The effect of salmon calcitonin against glutamate-induced cytotoxicity in the C6 cell line and the roles the inflammatory and nitric oxide pathways play.

Ahmet Sevki Taskiran,Merve Ergul

doi:10.1007/s11011-021-00793-6

Abstract

Recent evidence has shown that salmon calcitonin (sCT) has positive effects on the nervous system. However, its effect and mechanisms on glutamate-induced cytotoxicity are still unclear. The current experiment was designed to examine the effect of sCT on glutamate-induced cytotoxicity in C6 cells, involving the inflammatory and nitric oxide stress pathways. The study used the C6 glioma cell line. Four cell groups were prepared to evaluate the effect of sCT on glutamate-induced cytotoxicity. The control group was without any treatment. Cells in the glutamate group were treated with 10mM glutamate for 24h. Cells in the sCT group were treated with various concentrations (3, 6, 12, 25, and 50µg/mL) of sCT for 24h. Cells in the sCT + glutamate group were pre-treated with various concentrations of sCT for 1h and then exposed to glutamate for 24h. The cell viability was evaluated with an XTT assay. Nuclear factor kappa b (NF-kB), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), neuronal nitric oxide synthase (nNOS), nitric oxide (NO), cyclic guanosine monophosphate (cGMP), caspase-3, and caspase-9 levels in the cells were measured by ELISA kits. Apoptosis was detected by flow cytometry method. sCT at all concentrations significantly improved the cell viability in C6 cells after glutamate-induced cytotoxicity (p < 0.001). Moreover, sCT significantly reduced the levels of NF-kB (p < 0.001), TNF-α, and IL-6 levels (p < 0.001). sCT also decreased nNOS, NO, and cGMP levels (P < 0.001). Furthermore, it decreased the apoptosis rate and increased the live-cell rate in the flow cytometry (P < 0.001). In conclusion, sCT has protective effects on glutamate-induced cytotoxicity in C6 glial cells by inhibiting inflammatory and nitric oxide pathways. sCT could be a useful supportive agent for people with neurodegenerative symptoms.

Highlights

Glutamate, an excitatory neurotransmitter, is most commonly found in the central nervous system
Nuclear factor kappa b (NF-kB), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), neuronal nitric oxide synthase, nitric oxide (NO), cyclic guanosine monophosphate, caspase-3, and caspase-9 levels in the cells were measured by ELISA kits
In this study, increasing doses of salmon calcitonin (sCT) (3–50 μg/mL) on cell survival were performed in both control and glutamate-treated C6 cells

Summary

Introduction

An excitatory neurotransmitter, is most commonly found in the central nervous system It is found in many areas in the central nervous system, the most common areas are the cerebral cortex and hippocampus (Danbolt 2001). It acts as a primary neurotransmitter in some specific regions in the cerebellum such as granule cells. It has two groups of receptors: ionotropic and metabotropic. The basic mechanism in the pathogenesis of these diseases is excitotoxicity caused by excessive glutamate stimulation, which leads to excessive calcium ion influx into the cell (Matute et al 2006). Calcium entrance to the cell triggers mitochondrial dysfunction, and increases intracellular nitric oxide levels, which induces apoptosis mechanisms in the cell (Arundine and Tymianski 2003)

Methods

Results

Conclusion