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Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation. Carnosine has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by macrophages stimulated with LPS + IFN-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS).ESI-MS and NMR spectroscopy in a cell-free systemshowed the formation of multiple adducts (at differentratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (β-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages,the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of the pro-inflammatory cytokine IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio.

In this report, a microchip electrophoresis system with fluorescence detection was used for the quantitation of intracellular carnosine in untreated and stimulated macrophage cell lysates. Carnosine was derivatized with NDA/CN and separated from other endogenous amine reported in macrophage cells. Based on ME-LIF with standard addition, macrophages were estimated to contain a basal intracellular concentration of carnosine (0.079 ± 0.02 nmol per million cells). Carnosine is readily taken up by macrophages in cell culture. Incubation with 20 mM carnosine led to a 600-fold increase in intracellular carnosine compared to basal levels. Furthermore, we have shown that under pro-inflammatory conditions using LPS and IFN-gamma stimulation there is a further 3-fold increase in carnosine uptake in macrophage cells. This suggests that there is a mechanism through which macrophages increase the usage of carnosine during oxidative stress.

By using an in vitro model of AD, we showed that carnosine prevents Aβ-induced oxidative stress in BV-2 microglial cells by decreasing the expression of iNOS and NADPH oxidase and the concentrations of nitric oxide and superoxide anion. Carnosine is also able to decrease the secretion of pro-inflammatory cytokines, simultaneously rescuing IL-10 levels and increasing the synthesis and the release of TGF-β1. Carnosine, through its multimodal mechanism of action, might represent a new pharmacological tool to yield neuroprotection in AD.

By using an in vitro model of AD, we showed that carnosine prevents Aβ-induced oxidative stress in BV-2 microglial cells by decreasing the expression of iNOS and NADPH oxidase and the concentrations of nitric oxide and superoxide anion. Carnosine is also able to decrease the secretion of pro-inflammatory cytokines, simultaneously rescuing IL-10 levels and increasing the synthesis and the release of TGF-β1. Carnosine, through its multimodal mechanism of action, might represent a new pharmacological tool to yield neuroprotection in AD.

This study demonstrated that amylin (and beta-amyloid) might play an important physiological role in promoting the release of neuroprotective factors, namely HspB5 and VEGF, from endothelial cells of the capillary neurovasculature. The data presented in this study confirm the existence of a “cross-talk” between endothelial and neuron-like cells and the relevance of a physiological role of amylin and beta-amyloid, such as the ability to promote the release of neuroprotective factors from endothelial cells.

This study demonstrated that amylin (and beta-amyloid) might play an important physiological role in promoting the release of neuroprotective factors, namely HspB5 and VEGF, from endothelial cells of the capillary neurovasculature. The data presented in this study confirm the existence of a “cross-talk” between endothelial and neuron-like cells and the relevance of a physiological role of amylin and beta-amyloid, such as the ability to promote the release of neuroprotective factors from endothelial cells.