Recombinant Human Fibronectin Mediates Macrophage Polarization via NF-κB/TGF-β1 Pathway to Enhance Fibroblast Proliferation

This research aims to explore the molecular mechanism where recombinant human fibronectin (rhFN) regulates macrophage polarization and then affects fibroblast proliferation via the nuclear factor kappa B (NF-κB)/transforming growth factor β1 (TGF-β1) signaling pathway.

Macrophages RAW 264.7 were activated with LPS and subsequently treated with rhFN, followed by flow cytometry to assess macrophag polarization. Cytokine levels of interleukin (IL)-10 tumor necrosis factor alpha (TNF-α), IL-6, and Arg-1, as well as TGF-β1, were measured using enzyme-linked immunosorbent assay (ELISA). Fibroblast NIH 3T3 was cultured with macrophage-conditioned media (CM), and CCK-8, cell adhesion, and wound healing assays were used to evaluate their proliferation, adhesion, and migration capacities. Western blot was conducted to detect the changes of proteins related to TGF-β1/Smad2/3 and NF-κB signaling.

RhFN significantly promoted macrophage M2 polarization and increased TGF-β1 secretion while reducing pro-inflammatory cytokines TNF-α and IL-6, increasing IL-10 and Arg-1 levels. Fibroblasts cultured with rhFN-treated macrophage-CM showed increased Smad2/3 phosphorylation, causing improved proliferation, adhesion, and migration abilities. Inhibition of NF-κB signaling promoted an anti-inflammatory macrophage profile, while NF-κB activation partially reversed rhFN's effects on fibroblast function. Inhibition of TGF-β1 resulted in reduced fibroblast proliferation, adhesion, and migration abilities, confirming its pivotal role in rhFN-mediated effects.

RhFN modulates macrophage polarization through NF-κB inhibition and promotes fibroblast proliferation, adhesion, and migration via TGF-β1/Smad2/3 signaling.