High glucose-induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes

Abstract

Growing evidence indicates that skin injuries are a common complication of diabetes. However, the cellular and molecular mechanisms of high glucose (HG) environment trigger nitrosative stress-mediated inflammation and apoptosis in keratinocytes remains unknown. Here we investigated whether reactive nitrogen species (RNS) induced by HG environment restrain antioxidant activity, and mitochondrial dysfunction leading to inflammation, and apoptosis via stress signaling pathways in keratinocytes. Our results established that the HG environment enhanced the production of nitric oxide (NO) and peroxynitrite anion (ONOO−) by inducible NO synthase (iNOS) in keratinocytes. Overproduction of RNS in HG environment suppress the antioxidants activity leading to mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, decrease in mitochondrial transcription factor A(TFAM), increase in mitochondrial DNA (mtDNA) displacement loop (D-loop) and decrease in glycolytic flux concentration, which was attenuated by pharmacological inhibitors of NO/ONOO−, Nω-Nitro-l-argininemethyl ester hydrochloride (NAME)/hydralazine hydrochloride (Hyd.HCl). Excess production of RNS in HG environment restrained 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA were regulated by NO or ONOO−. Further, HG-induced RNA production caused an increase in the production of inflammatory mediators accompanied by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascade, lipid peroxidation (LPO), and protein carbonylation (PC) reactions followed by breakdown the cell-cell communication and apoptosis. Pre-treatment of cell with NAME/Hyd.HCl, diminished the expression of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3, inflammatory mediators, and attenuated apoptosis in keratinocytes. Together, our results indicated that excess production of RNS in HG environment triggered inflammation and apoptosis, mediated by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascades in keratinocytes.