Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury

Abstract

Withania somnifera has been used in traditional medicine for a variety of neural disorders. Recently, chronic neurodegenerative conditions have been shown to benefit from treatment with this extract. To evaluate the action of this extract on traumatically injured neurons, the efficacy of W. somnifera root extract as a neuroprotective agent was examined in cultured model neurons exposed to an in vitro injury system designed to mimic mild traumatic brain injury (TBI). Neuronal health was evaluated by staining with annexin V (an early, apoptotic feature) and monitoring released lactate dehydrogenase activity (a terminal cell loss parameter). Potential mechanisms underlying the observed neuroprotection were examined. Additionally, morphological changes were monitored following injury and treatment. Although no differences were found in the expression of the antioxidant transcription factor nuclear factor erythroid 2-like 2 (Nrf2) or other Nrf2-related downstream components, significant changes were seen in apoptotic signaling. Treatment with the extract resulted in an increased length of neurites projecting from the neuronal cell body after injury. W. somnifera extract treatment also resulted in reduced cell death in the model neuron TBI system. The cell death factor Bax was involved (its expression was reduced 2-fold by the treatment) and injury-induced reduction in neurite lengths and numbers was reversed by the treatment. This all indicates that W. somnifera root extract was neuroprotective and could have therapeutic potential to target factors involved in secondary injury and long-term sequelae of mild TBI.

Keywords: Ayurveda; SH-SY5Y; Withania somnifera; neurites; neuroprotection; traumatic brain injury.

Figure 1.

Time line that cultured neurons were exposed to treatment, in vitro injury, and assays. SH-SY5Y model neurons were plated on a Flexcell plate with a specialized silastic membrane. Cells were treated 24 h after plating. Sixteen hours posttreatment, 4 injuries consisting of a nitrogen gas pulse of 35.3 psi with a peak injury pressure of 6.0 were delivered to the cultured neurons once every hour. Assays were performed 2 h following the final injury.

Figure 2.

Withania somnifera root extract 20 μg/mL prevents SH-SY5Y death. W. somnifera root extract at a final concentration of 4, 20, and 100 μg/mL was added to the cultured neurons for 16 h prior to injury. Two hours after the final injury, cells were stained with annexin-V to determine apoptosis (*P < 0.0007).

Figure 3.

Viability of SH-SY5Y cells improves in the presence of Withania somnifera root extract. Following in vitro injury, lactate dehydrogenase (LDH) was measured in the media of culture neurons. Pretreatment with 20 μg/mL of W. somnifera root extract was sufficient to protect model neurons from cell death (*P < 0.001). veh., vehicle, Inj., injury.

Figure 4.

Nuclear factor erythroid 2-like 2 (Nrf2) antioxidant signaling was unchanged following treatment with Withania somnifera extract. Heat shock protein 70 (HSP70) messenger RNA (mRNA) was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and showed no significant increase following treatment. Total protein from Nrf2, HSP70, and heme oxygenase 1 (HMOX1) was measured by Western blot (a). Immunocytochemistry for Nrf2 expression following treatment did not indicate activation-associated translocation to the nucleus (b).

Figure 5.

A protein array was used to examine any change in factors involved in apoptotic signaling. Proteins were collected 2 h following the final injury. N = 3 biological replicates were used. Several proteins showed a trend with injury and treatment, however, the only proteins that were significantly changed were Bax (*P < 0.05) and 14-3-3, by antibody array analysis (a). With Western immunoblots (b and c), we observed a significant decrease in Bax expression following injury in the presence of treatment compared to vehicle alone (*P < 0.05; B). We did not observe any change in heat shock protein 90 (HSP90; c).

Figure 6.

Treatment with Withania somnifera root extract increased the length of neurites. Two hours postinjury, cells were stained with crystal violet and phase contrast images were taken (a). Automated tracing of neurites using neurite tracer showed that in the presence of W. somnifera, neurites were significantly longer (26,709 total cells were measured, P < 0.0001; b). In other experiments, relief contrast images were analyzed using a semi-automated method, which allowed analysis of additional parameters (c). After tracing 350 cells, semi-automated analysis indicated that postinjury, the maximum process length was significantly increased (*P < 0.0001) by the treatment (d), while the percentage of cells with processes did show a strong trend with treatment, and it was not significantly different (P < 0.11; e).