Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells.

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

Increasing NO level regulates apoptosis and inflammation in macrophages after 2-chloroethyl ethyl sulphide challenge.

Generation of nitric oxide (NO) in cellular compartments acts in a redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging superoxide anions (O2-), to form peroxynitrite (ONOO-) or acting as a signaling molecule, altering gene expression that triggers various physiological processes. However, the molecular mechanisms of macrophage activation and NO production leads to apoptosis and inflammation after 2-chloroethyl ethyl sulphide (CEES) exposure remains unclear. We showed that CEES exposure in macrophages increased the O2- production. Also CEES exposure transiently increases the NO production and ONOO- accumulation via expression of inducible NO synthase (iNOS). Simultaneously, CEES exposure caused a significant reduction in cellular antioxidants and modulate lipid peroxidation (LPO), and protein carbonylation (PC) reactions, which was correlated with the increased level of NO and ONOO- accumulation. Mechanistic studies showed the DNA damage, 8-oxoGDNA glycosylase (OGG1) down regulation and 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA, which was also confirmed by phosphorylation of ATM, ATR and H2A.X. Elevated levels of NO/ONOO- plays an important role in apoptosis, and alteration of cell cycle regulatory proteins in macrophages after CEES exposure. Moreover, CEES exposure to macrophage cells enhanced the transcriptional activities of inflammatory mediators such as TNFα, IL-1α, ICAM, CX3CL1, CCL8, and CXCL10, which were linked with NO/ONOO- accumulation. These results showed a mechanistic explanation of how NO/ONOO- cooperate to conduct apoptosis and inflammatory signals in macrophages after CEES challenged. Further, the protective effects of NO/ONOO- inhibitors may provide the basis for the development of a therapeutic strategy to counteract exposure to CEES.

Hyperglycemia-induced inflammation caused down-regulation of 8-oxoG-DNA glycosylase levels in murine macrophages is mediated by oxidative-nitrosative stress-dependent pathways.

High glucose-induced increase in production of reactive oxygen/nitrogen species (ROS/RNS) is recognized as a major cause of the clinical complications associated with diabetes. ROS/RNS apart from being redox agents, cause an unwanted severe physiological load to cells, also act as cellular messengers, and play a key role in activation of circulating macrophages. However, the molecular mechanisms of activation of macrophages by hyperglycemic conditions are currently unclear. In the present study, we report that high glucose (HG) causes a dramatic increase in the production of inflammatory cytokines and chemokines, at least in part through enhanced mRNA transcription. The increase in levels of inflammatory cytokines/chemokines corresponds to increased levels of ROS/RNS, which is accompanied by increased activities of Akt, ERK1/2, tuberin, down regulation of 8-oxoG-DNA glycosylase (OGG1), and increase in 8-hydroxydeoxyguanosine (8-OHdG) accumulation in DNA. Elevated levels of ROS/RNS are triggering alteration in antioxidants level, biomolecules damage, cell cycle dysregulation, and apoptosis in macrophage cells. Pretreatment of antioxidants caused decrease in the levels of ROS/RNS leads to an increase in the levels of antioxidants, decrease in biomolecules damage, alterations in Akt, ERK1/2, tuberin, upregulation of OGG1, and decrease in 8-OHdG accumulations in DNA. Further, antioxidants treatments inhibit the effects of HG on the transcriptional activity of cytokines and chemokines. Our results demonstrate that intracellular signaling pathways mediated by ROS/RNS are linked to each other by elevated glucose in macrophages activation leading to inflammation. These findings provide a mechanistic explanation of how ROS/RNS cooperate to conduct inflammatory intracellular signals in macrophages related complications in hyperglycemic conditions.