A synthetic peptide, derived from neurotoxin GsMTx4, acts as a non-opioid analgesic to alleviate mechanical and neuropathic pain through the TRPV4 channel

A synthetic peptide, derived from neurotoxin GsMTx4, acts as a non-opioid analgesic to alleviate mechanical and neuropathic pain through the TRPV4 channel

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ShaoXi Ke^a^,^b, Ping Dong^a^,^c, Yi Mei^a, JiaQi Wang^a, Mingxi Tang^d^,^e, Wanxin Su^a, JingJing Wang^a, Chen Chen^a, Xiaohui Wang^a, JunWei Ji^a, XinRan Zhuang^a, ShuangShuang Yang^a, Yun Zhang^a, Linda M. Boland^f, Meng Cui^g, Masahiro Sokabe^h^,ⁱ, Zhe Zhang^a^,^j^,^*, QiongYao Tang^a^,^j^,^*

Acta Pharmaceutica Sinica B | 2025, 15(3) : 1447 - 1462

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Acta Pharmaceutica Sinica B | 2025, 15(3): 1447-1462

• ORIGINAL ARTICLE •

A synthetic peptide, derived from neurotoxin GsMTx4, acts as a non-opioid analgesic to alleviate mechanical and neuropathic pain through the TRPV4 channel

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Affiliations

^aJiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou 221004, China

^bThe Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310007, China

^cRuijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 201801, China

^dDepartment of Pathology, Yaan People's Hospital (Yaan Hospital of West China Hospital of Sichuan University), Ya'an 625000, China

^eDepartment of Pathology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China

^fDepartment of Biology, University of Richmond, Richmond, VA 23173, USA

^gDepartment of Pharmaceutical Sciences, Northeastern University School of Pharmacy, Boston, MA 02115, USA

^hMechanobiology Laboratory, Nagoya University, Graduate School of Medicine, Nagoya 464-8601, Japan

ⁱHuman Information Systems Lab, Kanazawa Institute of Technology, Kanazawa 921-8501, Japan

^jNMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, Xuzhou Medical University, Xuzhou 221004, China

About Author:

E-mail addresses: Zhangzhe70@xzhmu.edu.cn (Zhe Zhang)

qiongyaotang@xzhmu.edu.cn (QiongYao Tang).

^†

These authors made equal contributions to this work.

Author contributions

ShaoXi Ke: Project administration, Methodology, Investigation, Formal analysis, Data curation. Ping Dong: Project administration, Methodology, Investigation, Formal analysis, Data curation. Yi Mei: Project administration, Methodology, Investigation, Formal analysis, Data curation. JiaQi Wang: Project administration, Methodology, Investigation, Formal analysis, Data curation. Mingxi Tang: Writing – review & editing, Methodology, Investigation, Funding acquisition, Data curation. Wanxin Su: Methodology, Formal analysis, Data curation. JingJing Wang: Project administration, Methodology, Formal analysis, Data curation. Chen Chen: Project administration, Methodology, Formal analysis, Data curation. Xiaohui Wang: Project administration, Methodology, Data curation. JunWei Ji: Project administration, Methodology, Data curation. XinRan Zhuang: Project administration, Methodology, Formal analysis, Data curation. ShuangShuang Yang: Project administration, Methodology, Data curation. Yun Zhang: Project administration, Methodology, Data curation. Linda M. Boland: Writing – review & editing, Project administration, Methodology, Investigation. Meng Cui: Writing – review & editing, Methodology, Data curation. Masahiro Sokabe: Writing – review & editing, Supervision, Methodology, Investigation. Zhe Zhang: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. QiongYao Tang: Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

doi: 10.1016/j.apsb.2024.12.028

Outline

Abstract

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Mechanical pain is one of the most common causes of clinical pain, but there remains a lack of effective treatment for debilitating mechanical and chronic forms of neuropathic pain. Recently, neurotoxin GsMTx4, a selective mechanosensitive (MS) channel inhibitor, has been found to be effective, while the underlying mechanism remains elusive. Here, with multiple rodent pain models, we demonstrated that a GsMTx4-based 17-residue peptide, which we call P10581, was able to reduce mechanical hyperalgesia and neuropathic pain. The analgesic effects of P10581 can be as strong as morphine but is not toxic in animal models. The anti-hyperalgesic effect of the peptide was resistant to naloxone (an μ-opioid receptor antagonist) and showed no side effects of morphine, including tolerance, motor impairment, and conditioned place preference. Pharmacological inhibition of TRPV4 by P10581 in a heterogeneous expression system, combined with the use of Trpv4 knockout mice indicates that TRPV4 channels may act as the potential target for the analgesic effect of P10581. Our study identified a potential drug for curing mechanical pain and exposed its mechanism.

Key words

Peptide / Pain / Non-opioid analgesic / Mechanical pain / TRPV4 / Tolerance addiction / Mechanosensitive channel

Cite this Article

ShaoXi Ke, Ping Dong, Yi Mei, JiaQi Wang, Mingxi Tang, Wanxin Su, JingJing Wang, Chen Chen, Xiaohui Wang, JunWei Ji, XinRan Zhuang, ShuangShuang Yang, Yun Zhang, Linda M. Boland, Meng Cui, Masahiro Sokabe, Zhe Zhang, QiongYao Tang. A synthetic peptide, derived from neurotoxin GsMTx4, acts as a non-opioid analgesic to alleviate mechanical and neuropathic pain through the TRPV4 channel[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (3) : 1447 -1462 . DOI: 10.1016/j.apsb.2024.12.028

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1.　Introduction

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Mechanical pain is often caused by maternal labor, surgery, burns, inflammation, and neuropathy, among many other pathological conditions. Despite the relevance of treating mechanical nociception, there is no effective treatment available for the debilitating and chronic forms of mechanical pain¹. In humans, inflammation and nerve injury can cause abnormal sensory processes that result in exaggerated responses to mechanical stimuli^2,3. Tissue damage can lead to hypersensitivities, such as allodynia and hyperalgesia, which are usually resistant to analgesics^4-6. Developing effective treatments to alleviate mechanical pain is a clinically important goal.

Many ion channels are known to mediate certain types of pain. For example, activation of the TRPV1 channel mediates inflammatory thermal hyperalgesia⁷. TRPM8 and TRPA1 mediate pain associated with cold^8-10 and several acid-sensing ion channels (ASIC) subtypes are suggested to be therapeutic targets in peripheral pain conditions^11-13. Recently, the transient receptor potential vanilloid type 4 (TRPV4) channel was suggested to play an essential role in the mechanical hyperalgesia associated with pronociceptive inflammatory mediators and peripheral neuropathic pain^14-17, and the mechanosensitive (MS) ion channel Piezo1/Piezo2 are also shown to mediate sensitivity to mechanical pain in mice³. TRPV4 channel is particularly interesting due to its involvement in the development of hyperalgesia in inflamed tissue¹⁸ and neuropathic pain symptoms¹⁵. This channel is expressed in sensory neurons and represents an innovative target to tackle pain signaling in models induced by trauma, surgery, chemotherapy, cancer, diabetes, and alcohol intake¹⁹. A promising approach to reducing mechanical hyperalgesia and nociceptor sensitization is to identify specific blockers for MS channels, the discovery of the targeting molecule is urgently needed for the treatment of mechanical pain.

Polypeptide toxins play a central role in understanding the physiological and pathophysiological functions in ion channel studies, they have the advantages of higher bioactivity, permeability, target potency, selectivity, and safety compared with small-molecule and protein drugs²⁰. In the field of pain, they led to important advances in basic research and even in clinical applications^12,21,22. The neuropeptide GsMTx4, from Tramutola spatulata, was reported to inhibit stretch-activated cationic currents in mammalian astrocytes, cardiomyocytes, and the central nervous system^23-26. This peptide was also shown to reduce mechanical hyperalgesia and neuropathic pain in rats, presumably through its inhibitory effects on certain mechanically sensitive ion channels^1,27. While this venom has potential clinical applications for treating hyperalgesia^1,27, the complex structure and folding make it a major barrier to drug development.

Structural analysis reveals that the six cysteines in the backbone of GsMTx4 form three cystine knots²⁸. These inhibitor cystine-knot (ICK) motifs are common features of venom toxins^28-30. Sequence comparison between the two mechanosensitive peptides (GsMTx4 and GsMTx2) and with other homologous ICK peptides (e.g., VSTX1 and hanatoxin) shows more similarity between GsMTx4 with other ICK toxins^29,30. However, none of the ICK peptides have been identified as MS channel blockers, suggesting that the channel-blocking actions of GsMTx4 may be explained by a specific feature of this toxin that is not shared with other peptides^28,30. Based on the similarity shared by the two MS channel toxins in the backbone folds in loop2 and loop3, we have identified a 17-residue short peptide Pept 01 (named P10581 in this study), which acts to mimic the functional roles of GsMTx4 in inhibiting a mechanosensitive BK (SAKca) channel through the modification of the mechanogating³⁰.

The primary aim of the present study was to examine the novel effect of P10581 on mechanical hyperalgesia in rodent models. We have demonstrated that this natural toxin-based peptide P10581 selectively produces effective effects in reducing mechanical hyperalgesia and neuropathic pain in a way that is different from morphine. Pharmacological inhibition of TRPV4 by P10581 in expressed HEK293T cells, combined with Trpv4-gene deficient mice, we also identified the potential target for P10581 in alleviating mechanical pain. Our findings may offer a new opportunity for the development of a novel non-opioid analgesic in the treatment of mechanical forms of pain.

2.　Materials and methods

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2.1.　 Ethics and experimental animals

All animal studies complied with the ARRIVE guidelines^31,32. Animal care and procedures were approved by the Ethics Committee of Xuzhou Medical University (Xuzhou, China). Experiments were performed on 180–220 g adult Sprague–Dawley (SD) rats (the Experimental Animal Center, Xuzhou Medical University, Xuzhou, China) or C57BL/6J (mixed population of male and female) or Bk^−/−, and Trpv4^−/− knockout mice, backcrossed with C57BL/6J mice for more than 10 generations (GemPharmatech Co., Ltd., China) were used to examine the role of BK/TRPV4 involvements in anti-hyperalgesic effect of peptide. Corresponding WT littermates (male and female) were used as genetic background controls. The genotype of the mice was confirmed by PCR using genomic DNA extracted from the mouse's tail as a template. The animals were housed in groups of 2–3 for rats or 2–5 for mice at a controlled temperature of 23 ± 1 ℃. All animals had free access to food and water under a constant 12 h light–dark cycle (lights on at 7:00 a.m.). On the day of behavioral testing, animals were acclimatized to the environment for at least 30 min before testing. All animal behavior experiments were performed at room temperature (23–25 ℃).

All animals were used only once to prevent drug or testing experience from confounding the study. We did not test the possible off-target effect. There were no significantly different effects in the anti-hyperalgesic effects of P10581 between males and females (Supporting Information Fig. S1).

2.2.　 Inflammatory pain model

Carrageenan (Carr.)-induced model of acute inflammatory pain: this was evoked by intraplate injection as described previously^1,12. In brief, Carr. was injected intradermally (i.d.) into the right hindpaw of rats to induce inflammation immediately after the baseline testing of the paw withdrawal threshold (PWT). Peptides were administered intradermally (i.d. 1200 ng/kg in 5 μL) or intraperitoneally (i.p. 270 μg/kg in 5 μL) 1.5 h after Carr. injection in the same location of the inflamed area to test their effects. The doses of Carr. used were 5 μL for i.d. (1%) for i.d. or 50 μL (2%) for i.p. peptide testing^1,27. Carr. was injected 30 min later after BL was measured.

CFA-induced model of chronic inflammatory pain: CFA (Complete Freund's Adjuvant) was injected (10 μL, intraplantar) into the plantar surface of the right hind paw^33,34. Peptide/drugs were administrated by subcutaneous injection as described previously³⁵. In brief, the unanesthetized mice in lightly restrained, a 30 G needle (attached to a microsyringe) was inserted through the skin, and the peptide/drug (100 μL volume) was injected into the subcutaneous space. The effects of short peptide P10581 (2 μg/kg) were compared in wild-type and Kcnma1/Trpv4-deficient mice 3–5 days after CFA injection. Behavioral tests were performed by an experimenter blinded to experimental conditions.

2.3.　 Never injury model of neuropathic pain

The neuropathic pain model of rats was established using the chronic constriction nerve injury (CCI) model³⁶. Briefly, after baseline mechanical thresholds (BL) were measured, rats were anesthetized with isoflurane. Under anesthesia, the left common sciatic nerve of the hindpaw was exposed at the mid-thigh level through the biceps femoris; silk thread was tied loosely around the sciatic nerve until a brief twitch was observed. Great care was taken to secure the ligatures so that the nerve was constricted, but the circulation was not interrupted; the skin was then sutured. The sham control group was subjected to the same surgical operations except for the nerve ligature³⁶. Gentamicin (10 mg/mL, 0.2 mL; Solarbio Science & Technology Co., Ltd., Shanghai, China) was injected at the rat's belly (i.p.) to prevent infection. From Day 6, a peptide (i.d. 1.2 μg/kg, in 5 μL) was administered each day, and paw withdrawal thresholds were measured 2 h after injection to determine the anti-hyperalgesic effects of the peptide. The short peptide P10581 reached the maximum/stable anti-hyperalgesic effects on rats from Day 7 after the surgery.

Rats that failed to respond to the mechanical stimuli after Carr. or CCI (before peptide/drug treatment) were excluded from the presentation and data analysis. The equivalent volumes of saline and/or morphine were used as the negative and positive controls, respectively. Morphine was injected 30 min prior to testing.

2.4.　 Rotarod test

The rotarod treadmills (ENV-577M, Med associates, St. Albans, VT, USA) were used to assess the motor coordination of rats¹². In brief, on the day before testing, rats were trained on a fixed-speed (4 rpm) protocol until they could stay for 30 s. On the same day, rats were placed on the dowel rotating accelerated from 4 to 10 rpm at a constant rate of 5 rpm. The motor coordination of rats was evaluated by the time until the mouse fell from the rod which was recorded as the latency to fall. The effects of the peptide on rat motor impairment were assessed for five days and injected each day.

2.5.　 Mechanical nociceptive threshold measurements

2.5.1.　 Randall–Selitto test

Mechanical nociceptive thresholds were assessed using the hind paw-withdrawal test with a Randall–Sellito analgesia meter (Ugo-Basile Biological Research Apparatus, Comerio-Varese, Italy)^1,27. Mechanical pressure was continuously increased to the dorsal surface of the affected hind paw by using a blunt conical probe in a Randall–Sillito test instrument until vocalization or a withdrawal reflex occurred while the rats were lightly restrained. The mechanical thresholds of the baseline (BL) were recorded as the mean of 3 measurements (with at least 5-min intervals). After pharmacological reagent injection, the mechanical threshold measurements for each paw were repeated five times at a 5-min interval and averaged to be considered an independent observation.

2.5.2.　 Von Frey hair test

Mechanical nociceptive thresholds were also assessed using Von Frey filaments (Stoelting)³⁷. Rats were placed in transparent plastic domes with a metal mesh floor to allow access to the plantar surface of hindpaws. The filament was pressed perpendicular to the plantar surface of the hindpaw with sufficient force applied to cause a slight bucking for 6 s. Sharply withdrawing or flinching immediately after the removal of the filament was considered as a positive response. The force (in grams) producing a 50% likelihood of withdrawal was determined by the “up-down” method³⁷.

2.6.　 Analgesic tolerance tests

2.6.1.　 Peptide/morphine tolerance

Acute tolerance of peptide/morphine was induced in rats by repeated injections of peptide/morphine (5 mg/kg, i.d.) at 2 h intervals as described previously³⁸. In the inflammatory pain model induced by Carr. (1%, 5 μL, i.d.), rats received six consecutive injections of morphine (i.d. 5 mg/kg) or peptide (i.d. 2 μg/kg, 5 μL) at 2 h intervals. In the CFA-induced inflammatory pain model, rats were subjected to Randall–Sellito tests 1 h after peptide/morphine injections. Normal saline (i.d. 5 μL) was used for the negative control.

2.6.2.　 Chronic peptide/morphine tolerance

Chronic tolerance was produced in rats by repeated injection of peptide/morphine (s.c., twice a day) for ∼9 days. Briefly, seven days after CCI, rats received 8–9 consecutive administrations of morphine (i.d. 5 mg/kg)^12,39 or peptide (i.d. 8 μg/kg) twice a day (9:00 am and 5:00 pm). Rats were subjected to Randall–Sellito tests 1 h after morphine/peptide injections in the morning. The same amount of normal saline (i.d. 50 μL) was used for negative control.

2.7.　 CPP test

Conditioned place preference (CPP) test to peptide/drug was assessed using identical conditioning boxes that consisted of two chambers (20 cm × 20 cm × 20 cm), one with vertical black-white, and the other with horizontal stripes. The two chambers have different floor textures and were connected with a white central compartment (6 cm × 6 cm × 6 cm) that was not paired during peptide/drug treatment. Prior to testing, mice were habituated for 2–3 days (6 h per day) to the room where the behavioral tests were conducted as described previously^40,41 and then returned to the cage. Mice exhibiting signs of increased anxiety, (i.e., shaking and/or vocalizations) received additional handling until these signs were reduced.

Peptide/drug CPP: before CPP training, the initial preference (pre-conditioning) of the mice for the two chambers was determined in a 15-min session. The mice were placed in the central chamber and were allowed access to the two compartments. Mice did not exhibit initial preference (bias) for the horizontal or vertical striped chamber and were randomly assigned to three groups (saline, morphine, and peptide). Subsequently, mice received eight conditioning sessions (four in the morning and four in the evening). During conditioning, mice in the peptide/drug groups received a saline injection (s.c.) in the morning and were immediately confined to one chamber (saline-paired conditioning chamber) for 30 min. Four hours later, mice in the peptide/morphine group received a peptide/morphine (s.c.) injection in the afternoon and were immediately confined to the other chamber (peptide/morphine-paired conditioning chamber) for 30 min. Mice for the saline group received saline in both conditioning sessions (in the morning and afternoon). The training was repeated for 4 days. 24 h after the final dose injection on Day 5, mice were placed in the central chamber and were allowed to access two chambers for 15 min. Place preference scores were calculated by subtracting the time spent on the drug-paired side during preconditioning from the time spent on the drug-paired side during postconditioning⁴¹.

2.8.　 Electrophysiology

Electrophysiological Macropatch Recording in HEK293: whole-cell patch-clamp recordings were performed in HEK293 to test the effect of peptide P10581 on the mechanosensitive TRPV4 currents induced by GSK101, the selective TRPV4 activator. TRPV4 channels were transiently transfected in HEK293 cells using Lipofectamine™ 2000 (Invitrogen). Trpv4-eGFP cDNA was a gift from the laboratory of Dr. Fan Yang (Zhejiang Medical University, Zhejiang, China), where eGFP was used to monitor TRPV4 expression. HEK293 cell cultures were performed as previously described⁴². The pClamp 10 (Molecular Devices) was used to drive stimulus protocols and data acquisition. Currents were filtered at 2 kHz and digitized at 10 kHz. Whole-cell voltage-clamp recordings were performed 24 h after transfection at room temperature (∼25 ℃), where minor TRPV4 currents were observed. These currents transfected in HEK293T cells could be activated by TRPPV4 selective activator GSK101, and inhibited by TRPV4 selective inhibitor GSK219. The recording solutions used were the same as used previously described. The pipette (intracellular) were (in mmol/L) are: 140 CsCl, 5 EGTA, 10 HEPES, 2 MgATP, 0.2 NaGTP titrated to pH 7.2 with CsOH, and the bath (extracellular) solution consisted of (in mmol/L): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, titrated to pH 7.4 with NaOH⁴². Ramps (from −100 mV to +100 mV, 400 ms) were continuously repeated every 5 s.

Electrophysiological macropatch recording in oocytes: Two-electrode voltage-clamp (TEVC) recordings^43,44 were used to test the effects of peptide P10581 on the TRPV4 channel induced by exposure to extracellular hypotonic solution. Xenopus laevis oocytes were prepared and injected using standard protocols^45-47. Trpv4 cDNA was amplified with PCR and subcloned into the Xenopus oocyte expression vector pXoom, 3′ of the T7 promoter. The cRNA was prepared using the Ambion mESSAGE mACHINE T7 kit. The cRNA was injected at 10–25 ng/oocyte and TEVC recordings were carried out 2 days after injection. The base bath solution contained 66 mmol/L KCl, 100 mmol/L sorbitol, 1.8 mmol/L BaCl₂, and 5 mmol/L K-HEPES, pH 7.2. 4α-Phorbol 12,13-didecanoate (4α-PDD; 3 μmol/L) was added directly to the bath⁴⁸. Sorbitol was omitted from the base solution to form the hypotonic solution⁴⁸. Data were analyzed using both pClamp10 and Origin software.

2.9.　 Statistical analyses

To examine the dose–response relationship of peptides on mechanical hypersensitivity, data are presented as the increases in paw withdrawal threshold (ΔPWT) or latency (ΔPWL) post peptide injection, as shown in Eq. (1):

ΔPWT=PWT （ Post-Drug ） -PWT （ Pre-Drug ） （ or ： ΔPWL=PWL （ Post-Drug ） -PWL （ Pre-Drug ） ）

(1)

For comparison of the anti-hyperalgesia effect of P10581 to GsMTx4, the IC₅₀ values (half-maximal inhibitory concentrations) for the anti-hyperalgesic effects of drugs were obtained by linear regression with the standard Hill equation^49,50. Data from patch-clamp recordings were analyzed with Clampfit 10 (Molecular Devices) and plotted with GraphPad Prism 9.0.0 (Dotmatics) or Grapher (Golden).

The data and statistical analysis complied with pharmacology's recommendations and requirements on experimental design and analysis in pharmacology⁵¹. All peptide/drug treatment on animal experiments and data analysis were conducted blindly. Results are presented as means ± standard error of mean (SEM). Statistical differences were calculated by a Student's t-test or a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P-value <0.05 was considered as statistically significant.

2.10.　 Drugs and chemicals

The peptide P10581 was commercially synthesized with a purity of >98% (Sangon Biotech, Shanghai, China). The full-length neuropeptide GsMTx4 was purchased from Alomone Labs (Jerusalem, Israel) or BioScience Inc. (TX, USA). All peptides were dissolved in distilled water to make a 5 or 10 mmol/L stock solution and stored at −80 ℃. The stock solution was diluted with normal saline freshly at the time of injection or patch clamp experiment. Other chemicals were purchased from Sigma–Aldrich unless otherwise noted.

3.　Results

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3.1.　 The effect of P10581 on inflammatory pain administrated intradermally as determined by Randall–Sellito test

Previously, we identified a short peptide based on the natural toxin GsMTx4, which we call P10581 in this study (Fig. 1A), mimics the action of GsMTx4 to inhibit SAKca channel³⁰. In this study, we comprehensively tested whether peptide P10581 alleviates mechanical pain as GsMTx4 does^1,27.

P10581 contains both loop2, loop3, hydrophobic residue Trp-7 and the positively charged residue Lys-8 in loop1 in GsMTx4 (Fig. 1A). MD simulations with the free-product run method present a concave face commonly found in neurotoxins^12,52 and has a shape similar to GsMTx4 (Fig. 1B)³⁰. To determine whether P10581 reduces pressure-evoked mechanical pain, rats were subjected to the Randall–Sellito test^1,27. The paw withdrawal threshold (PWT) was significantly reduced from 201.5 ± 4.5 g (baseline, BL) to 23.5 ± 5.3 g after Carr., indicating the significant hyperalgesia induced by Carr. Intradermal (i.d.) injection of P10581 at 1200 ng/kg significantly increased PWT to 85.2 ± 6.6 g at 1 h post-peptide administration, and this effect reached the maximum at 3 h after injection (PWT was 120.6 ± 6.6 g) and lasted for 7 h as long as we tested. This anti-hyperalgesic effect of P10581 is comparable with that of the full-length toxin GsMTx4 under the same conditions (Fig. 1C) and was no longer detectable 24 h after peptide administration. Besides, although morphine increased the baseline of PWT greatly, neither P10581 nor GsMTx4 affected the baseline mechanical nociceptive threshold when the hindpaws were not inflamed (Fig. 1D).

A low dose at 0.6 μg/kg of P10581 (i.d.) modestly increased the threshold of PWT, whereas the same dose of GsMTx4 had only a mild impact on PWT (Fig. 1E), consistent with the peptide P10581 having a greater potency or efficacy than GsMTx4 on mechanical pain (Fig. 1F). The analgesic effects of P10581 and GsMTX4 were dose-dependent (Fig. 1G). The IC₅₀, required to reach half of the maximum effect in reducing the mechanical pain, was significantly lower for P10581 when compared with GsMTx4 (Fig. 1G, insert), indicating a more potent analgesic effect for P10581 when compared with the natural toxin.

We next compared the analgesic effect of P10581 with that of morphine when administered intradermally. Intradermal injection of peptide (0.4 μg per rat) produced a substantial increase in PWT (Fig. 1H), which is comparable with that of morphine (i.d., 200 μg per rat) (Fig. 1H, right), at which morphine reversed PWT to baseline before Carr. We suggested that the analgesic effect of peptide P10581 could be as strong as morphine against mechanical pain when given intradermally.

3.2.　 Effect of P10581 on inflammatory pain administrated intraperitoneally as determined by Randall–Sellito test

We next assessed the anti-hyperalgesic effect of P10581 when administered intraperitoneally (i.p.). As shown in Fig. 2A, administration of neither P10581 nor GsMTx4 (270 μg/kg) affected the baseline mechanical nociceptive threshold when hindpaws were not inflamed. However, following inflammation induced by Carr., the same dose of P10581 significantly increased the PWT in the Randall–Sellito test (from 29.8 ± 4.2 g to 159.9 ± 11.7 g), indicating the effective analgesic effect of peptide on rats (Fig. 2B). The anti-hyperalgesic effect of P10581 showed a dose-dependent effect (Fig. 2C). We did not observe significant differences in analgesic effects between P10581 and GsMTx4 at 270 μg/kg, consistent with the observation in Fig. 2B. Nevertheless, the maximum analgesic effect for P10581 was significantly greater than that induced by GsMTx4 (Fig. 2D), suggesting a greater potency for P10581 when compared with GsMTx4.

We also compared the acute anti-hyperalgesic effect of P10581 with morphine when administrated intraperitoneally. i.p. administration of P10581 (i.p. 0.08 mg/rat) produced a large increase in PWT, which is comparable with that of morphine at a dose of 0.4 mg/rat (Fig. 2E and F), suggesting that the analgesic effect of P10581 may be as strong as morphine on mechanical pain when administrated intraperitoneally.

3.3.　 Analgesic effect of P10581 as determined by Von Frey hair test

To assess the effect of P10581 on inflammation-induced mechanical allodynia, we used a Von Frey hair test²⁷. As shown in Supporting Information Fig. S2, significant allodynia was observed after inducing inflammation with Carr., which resulted in a reduced PWT to the application of Von Frey hairs. When P10581 was administered intraperitoneally (i.p. 270 μg/kg, 50 μL), a significant increase in PWT was observed, which was not significantly different from that of GsMTx4 (Fig. S2A). Furthermore, local administration of P10581 (1200 ng/kg, i.d.) injected into the same side of the hindpaw inflamed by Carr., elicited a nearly complete block of Carr.-induced allodynia (Fig. S2B).

Taken together, these results suggest that P10581 reduces inflammation-evoked mechanical pain with an equivalent or greater potency than GsMTx4. The potent effect of P10581 can be as strong as morphine when given either intradermally or intraperitoneally.

3.4.　 Effect of P10581 on neuropathic pain

Neuropathic pain is induced by classic constriction injury of the sciatic nerve (CCI) model, which causes local neuropathy and can last for more than one month^36,53. After sciatic nerve injury, rats showed signs of neuropathic pain, which was determined by the mechanical PWT subjected to the Randall–Sellito test (Fig. 3A). P10581 (1800 ng/kg) significantly raised PWT each day when compared with the pre-treatment values. It reversed ∼41.2% of PWT (71.5 ± 3.2 g before vs. 128.3 ± 5.5 g after), indicating a significant anti-hyperalgesic effect for P10581 on neuropathic pain (Fig. 3B and C), which is comparable with that of GsMTx4 at this tested dose (Fig. 3D).

The anti-hyperalgesic effects of P10581 became more prominent with the increases in the dose tested on Days 6, 7, 8, 10, and 14 after CCI (Fig. 3E), consistent with the dose-dependent effects of the peptide on neuropathic pain. The lower efficacy for P10581 obtained on Day 6 after CCI (Fig. 3E–G) may arise from the inflammation induced by chronic nerve injury. When compared with GsMTx4, the effect of P10581 on neuropathic pain reached a higher level of analgesia (Fig. 3H), suggesting a greater potency for P10581 than GsMTx4 (Fig. 3I).

3.5.　 Peptide P10581 fails to reduce thermal or cold pain

We also investigated whether P10581 modulates hot or cold pain. To measure analgesia to hot pain we measured paw withdrawal latency (PWL) in rats following the application of a thermal laser¹. The time taken for rats to lick or lift hindpaws was recorded as an indication of hot pain. As shown in Supporting Information Fig. S3A, rats administered with either P10581 or GsMTx4 (i.d. 1200 ng/kg, 5 μL) did not change their PWL when compared to the saline group.

To determine whether P10581 modulates cold pain, the plantar surface of the rat hindpaw was subjected to the noxious cold using dry ice stimulation of the hind paw⁵⁴. Although cold stimuli significantly decreased the rat PWL, intradermal injections of neither P10581 nor GsMTx4 at 1200 ng/kg (i.d. 5 μL) affected PWL following cold stimulus (Fig. S3B). Indeed, we did not observe significant differences in PWL response to cold stimuli even with a higher dose (∼4800 ng/kg) of the peptide (Supporting Information Fig. S4).

In conclusion, peptide P10581 fails to reduce either thermal or cold pain. These results may suggest a selective action of this nature-based peptide on the mechanisms that induce mechanical hyperalgesia.

3.6.　 P10581 does not develop analgesic tolerance

Morphine is a powerful pain reliever, but also a potent inducer of tolerance^12,55. We tested whether P10581 induces analgesic tolerance on mechanical pain as morphine does.

In the acute inflammatory pain model induced by Carr., rats received six consecutive repeated injections of 2 μg/kg P10581 (i.d.) at 2 h intervals (Fig. 4A), which produced the maximum analgesic effect (Fig. 1). As shown in Fig. 4B, repeated injections of morphine led to a time-dependent decrease in PWT, consistent with the tolerance developed in rats^12,38, whereas, under the same conditions, repeated injections of P10581 did not show a reduction in the anti-hyperalgesic effects, suggesting a lack of development of acute tolerance to peripherally administrated P10581.

In the chronic inflammatory pain model induced by Complete Freund's Adjuvant (CFA): injection of CFA into mouse hindpaw elicits tissue edema and hypersensitivity to mechanical stimulation, which results in a decreased mechanical PWT in the Randall–Sellito test (Fig. 4C). Intrathecal injections (i.t.) of P10581 induced a substantial central analgesic effect (Fig. 4C, inset) in a dose-dependent manner (Supporting Information Fig. S5). To investigate whether P10581 develops tolerance in the inflammatory CFA pain model, rats were subjected to peptide twice a day (at 9:00 am and 5:00 pm) from Day 4 after CFA, for 7 days of consecutive repeated injections (total 14 injections) (Fig. 4D) with 2 μg/kg, which produced the maximum central anti-hyperalgesic effect in mice (Fig. S5). We found central repeated injection of peptide did not reduce the analgesic effects (Fig. 4E), indicating that P10581 did not evoke central analgesic tolerance in rats. In contrast, repeated i.t. injection of morphine completely abolished the analgesic effect. We concluded that the central analgesic effect of P10581 did not develop tolerance.

In the chronic constriction nerve injury model induced by CCI: from Day 8 after CCI, rats were subjected to peptide twice a day (i.d., at 9:00 am and 5:00 pm) for 9 consecutive days (Fig. 4F). As a positive control, repeated injections of morphine (5 mg/kg, i.d.) led to a time-dependent decrease in PWT, and completely lost its anti-hyperalgesic effect from Day 4 (Fig. 4G). Interestingly, further injection of P10581 (7.2 μg/kg, i.d.) overcame the tolerance induced by morphine, suggesting again that peptide P10581 evokes an anti-hyperalgesic effect by a mechanism that may be distinct from that by morphine. On the other hand, repetitive injections of P10581 (18 total) did not produce a significant reduction in PWT (Fig. 4H and I). In another independent test, IC₅₀ obtained for the analgesic effect of P10581 on Day 8 after CCI (before repeated injections) was not significantly different from that obtained on Day 16 (after 9 days of repeated injections, Fig. 4J and K), demonstrating again that peptide P10581 did not evoke tolerance on neuropathic pain.

3.7.　 P10581 does not produce CPP

Opioids are the mainstay of pain treatment in the current clinic. However, prolonged use of opioids is commonly accompanied by burdensome side effects⁵⁶. And the addiction produced is a severe crisis for public health. To examine whether P10581 produces addition, the standard conditioned place preference (CPP) model⁴¹ was conducted. First, we identified that P10581 does not affect motor behavior evaluated by the Rotarod test (Supporting Information Fig. S6). To assess whether P10581 produces CPP, mice were randomly divided into three groups (saline, morphine, and P10581) and were habituated for 3 days (6 h/day) to the experimental room. The initial preferences (pre-conditioning) for two chambers (horizontal or vertical) were determined in 15 min on Day 0 (T1, Fig. 5A). Then each mouse was handled twice daily (from Days 1–4) and received eight conditioning sessions (s.c., morning saline and afternoon peptide/drug). In each session, mice were constrained in the chamber for 30 min (e.g. saline in the horizontal chamber, and peptide/morphine in the paired vertical one). 24 h after final dose injection on the test day (Day 5), mice were placed in the central chamber and the preferences for two chambers (horizontal or vertical) were determined for 15 min (T2, Fig. 5A). We found that morphine-paired group (10 mg/kg) significantly increased the time spent in morphine-paired (vertical) chamber, where mice were confined for 30 min in each session after morphine injection (Fig. 5B and C, middle). In contrast, 2 μmol/L/kg P10581, which produces the maximum analgesic effect in mice, did not show significant place reference. The preference scores for the P10581-paired group did not exhibit a significant increase compared to the saline group, whereas the morphine-paired group significantly increased CPP scores (Fig. 5D). We concluded that peptide P10581 does not exert conditioned place preference as morphine does.

3.8.　 The effect of P10581 exerts potent resistance to naloxone

To determine the role of the μ-opioid receptor in this process of the analgesic effect of peptide, naloxone, an μ-opioid receptor antagonist, was given subcutaneously (2 μg/kg) 2 h after peptide/morphine was administered (to ensure the maximum analgesic effect produced by peptide). Although naloxone completely prevented the increase in PWTs induced by morphine, P10581 (i.d. 2 μg/kg) showed a large increase in PWTs either in the absence or presence of naloxone (Fig. 5E), suggesting that naloxone did not significantly prevent the analgesic effect of P10581 (Fig. 5F). We concluded that the short peptide may not act as opioid-dependent analgesia in mice.

3.9.　 TRPV4, rather than the BK channel, is required for the analgesic effect of P10581 in mice

BK channels are expressed in sensory neurons and mediate both inflammatory and neuropathic pain in mice. We first tested whether BK channels contribute to the analgesic effects of P10581 by using a BK channel opener and a BK conditional knockout (Kcnma1-cKO) mice. As shown in Supporting Information Fig. S7, BK channel opener NS1619 injected intrathecally (i.t. 10 μg/kg, 10 μL) did not alter the analgesic effect of P10581 administered intraperitoneally. In addition, the deletion of Kcnma1-gene (Cre-Bk-cKO) also did not affect the anti-hyperalgesic effect of P10581 (2 μg/kg, 100 μL) administered subcutaneously (Supporting Information Fig. S8). Thus, it is most likely that BK (including both SAKca and regular BK) channels are not the molecular target for the analgesic effect of P10581. These results are consistent with our previous report. Although P10581 acts as a potent inhibitor of mechanosensitive BK (SAKc) channels in the heart, it does not inhibit regular/canonical BK (e.g. mSlo1) that are wildly expressed in peripheral and central neurons and have been shown to lack mechanosensitivity³⁰.

The mechanosensitive TRPV4 has been implicated in pathological pain conditions, including various inflammatory and neuropathic pain, but not in cold hypersensitivity^27,54. Interestingly, GsMTx-4 inhibits the hyperalgesia for mechanical and hypotonic stimuli, to the same extent as treatment with oligodeoxynucleotides (ODN) antisense to Trpv4²⁷. We used the Trpv4-gene deficient (Trpv4-KO) mice to assess the involvement of TRPV4 channels in the analgesic actions of P10581 (Fig. 6A). The genotypes for Trpv4 KO mice were determined by PCR (Fig. 6A and B) using the primers (Table 1). Homozygous Trpv4 (Trpv4^−/−) mice showed a significantly improved mechanical PWT (BL) when compared with wild-type (WT) littermates, a hallmark of mechanical hyperalgesia (Fig. 6C). The difference observed in our experiments for the increased baseline to other reports, may arise from the different methods used for the tests of the mechanical hyperalgesia. This is possible as TRPV4 channel expression is increased in wild-type channels, it is sensitive to shell-stretch and contributes to mechanical hyperalgesia⁵⁷.

Injection of CFA decreased the mechanical PWT and developed a similar degree from Days 3–10 (after CFA) (Fig. 6C). Subcutaneous (s.c.) injection of P10581 (2 μg/kg) in WT (Trpv4^+/+) mice significantly increased PWT of the hind paws (Fig. 6D), confirming the peripheral analgesic effects of P10581 evoked on wild-type mice. The anti-hyperalgesic effect of the peptide lasted 3 h as long as we tested. Interestingly, the same dose of P10581 showed a reduced analgesic potency in Trpv4^+/− (Fig. 6D; at 1 h post-injection) and mostly abolished in Trpv4-deficient mice (Fig. 6E). In fact, P10581 was mostly ineffective on both Trpv4^+/− and Trpv4^−/− mice at 2 h (Fig. 6F) or 3 h (Fig. 6G) after injections, suggesting that the TRPV4 channel plays a necessary role in the anti-hyperalgesic effect developed by P10581.

Since morphine was equally effective in the anti-hyperalgesic effects among wild-type and Trpv4-deficient mice (Fig. 6H), it is most likely that P10581 produces its analgesic effect through a pain pathway or mechanism different from that accessed by morphine, consistent with the result that the analgesic effect of P10581 is resistance to naloxone (Fig. 5E and F).

3.10.　 The peptide P10581 inhibits the TRPV4 channel when heterologously expressed in HEK 239T or Xenopus oocytes

To further investigate the direct functional role of TRPV4 played in the analgesic actions of P10581, we examined the inhibitory effect of the peptide on TRPV4 by using whole-cell current recordings when they are heterologously expressed in HEK 239T cells. We observed some basal currents for TRPV4 channels under our recording conditions (∼25 ℃), it is possible as the TRPV4 channel can be partially activated at 24–27 ℃. Extracellular application of a potent and selective TRPV4 activator GSK101 elicited a robust increase in the currents, and this GSK101-evoked-TRPV4 current was completely inhibited by further application of peptide P10581 (5 μmol/L) in the intact cell from the extracellular side (bath solution) of the cell (Fig. 7A and B). The remaining (basal) currents were not further inhibited by the TRPV4 selective inhibitor GSK219, suggesting that non-mechanosensitive basal currents may arise from unknown endogenous currents in HEK293 cells.

It has been reported that the TRPV4 channel can be directly activated by mechanical force produced with hypotonicity⁴⁸, whereas GsMTx-4 markedly reduces nociceptive flinching in response to hypotonic stimulation. Thus, we asked whether P10581 could inhibit the TRPV4 current that is activated by hypotonicity. As shown in Fig. 7C and D, again, extracellular application of peptide P10581 (1 μmol/L) inhibited the hypotonically activated TRPV4 currents to the level of the basal current (before hypotonic solution application). Subsequent application of the TRPV4 selective inhibitor GSK219 did not further decrease the currents. The inhibitory effect of P10581 on TRPV4 was in a dose-dependent manner (Fig. 7E and F).

Taken together, these results demonstrate that peptide P10581 not only inhibits the TRPV4 currents activated by GSK101, but also abolishes TRPV4 currents that are activated by hypotonicity (as summarized in Fig. 7G). We propose that peptide P10581 may act as an inhibitor directly targeting the mechanosensitive TRPV4 channel to alleviate mechanical and neuropathic pain in the rodent pain model. Nevertheless, considering that P10581 showed a minor analgesic effect in TRPV4 knockout mice, we cannot rule out the possibility that other pain- and mechano-sensing ion channels (e.g., Piezo) have a minor contribution to the anti-hyperalgesic effect of P10581 (see discussion).

4.　Discussion

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In the present study, we examined the anti-hyperalgesic effects of a natural toxin-based peptide (P10581) in the rodent models of inflammatory and neuropathic pain and explored the involvement of TRPV4 channel as the potential target for the peptide actions. We found that peptide P10581 effectively reduced the mechanical hyperalgesia and neuropathic pain subjected to the Randall–Sellito or Von Frey hair test, while it did not affect mechanical nociceptive thresholds to thermal or cold stimuli. The anti-hyperalgesic effect of P10581 is resistant to the opioid antagonist (naloxone) and can be as strong as morphine, but it did not produce the side effects of opioid-induced, e.g., tolerance, addiction, or motor impairment. In addition, we found that the analgesic effect of P10581 was nearly absent in Trpv4-deficient mice, suggesting an essential role of TRPV4 in the therapeutic activity of the peptide. Further pharmacological inhibition of TRPV4 by P10581 when expressed in HEK 239T cells, suggested that this mechanosensitive channel may act as the direct target for the anti-hyperalgesic of peptide. These studies show that this short peptide may have considerable potential as a non-opioid analgesic in alleviating the form of mechanical pain, as well as in the treatment of Trpv4-driven diseases.

Natural products represent a reliable source of biologically active compounds that may have pharmacological potency and safety, and natural product-based drug design is regarded as one of the effective means for new drug development. The MS channel selective inhibitor GsMTx4 extracted from the venom of the tarantula Grammostola spatulate^26,30, showed substantial analgesic effects in reducing mechanical and neuropathic pain, presumably via some type of MS channel^1,27. GsMTx4-based peptide Pept 01 (referred to as P10581 in this study) has been identified as a mechanosensitive BK (SAK_Ca) channel inhibitor³⁰. In this study, we characterized the novel effects of this peptide in alleviating mechanical pain. In pain models, P10581 effectively attenuated mechanical hyperalgesia and neuropathic pain without significant efficacy on thermal and cold pain even with a considerably higher dose. It is most likely that this peptide acts as a novel analgesic selective to mechanical pain. The result that the P10581 exhibited a strong analgesic effect in reducing inflammation-, and constriction nerve injury-evoked mechanical pain, supports the idea that the regions between loop2 + loop3 in the natural toxin (Trp⁷–Lys²³ in GsMTx4) are responsible for its action on the pharmacological blockade on a mechanosensitive BK (SAKca) channel³⁰. Nevertheless, the result that the analgesic effects of P10581 were retained in Kcnma1-gene-lacking mice (Fig. S6), makes it unlikely that the anti-hyperalgesic effect of peptide functions through BK (including SAKca and regular BK) channel as a target. Although P10581 is a potent inhibitor of mechanosensitive BK (SAKc) channels in the heart, it does not inhibit non-mechanosensitive regular/canonical BK (e.g., mSlo1) that are wildly expressed in peripheral and central neurons³⁰, the result that the knockout of Bk has no impact on the analgesic effect of P10581 is consistent with the previous report.

Peptides possess low immunogenicity and high specificity, and having been increasingly developed as drugs for the treatment of various diseases^58,59. In this study, we also identified a target channel for the anti-hyperalgesis actions of P10581. The results that the analgesic effect of the peptide was mostly abolished in Trpv4-deficient mice suggest that the mechanosensitive TRPV4 channel is a potential therapeutic target for peptide action. The result that IC₅₀ for P10581 on TRPV4 is ∼5.2 nmol/L, is approximately consistent with the concentration (1200 ng/kg) we used in mice (that is 7.9 nmol/L, based on the molecular weight and blood volume in mice). Since this type of peptide acts as a gating modifier that targets mechano-gating of the MS channels by partitioning into the cell membrane^26,30,60, the local concentration of peptide accumulated on the cell membrane might be higher than the actual concentration used.

While P10581 analgesia was absent in Trpv4-deficient mice, morphine's analgesic effects were retained and not significantly altered by the deletion of Trpv4-gene, indicating a lack of a generalized defect in analgesic pathways for morphine in this mouse strain. It is most likely that peptide P10581 produces its anti-hyperalgesic effect via a different pathway or mechanism from that of morphine. As naloxone did not significantly prevent the analgesic effect of P10581, we suggest that the peptide may act as a non-opioid analgesic. This is pretty important as a non-opioid analgesic may hold promise to develop safer and non-addictive medications^61,62.

In addition to TRPV4, two GsMTx4-sensitive MS channels TRPC1 and TRPC6, expressed in dorsal root ganglion neurons (DRG), are known to cooperate with TRPV4 to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization²⁷. Although these two channels may have distinctive roles, one may assume that the outcome of the analgesic effect for P10581 in this study could also be explained by peptide acting on TRPV4 exerting analgesia of TRPC1/TRPC6-mediated noxious sensations. If TRPC1/TRPC6 were the direct analgesic target of P10581, P10581's effect would have been retained in Trpv4-gene-lacking mice. However, this was not the case, suggesting that TRPV4 is the key analgesic and pharmacological target for the anti-hyperalgesic effect of P10581. The minor anti-hyperalgesic effect (a spike, ∼15%) remained in Trpv4-deficient mice at 1 h after administration (Fig. 6E and F) suggests that other MS channels may contribute to the analgesic effect of peptide in mice. It has been shown that the mechanosensitive Piezo1/Piezo2 channel is sensitive to GsMTx4, and mediates inflammatory- and nerve injury-induced neuropathic pain in mice and humans^63,64. Thus, one possibility is that the minor anti-hyperalgesic effect of P10581 remaining in Trpv4-KO mice may arise from the inhibition of the Piezo1/Piezo2 channel. Indeed, shear stress can produce a fast and transient, but minor Ca²⁺ influx via Piezo1 in Trpv4 KO mice⁶⁵. Whether P10581 inhibits Piezo1/Piezo2 and/or other pain-sensing ion channels, such as TRPV1 or ASIC, needs to be further identified in future studies. The lack of the anti-hyperalgesic effect of P10581 (tested 2 h post-injection) in both Trpv4^+/−/Trpv4^−/− mice supports the conclusion that TRPV4 plays an essential role in the effect of peptide.

5.　Conclusions

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In conclusion, we discovered a short peptide derived from the natural toxin GsMTx4. This peptide, we call P10581, exhibited effective anti-hyperalgesic effects on mechanical forms of pain and these actions depend on the mechanosensitive TRPV4 ion channel. The anti-hyperalgesic effect of P10581 is comparable with morphine but does not show analgesic tolerance, addiction, or motor dysfunction as morphine. These findings offer new opportunities for the development of a non-opioid analgesic in the treatment of mechanical forms of pain.

References

Less

Park

, Kim

, Koo

, Cho

, Lee

, Kim

, et al. A tarantula spider toxin, GsMTx4, reduces mechanical and neuropathic pain. Pain 2008;137:208—17.

Costigan

, Scholz

, Woolf

. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 2009;32:1—32.

Murthy

, Loud

, Daou

, Marshall

, Schwaller

, Kuhnemund

, et al. The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice. Sci Transl Med 2018;10:eaat9897.

Yamada

, Fujita

, Hayano

, Hayakawa

, Baba

, Mochizuki

, et al. Increased expression of fibronectin leucine-rich transmembrane protein 3 in the dorsal root ganglion induces neuropathic pain in rats. J Neurosci 2019;39:7615—27.

Baron

, Binder

, Wasner

. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol 2010;9:807—19.

Woolf

, Salter

. Neuronal plasticity: increasing the gain in pain. Science 2000;288:1765—9.

Caterina

, Leffler

, Malmberg

, Martin

, Trafton

, Petersen Zeitz

, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306—13.

Jordt

, Bautista

, Chuang

, McKemy

, Zygmunt

, Hogestatt

, et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 2004;427:260—5.

McKemy

, Neuhausser

, Julius

. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 2002;416:52—8.

10.

Story

, Peier

, Reeve

, Eid

, Mosbacher

, Hricik

, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112:819—29.

11.

Deval

, Noel

, Gasull

, Delaunay

, Alloui

, Friend

, et al. Acid-sensing ion channels in postoperative pain. J Neurosci 2011;31:6059—66.

12.

Diochot

, Baron

, Salinas

, Douguet

, Scarzello

, Dabert Gay

, et al. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature 2012;490:552—5.

13.

Lee

JYP

, Saez

, Cristofori Armstrong

, Anangi

, King

, Smith

, et al. Inhibition of acid-sensing ion channels by dimina-zene and APETx2 evoke partial and highly variable antihyperalgesia in a rat model of inflammatory pain. Br J Pharmacol 2018;175:2204—18.

14.

Alessandri Haber

, Dina

, Yeh

, Parada

, Reichling

, Levine

. Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat. J Neurosci 2004;24:4444—52.

15.

Alessandri Haber

, Joseph

, Dina

, Liedtke

, Levine

. TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator. Pain 2005;118:70—9.

16.

Alessandri Haber

, Yeh

, Boyd

, Parada

, Chen

, Reichling

, et al. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 2003;39:497—511.

17.

Choi

, Yang

, Yoo

, Choi

, Lim

, Cho

, et al. TRPV4-mediated anti-nociceptive effect of suberanilohydroxamic acid on mechanical pain. Mol Neurobiol 2019;56:444—53.

18.

Alessandri Haber

, Dina

, Joseph

, Reichling

, Levine

. A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. J Neurosci 2006;26:3864—74.

19.

Rodrigues

, Ruviaro

, Trevisan

. TRPV4 role in neuropathic pain mechanisms in rodents. Antioxidants (Basel) 2022;12:24.

20.

Wang

, Deng

, Cheng

, Li

, Zhang

, et al. A novel and low-toxic peptide DR3penA alleviates pulmonary fibrosis by regulating the MAPK/miR-23b-5p/AQP5 signaling axis. Acta Pharm Sin B 2023;13:722—38.

21.

Schmidtko

, Lotsch

, Freynhagen

, Geisslinger

. Ziconotide for treatment of severe chronic pain. Lancet 2010;375:1569—77.

22.

Mazzuca

, Heurteaux

, Alloui

, Diochot

, Baron

, Voilley

, et al. A tarantula peptide against pain via ASIC1a channels and opioid mechanisms. Nat Neurosci 2007;10:943—5.

23.

, Xu

, Shen

, Wang

, Tang

, Du

, et al. The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating. J Biol Chem 2019;294:11892—909.

24.

Suchyna

, Tape

, Koeppe

2nd, Andersen

, Sachs

, Gottlieb

. Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers. Nature 2004;430:235—40.

25.

Lee

, Leddy

, Chen

, Lee

, Zelenski

, McNulty

, et al. Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage. Proc Natl Acad Sci U S A 2014;111:E5114—22.

26.

Suchyna

, Johnson

, Hamer

, Leykam

, Gage

, Clemo

, et al. Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels. J Gen Physiol 2000;115:583—98.

27.

Alessandri Haber

, Dina

, Chen

, Levine

. TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization. J Neurosci 2009;29:6217—28.

28.

Oswald

, Suchyna

, McFeeters

, Gottlieb

, Sachs

. Solution structure of peptide toxins that block mechanosensitive ion channels. J Biol Chem 2002;277:34443—50.

29.

Nishizawa

, Nishizawa

, Gnanasambandam

, Sachs

, Sukharev

, Suchyna

. Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations. Biochim Biophys Acta 2015;1848:2767—78.

30.

Zhou

, Li

, Xu

, Shen

, Tang

, Wang

, et al. Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechano-gate. J Biol Chem 2022;298:102326.

31.

Percie du Sert

, Hurst

, Ahluwalia

, Alam

, Avey

, Baker

, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMJ Open Sci 2020;4:e100115.

32.

Lilley

, Stanford

, Kendall

, Alexander

SPH

, Cirino

, Docherty

, et al. ARRIVE 2.0 and the British Journal of Pharmacology: updated guidance for 2020. Br J Pharmacol 2020;177:3611—6.

33.

Caceres

, Liu

, Jabba

, Achanta

, Morris

, Jordt

. Transient receptor potential cation channel subfamily M member 8 channels mediate the anti-inflammatory effects of eucalyptol. Br J Pharmacol 2017;174:867—79.

34.

Honda

, Shinoda

, Kondo

, Shimizu

, Yonemoto

, Otsuki

, et al. Sensitization of TRPV1 and TRPA1 via peripheral mGluR5 signaling contributes to thermal and mechanical hypersensitivity. Pain 2017;158:1754—64.

35.

Corder

, Tawfik

, Wang

, Sypek

, Low

, Dickinson

, et al. Loss of μ opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia. Nat Med 2017;23:164—73.

36.

Bennett

, Xie

. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87—107.

37.

Chaplan

, Bach

, Pogrel

, Chung

, Yaksh

. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55—63.

38.

Tian

, Zhang

, Fang

, Xu

, Grandy

, Han

. Endogenous orphanin FQ: evidence for a role in the modulation of electroacupuncture analgesia and the development of tolerance to analgesia produced by morphine and electroacupuncture. Br J Pharmacol 1998;124:21—6.

39.

Deng

, Chen

, Pan

. α2δ-1-Bound N-methyl-D-aspartate receptors mediate morphine-induced hyperalgesia and analgesic tolerance by potentiating glutamatergic input in rodents. Anesthesiology 2019;130:804—19.

40.

Portugal

, Gould

. Nicotine withdrawal disrupts new contextual learning. Pharmacol Biochem Behav 2009;92:117—23.

41.

Portugal

, Al Hasani

, Fakira

, Gonzalez Romero

, Melyan

, McCall

, et al. Hippocampal long-term potentiation is disrupted during expression and extinction but is restored after rein-statement of morphine place preference. J Neurosci 2014;34:527—38.

42.

Bang

, Yoo

, Yang

, Cho

, Hwang

. Nociceptive and proinflammatory effects of dimethylallyl pyrophosphate via TRPV4 activation. Br J Pharmacol 2012;166:1433—43.

43.

Tang

, Zhang

, Xia

, Ren

, Logothetis

. Phosphatidylinositol 4,5-bisphosphate activates Slo3 currents and its hydrolysis underlies the epidermal growth factor-induced current inhibition. J Biol Chem 2010;285:19259—66.

44.

, Zhang

, Gan

, Zhang

, Shen

, Guo

, et al. Identification of the acid-sensitive site critical for chloral hydrate (CH) activation of the proton-activated chloride channel. J Neurosci 2023;43:526—39.

45.

Tang

, Zhang

, Xu

, Wang

, Chen

, Logothetis

, et al. Epilepsy-related slack channel mutants lead to channel over-activity by two different mechanisms. Cell Rep 2016;14:129—39.

46.

Tang

, Zeng

, Lingle

. Closed-channel block of BK potassium channels by bbTBA requires partial activation. J Gen Physiol 2009;134:409—36.

47.

, Lv

, Zhao

, Wang

, Shen

, Li

, et al. Identification of sodium- and chloride-sensitive sites in the slack channel. J Neurosci 2023;43:2665—81.

48.

Loukin

, Zhou

, Su

, Saimi

, Kung

. Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force. J Biol Chem 2010;285:27176—81.

49.

Tang

, Zhang

, Meng

, Cui

, Logothetis

. Structural determinants of phosphatidylinositol 4,5-bisphosphate (PIP2) regulation of BK channel activity through the RCK1 Ca²+ coordination site. J Biol Chem 2014;289:18860—72.

50.

Wang

, Zhong

, Du

, Zhang

, Guo

, Liu

, et al. Cloning and characterization of the rat Slo3 (K_Ca 5.1) channel: from biophysics to pharmacology. Br J Pharmacol 2020;177:3552—67.

51.

Curtis

, Alexander

, Cirino

, Docherty

, George

, Giembycz

, et al. Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Br J Pharmacol 2018;175:987—93.

52.

Escoubas

, De Weille

, Lecoq

, Diochot

, Waldmann

, Champigny

, et al. Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels. J Biol Chem 2000;275:25116—21.

53.

Chen

, Wang

, Li

, Wu

, Zhao

, Liu

, et al. Rhodojaponin VI indirectly targets Cav2.2 channels via N-ethylmaleimide-sensitive fusion protein to alleviate neuropathic pain. Acta Pharm Sin B 2023;13:1326—36.

54.

Ehlers

, Sadler

, Stucky

. Peripheral transient receptor potential vanilloid type 4 hypersensitivity contributes to chronic sickle cell disease pain. Pain 2023;164:1874—86.

55.

Bohn

, Gainetdinov

, Lin

, Lefkowitz

, Caron

. μ-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 2000;408:720—3.

56.

Fraczek

, Ferraiolo

, Hermans

, Bujalska Zadrozny

, Kasarello

, Erdei

, et al. Novel opioid-neurotensin-based hybrid peptide with spinal long-lasting antinociceptive activity and a propensity to delay tolerance development. Acta Pharm Sin B 2020;10:1440—52.

57.

Swain

, Romac

, Vigna

, Liddle

. Piezo1-mediated stellate cell activation causes pressure-induced pancreatic fibrosis in mice. JCI Insight 2022;7:e158288.

58.

Zhu

, Chen

, Paul

, Lu

, Wu

, Qi

. Oral delivery of proteins and peptides: challenges, status quo and future perspectives. Acta Pharm Sin B 2021;11:2416—48.

59.

Wang

, Shen

, Li

, Xie

, Lu

, Wang

, et al. Efficacy of inverso isomer of CendR peptide on tumor tissue penetration. Acta Pharm Sin B 2018;8:825—32.

60.

Bode

, Sachs

, Franz

. Tarantula peptide inhibits atrial fibrillation. Nature 2001;409:35—6.

61.

Alsaloum

, Higerd

, Effraim

, Waxman

. Status of peripheral sodium channel blockers for non-addictive pain treatment. Nat Rev Neurol 2020;16:689—705.

62.

Perez de Vega

, Ferrer Montiel

, Gonzalez Muniz

. Recent progress in non-opioid analgesic peptides. Arch Biochem Biophys 2018;660:36—52.

63.

Szczot

, Liljencrantz

, Ghitani

, Barik

, Lam

, Thompson

, et al. PIEZO2 mediates injury-induced tactile pain in mice and humans. Sci Transl Med 2018;10:eaat9892.

64.

Lee

, Nims

, Savadipour

, Zhang

, Leddy

, Liu

, et al. Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis. Proc Natl Acad Sci U S A 2021;118:e2001611118.

65.

Swartz

, MacKinnon

. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels. Neuron 1997;18:675—82.

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Year 2025 volume 15 Issue 3

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doi: 10.1016/j.apsb.2024.12.028

Receive Date：2024-07-23
Online Date：2026-09-18

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Received：2024-07-23
Revised：2024-10-20
Accepted：2024-11-14

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