Effect of citicoline on cell viability in lipopolysaccharide-stimulated human microglial cells

Neuroinflammation is a key pathological response of the central nervous system (CNS) and contributes to the progression of several neurodegenerative disorders. Microglia, as resident immune cells of the CNS, play a central role in this process and release pro-inflammatory mediators following stimulation by inflammatory triggers such as lipopolysaccharide (LPS) [1]. Citicoline (cytidine-5′-diphosphocholine or CDP-choline) is an endogenous molecule essential for the biosynthesis of phosphatidylcholine, a principal constituent of neuronal cell membranes. It has also demonstrated neuroprotective effects by modulating oxidative stress and inflammation [2]. These properties suggest citicoline may influence cellular responses during inflammation. This study investigated the effect of citicoline on cell viability in LPS-induced HMC3 human microglial cell line using the MTT assay, with both citicoline cytotoxicity and impact following LPS stimulation assessed. In this study, HMC3 cells were cultured in EMEM supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37°C in a humidified incubator with 5% CO₂. The groups were organized as follows: control, LPS, citicoline, and LPS + citicoline. Prior to citicoline administration, cells were seeded into 96-well plates at a density of 5x10³ cells per well. After seeding, LPS (10 µM) was applied to the cells, followed by citicoline at concentrations of 10, 50, 100, 500, and 1000 µM for 24 hours [3]. After treatment, 1% MTT reagent was added to each well, and the plate was incubated at 37°C for 3 hours. Thereafter, the MTT-containing medium was removed from each well without disrupting the cell clusters, and DMSO was added to each well to dissolve the formazan crystals. After all crystals were dissolved, absorbance was measured at 570 nm using a microplate reader. Each concentration and condition was tested in at least three wells. Statistical comparisons were performed using one-way ANOVA. A p-value less than 0.05 was considered statistically significant. Citicoline treatment (without LPS) did not induce a statistically significant change in MTT-derived cell viability in HMC3 cells after 24 hours, indicating no apparent cytotoxic effect within the tested concentration range. In the 24-hour post-treatment MTT assay, LPS exposure increased the MTT signal relative to the control group, suggesting enhanced metabolic activity under inflammatory stimulation. After LPS exposure, post-treatment with citicoline further elevated MTT-derived cell viability/metabolic activity, with the most pronounced effect observed at 500 µM. Compared with the control group, cell viability increased in all treatment groups. Following LPS treatment, significant increases in cell viability were observed at 50 µM and 100 µM doses compared with the control group (*p < 0.05), while a more pronounced increase was detected at the 500 µM dose (**p < 0.01). These findings suggest that post-treatment with citicoline may enhance metabolic activity in HMC3 cells under LPS-induced inflammatory conditions. The most prominent effect was observed at 500 µM, suggesting a potential concentration-dependent cellular response. Further investigations employing complementary viability and inflammatory marker assays are required to determine whether this effect is attributable to cytoprotection, metabolic modulation, or anti-inflammatory activity. This study constitutes an initial phase of our ongoing research, with additional analyses presently in progress.

References

1. García-Domínguez M. (2025). Neuroinflammation: Mechanisms, dual roles, and therapeutic strategies in neurological disorders. Current Issues in Molecular Biology, 47(6), 417. https://doi.org/10.3390/cimb47060417
2. Cavalu S., Saber S., Ramadan A., Elmorsy E.A., Hamad R.S., Abdel-Reheim M.A., Youssef M.E. (2024). Unveiling citicoline’s mechanisms and clinical relevance in the treatment of neuroinflammatory disorders. FASEB J, Sep 15;38(17):e70030. https://doi.org/10.1096/fj.202400823R
3. Matteucci A., Varano M., Gaddini L., Mallozzi C., Villa M., Pricci F., Malchiodi-Albedi F. (2014). Neuroprotective effects of citicoline in in vitro models of retinal neurodegeneration. International journal of molecular sciences, 15(4), 6286-6297. https://doi.org/10.3390/ijms15046286