Neuroprotective Effects of Kuromanin Chloride (Anthocyanin) Against Glutamatergic Excitotoxicity in Neuroblastoma Cells

• ¹ United Arab Emirates University, United Arab Emirates

The increase in prevalence of neurodegenerative diseases in the modern society is imposing several health, social and economic burden. An estimated 6.8 million people die from neurological diseases worldwide every year. Studies have also revealed that out of the one billion people who are effected by neurological disorders, 24 million are suffering from neurodegenerative disorders such Alzheimer’s and other dementias. Neurodegeneration is the process of deterioration and ultimately the death of neuronal cells caused by several factors such as oxidative stress, inflammatory changes, mitochondrial dysfunction, excitotoxicity, accumulation of iron and brain proteins. Previous studies on animal models of neurodegenerative diseases have shown the possible role of chronic excitotoxicity in these diseases. One of the major cause of excitotoxicity is by the over-activation of the glutamatergic system leading to glutamate toxicity and neurodegeneration. L-glutamate is an important neurotransmitter that is involved in almost all functions of the nervous system. Till date, no complete cure has been developed for neurodegenerative diseases. Current treatments are mainly palliative and hence keeps the patients from becoming worse while the pathology of the disease is still incurable. Neuroprotection broadly refers to the various strategies and mechanisms employed to preserve the structure and function of neurons and to protect them from neurodegeneration and nerve injury caused by various agents and diseases. Current methods of neuroprotection do not reverse the damage that have occurred but can protect further damage and slow down degeneration. Neuroprotection maybe achieved by several mechanisms such as free radical trapping, apoptosis inhibition, inhibiting inflammation and preventing excitotoxicity. In this study, commercially available L-glutamic acid monosodium (MSG) was used as a source for inducing excitotoxicity. MSG is the most abundant naturally occurring amino acid and is seen in almost any food products in stores including baby food, cosmetics, processed meat, dietary supplements, vaccines, etc. Studies have shown the toxic side-effects of excess MSG exhibited as anomalies of respiratory, nervous, digestive, metabolic and circulatory systems in both animal and human models and as a potent neurotoxin that activates the neurodegenerative pathways in the brain along with genotoxic effects such as altering the expression of various metabolites in oxidative stress pathway. This study focused on the neuroprotective effects by kuromanin chloride against glutamatergic excitotoxicity in neurodegenerative diseases. Kuromanin chloride also known as cyanidin-3-O-glucoside is the major form of anthocyanin that is found in most plants. Anthocyanin is a plant pigment that belong to the phenolic group and is responsible for the purple, blue and red colors of fruits and vegetables. They are seen naturally in the glycosylated form and are strong anti-oxidants with a range of beneficial usage ranging from natural dye and food colorant to medicinal values such as anti-inflammatory, anti-microbial, antidiabetic, anticancer and neuroprotection. Studies are exploring the possibility of replacing synthetic food colors by the natural alternatives such as anthocyanin. Studies have revealed that kuromanin chloride (cyanidin-3-O-glucoside) is the most bioavailable compared to other anthocyanin components making it a potent nutraceutical and pharmaceutical ingredient. The action of anthocyanin in reducing the risk of a disease is believed to be either by direct mechanisms such as free-radical scavenging or indirect mechanisms such as downregulating apoptosis and cell proliferation pathways. The aim of this study was to analyze various altered cellular events in neuroblastoma cell line SH-SY5Y caused by glutamatergic excitotoxicity induced by L-glutamic acid monosodium. Cells were exposed to kuromanin chloride prior to excitotoxicity induction to show its neuroprotective effect. SH-SY5Y cell line is a human neuroblastoma cell line derived from bone marrow that have dopaminergic (DAergic) characteristics and have been used as in-vitro model for neurodegenerative diseases such as Parkinson’s Disease (PD). Various cellular parameters were assessed to monitor the effect of kuromanin chloride against MSG toxicity and the effect of MSG and kuromanin chloride alone on the cells. Namely, Lactate Dehydrogenase (LDH) assay which measures the quantity of LDH released as a result of cell lysis was performed to analyze the extent of cytotoxic effects of the compounds alone and in combination. Next, a modified MTT assay which measures the viable cells was done to assess the effect of these compounds on cell proliferation. This was then followed by quantitative real time PCR (qRT-PCR) where differential expression of genes involved in neuronal survival and maintenance such as BDNF and DNA damage response such as BRCA1 was studied. Further, the differential expression of these genes were confirmed at the protein level to visualize the up or downregulation of protein expression. Morphology of cells that are neuroprotected by kuromanin chloride versus the cells exposed to glutamatergic excitotoxicity without neuroprotection was also studied. Experimental data showed that MSG had a significant cytotoxic effect on cells at a concentration of 50mM which was also seen as a decline in cell proliferation at the same concentration. However, treating cells with kuromanin chloride alone showed that it had least cytotoxic effects at 10µM while higher concentrations showed higher cytotoxicity. These findings were supported by similar results in cell proliferation assay where there were maximum cells at 10µM. Gene expression data revealed that BDNF was highly upregulated in cells undergoing excitotoxicity probably in an effort by the cells to survive. Significant findings in this study was enlightening about the neurotoxic effects of MSG and the use of a naturally occurring compound kuromanin chloride as a potential neuroprotective agent to prevent neurodegeneration caused by acute or chronic excitotoxicity. This have paved path for future advanced studies to elucidate the precise molecular mechanism of neuroprotection exerted by kuromanin chloride. Hence, known about the possible role of chronic ecxitotoxicity in neurodegenerative diseases, kuromanin chloride is a putative neuro-protective agent that can be developed to a potential therapy to help protect neurons from neurodegeneration hence prolonging the onset of neurodegenerative diseases or for preventing further damage of neurodegeneration in patients suffering from various neurodegenerative diseases.