Ashwagandha and Its Active Ingredient, Withanolide A, Increase Phosphorylation of TrkB in Cultured Hippocampal Neurons

Abstract

Aims: To primary rat embryonic hippocampal neurons in culture, ashwagandha or one of its active ingredients, withanolide A were added in the presence or absence of nutrient supplementation and then assayed for activity of the BDNF receptor, TrkB. Study Design:  Primary hippocampal neurons were cultured and grown in nutrient-rich or nutrient-poor medium.  Ashwagandha or withanolide A were then be added to both types of media with or without an inhibitor of TrkB or either the PI-3K or MAPK pathway. Place and Duration of Study: Department of Biological Sciences, California State University, Los Angeles, CA, USA, between July 2021 and August 2022. Methodology: Rat embryos were removed by cesarean section from mother rats at 18 days’ gestation and the hippocampi of the former dissected, plated into culture dishes, and treated with the appropriate drug(s) (see Study Design above).  After 4 days, neurons were harvested for Western blotting.  Optical density of Western blot bands were quantified and statistically analyzed in a 2-way ANOVA, using a level of statistical significance at P < .05. Results: Under normal conditions (with N2 supplement), ashwagandha, but not withanolide A, increased phospho-TrkB immunoreactivity when compared to the effects of vehicle (controls, F(11, 24) = 22.48, P < .001), although withanolide A did not quite reach statistical significance (P = .069) when compared to that of the controlled condition.  Likewise, under nutrient-deprived conditions, both ashwagandha and withanolide A also increased phosphorylation of TrkB when compared to that of vehicle-nutrient-deprived conditions (P < .0001). The same results were obtained in the presence of inhibitors of TrkB itself and the PI-3K (ashwagandha, P < .001; withanolide A, P < .001) and MAPK (ashwagandha, P = .027; withanolide A, P = .045) pathways. Conclusion: Ashwagandha or withanolide A activates TrkB, in nutrient-deprived hippocampal neurons, underscoring its role in neuronal survival signaling.