The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells

Francesca Felicia Caputi,Sanzio Candeletti,Sanjay Kasture,Elio Acquas,Patrizia Romualdi,Stefania Ruiu

doi:10.1186/s12906-017-2065-9

Abstract

BackgroundBehavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine’s analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of μ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells.MethodsA range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated.ResultsData analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation.ConclusionPresent in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

Highlights

Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine’s analgesic effect; little is known about the underpinning molecular mechanism(s)
Phytochemical analysis of WSE high-performance liquid chromatography (HPLC)-fingerprint analysis of WSE indicated the presence of the following withanolides: withanoside-IV, withanoside-V, withaferin-A, 12-deoxy withastramonolide, withanolide-A, and withanolide-B (Fig.1a and 1b)
MTT assay for cell viability SH-SY5Y cells exposed to WSE showed not significant alterations of cell survival following 5 h (Table 4)

Summary

Introduction

Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine’s analgesic effect; little is known about the underpinning molecular mechanism(s). Its increasing attractiveness is mostly due to the antiinflammatory [1, 8] and anti-cancer [1, 6] properties, and to a number of central effects related to stress [8], anxiety [16] and neurodegenerative disorders [8, 17]. In this frame, it is worth noting that Withanolides and Withaferin A, abundantly present in Withania somnifera roots, have been reported to interact with cholinergic mechanisms [18] and with Nuclear factor-κB [19–21]. We assessed the binding affinities for a number of receptors [23, 25] and we found that WSE shows moderate affinities for GABAA and GABAB (13 and 130 μg/ml, respectively) as well as for opioid [μ (MOP): 385 μg/ml; δ: 166 μg/ml; κ: 775 μg/ml] receptors

Methods

Results

Discussion

Conclusion