The Influence of Vitamin D Treatment on the Inducible Nitric Oxide Synthase (INOS) Expression in Primary Hippocampal Neurons

Abstract

Introduction: Neurodegeneration is a process that is characterized by the loss of neuronal structure and function and eventually ends with neuronal death. An elevated level of inducible nitric oxide synthase (iNOS) is suggested to accompany this process by inducing oxidative and nitrosative damage. Vitamin D is reported to protect glial cells against neurotoxicity via suppressing iNOS synthesis. Though there was no data about whether iNOS is regulated by vitamin D in hippocampal neurons. In this study our aim was to determine any alteration in iNOS expression of hippocampal neurons in response to vitamin D treatment.

Method: Twenty four and 48 hours of vitamin D treatments were performed on primary hippocampal neuron cultures that were prepared from Sprague dawley rat embryos (E18). The alterations in the iNOS mRNA expression were determined with quantative real time polymerase chain reaction (qRT-PCR). The cytotoxicity levels of each group were investigated by the measurement of lactate dehydrogenase (LDH) that is released to culture medium.

Results: No difference was observed between groups in 24 hours of treatment regarding the iNOS expression. Though the iNOS mRNA level of vitamin D treated group was significantly lower than that of control group on the 48th hours of treatment (p<.001). Vitamin D treatment also attenuated the LDH release which is an indicator of cytotoxicity (p<.001).

Conclusion: Our results indicated that vitamin D has the potential to prevent oxidative damage by suppressing iNOS expression.

Keywords: Alzheimer’s disease; inducible nitric oxide synthase (iNOS); primary hippocampal neuron culture; vitamin D.

Figure 1

Phase-contrast image of seven-day-old primary hippocampal neurons ×20

Figure 2. Seven-day-old primary hippocampal neurons

Neurons are green (FITC-labeled PAN neuronal marker antibody), glia are red (Texas Red-labeled GFAP antibody), the nuclei are blue (DAPI). Glia ratio in cultured cells was 20%. ×40.

Figure 3. iNOS mRNA expression of 24 and 48 hours vitamin D treated hippocampal neurons

There was no statistically significant difference between vitamin D treated group and control group in 24 hours of treatment (p>0.05). iNOS mRNA level was significantly decreased in vitamin D treated group in 48 hours of treatment when compared with control group (*p<.001). Data were given as mean and SD.