Korean J Physiol Pharmacol.  2011 Dec;15(6):353-361. 10.4196/kjpp.2011.15.6.353.

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation

• Bhuiyan MI
• Kim HB
• Kim SY
• Cho KO

• KMID: 2285422
• DOI: http://doi.org/10.4196/kjpp.2011.15.6.353

Abstract

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50microM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

Keyword

Cyanidin-3-glucoside; Oxygen-glucose deprivation; Morus alba L.; Neuroprotection; Glutamate

MeSH Terms

Animals
Anthocyanins
Cell Death
Cell Membrane
Fluorescence
Fruit
Glucosides
Glutamic Acid
Hand
Membrane Potential, Mitochondrial
Membranes
Morus
Neurons
Neuroprotective Agents
Rats
Anthocyanins
Glucosides
Glutamic Acid
Neuroprotective Agents

Figure

• 

  Fig. 1 Double fluorescent immunocytochemistry of DIV-7 rat primary cortical neurons in serum-free Neurobasal medium supplemented with B27. The cells were identified as astrocytes using an anti-GFAP antibody (red; A) or as neurons using an anti-NeuN antibody (green; B). Astrocyte contamination was <1% in this serum-free culture condition.

• 

  Fig. 2 The experimental design of the OGD-model in rat primary cortical neurons (A). OGD-duration-dependent neuronal cell death (B). DIV-7 rat primary cortical neurons were transiently placed in an anaerobic chamber for the indicated duration (1~4 h), and neuronal injury was assessed using LDH release assay 24 h following the simulated reperfusion. The values are given as the mean±S.E.M. of at least four independent experiments with different culture batches; *p<0.01 compared with the sham.

• 

  Fig. 3 The neuroprotective effect of mulberry C3G fraction against OGD-induced neuronal cell death. DIV-7 rat primary cortical neurons were exposed to OGD for 3.5 h with or without mulberry C3G fraction. Cell survival and death were measured 24 h following simulated reperfusion using the MTT reduction (A) and LDH release assays (B), respectively. The values are given as the mean±S.E.M. of at least six independent experiments. #p<0.01 compared with the sham. *p<0.05 and **p<0.01 compared with OGD without mulberry C3G fraction.

• 

  Fig. 4 The effects of different glutamate concentrations and culture durations on glutamate-induced neurotoxicity. Sister cultures of rat primary cortical neurons cultured for different periods of time were incubated with different concentrations of glutamate for 10 min. The resultant neuronal injury was assessed using the LDH release assay 24 h following the glutamate exposure. The values are given as the mean of at least four independent experiments with different culture batches.

• 

  Fig. 5 The effects of mulberry C3G fraction on glutamate-induced neurotoxicity. DIV-9 cells were exposed to 50µM of glutamate with or without mulberry C3G fraction. Cell survival and death were measured 24 h following glutamate exposure using the MTT reduction (A) and LDH release assays (B), respectively. The values are given as the mean±S.E.M. of at least four independent experiments with different culture batches. #p<0.01 compared with the sham. *p<0.05 compared with glutamate without mulberry C3G fraction treatment.

• 

  Fig. 6 The effects of mulberry C3G fraction on MMP maintenance in primary cortical neurons subjected to OGD. DIV-7 cells were exposed to OGD for 3.5 h with or without mulberry C3G fraction treatment. Following 24 h of simulated reperfusion, the MMP was measured using the fluorescent dye rhodamine-123. Representative fluorescent images of sham-treated cells (A), cells subjected to OGD only (B) and cells subjected to OGD with 5µg/ml of mulberry C3G fraction (C). The scale bar is 100 mm. The bar graph (D) summarizes the fluorescence measurements from different groups. The values are given as the mean±S.E.M. of at least three independent experiments with different culture batches. #p<0.01 compared with the sham. *p<0.05 compared with OGD without mulberry C3G fraction treatment.

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