Cholesterol hydroperoxides (ChOOHs) are included as lipid peroxidation products in the skin exposed to ultraviolet (UV) light irradiation. They may exert physicochemical actions affecting biomembrane rigidity because cholesterol is one of the major components of cell membranes. We investigated the distribution of isomeric ChOOHs in heterogeneous cell membranes with different lipid profiles using mouse fibroblast NIH-3T3 cells as a model of the dermis. Before and after UVA irradiation in the presence of hematoporphyrin, cell membranes were partitioned to microdomains (lipid rafts and caveolae) containing a higher amount of cholesterol and non-microdomains (containing a lower amount of cholesterol) by ultracentrifugation. By a combination of diphenylpyrenylphosphine-thin-layer chromatography blotting analyses and gas chromatography-electron ionization-mass spectrometry/selected ion monitoring analyses, ChOOH isomers were determined as their trimethylsilyloxyl derivatives. Cholesterol 5?-, 7?- and 7?-hydroperoxide were found as isomeric ChOOHs before irradiation. The amounts of the three ChOOH isomers increased significantly after photoirradiation for 2h. No difference was observed between microdomains and non-microdomains with regard to the ratio of the amounts of isomeric ChOOHs to that of cholesterol, suggesting that these ChOOH isomers were distributed equally in both parts depending on cholesterol content. When cells were treated with a purified mixture of ChOOH isomers, cell membranes incorporated ChOOHs into microdomains as well as non-microdomains evenly. Cellular matrix metalloproteinase-9 (MMP-9) activity was elevated by treatment with the purified mixture of ChOOH isomers. These results strongly suggest that ChOOHs accumulate in cell membranes irrespective of the heterogeneous microstructure and promote MMP activity if dermal cells are exposed to photodynamic actions.
Lactoferrin (Lf), a multifunctional glycoprotein, is known to activate dermal fibroblasts. Enhancing percutaneous absorption without decreasing the activity of Lf is critical in making the dermal administration of Lf beneficial. Sophorolipid (SL), a glycolipid-type biosurfactant, is known to form assemblies that may elevate the efficiency of the transdermal delivery of active ingredients. Here, we investigated the role of SL in the transdermal absorption of bovine Lf (bLf) and the effect of SL on the bLf activity on dermal fibroblasts. Transdermal absorption of bLf through a model skin was enhanced by 1.3-fold to 1.7-fold when SL was added. The effects of SL on the bLf activities on dermal fibroblasts were examined by cell proliferation activities and by gene expression levels of elastic fiber components, collagen IV, and hyaluronan synthases, revealing that SL did not depress the effect of bLf to any extent. Instead, the tropoelastin gene expression was upregulated ~60-fold by bLf alone, which was further increased to ~160-fold by bLf and SL together, suggesting a significant synergism between bLf and SL. Protein levels of elastin, assessed by immunohistochemistry, correlated well with the results of gene expressions. These results indicate the feasibility of the transdermal administration of bLf with SL.
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