ALA-PDT promotes the death and contractile capacity of hypertrophic scar fibroblasts through inhibiting the TGF-81/Smad2/3/4 signaling pathway

Background: Hypertrophic scars, an abnormal wound-healing response to burn injuries, are characterized by massive fibroblast proliferation and excessive deposition of extracellular matrix and collagen. 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is a promising therapy for hypertrophic scar, details of the mechanisms remain to be elucidated. In this study, we aimed to investigate the molecular mechanisms involved in ALA-PDT against hypertrophic scar fibroblasts.Methods: The morphologies of hypertrophic scar fibroblasts (HSFs) treated with ALA-PDT were observed under a light microscopy. The viability of HSFs was detected using the CCK-8 assay. HSFs-populated collagen gel contraction assays were conducted to examine the fibroblast contractility and the cytotoxicity of HSFs in 3D collagen tissues were observed using confocal microscopy. The effect of ALA-PDT on TGF-81/Smad2/3/4 signaling pathway activation and effector gene expression were verified by immunoprecipitation, western blot and real-time quantitative PCR analysis.Results: We observed significant changes in cell morphology after ALA-PDT treatment of HSFs. As ALA concentration and light dose increased, the viability of HSFs significantly decreased. ALA-PDT can significantly alleviate the contractile capacity and promote the death of HSFs induced by TGF-81 treatment in a threedimensional collagen culture model. TGF-81 treatment of HSFs can significantly induce phosphorylation of Smad2/3 (p-Smad2/3) in whole cells, as well as p-Smad2/3 and Smad4 proteins into the nucleus and increase the mRNA levels of collagen 1/3 and a-SMA. ALA-PDT hampers the TGF-81-Smad2/3/4 signaling pathway activation by inducing K48-linked ubiquitination and degradation of Smad4.Conclusions: Our results provide evidence that ALA-PDT can inhibit fibroblast contraction and promote cell death by inhibiting the activation of the TGF-81 signaling pathway that mediates hypertrophic scar formation, which may be the basis for the efficacy of ALA-PDT in the treatment of hypertrophic scars.