Protocol for Studying Honokiol Regulation of Transforming Growth Factor Beta 1/Smad Signaling in Ultraviolet...

Honokiol (HNK), a key component derived from Magnolia officinalis, has the potential to treat skin diseases. At present, the impact and mechanism of action of HNK on Ultraviolet A (UVA)-induced photodamage in the skin are unclear. A photo-aging damage model of HDF cells and hairless mouse skin was constructed by UVA induction. The impact of HNK on the viability of normal HDF cells and UVA-induced HDF cells was examined through the Cell viability assay. SOD, GSH, CAT, and MDA levels in HDF cells were measured using different kits, and ROS levels were measured using DCFH-DA and Dihydroethidium. SA-β-galactosidase and TUNEL staining detected senescence and apoptosis in HDF cells, and flow cytometry assessed cell cycle progression. Histopathological damage to the skin was evaluated through pathological staining. In addition, immunofluorescence and Western blot were used to assess Ki67, collagen, matrix metalloproteinase (MMP), and TGF-β1/Smad pathway-associated protein levels. HNK (2.5-10 µM) treatment had no adverse impact on normal HDF cell viability but attenuated UVA-induced HDF cell damage, reducing oxidative stress and apoptosis. UVA induced senescence of HDF cells, induced S-phase arrest, downregulated Ki67, decreased collagen content, and increased MMP expression, while HNK effectively attenuated these UVA-induced abnormalities. In addition, UVA caused deeper wrinkles, decreased water content and collagen content, and increased epithelial thickness in mouse skin, whereas subcutaneous injection of HNK attenuated pathological damage to skin tissue and increased water and collagen content. Furthermore, HNK activated the TGF-β1/Smad pathway, and the TGF-β1 inhibitor SB431542 impaired the protective effect of HNK against photo-aging damage in HDF cells. In conclusion, HNK has the potential to be a drug for ameliorating photo-aging damage by reducing MMP expression through activation of the TGF-β1/Smad pathway, thereby inhibiting UVA-induced photo-aging in HDF cells and mouse skin.