Synergistic Neuroprotection by Cannabis sativa and Tilia × viridis: Attenuation of Hippocampal Neurons Glutamate-Induced Oxidative Stress and LPS-Driven Microglial Inflammation

Abstract Throughout history, Cannabis sativa has been linked to the therapeutic management of epilepsy and Tilia × viridis has a tradition of use as a sedative. This study aimed to evaluate the protective effect of an ethanolic extract of C. sativa (CSRD), an aqueous extract of T. × viridis (TE), and their combination against oxidative stress induced by glutamate in a murine hippocampal neuronal (HT-22) cell line, as well as their anti-inflammatory activity in male Wistar ratsʼ microglial cells stimulated with LPS. A phytochemical analysis was also conducted. Glutamate-induced reactive oxygen species (ROS) were quantified using 2′,7′-dichlorodihydrofluorescein diacetate via fluorescence microscopy. Cell viability was assessed using the MTT assay. Distinct microglial cell phenotypes were identified via immunofluorescence. Extracts partially reversed glutamate-induced loss of cell viability (52% to 200% for CSRD; 22% to 82% for TE). Their combination produced a greater effect, reversing glutamate-induced toxicity by 133% to 284% and fully restoring cell viability to control levels. Moreover, the combined treatment reduced intracellular ROS levels (52% to 58%). Notably, the combination also exhibited the most pronounced anti-inflammatory effects, significantly reducing the proportion of reactive phenotype 1 cells, while increasing the population of anti-inflammatory phenotype 2 cells and preserving the trophic phenotype 3 subpopulation. In conclusion, this study not only validates the ethnobotanical uses of C. sativa and T. × viridis but also reveals a potent synergy when combined. This provides a strong foundation for the development of phytomedicines with translational potential for managing complex pathologies like epilepsy or neuroinflammation associated with neurodegenerative diseases.