Desensitization of TRPV1 Involved in the Antipruritic Effect of Osthole on Histamine-Induced Scratching Behavior in Mice

Osthole has been isolated from the fruits of Cnidium monnieri (L.) Cusson, which has been used in Chinese traditional medicine to treat pruritic disorders for a long time. However, the antipruritic mechanism of osthole is not fully understood. In the present study, using calcium imaging, molecular docking, and animal scratching behavior, we analyzed the pharmacological effects of osthole on transient receptor potential vanilloid 1 (TRPV1). The results showed that osthole significantly induced calcium influx in a dose-dependent manner in dorsal root ganglion (DRG) neurons. Osthole-induced calcium influx was inhibited by AMG9810, an antagonist of TRPV1. Osthole and the TRPV1 agonist capsaicin-induced calcium influx were desensitized by pretreatment with osthole. Furthermore, molecular docking results showed that osthole could bind to TRPV1 with a hydrogen bond by anchoring to the amino acid residue ARG557 in the binding pocket of TRPV1. In addition, TRPV1 is a downstream ion channel for the histamine H1 and H4 receptors to transmit itch signals. Osthole attenuated scratching behavior induced by histamine, HTMT (histamine H1 receptor agonist), and VUF8430 (histamine H4 receptor agonist) in mice. These results suggest that osthole inhibition of histamine-dependent itch may be due to the activation and subsequent desensitization of TRPV1 in DRG neurons.


Introduction
e transient receptor potential vanilloid member 1 (TRPV1) channel is a well-known subfamily the TRP ion channel family, comprising nonselective cation ion channels [1], expressed on a large subset of cutaneous and sensory neurons in the dorsal root ganglion (DRG) that responds to noxious stimulation. TRPV1 plays a role in mediating acute and chronic itch stimulated by histamine and other pruritogens, such as bradykinin, IL-31, and LTB4 [2,3]. Injecting trypsin into TRPV1 -/mice resulted in a significant reduction in scratching behavior [4]. Histamine activates H1R and H4R in DRG neurons via the PLCβ/PKC pathway and/or the PLA2/lipoxygenase (LO) pathway and finally coupled to TRPV1 [5][6][7].
Previous studies have demonstrated that both antagonists and agonists of TRPV1 can lead to pain or itch relief through inactivation and chronic desensitization. Topically applied capsaicin (0.025%) can effectively treat moderate and severe pruritus induced by psoriasis [8,9] and exert moderate restraint effects on itch responses induced by histamine, substance P, and PAR-2 agonist [10]. Furthermore, 8% topical capsaicin patches also have a therapeutic effect in chronic pruritus [11,12], thus making targeting TRPV1 an attractive strategy for itch therapy.
In this study, we found that osthole can activate TRPV1 in DRG neurons and is inhibited by AMG9810 (an antagonist of TRPV1). Furthermore, the scratching behavior induced by histamine, HTMT, and VUF8430 restrained by osthole may be due to desensitization of TRPV1.

Animals.
A total of 60 male and female C57BL/6 mice aged 8 weeks were used for itch behavioral testing and calcium imaging. e animals were housed in a temperature-controlled room (22 ± 2°C) under a 12-hour light/dark cycle, with free access to food and water. All experiments were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee of Yangzhou University, Guangxi University of Chinese Medicine (Ethics license DW20181220-153), and Nanjing University of Chinese Medicine (Ethics license ACU190601, 20,190,605).

Calcium
Imaging. Dorsal root ganglia were dissociated and cultured from mice for 16-18 h. For calcium imaging experiments, cells were loaded with fura-2-acetomethoxyl ester (Molecular Probes) in HBSS solution for 30 min in the dark at room temperature, 26°C [25]. After washing three times, cells were imaged at 340 nm and 380 nm excitation to detect intracellular free calcium. Normal solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM D-(+)-glucose, and pH 7.4 with NaOH. Baseline readings were taken at 20 s before applying osthole or capsaicin to DRG neurons.

Molecular Docking.
Interaction between osthole and TRPV1 was analyzed in silico using molecular docking by AutoDock 4.2.6.

Molecular and Protein Structure Preparation.
e three-dimensional structure of the target TRPV1 protein was obtained from the Protein Data Bank (5IRZ) (PDB, https://www.rcsb.org/). e TRPV1 ligands phosphatidylinositol and osthole were used in the MOPAC program to optimize the molecular structure and calculate the AM1 atomic charge. All protein and molecular structures were prepared using AutoDock Tools 1.5.6, and the corresponding pdbqt file was generated for docking preparation.

Molecular Docking
Method. AutoDock 4.2.6 software was used to perform the molecular docking experiments [26]. First, the docking grid box sizes were 50 × 50 × 60 for TRPV1 with a default spacing of 0.375Å, and the x, y, z center was (126.562 135.846 104.054). Second, the grid point energy of the docking area was calculated using the Auto-Grid program.
e Lamarckian genetic algorithm (LGA) method was employed for docking experiments. e number of GA runs was 100, the population size was 150, and the maximum number of iterations. e maximum number of evaluations was 25000000. All other parameter values were set to the default for AutoDock 4.2.6. All calculations were performed using the MolDesigner molecular simulation platform. All results were analyzed using PyMol. PoseView created a two-dimensional schematic representation [27]. e three-dimensional structure of the protein and ligand was created using PyMol. Chimera created a hydrophobic surface schematic representation.

Scratching Behavior Assays.
To measure scratching behavior, the model was described by Kuraishi et al. [28]. Mice were randomized into two groups: the osthole group and the control group. e rostral and dorsal necks of mice were clipped and depilated with an electric hair clipper two days before the experiments. Mice were placed in a transparent plastic box (4.5 × 4.5 × 7 inches) for approximately 30 min to avoid acclimatization before each experiment. e animals in the osthole group were subcutaneously injected with 1 μM osthole in a volume of 100 μl, as the control group animals received 100 μl of equivalent vehicle solution (DMSO, 1‰) without osthole. Histamine or histamine H1 receptor agonist HTMTand histamine H4 receptor agonist VUF 8430 were injected into the nape of the mouse neck 30 min later. One scratch response was defined as the lifting of the hind limb towards the injection site. In all experiments, videotapes were played back, and the number of scratch bouts was counted at 5 min intervals for 30 min by an investigator blinded to the treatments. e final concentrations of DMSO or water did not exceed 0.5%.

Statistical Analysis.
All data were expressed as mean-± S.E.M. Differences among the groups were evaluated by the t-test and one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. A result with P ≤ 0.05 was considered "significantly different."

Osthole Bound to TRPV1 In Silico.
In our previous study, we demonstrated that osthole inhibits histamine-dependent itch via the modulation of TRPV1 function [24]. To further determine the ligand role of osthole on TRPV1, we investigated the interaction between osthole and TRPV1 by molecular docking in silico ( Figure 4). As shown in Figures 4(a) and 4(b), osthole interacted with TRPV1. e dominant conformation of osthole binding to TRPV1 is located in the hydrophobic pocket of TRPV1. In addition, the binding pattern analysis showed that osthole links with TRPV1 between the S4 and S4-S5 linkers in the binding pocket. Furthermore, osthole formed a hydrophobic interaction with the amino acid residues ALA566, VAL567, and GLN700 (as shown in Figures 4(d) and 4(e)). Osthole formed a strong hydrogen bond interaction with the hydroxyl of ARG557 (1.8-2.0Å), which is an important residue for opening the TRPV1 channel. e binding energy of osthole was −7.97 kcal/mol, which was slightly higher than that of TRPV1 agonist resiniferatoxin (−11.70 kcal/mol). ARG557 also formed a hydrogen bond with GLU570. e S4-S5 linker was pulled away from the central axis of the TRPV1 ion channel by the hydrogen bond of osthole with ARG557 and the hydrogen bond of ARG557 with GLU570.

Osthole Desensitized Capsaicin-Induced Calcium Influx.
To test whether osthole could desensitize TRPV1, we examined the response of neurons to capsaicin with or without pretreatment with 10 μM or 30 μM osthole ( Figure 6). e results showed that a single application of 0.1 μM capsaicin induced robust increase in intracellular calcium, and the fluorescence intensity of the DRG neurons was 0.72 ± 0.04 (26.23% positive cells in total cells, n � 3 animals). Although pretreatment with 10 μM osthole did not significantly affect the capsaicin reaction, the fluorescence intensity of the DRG neuron response to capsaicin was 0.69 ± 0.05 (6.36% positive cells in total cells, n � 3 animals) (Figure 6(a)). However, pretreatment with 30 μM osthole significantly decreased the capsaicin response to 0.57 ± 0.03 (7.50% positive cells in total Evidence-Based Complementary and Alternative Medicine cells, n � 3 animals, * P < 0.05, one-way ANOVA followed by Dunnett's test versus capsaicin treated alone) (Figure 6(b)). In addition, after pretreatment with capsaicin, the DRG neuron response to capsaicin again was reduced (0.26 ± 0.06 vs. 0.72 ± 0.04, n � 3 animals, * * * P < 0.001, one-way ANOVA followed by Dunnett's test versus capsaicin treated alone) (Figure 6(c)). e data showed that osthole might dose-dependently desensitize capsaicin-induced calcium influx, which can activate and subsequently desensitize the TRPV1 channel ( Figure 6(d)).

Discussion
Itchiness is a common symptom or abnormal sensation in atopic dermatitis, urticaria, and cholestasis, and it is involved in many signaling pathways [30]. ese pathways can be subdivided into histamine-dependent such and histamineindependent signaling pathways, such as Mas-related G protein-coupled receptors (Mrgprs) and PAR-2 receptors [31]. TRPV1 is a key downstream ionic channel and a target for the treatment of histamine-dependent diseases [32]. Traditional Chinese medicine or natural herb compounds have been used to treat itch or itchy diseases for a long time [33]. It is valuable to identify active ingredients in traditional Chinese medicine for the treatment of pruritus diseases. is study aimed to investigate the antipruritic molecular mechanisms of osthole, the active component of C. monnieri, by active TRPV1.

Evidence-Based Complementary and Alternative Medicine
In this study, it is shown that osthole could repeatedly activate the same DRG neurons (Figure 2), although the neurons were weakly desensitized using osthole for the third time. Furthermore, neurons activated by osthole were almost a subpopulation of small-sized isolated DRG neurons in a dose-dependent manner (Figure 3). In our previous study, we demonstrated that osthole attenuated the scratching behavior induced by histamine via the modulation of the TRPV1 channel [24]. ese results suggest that TRPV1 may be an important molecule channel for osthole to treat pruritic diseases.
Previous research has reported that the function of TRPV1 agonists seems to be mainly affected by the presence of residues Tyr511, Ser512, Leu515, Phe543, Met547, r550, Lys571, Arg 557, and Glu570 in a homologous model [34][35][36]. Using molecular docking, we found the osthole hydroxyl on Arg557, which is in accordance with a previous study (Figure 4). e binding pocket of TRPV1 comprises three parts. Several hydrophobic residues, including Leu518, Leu547, Phe554, Leu663, and Leu670 formed the upper part of the binding pocket; Tyr511, Met514, and r550 formed the middle part; and two charged residues, Glu570 and Arg557, formed the bottom part [36]. Osthole interacting with the hydroxyl hydrophobic residue Arg557 in the bottom part of the binding pocket may open the channel. However, Wang et al. found that evodiamine as a TRPV1 agonist interacted with the binding pocket formed by Ser510, Tyr511, Leu515, Tyr555, Met568, Ile569, Glu570, and Lys571 [37]. In summary, osthole can activate TRPV1 by forming a hydrogen bond interaction with hydrophobic residues.
In the calcium imaging assay, osthole-induced intracellular calcium increase and the percentage of activated neurons were inhibited by the TRPV1 antagonist AMG9810 ( Figure 5). It has been suggested that osthole is an agonist of TRPV1. However, the osthole-induced intracellular calcium influx cannot be completely blocked by AMG9810. is may be because osthole activates the other TRP channels. In other studies, osthole inhibited atopic dermatitis chronic itch by directly downregulating TSLP production from keratinocytes or activating warm-temperature Ca 2+ -permeable TRPV3 channels [19,38]. In addition, the inhibition of TRPV3 by osthole significantly attenuated the scratching behavior induced by either acetone-ether-water (AEW) or histamine [18].
is indicates that TRP channels play an important role in the pharmacological functions of osthole.
Pharmacological desensitization of receptors is essential for reducing neuronal activity. Low-dose capsaicin topical patches have been widely used in treating pain and pruritus diseases through TRPV1 desensitization in clinical settings [8,9,11,12,39]. e repeated application of the TRPV1 agonist capsaicin or natural compounds such as evodiamine from Evodia rutaecarpa Bentham [40], imperatorin from Angelica dahurica root [41], and camphor from Cinnamomum camphora [42] to DRG neurons can induce desensitization responses.
In this study, the robust calcium influx induced by capsaicin was significantly different after treatment with or without osthole ( Figure 6). e abovementioned results showed that the osthole is an agonist of TRPV1 through interaction with the hydroxyl hydrophobic residue Arg557 in the bottom part of the binding pocket, which the antagonist TRPV1 (AMG9810) can also inhibit. Interestingly, calcium influx was induced by capsaicin-desensitized pretreatment with osthole or capsaicin ( Figure 6). However, the desensitization effect of osthole was weaker than that of capsaicin ( Figure 6). is proved that osthole, an agonist of TRPV1, can exert its pharmacological action by activating and subsequently desensitizing TRPV1. TRPV1 is one of the main targets for treating histamine-dependent itch [1], and drugs that can desensitize TRPV1 may have an inhibitory effect on histamine-dependent itch [40,41]. e behavior results shown in Figure 7 are consistent with this theory. Scratching behavior induced by histamine and histamine receptor agonists was inhibited by osthole ( Figure 7). In our previous study, osthole also inhibited the increase in intracellular calcium caused by histamine and histamine Osthole also inhibited the VUF8430-induced scratching behavior in mice (n � 6) (e, f ) ( * P < 0.05, pair t-test). 8 Evidence-Based Complementary and Alternative Medicine receptor agonists [24]. Combining all the results mentioned above, we speculate that osthole inhibits histamine-dependent itch in which TRPV1 may play a role.

Conclusions
In summary, the results demonstrated that osthole could activate DRG neurons, and the molecular docking results show that osthole can be coupled to TRPV1 with the hydrophobic residue Arg557 as an agonist. Indeed, the neurons activated by osthole were inhibited by AMG9810, and osthole could desensitize TRPV1. Furthermore, osthole inhibited the histamine-dependent scratching behavior.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.