Asiri 2017 Thesis
|Asiri A (2017) Effects of asiatic acid on neurite outgrowth in Neuro-2a cells. Master's Thesis p42.|
Abstract: Recently, medicinal plants from ancient Ayurvedic medicine have provided clues to the discovery of novel therapeutics for various diseases. In Ayurvedi c medicine, a common Indian plant, Centella asiatica is highly regarded as a "rasayana" or nerve tonic. The Centella extract is used to ward off age-related dementia and to increase memory and intelligence. The mechanism by which Centella improves memory and learning and reduces the risk of dementia is unclear.
We recently tested the effects of asiatic acid, the main active component of Centella, on neuronal growth. We hypothesized that asiatic acid will promote neuronal growth and neurite network formation. To test this hypothesis, we examined the effects of asi atic acid on neuronal growth in murine neuroblastoma cells, Neuro2a. Neuro2a cells were cultured for 24 hours in DMEM medium containing lO mM glucose and 10% FBS in six-well plates at a concentration of 200,000 cell s/well. The cells were further cultured for 72 hours in DMEM containing 10 mM glucose and with either 1 μM asiatic acid in ethanol or ethanol alone (vehicle). Cells were photographed, and neurite outgrowth quantified using NeuronJ software. The results revealed that asiatic acid treatment significantly increased the percentage of cells bearing neurites as compared to neurons grown in medium alone. In addition, asiatic acid treatment increased neurite extension and combined length of neurites. To investigate the impact of asiatic acid on bioenergetics in Neuro2a cells, we first analyzed the electron transport chain of the mitochondria via respirometry. The respiration rates of Neuro2a cells cultured in medium containing asiatic acid was significantly (p< 0.05) higher than cells grown in medium containing vehicle alone. Also, western blot analyses were used to examine if asiatic acid could increase the mitochondrial complex. The result showed that Asiatic acid increased complex 1, 2, 3, and 4. In addition, we examined if asiatic acid would increase oxidative phosphorylation instead of glycolysis that results in l actate production. The results indicated that Neuro2a cells treated for 24 hours with 1 μM of asiatic acid induced less lactate as compared to Neuro2a with ethanol alone (vehicle). Also, the MTT assay was used to detect the viable cells in Neuro2a cells treated with either l μM asiatic acid or ethanol alone (vehicle) for two days. The result shows that Neuro2a cells treated with asiatic acid showed increased cell viability as compared to Neuro2a exposed to ethanol alone. Finally, the effect of asiatic acid on cell proliferation was examined using standard trypan blue staining. The data revealed that doubling time was significantly slower in cells cultured in presence of asiatic acid as compared to cells grown in vehicle (ethanol) alone (p< 0. 05).
Together these results suggest that asiatic acid is neurotrophic. This effect may explain the beneficial role of Centella asiatica extract on learning and memory and in preventing neurological disorders.
Labels: MiParea: Respiration, Pharmacology;toxicology Pathology: Cancer
Organism: Mouse Tissue;cell: Nervous system Preparation: Intact cells Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase
Coupling state: LEAK, ROUTINE, ET