Context: Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown.
Objective: The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation.
Materials and methods: The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200 μg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 μg/mL). The effects of EAP (37.5, 75, and 150 μg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors.
Results: The results showed that EAP (50, 100, and 150 μg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway.
Discussion and conclusion: The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3K→Rac→PAK→MEK signaling pathway.
Keywords: ERK; NIH3T3; PI3K; signaling pathway.