PI3K/AKT-associated fibroblast activation by native Argiope anasuja spidroin for cutaneous wound healing: An in vitro approach

Abstract

Spidroins, the main structural protein constituents of spider silk, show excellent mechanical strength, durability and biocompatibility; therefore, they are potentially useful biomaterials for medical uses. Nevertheless, the molecular mechanisms that underlie their wound-healing activity are not well understood. The present study has been designed to investigate the in vitro wound-healing potential of the Native Argiope anasuja orb-web spidroin protein in NIH/3T3 fibroblasts and the underlying molecular mechanisms. Cell viability was measured by MTT assay, and fibroblast migration and re-epithelialization were determined by scratch wound assay under 0% FBS, 10% FBS, 1× PBS vehicle control and 5% spidroin treatment. In the spidroin-treated group, fibroblast migration was enhanced, and wound closure was complete within 36 h when compared to the control groups. Quantitative real-time PCR (qRT-PCR) analysis demonstrated an increased in VEGFR2, PI3K and AKT gene expression in spidroin-treated cells, indicating the activation of angiogenic and proliferative signalling pathways. Western blot analysis also verified activation of the PI3K/AKT signalling pathway, as indicated by an 8.5-fold increase in the expression of PI3K, a 1.2-fold increase in the expression of AKT, and a 2.8-fold increase in the expression of phosphorylated AKT (p-AKT) compared with controls. Immunofluorescence studies also revealed increased fluorescence intensity of VEGFR2, PI3K and AKT in the treated cells. In total, these results indicate that native Argiope anasuja spidroin induces fibroblast proliferation and migration through involvement of VEGFR2-associated PI3K/AKT signalling pathways, which illustrates its therapeutic potential in wound healing and tissue regeneration.