N-Acetyl-L-cysteine effects on oxidative stress-induced high glucose-cultured human dermal fibroblasts

Abstract

BackgroundWound healing in diabetic patients is delayed, but the mechanism of this impairment is unclear. We sought to investigate molecular changes in human dermal fibroblasts (HDFs) in a high-glucose state and improving effect of N-acetyl-L-cysteine (NAC). MethodsHDFs were cultured in 5.5, 25, 50, and 75 mM glucose concentrations for 72 h. Cell proliferation and migration were examined via in vitro scratch and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assays. Cytotoxicity was determined through the measurement of lactate dehydrogenase (LDH). The level of intracellular reactive oxygen species (ROS) was also measured in the HDFs. Oxidative stress markers of nuclear factor erythroid 2 (NFE2)- related factor 2 (NRF2), Catalase (CAT) and Glutathione (GSH) were quantified with Real time polymerase chain reaction (PCR). The antioxidant effect of NAC, 1 mM was examined to evaluate the involvement of ROS and LDH and oxidative markers in the glucose effects on the HDFs. ResultsThe in vitro scratch assay showed that high glucose concentrations significantly reduced fibroblast migration and proliferation at 12, 24, and 48 h. The MTT assay revealed a decline in cell viability in 50 and 75 mM glucose concentrations. A significant increase in LDH and ROS levels was observed in the HDFs incubated in 50 and 75 mM glucose concentrations after 72 h. mRNA level of Nrf2, CAT and GSH were downregulated. The antioxidant addition of NAC reduced the inhibitory effect of the high-glucose state on the migration and proliferation of the HDFs. ConclusionsA high-glucose state impairs the in vitro proliferation and migration of HDFs and may, therefore, induce increased oxidative stress and cellular dysfunction. The antioxidant effect of NAC ameliorates the damaging impact of a high-glucose state.