Curcumin enhances neuronal survival in N-methyl-d-aspartic acid toxicity by inducing RANTES expression in astrocytes via PI-3K and MAPK signaling pathways

Object

Neuroinflammation, which is characterized by the overproduction of cytokines and chemokines, plays an important role in neurodegenerative diseases, especially in Alzheimer's disease (AD). In the brain, chemokines are predominantly released by astrocytes and microglias. Expression of RANTES, as well as other cytokines, is involved in the inflammatory cascade that contributes to neurodegeneration in AD. Expression of RANTES may also have a neuroprotective effect. We sought to investigate whether curcumin exhibited neuroprotective and antioxidant activity via enhanced RANTES expression by astrocytes in cortical neuron cultures. We evaluated the neuroprotective and anti-neurodegenerative effects of curcumin in NMDA toxicity and in long-term cultures.

Methods

Pregnant female Sprague-Dawley (SD) rats were used for primary culture of cortical neurons, and neonatal 0- to 2-day-old SD rats were used for primary culture of astrocytes. Cultured astrocytes were conditioned with curcumin to prepare astrocyte-conditioned medium (ACM). Real-time polymerase chain reaction was performed to assess RANTES and iNOS mRNA expression in astrocytes following curcumin treatment. ELISA was used to detect astrocyte-secreted RANTES protein in ACM with curcumin treatment. JAK/STAT, PI-3K, PKC and MAPK inhibitors were used to ascertain whether the effects of curcumin involved these signaling pathways. To evaluate the effects of curcumin-enhanced astrocytes on neuronal survival, cultured cortical neurons treated or untreated with NMDA were incubated in ACM with or without curcumin treatment. Long-term culture (15 days in vitro, DIV) was performed to investigate the effects of curcumin-treated astrocytes on the survival of cultured cortical neurons. Neuronal survival rate was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay (for cell viability), and the lactate dehydrogenase (LDH) release assay (for cell death).

Results

We demonstrated that curcumin enhanced RANTES expression in primary cultured astrocytes, and that this effect was related to activation of PI-3K and MAPK signaling pathways. We found that curcumin inhibited iNOS expression in primary cultured astrocytes in non-stressed condition. We also found that neurons exposed to NMDA and cultured with curcumin treated ACM, which characteristically exhibited elevated RANTES expression showed higher level of cell viability and lower level of cell death. Using a small interfering RNA (siRNA) knockdown model, we found evidence that the basal level of RANTES expression in non-stimulated astrocytes provided neuroprotection.

Conclusion

We postulate that the enhanced neuronal survival by curcumin treatment in NMDA toxicity and long-term cultures was in part attributable to elevated astrocyte-derived RANTES expression via activation of PI3K/MAPK signaling pathways.