Korean J Pain 2021; 34(3): 262-270
Published online July 1, 2021 https://doi.org/10.3344/kjp.2021.34.3.262
Copyright © The Korean Pain Society.
1Department of Biological Sciences, Faculty of Science, The University of Jordan, Amman, Jordan
2Department of Pharmaceutical Sciences, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman, Jordan
3Pharmacological and Diagnostic Research Center, Al-Ahliyya Amman University, Amman, Jordan
4Department of Medical Laboratory Sciences, Faculty of Allied Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan
Correspondence to:Sahar Majdi Jaffal
Department of BiologicalSciences, Faculty of Science, The University of Jordan, Amman 11942, Jordan
Handling Editor: Sang Hun Kim
Received: January 2, 2021; Revised: April 7, 2021; Accepted: April 8, 2021
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel implicated in pain sensation in response to heat, protons, and capsaicin (CAPS). It is well established that TRPV1 is involved in mechanical allodynia. This study investigates the effect of Ononis spinosa (Fabaceae) in CAPS-induced mechanical allodynia and its mechanism of action.
Methods: Mechanical allodynia was induced by the intraplantar (ipl) injection of 40 μg CAPS into the left hind paw of male Wistar rats. Animals received an ipl injection of 100 μg O. spinosa methanolic leaf extract or 2.5% diclofenac sodium 20 minutes before CAPS injection. Paw withdrawal threshold (PWT) was measured using von Frey filament 30, 90, and 150 minutes after CAPS injection. A molecular docking tool, AutoDock 4.2, was used to study the binding energies and intermolecular interactions between O. spinosa constituents and TRPV1 receptor.
Results: The ipsilateral ipl injection of O. spinosa before CAPS injection increased PWT in rats at all time points. O. spinosa decreased mechanical allodynia by 5.35-fold compared to a 3.59-fold decrease produced by diclofenac sodium. The ipsilateral pretreatment with TRPV1 antagonist (300 μg 4-[3-Chloro-2-pyridinyl]- N-[4-[1,1-dimethylethyl] phenyl]-1-piperazinecarboxamide [BCTC]) as well as the β2-adrenoreceptor antagonist (150 μg butoxamine) attenuated the action of O. spinosa. Depending on molecular docking results, the activity of the extract could be attributed to the bindings of campesterol, stigmasterol, and ononin compounds to TRPV1.
Conclusions: O. spinosa alleviated CAPS-induced mechanical allodynia through 2 mechanisms: the direct modulation of TRPV1 and the involvement of β2 adrenoreceptor signaling.
Keywords: Butoxamine, Capsaicin, Fabaceae, Hyperalgesia, Molecular Docking Simulation, Neuralgia, Ononis, Pain, Stigmasterol, TRPV Cation Channels.
Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in inducing the symptoms of pain in response to heat, protons, and capsaicin (CAPS) . It is expressed in the dorsal root ganglia (DRGs), the cell bodies of the nociceptors extending to Aδ- and C-fibers . It is well established that the activation of this channel is responsible for mechanical allodynia  and thermal hyperalgesia . Several inflammatory mediators modulate TRPV1 indirectly through binding to their receptors and formation of second messengers that activate TRPV1, leading to its phosphorylation [5,6]. This stimulation increases the probability of activating the open state of this channel or its translocation to the plasma membrane [5,6].
Despite the fact that there are several analgesics in the market, many of them have side effects . Accordingly, there is an increasing need for the development of new and safe drugs.
Mechanical allodynia is defined as a painful sensation that results from innocuous stimuli such as touch . It can be produced by the intraplantar (ipl) injection of CAPS (the pungent ingredient in chili pepper and the agonist of TRPV1 channel) into the hind paw of animal [2,3]. The nociceptive flexion reflex is quantified by applying an increasing mechanical force into the rat’s hind paw . Since previous studies have not examined the effect of
Butoxamine hydrochloride, a selective β2 adrenoreceptor antagonist, was purchased from Sigma-Aldrich, St. Louis. Diclofenac sodium, a non-steroidal anti-inflammatory drug (NSAID), was brought from Novartis, El-Salvador. CAPS, a TRPV1 agonist, and BCTC [4-[3-Chloro-2-pyridinyl]-
All procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and comply with the guidelines of the International Association for the Study of Pain. Ethical approval (No. AAU-2/4/2018) for conducting the work was obtained from Al-Ahliyya Amman University. Male Wistar rats (250-300 g) were brought from the animal house at Al-Ahliyya Amman University, Jordan, and were kept at 23 ± 2°C with an alternating 12 hour light-dark cycle. Food and water were provided
The animals were divided into 8 groups (with 8 rats in each group). Mechanical allodynia was induced by the ipl injection of 40 µg CAPS into the left hind paw of the animals. Group I received ipl injection of vehicle only (control group). Group II received ipl injection of CAPS. Group III received ipl injection of 100 µg plant extract into the left hind paw 20 minutes before the ipl injection of CAPS. Group IV, the positive control group, received 2.5% diclofenac sodium 20 minutes prior to CAPS injection. The dose and time of injecting diclofenac sodium was chosen pursuant to the study of Nozadze et al. . Groups V and VI received 300 µg BCTC and 150 µg butoxamine 30 minutes prior to the injection of plant extract, respectively followed by CAPS injection. Groups VII and VIII received the antagonists BCTC and butoxamine 30 minutes before CAPS injection, respectively. All drugs, vehicle, and plant extract were injected in a volume of 100 µL/paw into the left paw of the rats.
The experimental rats were allowed to acclimatize for 1 hour in wire mesh-floor cages covered with perforated lids. Baseline withdrawal thresholds were assessed in the first day to ensure that the animals have equivalent baseline readings before starting the test. Paw withdrawal threshold (PWT) of the ipsilateral (left hind paw) and the contralateral (right hind paw) hind paws were measured using handmade von Frey filament (pursuant to Ferrier et al. ) 30, 90, and 150 minutes after CAPS injection. Briefly, a thin filament was used to press against the plantar surface of the animal’s hind paw. The filament was pressed once against the hind paw of the animal at each measured time point. A sharply withdrawn paw was regarded as a positive response. A cut-off mass unit of 80 g was used in this experiment according to Ferrier et al. . Also, the behavior of animals was observed and recorded.
The X-ray crystallographic structure of TRPV1 (PDB ID: 5IS0) was selected from Protein Data Bank (https://www.rcsb.org/) . The crystal structure is co-crystallised with the TRPV1 antagonist, capsazepine . The molecular docking tool, AutoDock 4.2 (The Scripps Research Institute, La Jolla, CA) was used to study the intermolecular interactions and binding energies of the proposed compounds found in
The two dimensional (2D) chemical structures of the major compounds found in
The X-ray crystallographic structure of TRPV1 (PDB ID: 5IS0) was downloaded from the Protein Data Bank. According to the co-crystallised ligand (capsazepine), the intermolecular interactions involved chains B and C, while chains D and E were removed from the crystal structure prior to docking. Polar hydrogens as well as Kollman charges were added on the amino acids. The charged protein was solvated using addsol utility in AutoDockTools.
In Abbas et al. , the compounds in the methanolic leaf extract of
The normality test was conducted for all groups using Shapiro–Wilk test. The statistical significance of differences between groups was assessed by one-way analysis of variance, followed by Tukey’s post-hoc test using GraphPad Prism version 7 (GraphPad Software, San Diego, CA).
The injection of CAPS into the left hind paw of rats induced mechanical allodynia at all-time points (measured 30, 90, or 150 min after CAPS injection) and decreased the PWT significantly compared to control group that received the vehicle (Fig. 1). Pre-injecting 100 µg
All the proposed compounds in this study were successfully docked against TRPV1 (Table 1). The compounds that had the lowest binding energies are listed in Table 2 and Fig. 3. These compounds include campesterol, stigmasterol, and ononin with –11.73, –11.02, and –8.91 kcal/mol binding energies, respectively. The results showed a conserved intermolecular interaction in these compounds, as well as the TRPV1 antagonist, capsazepine. The amino acids Leu515 and Leu553 were found to participate in the hydrophobic interaction with campesterol, stigmasterol, ononin, and capsazepine. Importantly Arg557 was shown to participate in the hydrogen bond interactions with previously mentioned compounds, except with capsazepine. Interestingly, the free energy of binding for the docked compounds is greatly affected by the number of residues that participate in hydrophobic interactions.
It is well established that the activation of TRPV1 channel is responsible for mechanical allodynia . In this study, the anti-allodynic activity of
Ipsilateral injection of
Interestingly, previous studies have investigated the interaction between β2 adrenoreceptors and TRPV1 [25,26]. It is well established that β-adrenoreceptors are coupled to G stimulatory protein that enhances adenylate cyclase activity and intracellular cyclic adenosine monophosphate (cAMP), leading to the activation of protein kinase A, PKA (a cAMP-dependent protein kinase) . Therefore, it is expected that β2 adrenoreceptor blockers will deactivate PKA, thereby reducing TRPV1 activity and its availability in the plasma membrane, finally elevating PWT in the CAPS-induced mechanical allodynia model. This may explain the findings in the present study in which a β2 blocker (butoxamine) decreased the anti-allodynic action of
Notably, there are several receptors in the noradrenergic system whereby the effect of the noradrenergic system depends on the subtype of the receptors involved. Noradrenaline reduced the amplitudes of the inward CAPS currents in DRG neurons by either yohimbine (an antagonist for α2 adrenergic receptor) or propranolol (a β adrenoreceptor antagonist) . Previous reports showed the involvement of PKA and protein kinase C (PKC) in the inhibitory effect of α2 adrenergic receptors on TRPV1 . However, it was found that α2 adrenergic receptors inhibited the function of TRPV1 in DRG neurons in a mechanism that involves calmodulin-dependent kinase II .
Earlier reports showed the involvement of β2 adrenoreceptors in pain modulation, generally. In more detail, inhibition of catechol-O-methyltransferase, the enzyme that degrades catecholamines, increased pain sensitivity through activation of both β2- and β3-adrenergic receptors . Also, epinephrine caused mechanical hyperalgesia mediated by β adrenoreceptor and an
Importantly, the anti-nociceptive effect of
The results of the behavioral assay used in this study showed the efficacy of
The present study sheds the light on the importance of
This work was published with the support of Al-Ahliyya Amman University, Amman, Jordan. The authors acknowledge the University of Jordan.
Sahar Majdi Jaffal: Investigation, Writing/manuscript preparation, Analysis for results; Belal Omar Al-Najjar: Investigation; Manal Ahmad Abbas: Investigation.
No potential conflict of interest relevant to this article was reported.
No funding to declare.