Neuroprotective effects induced by citicoline/coenzyme Q10 fixed combination in rat CTX-TNA2 astrocytes exposed to oxidative stress

Abstract

The present study aimed to investigate the rationale and efficacy of testing endogenous substances widely used as dietary supplement such as citicoline, coenzyme Q10 (CoQ10) and a fixed combination of them in countering the oxidative stress and neurotoxicity occurring in neurological diseases. Rat CTX-TNA2 astrocytes, which have considerable antioxidant potential and could represent a key target for neurotherapies were selected as in vitro model to conduct the experiments. The efficacy of citicoline and coenzyme Q10 (1 nM-10 μM), with their fixed combination, were assayed in rat astrocytes either in basal condition or after challenging the cells with hydrogen peroxide in order to evaluate the biocompatibility of treatments. The gene expression of B-Cell Lymphoma protein 2 (BCL-2), BCL-2 Associated X (BAX), Superoxide dismutase 2 (SOD2), Cardiolipin Synthase 1 (CRLS1), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) involved in neurodegenerative diseases and neuroinflammation were investigated in CTX-TNA2 cells. Furthermore, in the same condition, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in astrocytes. Neither citicoline, nor coenzyme Q10 significantly altered astrocytes cell viability; thus, suggesting the biocompatibility of single ingredients and fixed combination in the concentration range considered for the study. Moreover, each compound tested alone or in combination were effective in inhibiting the hydrogen peroxide-induced gene expression of BAX, and SOD2, in inducing the gene expression of BCL-2 and CRLS1 and in reducing apoptosis. The blunting effects induced by the abovementioned treatments on hydrogen-peroxide induced apoptosis was also confirmed by TUNEL assay that demonstrated the capability of citicoline, CoQ10, and their combination to reduce the ratio TUNEL positive nuclei/total nuclei, a reliable marker of apoptosis. Additionally, citicoline, CoQ10, and their association were effective in inhibiting the hydrogen peroxide-induced NFkB, TNFα, and IL-6 gene. In parallel, there was an inhibition of both TNFα and IL-6 gene expression in basal condition. The co-administration of citicoline/coenzyme Q10 was overall more effective than individual ingredients. The present findings support the beneficial and synergistic effects of citicoline and coenzyme Q10 in fixed combination in reducing oxidation, and in stimulating neuroprotection in rat CTX-TNA2 astrocytes.