Study of rutin effect on neural cells in a glutamatergic excitotoxicity model.
Flavonoids; glutamatergic excitotoxicity; neurodegenerative diseases; neuroprotection.
Rutin is one of the most common used polyphenolic compounds described in the literature, with great scientific prominence due to its interesting pharmacological activities for of human health, including its neuroprotective potential. In the context of neurodegenerative diseases, glutamatergic excitotoxicity has been identified as an important mechanism of neuronal death associated with progression of these diseases. Therefore, this study aimed to investigate the rutin neuroprotective activity for neural cells under cytotoxic glutamate action. For this, cells of the PC12 strain were subjected to different treatments: direct treatment with rutin (0.5 - 1 μM) and /or glutamate (10 - 60 mM); indirect treatment using conditioned medium obtained from brain organotypic culture of Wistar rat (p7-p9) treated with rutin (0.5 μM) and glutamate (60 mM). In addition, we also treated primary culture of mesencephalic neurons/glial cells with 0.5 μM rutin. After 24 hours of direct or indirect treatment of PC12 cells, we performed cell viability analyzes from the Trypan Blue and Propidium Iodide exclusion test. In addition, we performed morphological analyzes after Rosenfeld staining for PC12 cells and for primary culture of mesencephalic neurons/glial cells. The results obtained with the cell viability test demonstrated that rutin was not toxic to PC12 cells. Glutamate, in turn, induced a toxic effect in PC12 cells in a concentration-dependent manner, and the concentration at 60 mM was able to cause almost 100% of death to the cells in culture. Regarding the direct neuroprotection assays, it was shown that, compared to the control group that presented 2% death, rutin was not able to protect PC12 cells against glutamate toxicity at high concentrations (30 and 60 mM) inducing about 20% and 100% death respectively. On the other hand, conditioned media from brain organotypic culture treated with rutin- (0.5 μM) + glutamate (60 mM) induced low toxicity in PC12 cultures, more specifically only 8% of cells died with treatment, whereas direct treatment presented 100% dead cells. Regarding morphological changes, indirect treatment with rutin induced a characteristic change in neuronal differentiation for approximately 26% of cells in culture, unlike direct treatment which induced 2.5%. These morphological changes related to the increase of branch points and the increase of neurite length were observed in PC12 cells and in primary culture of mesencephalic neurons/glial cells. These results indicate that the neuroprotective and morphogenic effects of rutin may be associated with modulation of glutamate metabolism by astrocytes and/ or release of soluble neuroprotective and inductors of neural differentiation factors.