The Study Of Roles And Signaling Pathway Of Earthworm Active Protein In Fibroblast Proliferation

Fibroblasts play a vital role in tissue regeneration, restoration and reconstruction in wound healing, which could secrete extracellular matrix such as collagen and growth factors to stimulate wound healing. In recent years, the earthworms are widely used in Chinese herbal medicine to remove stasis and stimulate wound-healing,and many researches demonstrate that the earthworm extracts may promote wound healing. However, Exactly how earthworms act on the wound and promote tissue survival remain unknown. In this work, we used fresh earthworms as materials and summarized the best approaches of earthworm reactive protein (EAP) process optimization according to the proliferation-promoting action.,then investigated the effects of EAP on mouse embryonic fibroblast NIH/3T3cells, also investigated the molecular mechanisms by which EAP promotes the NIH/3T3cell proliferation. The main methods and results are as follows:1.Extraction process optimization and purification of earthworm proteinBased on preliminary studies, using the MTT screening method, we examined how the salting and desalting conditions affected activities of the earthworm protein extraction, then we examined the effects of solvent, temperature and salting conditions on activity of earthworm protein by Orthogonal Design and,at last, we determined the optimal extraction process, that is:first, we added twice the amount of phosphate buffer solution to fresh earthworms, and homogenate in ice bath,4℃allowed to stand for4hours, then centrifuged them for25min; second, we took the supernatant to salt out until50%saturation,4℃allowed to stand for2h,and then centrifuged25min; last, we took the supernatant and used a hollow fiber membrane module of0.2μm to remove macromolecules, and then desalted by ultrafiltration with a hollow fiber membrane module of20K-50K molecular weight cutoff. Next, we obtained EAP in accordance with the separation and purification processes--DEAE ion exchange chromatography, S200gel filtration chromatography and superdex75PG gel filtration chromatographySDS-polyacrylamide gel electrophoresis analysis of the EAP showed that the protein was a single electrophoretically pure protein, and molecular weight was58.84kDa.Total carbohydrate was determined by the phenol sulfuric acid colorimetric method with D-glucose as a standard at490nm, amino acids composition was determined by HPLC-ELSD analysis.the content was as follows:molecular weight58.84kDa,polysaccharide content13.77%. the protein isoelectric point about4, containing11kinds of known and a variety of unknown compositions. Finally, it was a new protein by MALDI-TOF mass spectrographic analysis.2.The effect of EAP to cell proliferation and migration characteristics of NIH3T3First, detect the activity of NIH3T3cells treated with different concentrations of EAP by the MTT and Brdu incorporation assay. The results showed that EAP could increase DNA replication of NIH3T3fibroblasts to promote their proliferation by a dose-dependent manner. The cell cycle was detected by fkow cytometry and the result showed that EAP could be able to increase the percentage of S phase fraction; The following western blotting results showed that expression level of CyclinD1protein which regulate the G1/S phase test point was significantly increased. Finally, the results of cell scratch experiments and the trans well chamber showed that EAP promoted the migration of NIH3T3cell in a dose-dependent manner.3, Possible mechanism leading to the proliferation of NIH3T3cells by EAPFirst, different signaling pathway inhibitors were used to study the signaling pathways involved in the process of EAP promoting cell proliferation. MTT assay and Western blotting results showed that the MEK inhibitors PD98059and U0126pretreatment could significantly inhibit cell proliferation and block the EAP activated ERK phosphorylation. Flow cytometry and western blotting results showed that U0126pretreatment significantly blocked cell cycle progression and the expression levels of Cyclin D1protein. Then MTT assay showed that B-Raf and Raf-1inhibitors pretreatment had no significant effect on cell proliferation.So PI3K inhibitor LY294002pretreatment was detected by MTT and Western blotting, showing that it significantly inhibited cell proliferation and suppressed the Akt and ERK phosphorylation levels elevated caused by the EAP, which suggested that PI3K is also involved in the EAP-inducing proliferation of NIH3T3cells.In summary, we believe the MEK/ERK signaling pathway and PI3K signaling pathway were involved in EAP-promoting cell proliferation, it could cause elevated CyclinD1protein expression and promote cell cycle from G1to S phase, and the PI3K signaling pathway may be the upstream of MEK/ERK.The above results provide evidence to further understanding EAP-promoting NIH3T3cell proliferation potential mechanism, and it was able to promote the development of burning drugs, providing references for the mechanism of burning drugs in clinical, which is vital to the rational application of traditional Chinese medicine earthworms as material.