Isorhamnetin inhibits hypertrophic scar formation through TGF-ß1/Smad and TGF-ß1/CREB3L1 signaling pathways.

ABSTRACT

Background: Hypertrophic scar (HS) is a common fibrotic skin disease that occurs secondary to burns or injuries. The activation of the TGF-ß signaling pathway contributes immensely to HS formation. Isorhamnetin (ISO) is a type of flavonoid compound that exerts an antifibrotic effect via TGF-ß signaling suppression. However, whether ISO can inhibit HS formation via TGF-ß signaling is yet to be elucidated. This study aimed to examine the influence of ISO on HS pathogenesis and TGF-ß signaling, especially the downstream molecules and networks of TGF-ß signaling that facilitate HS formation. Methods: Hypertrophic scar fibroblasts (HSFBs) were isolated from human HS tissues. The in vitro proliferation, migration, contractile ability, cell cycle, and apoptosis of HSFBs after ISO treatment were determined using cell viability assay, EdU staining, wound healing assay, collagen gel contraction assay, and flow cytometry. The expressions of genes and proteins involved in TGF-ß signaling and its downstream molecules in ISO-treated HSFBs were determined using quantitative PCR (qPCR), immunofluorescence, and western blotting. In vivo, a rabbit HS model was established, and the effects of ISO on rabbit HS formation were investigated using histological analysis, immunohistochemical staining, and qPCR. Results: In vitro studies indicated that ISO treatment suppressed the proliferation, migration, and contractile ability of HSFBs; attenuated the expressions of COL Ⅰ, COL Ⅲ, and α-SMA; and inhibited TGF-ß1 signaling-induced activation of HSFBs by decreasing the levels of phosphorylated Smad2/3 and cleaved CREB3L1 in a dose-dependent manner. Furthermore, ISO augmented apoptosis and G2 phase cell cycle arrest of HSFBs by upregulating the expressions of the proapoptotic proteins Bax and cleaved caspase-3 and downregulating the expression of the antiapoptotic protein Bcl-2. In vivo studies revealed that ISO ameliorated HS formation in the rabbit ear by lowering the scar elevation index, attenuating the collagen density, facilitating the regular arrangement of collagen fibers, and downregulating the expressions of TGF-ß1, CREB3L1, COL Ⅰ, COL Ⅲ, and α-SMA. Conclusions: ISO suppressed HS pathogenesis by dampening TGF-ß1/Smad and TGF-ß1/CREB3L1 signaling pathways, which suggests that it may serve as a candidate inhibitor of TGF-ß1 signaling and a promising anti-HS drug with a high therapeutic potential.