Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.


Introduction
The warmth-sensitive and Ca 2+ -permeable transient receptor potential vanilloid 3 (TRPV3) is robustly expressed in the skin, playing a critical role in skin sensation, skin barrier formation, hair growth, and vasodilation [1][2][3].The gain-of-function mutations in the TRPV3 channel not only lead to rodent hairlessness and dermatitis but also cause human-inherited skin disease Olmsted syndrome, characterized by keratoderma, dermatitis, hair loss, and severe itch [4][5][6].In the lesional epidermis of atopic dermatitis patients and pruritic burn scars, the expression of TRPV3 is markedly upregulated [7][8][9].Suppression of TRPV3 channel function by gene knockout and pharmacological inhibitors has been shown to alleviate dermatitis and pruritus [10][11][12].These investigations highlight the potential therapeutic value of pharmacological inhibition of TRPV3 for the therapy of skin diseases.
Natural caffeic acid (CA) and its analogues, such as caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), are phenolic compounds found in fruits, propolis, coffee, and many medicinal plants, and they exhibit a wide range of biological activities, including anti-inflammatory, anti-pruritic, anti-microbial effects and protection against ultraviolet irradiation [25][26][27][28].However, the molecular mechanism underlying the effects of these caffeoyl analogues remains largely unknown.It is also of interest that many natural TRPV3 inhibitors, including forsythiaside B, verbascoside, isochlorogenic acid A, and isochlorogenic acid B, contain caffeioyl groups (Figure 1).This suggests that caffeoyl analogues likely act on TRPV3 to exert their biological activities.To test this hypothesis, we selected three natural caffeoyl analogues that mainly differed in the substituent of acrylic acid at the fourth position of the phenyl ring and tested their effects on the inhibition of TRPV3.In this study, we found three caffeoyl analogues that inhibit TRPV3 currents in concentration-and structure-dependent manners.CA specifically inhibits the TRPV3 channel over other thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8.CA binds to the pocket formed by the Pore-loop and the S6, and it also effectively alleviates skin inflammation.These findings indicate that CA could be developed as a lead or an agent to improve TRPV3-related skin diseases.

Concentration-and Structure-Dependent Inhibition of hTRPV3 Currents by Caffeoyl Analogues
To assess the effects of caffeoyl analogues on TRPV3 channels, we carried out whole-cell current recordings of TRPV3 currents expressed in human embryonic kidney (HEK) 293T cells.We started testing the overexpression of TRPV3 in HEK293T cells 24 h after transient transfection of human TRPV3 cDNA.Consistent with the previous literature [29], TRPV3 protein is abundantly expressed in HEK293T cells transiently transfected with hTRPV3 (Figure S1).We proceeded to test the effect of caffeic acid (CA) and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), on hTRPV3.The perfusion of CA at concentrations ranging from 1 to 1000 µM resulted in the concentration-dependent inhibition of hTRPV3 currents activated by agonist 50 µM 2-APB with IC 50 values of 102.1 ± 19.7 µM (Figure 2A,D).CAPE and CAMC inhibited whole-cell TRPV3 currents induced by 2-APB (50 µM) in a concentration-dependent manner with the right-shifted IC 50 values of 276.7 ± 41.9 µM and 409.8 ± 57.9 µM, respectively (Figure 2B-D).These results demonstrate the concentration-and structure-dependent inhibition of hTRPV3 currents by CA and its analogues CAPE and CAMC.

Inhibition of Single TRPV3 Channel by Caffeic Acid
To further confirm the inhibitory effect of CA on the TRPV3 channel, we recorded the single channels in an inside-out configuration.3A-D).These results confirm that CA acts directly on individual TRPV3 channels by reducing the channel open probability and open frequency without altering its unitary conductance.

Identification of TRPV3 Residues Crucial for Caffeic Acid Binding
To further validate the binding pocket between CA and TRPV3, we utilized Schrödinger's Glide model for molecular docking of CA and its analogues CAPE and CAMC into the cryo-EM structure of mTRPV3 (PDB: 6DVY).The docking predicted that CA, CAPE, and CAMC are confined in the central cavity pocket formed by the Pore-loop and the S6 segment (Figure 5A), consistent with our previous finding for caffeoyl analogues isochlorogenic acid A and isochlorogenic acid B binding to TRPV3 [22].CA is mainly recognized by the residues T636, I637, F666, L669, and L670, with a docking score of −5.4.Within this pocket, two hydroxyl groups of the phenyl ring and acrylic acid of CA bind to the residue T636 from two p-loops through two hydrogen bonds.CAPE is recognized by the residue T636 through two hydrogen bonds with a docking score of −5.1, while CAMC is recognized by the residue I637 through one hydrogen bond with a docking score of −4.8 (Figure 5B).
To further validate the key residues critical for CA binding to TRPV3, we performed site-directed mutagenesis on the residues, including T636, I637, F666, L669, and L670.Whole-cell recordings showed that mutating T636, I637, and L669 to alanine significantly diminished TRPV3 inhibition by CA compared to WT channel currents.Conversely, the mutations F666A and L670A showed no significant difference in sensitivity to CA-mediated TRPV3 inhibition compared to the WT (Figure 5C-E).We also determined the IC 50 values of CA for T636A, I637A, F666A, and L669A mutant channels.Among the determined IC 50 values, CA exhibited the lowest potency with an IC 50 of 1000 µM for the T636A mutant, approximately 10-fold less potent than the WT (Figure 5F).Mutating T636 into alanine led to reduced potencies of CAPE with IC 50 values of 506.3 ± 57.3 µM.Conversely, the T636A mutant showed no significant alteration in sensitivity to CAMC-mediated TRPV3 inhibition with IC 50 of 416.7 ± 45.1 µM, as compared to the IC 50 of 409.8 ± 57.9 µM in WT.In contrast, mutating I637 into alanine (I637A) led to reduced potencies of CAMC with an IC 50 value of 522.0 ± 43.8 µM (Figure 5G).These results demonstrate the critical role of the T636 residue in the pocket for CA binding to the TRPV3 channel.

Caffeic Acid Alleviates Skin Inflammation Induced by Skin Sensitizer Carvacrol
Overactive TRPV3 function by skin sensitizer carvacrol causes TRPV3-mediated cutaneous inflammation [11,21].We, therefore, generated a mouse model of dorsal skin inflammation induced by topical applications of skin sensitizer carvacrol at 2% concentration for 5 consecutive days (Figure 6A).Topical carvacrol resulted in a time-dependent development of dorsal skin inflammation as compared with the vehicle control (vehicle: 30% ethanol and 70% saline).In contrast, subcutaneous injections of CA at different concentrations (0.1 and 1 mM) alleviated the skin inflammation and significantly reduced the dermatitis scores, as compared with the carvacrol model group (Figure 6B,C).We further carried out histological examinations of dorsal skin tissue sections and found that subcutaneous injections of CA at different concentrations (0.1 and 1 mM) reduced epidermal thickness induced by carvacrol, as compared with the carvacrol model group (Figure 6B,D), which are consistent with above phenotypic observations.These results indicate that CA alleviates TRPV3-mediated skin inflammation induced by the skin sensitizer carvacrol.

Animals
Adult male C57BL/6J mice (aged 6-8 weeks, weighing 20 ± 3 g) purchased from Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).All experimental mice were acclimated for at least one week before experiments for their adaptation to new experimental environment where the temperature was maintained at 22 ± 2 • C with 12 h light and dark circulation per day.Mice had free access to water and food.All in vivo experimental protocols are approved by the Institutional Animal Care and Use Committee of Qingdao University Health Science Center (protocol code QDU-AEC-2023092), approval date 10 March 2023 (Qingdao, China).

Electrophysiological Recordings
Patch clamp recordings were conducted using an EPC10 amplifier powered and analysed by Patchmaster software (HEKA Harvard, Holliston, Church Hill, TN, USA).The borosilicate glass pipettes were fabricated using a vertical micropipette puller (PC-100, Narishige, Tokyo, Japan) to achieve the tip resistances of 3-5 MΩ for whole-cell recording and 5-10 MΩ for single-channel recording when filled with pipette solution.Pipette solution and bath solution both contained (in mM) 130 NaCl, 3 HEPES, and 0.2 EDTA (pH = 7.4) [14].Whole-cell patch clamp recording showed that the membrane potential was maintained at 0 mV; currents were recorded at a slope voltage ramp from −100 to +100 mV for 500 ms and analysed at ±80 mV.For inside-out patch recordings of single channels, membrane potential was held at 0 mV before a voltage of −60 mV to monitor the opening of single channels.Single channel currents were sampled at 10 kHz and filtered at 2.0 kHz.All recordings were conducted at room temperature of 22 ± 2 • C, and data were analysed using Igor Pro (Wave-metrics, Lake Oswego, OR, USA) and Origin 8.6 (OriginLab, Northampton, MA, USA).

Molecular Docking and Site-Directed Mutagenesis
Molecular docking was performed using Schrödinger Glide (Maestro software suite 2015, Schrödinger, New York, NY, USA).Small molecules CA, CAPE, and CAMC were drawn using ChemBioDraw Ultra 14.0 (Cambridge Soft, Cambridge, MA, USA) and opti-mized for docking using the built-in program Ligprep in Maestro.The TRPV3 EM-structure (PBD code: 6DVY) was obtained from the Protein Data Bank and docked using the standard docking module SP according to Maestro's standard procedure.The binding pocket between ligand and TRPV3 was selected using Glide based on the reported inhibitor binding sites for TRPV3 and ranked based on the obtained scores.All TRPV3 site-directed mutations were generated using the Mut Express II Fast Mutagenesis Kit V2 according to the manufacturer's instructions.All mutants were confirmed by sequencing for correct generation of mutation.

Mouse Models of Dermatitis Induced by Skin Sensitizer Carvacrol
Mice were placed in a gas anaesthesia device (SurgiVet, Smiths Medical, Minneapolis, MN, USA) for anaesthesia before the hair on the back was removed with a razor, followed by the application of an appropriate amount of hair removal cream to gently shave off the remaining hair.Carvacrol (2%) was topically applied to mouse dorsal skin once a day for 5 consecutive days.Different concentrations of CA (0.1, 1 mM) in 100 µL were prepared in saline solution before subcutaneous injection into the mouse dorsal skin 30 min before topical application of 2% carvacrol once a day for 5 days.

Histological Sections of Skin Tissues
Mouse dorsal skin tissues were dissected using scissors and forceps immediately after sacrifice and fixed with 4% paraformaldehyde overnight at 4 • C before being dehydrated in ethanol and embedded in paraffin.Paraffin-embedded tissues were sectioned and stained with haematoxylins and eosin (H&E).Stained sections were observed using bright-field microscopy (ECLIPSE Ti-S, Nikon, Tokyo, Japan) and CCD-camera (DS-Ri2, Nikon, Tokyo, Japan).

Statistical Analysis
All data are expressed as the mean ± standard deviation (SD).Unpaired t-test and oneway and two-way ANOVA followed by multiple-comparison test were used to evaluate statistical significance using GraphPad Prism 7.0 software (La Jolla, CA, USA).A value of p < 0.05 is considered statistically significant.

Discussion
By analysing the chemical structures of known TRPV3 inhibitors, we find that many natural inhibitors contain caffeioyl groups.In this study, we selected caffeic acid (CA) and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), and tested their inhibitory effects on TRPV3 currents.These three caffeoyl chemicals are confined in the same binding pocket, and they share a common caffeic acid backbone but differ in their structural modifications.The molecular docking predicts that hydroxyl groups on CA and its two analogues are the key groups forming hydrogen bonds with TRPV3.CA with a free carboxylic acid group and two hydroxyl groups on the phenyal ring exhibits about three-or four-fold better potency than CAPE esterified with the hydroxyl group of phenethyl alcohol and CAMC esterified with a methyl group.CA (IC 50 = 102.1 ± 19.7 µM) appeared to inhibit TRPV3 in low potency as compared to caffeoyl analogues, such as forsythiaside B (IC 50 = 6.7 ± 0.7 µM) [18], verbascoside (IC 50 = 14.1 ± 3.3 µM) [19], isochlorogenic acid A (IC 50 = 2.7 ± 1.3 µM), and isochlorogenic acid B (IC 50 = 0.9 ± 0.3 µM) [22], which could be related to the low number of hydroxyl groups.This structure-activity relationship may serve as a hint for further improvement by chemical modifications of CA for its derivatives.
In this study, our findings show that the F666A mutation showed no significant difference in sensitivity to CA-mediated TRPV3 inhibition compared to the WT type (Figure 5E,F).This mechanism of action of CA is distinct from TRPV3 inhibitors such as dyclonine, scutellarein, isochlorogenic acid A, and isochlorogenic acid B that primarily act on the channel residue F666 located at the S6-helix and critical for TRPV3 channel gating [13,21,22].This may explain the worse inhibitory potency of CA with IC 50 of approximately 100 µM and may serve as a strategy for enhanced potency of CA derivatives by increasing hydrogen bond interactions between the residue F666 of TRPV3 and CA derivatives.
Natural CA is a plant-derived compound with a wide range of pharmacological activities such as anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial effects [25, [30][31][32][33].CA has also been reported to inhibit histamine-induced intracellular calcium increase in the H1R/TRPV1 pathway and chloroquine-induced responses in the MRGPRA3/TRPA1 pathway, as well as to reduce scratching behaviour in mice induced by multiple itchy compounds.However, the effects of CA directly targeted the TRP channel have not been evaluated using patch clamp recordings [27].In this study, we identified CA that selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPV1 and TRPA1, using whole-cell and single-channel recordings.TRPV3 participates in signalling pathways associated with acute and chronic itch [34][35][36], making it more likely to be a molecular target of CA.In addition, numerous studies have reported that CA alleviates ultraviolet (UV)-induced skin injury by regulating multiple signalling pathways [37][38][39][40].Recently, our laboratory has also reported the significant role of TRPV3 in UVB-induced skin damage [41].We speculate that CA may prevent UV-induced inflammation and photocarcinogenesis by inhibiting TRPV3.However, further validation is needed to support this hypothesis.
In this study, the high concentration of CA and its two analogues was used in vitro and in vivo experiments.To make sure all the results are not influenced by toxicity, we evaluated the cellular toxicity of three caffeoyl chemicals in HEK293T cells.Consistent with the previous literature [27], the high concentration of CA (1 mM) treatment on HEK293T cells did not cause any noticeable cytotoxicity (Figure S2).
In conclusion, we demonstrate that three caffeoyl analogues inhibit TRPV3 channels in concentration-and structure-dependent manners.CA containing only one caffeioyl group inhibits the TRPV3 channel and alleviates skin inflammation.CA as the TRPV3 inhibitor not only explains the mechanistic insights into the anti-pruritic and anti-inflammatory effects of CA but also provides a lead for further optimization and identification of specific TRPV3 channel inhibitors.

Figure 3 .
Figure 3. Reduction of hTRPV3 single-channel open probability by caffeic acid.(A) Left panel, representative single-channel current traces recorded at −60 mV in inside-out configurations before and after the addition of different TRPV3 agonists (50 µM 2-APB or 300 µM carvacrol) and coapplication with 500 µM caffeic acid (CA).All-point amplitude histograms of single-channel currents in 5 s were shown in their right panels.Dotted lines indicate the closed channel state (C) and the opened channel state (O), respectively.(B) Summary of the average open probability (P OPEN ) values of hTRPV3 single channel in the presence of control (circle), different TRPV3 agonists (square) and co-application with CA (triangles) (n = 5, **** p < 0.0001, by unpaired t test).(C) Summary of hTRPV3 single-channel open frequency (Freq) after exposure to control (circle), different TRPV3 agonists (square) and co-application with CA (triangles) (n = 5, **** p < 0.0001, by unpaired t test).(D) Summary of hTRPV3 single channel conductance after exposure to different TRPV3 agonists (square) and co-application with CA (triangles) (n = 5, ns, no significance, by unpaired t test).Data are expressed as the mean ± SD.

Figure 6 .
Figure 6.Attenuation of dermatitis induced by skin sensitizer carvacrol by caffeic acid.(A) Schematic drawing of experimental procedures for generation of the mouse dermatitis model of dorsal skin by topical applications of skin sensitizer carvacrol (2%) and subcutaneous injections of caffeic acid (CA) at different concentrations (0.1, 1 mM) for 5 consecutive days.(B) Phenotypic features and histologic images of H&E staining of dorsal skin tissue sections before and after topical applications of carvacrol (2%) for 5 consecutive days with and without subcutaneous injections of different concentrations CA.Scale bar = 100 µm for histologic images.(C) Dermatitis scores of mice in different groups treated with or without CA at different concentrations for 5 consecutive days from B (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by two-way ANOVA, followed by the Bonferroni's test).(D) Summary of epidermal thickness of mouse dorsal skin sections (n = 3, **** p < 0.0001, by one-way ANOVA, followed by the Dunnet's test).Data are expressed as the mean ± SD.

:
The expressions of TRPV3 proteins in HEK293T cells transiently transfected with human TRPV3 cDNA; Figure S2: Effects of caffeoyl analogues on cell viability of HEK293T cells.Author Contributions: Conceptualization, G.Z. and X.S.; methodology, Y.Q. and S.M.; formal analysis, G.Z. and L.W.; writing-original draft preparation, G.Z.; writing-review and editing, X.S. and K.W.; funding acquisition, X.S. and K.W.All authors have read and agreed to the published version of the manuscript.Funding: This study was supported by the Natural Science Foundation of Shandong Province [Grant ZR2023MH081] (to X.S.), the National Natural Science Foundation of China [Grant 81903734] (to X.S) and [Grant 81973299] (to K.W.) and by the Ministry of Science and Technology of China [Grant 2018ZX09711001-004-006] (to K.W.).Institutional Review Board Statement: The animal study protocol was approved by the Institutional Review Board of Animal Care and Use Committee of Qingdao University Health Science Center (protocol code QDU-AEC-2023092), approval date 10 March 2023.Informed Consent Statement: Not applicable.