Evodiamine suppresses capsaicin-induced thermal hyperalgesia through activation and subsequent desensitization of the transient receptor potential V1 channels

Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.


Introduction
The transient receptor potential channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor, is expressed by a subset of the small-sized dorsal root ganglion (DRG) or trigeminal ganglia neurons [1]. TRPV1 is activated by capsaicin, protons, or noxious heat (with a thermal threshold [43°C), which causes pain in vivo [1,2]. TRPV1 selective antagonists can inhibit mechanotransmission in primary sensory neurons following inflammation [3]. In addition, TRPV1 can be sensitized by proinflammatory agents such as prostaglandins, bradykinin, adenosine triphosphate (ATP), protease activated receptors (PAR) 2, nerve growth factor (NGF), and tumor necrosis factor alpha (TNF-a), which directly or indirectly modulate the channel protein or the probability of channel opening by stimuli [4][5][6][7][8]. Analyses of mice lacking TRPV1 have shown that it is essential for selective modalities of pain sensation as well as tissue injury-induced thermal hyperalgesia [9]. Thus, TRPV1 functions as a molecular integrator of painful stimuli in which each stimulus sensitizes the channel to other stimuli, with the end result that TRPV1 acts as a molecular amplifier in the sensory neuron. These insights have renewed the interest in TRPV1 as an important site of analgesia.
Evodiae fructus (EF) is one of the most popular and multi-purpose herbs traditionally used in China (known as Wu-Zhu-Yu) and Japan (Goshuyu) for the treatment of headaches, abdominal pain, difficult menstruation, vomiting, diarrhea, and other diseases [10]. Evodiamine (Fig. 1), a natural indole alkaloid, is the major bioactive constituent of EF. Evodiamine has been reported to possess multiple biological effects such as antinociceptive, antiinflammatory, antineoplastic, antidiabetic, and thermoregulatory [11][12][13][14][15]. However, the molecular mechanism underlying evodiamine's effect on antinociception remains unclear. Pearce and coworkers have characterized evodiamine as an agonist for rat TRPV1 expressed heterologously in CHO cells using calcium uptake analysis [16]. However, whether evodiamine directly excites TRPV1 in sensory neurons has not been clarified yet. Since activation of TRPV1 may produce a nociceptive response, it is of interest whether and how evodiamine exerts its antinociceptive effect through TRPV1 activation, and this is important for explaining the effect of EF in pain-related diseases.
To the best of our knowledge, we report for the first time that evodiamine activates TRPV1 in sensory neurons. Our patch-clamp analyses indicate that the antinociceptive effect of evodiamine may be attributed to the evodiamineinduced activation and subsequent desensitization of TRPV1.

Materials and methods
Mammalian cell culture HEK 293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamax, penicillin, and streptomycin. HEK293 cells were transfected with 1 lg human TRPA1 (hTRPA1) cDNA or rat TRPV1 (rTRPV1) cDNA using Lipofectamine LTX and PLUS Reagent (Invitrogen, Carlsbad, CA, USA). An enhanced green fluorescence protein reporter plasmid (BD Biosciences, San Jose, CA, USA) was cotransfected with the TRP channels. The hTRPA1 and rTRPV1 cDNAs were generous gifts from Prof. Makoto Tominaga (Okazaki Institute of Integrative Bioscience, Okazaki, Japan). For primary cultures of DRG neurons, DRGs were collected from adult Sprague-Dawley rats (100-150 g) using sterile techniques and placed in ice-cold Earle's Balanced Salt Solution (EBSS, Sigma-Aldrich Co. LLC, St Louis, MO, USA). The adhering fat and connective tissue were removed, and each DRG preparation was immediately placed in a medium consisting of 2 ml EBSS and 1.25 mg/ml collagenase P (Sigma-Aldrich Co. LLC, St Louis, MO, USA) and kept at 37°C for 60 min with occasional agitation. After dissociation of the DRG cells, the cell suspension was centrifuged for 5 min at 250 9 g, and the cell pellet was resuspended in EBSS supplemented with 10 % FBS, 2 mM glutamax and glucose, 1 9 MEM vitamin, penicillin, and streptomycin. Recombinant rat NGF (100 ng/ml, Sigma-Aldrich Co. LLC, St Louis, MO, USA) was added to the medium.

Calcium imaging
Ratiometric calcium imaging was performed using an Olympus fluorescence microscope (IX 70, Olympus) equipped with an Orca-ER digital CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Dual images (340 and 380 nm excitation, 510 nm emission) were collected, and pseudocolor ratiometric images were monitored every 5 s during the experiment using a ratio imaging system (AQUACOSMOS/Ratio, Hamamatsu Photonics, Shizuoka, Japan). Rat DRG neurons were cultured on poly-L-lysinecoated glass coverslips for 18-24 h and were subsequently loaded with 2 lM Fura-2 acetoxymethyl ester (Fura-2 AM) for 40 min at 37°C. DRG neurons in one randomly selected microscopic field (10-30 cells) on one glass coverslip were measured.

Electrophysiology
Whole-cell patch-clamp recordings were performed 2 days after transfection of HEK293 cells with hTRPA1 cDNA or 1 day after transfection with rTRPV1 cDNA or DRG neuron culture. Voltage-clamp experiments were performed at a -60 mV holding potential, and recordings were sampled at 5 kHz and filtered at 2 kHz. In all tests, agonists were applied until the evoked currents underwent desensitization. In all experiments, the current magnitude was quantified by the peak current amplitude. The standard bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, and 10 mM glucose, pH 7.4 (adjusted with NaOH). The pipette solution contained 140 mM KCl, 2 mM MgCl 2 , 0.5 mM CaCl 2 , Fig. 1 Chemical structure of evodiamine 5 mM Mg-ATP, 5 mM EGTA, and 10 mM HEPES, pH 7.2 (adjusted with KOH). All patch-clamp experiments were performed at room temperature (RT, *25°C). The solutions containing drugs were applied to the chamber (1 ml) by a gravity system at a flow rate of 3-4 ml/min.

Behavior studies
Twenty male adult Sprague-Dawley rats (200-250 g) were used for the behavioral analyses. After adaptation, each rat received an intraplantar injection of 50 ll evodiamine (100 lM in 5 % DMSO and 0.5 % Tween-20 in saline) or vehicle (5 % DMSO and 0.5 % Tween-20 in saline) into the left hind paw. At 10 min after injection, rats received an intradermal injection of 50 ll capsaicin (220 lM, 0.5 % Tween-20 in saline) or vehicle (0.5 % Tween-20 in saline) in the same area as the evodiamine injection. For the thermal hyperalgesia analysis, the response latencies to a radiant paw heating were measured after injection of capsaicin at 0, 10, 30, 60, and 120 min using the plantar test (Ugo Basile, Comerio, Italy) as described previously [6]. Briefly, a radiant heat source beneath a glass pane was aimed at the planter surface of the hindpaw. Two latency measurements were taken for each ipsilateral hindpaw in each test session. The hindpaws were tested alternately, with 5-min intervals between consecutive tests. The two latency measurements per side were averaged. The heat stimulus was terminated after a withdrawal response or after 30 s to avoid skin damage. The ratio of the recording at 10, 30, 60, or 120 min after injection to that before the injection in each rat was used for statistical analysis. A researcher who was unaware of the treatment group performed all the behavioral experiments. All procedures involving the care and use of animals were approved by the Hyogo University of Health Sciences Committee on Animal Research and were carried out in accordance with the NIH guidelines for the care and use of laboratory animals.

Plant material and extract preparation
EF (produced in Jiangxi Province, China, lot no. 003807001), the fruit of Evodia rutaecarpa Bentham, E. officinalis Dode, or E. bodinieri Dode, was purchased from Tochimoto Tenkaido Co., Ltd. (Osaka, Japan) and kept in the laboratory of Hyogo University of Health Sciences at room temperature. The EF was extracted with MeOH and evaporated in vacuo to obtain MeOH extract.

Statistical analysis
All results are expressed as mean ± SEM. An unpaired t test was used to compare the electrophysiological data between the two groups. Two-way repeated measures ANOVA followed by Fisher's PLSD was applied to the behavioral data. A difference was accepted as significant if the probability was \5 % (p \ 0.05).

Results and discussion
Evodiamine activated TRPV1 in sensory neurons Using calcium imaging, we found that either the extract of EF or evodiamine caused a rapid elevation in intracellular calcium levels in a subpopulation of small-sized isolated rat DRG neurons; almost all of the evodiamine-sensitive neurons also responded to capsaicin (Fig. 2a-c). In the whole-cell patch clamp recording, evodiamine induced robust inward currents in the capsaicin-sensitive DRG neurons (Fig. 2d). The evodiamine-induced intracellular calcium response was completely blocked by capsazepine (Fig. 3a, b). These data suggested that the traditional herbal medicine EF might directly excite primary sensory neurons through the evodiamine-induced activation of TRPV1, providing the first evidence for evodiamine-induced sensory neuron activation. A previous study has reported that evodiamine induced calcium uptake in TRPV1-expressing CHO cells, which showed the same interaction of evodiamine and TRPV1 but in heterologous cells [16]. In the present study, capsazepine completely blocked the evodiamine-induced calcium influx, suggesting that evodiamine may bind to TRPV1 at the same binding site with capsaicin. This idea is also supported by other studies [16,17].

Evodiamine activated and subsequently desensitized TRPV1 in heterologous HEK cells
To further determine the agonistic action of evodiamine on TRPV1, we examined the evodiamine-activated currents in TRPV1-or TRPA1-transfected HEK293 cells. Evodiamine (10 lM) did not evoke any current in untransfected or TRPA1-transfected HEK293 cells as measured by voltage clamp recordings (data not shown and Fig. 4a, b).
Pharmacological desensitization of receptors is a fundamental approach for reducing neuronal activity. We thus The horizontal bars indicate the duration of the compound application. Note that capsazepine completely blocks the EVO-induced calcium influx. All of the neuron that responds to KCl (50 mM) is used for analysis in calcium imaging. EVO evodiamine, CPZ capsazepine examined the effects of pretreatment with evodiamine on the capsaicin-induced currents in TRPV1-expressing HEK293 cells. We found that a single application of 100 nM capsaicin (without evodiamine pretreatment) induced robust inward currents with a current density of -327.4 ± 61.3 pA/pF (n = 12), which were significantly inhibited by pretreatment with 1 lM evodiamine (-132.5 ± 16.2 pA/pF; n = 6; p \ 0.05 vs. capsaicin) (Fig. 5a, b). These observations indicated that pretreatment with evodiamine could desensitize the channel activity of TRPV1 to capsaicin. Evodiamine has been suggested to sensitize and desensitize the capsaicin-sensitive sensory afferents in mice [18]. Here, we first demonstrated at the cellular level that evodiamine could activate and then subsequently desensitize the TRPV1 channel.

Evodiamine reduced capsaicin-induced thermal hyperalgesia
Activation of TRPV1 by pungent natural products suggests a nociceptive role for TRPV1. Desensitization of TRPV1 channels shows a therapeutic value in pain relief [19]. Several TRPV1 agonists have been tried for pain therapy in the clinical practice [20,21]. Due to its agonism, evodiamine activated and then desensitized TRPV1 in our present in vitro experiments. To further validate whether evodiamine application in vivo can also suppress the TRPV1-mediated pain behavior, we administered intraplantar injections of evodiamine followed by capsaicin injections to Sprague-Dawley rats and recorded their behavior toward capsaicin-induced thermal hyperalgesia. Consistent with our previous studies [6,22], the capsaicin injection induced a significant and rapid thermal hyperalgesia at the site of injection that vanished 2 h post injection. Interestingly, the injection of evodiamine (50 ll, 100 lM in 5 % DMSO, and 0.5 % Tween-20 in saline) itself did not cause acute nocifensive behavior such as paw lifting, flinching, or licking. This lack of an irritant effect of evodiamine may be due to its low potency and efficacy for activating TRPV1. At 10 min after evodiamine or vehicle treatment, capsaicin was injected into the same area of the hind paw. As expected, the pretreatment with evodiamine induced a significant decrease in the withdrawal latency of the hind paw 30 min after the intraplantar capsaicin injection (Fig. 6).

Conclusion
In this study, we demonstrated that evodiamine strongly desensitized TRPV1 channels resulting in the suppression of the capsaicin-induced thermal hyperalgesia response after a tonic pre-application with evodiamine. To our knowledge, these results identify for the first time that the mechanism underlying the analgesic effect of EF or evodiamine may be the activation and subsequent desensitization of TRPV1 in sensory neurons.  Fig. 6 Evodiamine suppresses capsaicin-induced thermal hyperalgesia in rats. The graph shows the pain response (withdrawal latency to radiant heat) to the subcutaneous application of capsaicin with or without pretreatment of evodiamine. Animals were pretreated subcutaneously with evodiamine (100 lM, 50 ll) or vehicle (5 % DMSO and 0.5 % Tween-20 in saline) 10 min before the capsaicin (220 lM, 50 ll) or vehicle (0.5 % Tween-20 in saline) injection. The withdrawal latencies of rats at each time point were normalized to their baseline values (obtained before the application). EVO ? VEH evodiamine (100 lM, 50 ll) with vehicle (0.5 % Tween-20 in saline) injection, VEH ? CAP vehicle (5 % DMSO and 0.5 % Tween-20 in saline) with capsaicin (220 lM, 50 ll) injection, EVO ? CAP evodiamine (100 lM, 50 ll) with capsaicin (220 lM, 50 ll) injection. CAP capsaicin, EVO evodiamine. Asterisk p \ 0.05, EVO ? CAP versus VEH ? CAP, two-way repeated ANOVA followed by Fisher's PLSD. Six rats were used in the EVO ? VEH group; seven rats were used in VEH ? CAP or EVO ? CAP group