no significant effect on the normal growth of NIH3T3 cells,and the cell morphology did not change significantly.Conclusion:(1)The protein identification results show that the active protein components contain multiple functional proteins.The Cytochrome c oxidase has the function of oxidative phosphorylation,which plays an important role in the energy metabolism of cells.Fibrinolytic protease have antibacterial and anti-inflammatory effects and Lysozyme have the function of sterilization,which may accelerate wound healing by promoting exudate absorption and cell proliferation stages.In addition,Keratin,type Ⅱ cytoskeletal 1 protein has the skin barrier function,which also could regulate inflammation and angiogenesis.Heat shock protein 70 protein has anti-apoptosis effect and the secretion of the protein will be increased by external stimulation.Heat shock protein 90 is involved in the regulation of Ras/Raf/MEK/ERK signaling pathway,and the downstream pathway ERK of this pathway can directly regulate cell proliferation and differentiation.(2)The DL3 can promote the proliferation of fibroblasts.According to the protein identification results,we infer that it may be achieved by regulating Ras/Raf/MEK/ERK signaling pathway.(3)Combined with the protein identification results,we infer that Cytochrome c oxidase subunit 1,Fibrinolytic protease P-Ⅲ-1 and Lysozyme may be produced by the Eisenia fetida when it was broken.The proteins in the tail-amputated Pheretima guillelmi were Cytochrome c oxidase subunit 1 and Antimicrobial peptide lumbricin-PG,which were similar to those of Eisenia fetida,and Cytochrome c oxidase subunit 1 was the only protein identified in both DL3 and HL3.As for the specific role of each active protein in wound repair remains to be further explored. |
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