A scophthalmus TRPV1 inhibiting peptide, preparation method and application

Abstract

The application discloses a sciaenops ocellatus TRPV1 inhibiting peptide, a preparation method and application, belongs to the polypeptide field, and the amino acid sequence of the sciaenops ocellatus TRPV1 inhibiting peptide is RLF.The sciaenops ocellatus TRPV1 inhibiting peptide, the preparation method and the application take the skin of sciaenops ocellatus as starting raw material, and the amino acid sequence of the sciaenops ocellatus TRPV1 inhibiting peptide is successfully obtained through a specific process.Experiment verification shows that the prepared sciaenops ocellatus TRPV1 inhibiting peptide shows excellent inhibiting effect on TRPV1.The inhibiting peptide can be used for preparing a TRPV1 antagonist, the antagonist can play an anti-inflammatory regulation role through multiple channels, and can effectively down-regulate skin microvascular hyperreactivity.

Description

Technical Field

[0001] This invention belongs to the field of polypeptides, and particularly relates to a pufferfish TRPV1 inhibitory peptide, its preparation method, and its application. Background Technology

[0002] The skin is the largest defense organ in the human body, and an imbalance between its barrier function and neuroimmunity is the pathogenesis of sensitive skin. Transient Receptor Potential Vanilloid 1 (TRPV1), a multimodal, calcium-permeable, non-selective cation channel, is widely distributed in keratinocytes and sensory nerve endings in the skin. It mediates the activation of calcium receptors by stimuli such as capsaicin, high temperature (>43℃), and low pH (<5.9). 2 TRPV1 influx triggers a neurogenic inflammatory response. Studies have shown that TRPV1 plays a crucial role in sensitive skin; its aberrant activation leads to the release of pro-inflammatory factors (such as IL-6 and TNF-α), the secretion of neuropeptides (substance P, CGRP), and vascular endothelial hyperreactivity, resulting in skin erythema, burning, and barrier function impairment. However, existing TRPV1 antagonists suffer from low transdermal absorption efficiency and long-term use can cause skin desensitization. Therefore, there is a need to develop novel inhibitors that combine high efficacy, safety, and multi-target regulatory properties.

[0003] Peptide compounds have become a hot topic in the development of pain-relieving active substances in recent years due to their advantages such as small molecular weight, strong targeting, and high biocompatibility. Currently, APHC peptides from sea anemones are the first reported peptide antagonists that bind to the TRPV1 channel, showing a strong inhibitory effect on capsaicin-induced TRPV1 activation. In vivo studies have shown that APHC peptides have significant analgesic effects in different pain models. A novel TRPV1-targeting peptide (TIP) has also been discovered. TIP can effectively inhibit capsaicin-induced calcium influx and TRPV1 activation. In vivo, TIP attenuates UV-induced erythema and the expression of inflammatory factors in human skin, providing a treatment method for UV-induced inflammation and photoaging. However, current research on TRPV1 inhibitors is extremely limited, focusing primarily on fish. Summary of the Invention

[0004] The purpose of this invention is to provide a pufferfish TRPV1 inhibitory peptide, its preparation method, and its application, so as to overcome at least one of the above-mentioned defects in the prior art.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] The present invention provides a pufferfish TRPV1 inhibitory peptide, the amino acid sequence of which is: RLF.

[0007] The present invention also provides a method for preparing the pufferfish TRPV1 inhibitory peptide as described above, comprising the following steps:

[0008] S1. Extraction of enzymatically hydrolyzed polypeptides from pufferfish skin

[0009] Pufferfish skin was hydrolyzed using alkaline protease. After hydrolysis, the enzyme was inactivated. Then, polypeptides with a molecular weight of no more than 1 kDa were screened from the hydrolysate and freeze-dried to obtain pufferfish skin hydrolysate polypeptides.

[0010] S2. Sequence Identification and Preliminary Screening

[0011] Mass spectrometry was used to determine the enzymatic hydrolysis peptides from pufferfish skin. The results were analyzed using mass spectrometry software. Multiple non-repetitive peptide sequences were screened out based on the criteria of confidence level -10lgP > 20 and number of amino acids < 10.

[0012] S3, Virtual Filtering

[0013] The peptide sequences selected in step S2 are molecularly docked with the TRPV1 receptor using software to screen out peptide sequences with strong binding ability to the TRPV1 receptor.

[0014] S4, polypeptide synthesis

[0015] The polypeptide sequences screened in step S3 were synthesized in a solid phase to obtain the pufferfish TRPV1 inhibitory peptide.

[0016] Preferably, before step S1, step S0 is further included: first, the pufferfish skin is cut into small pieces, and then the cut pieces of fish skin are freeze-dried; then, the freeze-dried fish skin is soaked in NaCl solution and stirred continuously for 12-36 hours, and the NaCl solution is replaced every 6-12 hours, with a mass concentration of 6-12%; after the fish skin is soaked, it is washed with distilled water and filtered with nylon yarn, and this process is repeated 3-5 times.

[0017] Preferably, in step S1, the conditions for alkaline protease hydrolysis of pufferfish skin are: solid-liquid ratio 1:10, enzyme dosage 8000 U / g, hydrolysis temperature 50℃, hydrolysis pH 9.0, and hydrolysis time 4h.

[0018] Preferably, in step S1: the enzymatic hydrolysate is first microfiltered through a ceramic membrane, and then separated into polypeptides with a molecular weight cutoff of no more than 1 kDa through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa.

[0019] Preferably, the mass spectrometry determination of pufferfish skin enzymatic hydrolysate peptides in step S2 is specifically as follows: Pufferfish skin enzymatic hydrolysate peptides are dissolved in solvent A to obtain sample peptides; 1 μL of sample peptide is loaded onto a 25 cm analytical column, and separation is initiated from 2% buffer B with a 60-minute gradient, gradually increasing to 35% over 47 minutes, then to 100% over 1 minute, and held for 12 minutes; the column current is maintained at 300 nL / min, and the column temperature is 40 °C; the electrospray voltage is set to 2 kV; the mass spectrometer is operated in data-dependent acquisition mode, automatically switching between MS and MS / MS modes; a full scan mass spectrum is obtained on an Orbitrap with a resolution of 70,000; the automatic gain control target is 3e6, and the maximum injection time is 50 ms; precursor ions are selected and introduced into the collision cell for high-energy collision dissociation; fragmentation is performed, and the normalized collection energy is 28%; the MS / MS resolution is set to 17,500, the automatic gain control target is 1e5, the maximum injection time is 45 ms, and the dynamic exclusion time is 30 s.

[0020] Preferably, in step S2: solvent A is an aqueous solution of formic acid with a mass concentration of 0.1%, and buffer B is an aqueous solution containing 80% ACN and 0.1% FA.

[0021] Preferably, the crystal structure of the TRPV1 receptor is 8GFA.

[0022] The present invention also provides the application of the above-mentioned pufferfish TRPV1 inhibitory peptide in the preparation of TRPV1 antagonists.

[0023] Preferably, the pufferfish TRPV1 inhibitory peptide is used for one or more of the following: a. inhibiting TRPV1 protein expression, b. downregulating the release of pro-inflammatory factors, c. upregulating the level of matrix metalloproteinase inhibitor TIMP-1, and d. inhibiting the secretion of ET-1 and ICAM-1 in vascular endothelial cells.

[0024] The beneficial effects of this invention are as follows:

[0025] Using pufferfish skin as the starting material, the amino acid sequence of a pufferfish TRPV1 inhibitory peptide was successfully obtained through a specific process. Experimental verification showed that the prepared pufferfish TRPV1 inhibitory peptide exhibited excellent inhibitory effects against TRPV1. This inhibitory peptide can be used to prepare a TRPV1 antagonist, which can exert anti-inflammatory regulatory effects through multiple pathways, effectively downregulating the hyperreactivity of skin microvessels. The pufferfish TRPV1 inhibitory peptide showed significant efficacy in soothing the skin and regulating neuroimmune function. This invention provides a new strategy for developing functional ingredients for sensitive skin, and is expected to contribute to the upgrading and innovation of sensitive skin care products. Attached Figure Description

[0026] Figure 1This is a 3D diagram of the T21 and TRPV1 docking composite mode of the present invention.

[0027] Figure 2 This is a 2D diagram of the docking composite mode of T21 and TRPV1 of the present invention.

[0028] Figure 3 This is the RAW 264.7 cytokine array diagram of the present invention (each pair of dots represents two parallels of each cytokine or chemokine).

[0029] Figure 4 This is a heatmap of the expression of 40 cytokine genes in this invention.

[0030] Figure 5 This is a diagram showing the inhibitory effect of T21 on ET-1 expression in this invention.

[0031] Figure 6 This is a diagram showing the inhibitory effect of T21 on ICAM-1 expression in this invention.

[0032] Figure 7 This is a graph showing the expression level of TRPV1 protein detected by the Western Blot method of this invention.

[0033] Figure 8 This is a diagram showing the effect of T21 of the present invention on the expression of TRPV1 protein in HaCaT cells induced by CAP.

[0034] Figure 9 This is a graph showing the effect of the present invention T21 on the mortality rate of zebrafish.

[0035] Figure 10 This is a fluorescence image of neutrophils in zebrafish treated with T21 according to the present invention.

[0036] Figure 11 This is a graph showing the effect of the present invention T21 on the number of neutrophils in zebrafish.

[0037] Figure 12 This is the zebrafish path diagram of the present invention.

[0038] Figure 13 This is a diagram showing the effect of the present invention T21 on the total distance traveled by zebrafish.

[0039] Figure 14 This is a diagram showing the effect of the present invention T21 on the rotational frequency of zebrafish.

[0040] Figure 15 This is a diagram showing the effect of the present invention T21 on the activity state of zebrafish. Detailed Implementation

[0041] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0042] This embodiment provides a pufferfish TRPV1 inhibitory peptide with the amino acid sequence: RLF.

[0043] The present invention also provides a method for preparing the pufferfish TRPV1 inhibitory peptide as described above, comprising the following steps:

[0044] Step SO: Fish skin pretreatment

[0045] In this embodiment, the pufferfish used is the dark-spotted pufferfish. First, the pufferfish skin is cut into small pieces using a meat slicer. Then, the cut skin pieces are freeze-dried to remove moisture and vacuum-packed for preservation. Next, the freeze-dried skin is soaked in a 9% NaCl solution with continuous stirring for 24 hours, with the NaCl solution replaced every 12 hours. After soaking, the skin is washed with distilled water and filtered through nylon mesh, repeated three times to remove non-collagenous proteins.

[0046] S1. Extraction of enzymatically hydrolyzed polypeptides from pufferfish skin

[0047] The optimal conditions for enzymatic hydrolysis of pufferfish skin using alkaline protease (B8360, Solarbio) were: solid-liquid ratio 1:10, enzyme dosage 8000 U / g, hydrolysis temperature 50℃, hydrolysis pH 9.0, and hydrolysis time 4 h.

[0048] After enzymatic hydrolysis, the enzyme was inactivated for 10 minutes. The hydrolysate was first passed through a ceramic membrane for microfiltration, and then separated into polypeptides with a molecular weight cutoff of 1 kDa through an ultrafiltration membrane. After vacuum freeze-drying, the polypeptides were stored at -20°C to obtain enzymatic hydrolysed pufferfish skin polypeptides.

[0049] S2. Sequence Identification and Preliminary Screening

[0050] Mass spectrometry was used to determine the enzymatic hydrolysis peptides from pufferfish skin. The results were analyzed using mass spectrometry software. Multiple non-repetitive peptide sequences were screened out based on the criteria of confidence level -10lgP > 20 and number of amino acids < 10.

[0051] Specifically, peptide sequences were identified by Nano-HPLC-MS / MS. The peptides were redissolved in solvent A (A: 0.1% formic acid aqueous solution) to obtain sample peptides, which were then analyzed using an Orbitrap Q-Exactive Plus system with an EASY-nanoLC 1200 (Thermo Fisher Scientific, MA, USA). 1 μL of sample peptide was loaded onto a 25 cm analytical column (75 μm inner diameter, 1.9 μm resin (Dr Maisch)). Separation was initiated with a 60-min gradient from 2% buffer B (80% ACN (acetonitrile), 0.1% FA (formic acid)), gradually increasing to 35% at 47 min, then to 100% at 1 min, and held for 12 min. The column flow rate was maintained at 300 nL / min, and the column temperature at 40 °C. The electrospray voltage was set to 2 kV. The mass spectrometer was operated in data-dependent acquisition (DDA) mode and automatically switched between MS and MS / MS modes. Full-scan mass spectra (m / z 200-1800) were obtained on an Orbitrap at a resolution of 70,000. Automatic gain control (AGC) target was 3e6, and the maximum injection time was 50 ms. Precursor ions were selected for high-energy collisional decomposition (HCD) in the collision cell, with a normalized collection energy of 28%. MS / MS resolution was set to 17,500, AGC target was 1e5, maximum injection time was 45 ms, and dynamic exclusion time was 30 s.

[0052] The peptide sequences were identified using nano-HPLC-MS / MS. In the peptide results, the -10lgP index represents the confidence level of the corresponding spectrum identification. The larger the value, the better the matching result. Based on the mass spectrometry analysis results, peptides with a confidence level of -10lgP > 20 and an amino acid count < 10 were screened, and 190 peptides were initially screened.

[0053] S3, Virtual Filtering

[0054] The peptide sequences selected in step S2 are molecularly docked with the TRPV1 receptor using software to identify peptide sequences with strong binding affinity to the TRPV1 receptor.

[0055] Specifically, based on the mass spectrometry analysis results, peptides were initially screened using the confidence level of the spectral identification (-10lgP) and the number of amino acids. TRPV1 (PDB: 8GFA) was selected as the receptor protein, and its 3D structure was downloaded from the RCSB PDB database (https: / / www.rcsb.org). Before virtual screening, MOE 2022 software was used to remove unwanted water molecules from the receptor protein, add hydrogen, add charge, and optimize energy. The peptides were plotted as small molecules as ligands using Discovery Studio 2019 Client software to determine the active pocket of TRPV1, and the location of the active pocket (X: 108.603; Y: 79.704; Z: 88.011). Virtual molecular screening was performed using DOCK 6.9, and the lead peptide sequences with the highest docking scores were selected based on the Grid Score and binding mode.

[0056] S4, polypeptide synthesis

[0057] The precursor polypeptide sequences selected in step S3 were synthesized in a solid phase, and the precursor polypeptide sequences with good TRPV1 inhibition effects were screened to obtain the pufferfish TRPV1 inhibitory peptide RLF(T21).

[0058] The following experiments will verify the screening of the seed peptide RLF(T21).

[0059] 1. Experimental Materials and Methods

[0060] 1.1 Molecular docking

[0061] TRPV1 is the receptor protein, and T21 is the ligand. Using Discovery Studio 2019 software, the TRPV1 protein was dehydrated, protonated, hydrogenated, and charged. The 2D structure of the peptide was drawn using ChemDraw 20.0, and the 3D structure was drawn using Chem3D, with energy minimization performed. The TRPV1 pocket location was determined in Discovery Studio. TRPV1 was then Libdock-coupled with the peptide ligand. The best-scoring conformation was output, and the intermolecular interactions were visualized and analyzed using Discovery Studio 2019.

[0062] 1.2 Molecular Dynamics

[0063] Molecular dynamics simulations of the D chain of the 8GFA protein were performed using Amber 20: after deleting the ABC chain and water molecules and repairing the structure, the protein was treated with an AMBERff19SB force field. Small peptides were hydrogenated and optimized for charge, and then parameterized using a GAFF force field and AM1-BCC charge. The system was constructed using an OPC water model to create a truncated octahedral solvent box. A buffer was added, and NaCl was added to neutralize the charge. Energy minimization was performed stepwise (constraining heavy atoms → all-atom relaxation to energy convergence). Stepwise equilibrium was achieved through a 0.02 ns NVT heating to 300 K, a 200 ps heavy atom-constrained NPT equilibrium, and a 1 ns free NVT relaxation. Finally, a 100 ns production simulation (300 K, 2 fs step size) was performed, using Langevin temperature control, SHAKE algorithm constraints, and periodic boundary conditions.

[0064] 1.3 Mouse Cytokine Array Analysis

[0065] The expression of 40 cytokines / chemokines in the supernatant of Raw264.7 cell culture was detected using the Proteome Profiler™ antibody array kit (ARY006, R&D Systems). The experimental procedure was as follows: Cell supernatants from each treatment group were collected and processed according to the kit instructions (300 μL of supernatant per sample). The reagents were first warmed to room temperature, and the array membrane was blocked for 2 hours. Simultaneously, the detection antibodies were co-incubated with the supernatant at room temperature for 1 hour, and then transferred to the membrane for overnight binding at 4°C. The next day, the cells were washed three times (≥10 min each time) with washing buffer, incubated with HRP-streptavidin at room temperature for 2 hours, washed again, and then developed using a chemiluminescent reagent. Signals were acquired using a chemiluminescence imaging system, and the integrated optical density was quantitatively analyzed using ImageJ software.

[0066] 1.4 HUVEC cell stimulation response

[0067] Take cells in the logarithmic growth phase, at 2 × 10⁻⁶ 5 CFU / mL was seeded into 24-well plates and cultured overnight. The supernatant was discarded. Different concentrations of T21 (50, 100, 200, 400 μM) were added to each well and the plates were incubated for 6 h. Then, LPS (10 μg / mL) was added, and the plates were incubated for a total of 24 h. The cell culture supernatant was collected, and the expression levels of ET-1 and ICAM-1 were detected according to the ELISA kit instructions. The inhibition rate was calculated.

[0068] Inhibition rate = (expression level of LPS group - expression level of peptide group) / (expression level of LPS group - expression level of negative control group) × 100%.

[0069] 1.5. Western blot analysis of T21-induced TRPV1 protein expression in HaCaT cells.

[0070] After T21 pretreatment for 6 h, the protein was co-cultured with CAP for 24 h. After washing with DPBS, the protein was lysed on ice with RIPA lysis buffer for 1 h, centrifuged at 4 °C, and the supernatant was collected. Protein concentration was determined using the BCA method. An equal volume of protein was mixed with Loading Buffer (1:3) and denatured at 95 °C for 5 min. The protein was transferred to PVDF membrane via SDS-PAGE (10 μL / lane) and blocked with 5% skim milk powder-TBST for 2 h. TRPV1 (A00128, BOSTER, Wuhan, China) and GAPDH (CL594-6004, Abclonal, Wuhan, China) antibodies were prepared according to the manufacturer's instructions and incubated overnight at 4 °C. After washing with TBST, the membrane was incubated with HRP secondary antibody at room temperature for 1 h. Final washing with TBST was performed, and the membrane was developed with ECL reagent (K-12045-D50, Advansta, San Jose, CA, USA). The band gray values ​​were quantitatively analyzed using ImageJ.

[0071] 1.6. Determination of Zebrafish Embryo Survival Rate

[0072] Wild-type zebrafish embryos of strain AB, 24 hours post-fertilization (24 hpf), were used to assess the toxicity of T21 using standard E3 medium. Six concentration gradients (0, 100, 200, 300, 400, and 500 μM) were established for each compound. Thirty zebrafish embryos were used per well, and the medium containing the corresponding concentration of compound was changed twice daily for continuous exposure until 3 days post-fertilization (dpf). Embryo viability was recorded every 24 hours during the observation period to determine the highest non-toxic concentration. The experiment was repeated three times.

[0073] 1.7. Neutrophil Experiment in Zebrafish

[0074] An inflammation model was constructed using transgenic fluorescent neutrophil zebrafish (Tg mpeg1:EGFP, 2dpf) bred through natural mating. Inflammatory responses were induced by sodium dodecyl sulfate (SLS). The experiment included a normal control group, a model group (200 μM SLS), and a positive control group (1000 μM dipotassium glycyrrhizate + 200 μmol / L SLS). Multiple concentration treatment groups (T21, 1, 5, and 25 μM) were also included, with 30 zebrafish in each group. All treatment groups were exposed to 200 μM SLS and treated at 28°C in the dark for 24 hours. Afterward, images were taken and data collected under a fluorescence microscope to quantitatively analyze the number of neutrophils aggregated in the spinal cord and abdominal region of the zebrafish.

[0075] 1.8 Zebrafish Behavioral Experiment

[0076] An inflammation model was established in 2dpf AB strain zebrafish using 1000 μM SLS. The experiment was divided into a normal control group (untreated), a model group (1000 μM SLS), and a T21 peptide treatment group (1 μM + 1000 μM SLS). All groups were continuously exposed at 28 ± 0.5℃ in the dark for 72 h. After exposure, the total distance traveled, rotation frequency, and activity level within 30 min were measured using a zebrafish behavior analysis system. The data acquisition parameters were set as follows: activity level below 20% indicates a quiescent state, 20%-60% indicates an active state, and above 60% indicates a frenzied state.

[0077] 2. Results

[0078] 2.1 Interaction Analysis between T21 and TRPV1

[0079] T21 is bonded together through hydrogen bonding, electrostatic interactions, salt bridging, and hydrophobic interactions. Figure 1 It forms 6 hydrogen bonds with ASN438, PHE488, ARG491, TYR554, TYR555, and GLU513; two electrostatic interactions with TYR554 and GLU513; one salt bridge interaction with GLU513; and four hydrophobic interactions with PHE435, VAL441, TYR554, and TYR555. The binding energy ΔG of T21 docking with TRPV1 is -4.86 kcal / mol.

[0080] like Figure 2 As shown, in the early stages of the simulation, the amplitude of the complex system was relatively large, which is a normal conformational adjustment phase where molecules explore and adapt to the new environment. As the simulation progressed, different complex systems exhibited different stability characteristics. The TRPV1-T21 complex reached a stable state after 18 ns. At the end of the 100 ns simulation, the amplitude of the TRPV1-T21 complex was less than... This result indicates that the complex system has reached an equilibrium state, meaning that the molecules gradually stabilized during the simulation and formed a relatively stable structure. At the same time, this result also indirectly verifies the correctness of the molecular docking process, indicating that T21 and TRPV1 formed a relatively tight bond.

[0081] 2.2 Effects of T21 on the expression levels of cytokines in RAW 264.7 cells

[0082] from Figure 3-4As can be seen, compared with the normal group, the LPS group significantly upregulated the expression levels of ICAM-1, IL-1ra, IL-6, CCL3, CCL5, and TNF-α cytokines; and slightly upregulated the expression levels of G-CSF, GM-CSF, IL-23, CXCL10, CXCL2, and TIMP-1 cytokines. After incubation with T21, the expression levels of G-CSF, GM-CSF, ICAM-1, IL-1ra, IL-6, IL-23, CCL3, CXCL2, CCL5, and TNF-α cytokines were downregulated; CXCL10 and CXCL2 showed almost no change; and the expression level of TIMP-1 cytokines was significantly upregulated.

[0083] 2.3T21 reduces the irritant response of HUVECs.

[0084] like Figure 5 As shown, stimulation of HUVEC cells with 10 μg / mL LPS for 24 h significantly increased ET-1 expression from 2.14 pg / mL to 3.0 pg / mL. Pretreatment with T21 for 6 h followed by co-culturing with LPS for 24 h resulted in a dose-dependent reduction in ET-1 expression. At a T21 concentration of 50 μM, ET-1 expression decreased to 2.74 pg / mL, with an inhibition rate of 29%; when the T21 concentration was increased to 400 μM, ET-1 expression decreased to 2.33 pg / mL, with an inhibition rate as high as 78%, a statistically significant difference.

[0085] Similarly, in Figure 6 In the experiment, the expression level of ICAM-1 increased from 0.42 pg / mL to 0.59 pg / mL upon LPS stimulation. After pretreatment with T21, T21 dose-dependently reduced the expression level of ICAM-1. When T21 was 50 μM, the expression level of ICAM-1 decreased to 0.53 pg / mL, with an inhibition rate of 33%; when T21 was 400 μM, the expression level of ICAM-1 decreased to 0.50 pg / mL, with an inhibition rate of 57%, showing a significant difference.

[0086] 2.4T21 inhibits the expression of TRPV1 protein in HaCaT cells.

[0087] Western blot analysis revealed the regulatory role of T21 on CAP-induced TRPV1 channel protein expression in HaCaT cells. Experimental data showed that after 24 hours of stimulation with 20 μM CAP, TRPV1 protein expression in HaCaT cells was significantly increased compared to the control group. Figure 7-8As shown, treatment with T21 at concentration gradients of 50-400 μM resulted in a significant dose-dependent inhibitory effect on TRPV1 expression levels. This result indicates that T21 can effectively inhibit CAP-induced neurogenic inflammatory responses by targeting and regulating the TRPV1-mediated calcium signaling pathway.

[0088] Effects of 2.5T21 on zebrafish neutrophils and behavior

[0089] like Figure 9 As shown, at a concentration of 50 μM, the mortality rate in group T21 reached 33%. Therefore, the concentration range for subsequent zebrafish experiments was selected as 0-25 μM, at which the survival rate of zebrafish embryos remained at 90%. Figure 10 As shown, the number of neutrophils infiltrating the epidermis of zebrafish in the blank control group was (16.4±7.0) cells / field (n=30), which significantly increased to 50.1±18.1 after 24 h of stimulation with 1000 μM SLS. After the addition of T21, it was shown that T21 stably inhibited the number of neutrophils from 23.7±15.9 to 31.5±15.1 within the range of 1-25 μM, with an inhibition rate of 72.7%-78.3%.

[0090] like Figure 10 and 11 As shown, the number of neutrophils infiltrating the epidermis of zebrafish in the blank control group was (16.4±7.0) cells / field (n=30), which significantly increased to 50.1±18.1 after stimulation with 1000μM SLS for 24h. After adding four lead peptides, the results showed that T1 reduced the neutrophil count to 20.3±9.4 at 1 μM (inhibition rate 88.2%) and increased it to 24.9±10.9 at 5 μM (inhibition rate 74.8%). T11 reduced the neutrophil count to 26.0±13.5 at 1 μM (inhibition rate 71.6%) and abnormally increased it to 36.1±20.5 at 5 μM. T22 reduced the neutrophil count to 23.7±13.8 at 1 μM (inhibition rate 78.1%) and surged to 55±21.9 at 5 μM, exhibiting significant concentration toxicity. T21 stably inhibited the neutrophil count in the range of 23.7±15.9 to 25.6±10.8 within the 1-5 μM range (inhibition rate 72.7%-78.3%). The results showed that the anti-inflammatory titers of the four peptides differed significantly at 1 μM; therefore, 1 μM was chosen as the uniform concentration for subsequent experiments.

[0091] The Zebralab video tracking system was used to quantitatively analyze the movement behavior characteristics of zebrafish larvae (5 dpf). Figure 12 ).like Figure 13As shown, the cumulative movement distance of zebrafish in the blank control group was 51.3±9.5 mm during the 30-minute observation period; however, after SLS stimulation, the total movement distance of zebrafish significantly increased to 2238.2 mm±192.6 mm. With the addition of T21, the movement distance was only 926.3±69.2 mm, with an inhibition rate as high as 60.0%.

[0092] Based on the zebrafish rotation frequency results ( Figure 14 The rotation frequency in the blank control group was 17.3±2.0 times / minute; compared with the blank group, the frequency in the SLS model group was significantly increased to 100.7±4.6 times / minute; after adding T21, the rotation frequency decreased to 53.5±3.9 times / minute, with an inhibition rate as high as 56.6%.

[0093] Regarding the proportion of zebrafish activity time, such as Figure 15 As shown, the proportion of still time in the T21 group reached 97.2±1.8%, significantly higher than the 94.5±3.1% in the model group; the proportion of active movement time in the T21 group decreased to 2.3±0.9%, a 52.1% reduction compared to the 4.8±1.6% in the model group; and the proportion of abnormal activity time in the T21 group was 0.5±0.2%, an 83.3% reduction compared to the 3.0±0.7% in the model group. The results indicate that in the experimental group treated with T21, zebrafish exhibited shorter periods of agitation and sustained activity, and longer periods of stillness, suggesting that T21 treatment effectively reduces the stimulation of zebrafish by SLS.

[0094] This embodiment also provides the application of the above-mentioned pufferfish TRPV1 inhibitory peptide in the preparation of TRPV1 antagonists.

[0095] The pufferfish TRPV1 inhibitory peptide is used for one or more of the following expressions:

[0096] a. Inhibit TRPV1 protein expression.

[0097] b. Downregulates the release of pro-inflammatory factors.

[0098] c. Upregulates the level of matrix metalloproteinase inhibitor TIMP-1.

[0099] d. Inhibit the secretion of ET-1 and ICAM-1 in vascular endothelial cells.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of pufferfish TRPV1 inhibitory peptide in the preparation of TRPV1 antagonists, characterized in that: The amino acid sequence of the pufferfish TRPV1 inhibitory peptide is: RLF.

2. The application according to claim 1, characterized in that: The pufferfish TRPV1 inhibitory peptide includes one or more of the following applications: a. Inhibit TRPV1 protein expression; b. Downregulate the expression levels of G-CSF, GM-CSF, IL-6, IL-23, CCL3, CXCL2, CCL5, and TNF-α cytokines; c. Upregulates the level of the matrix metalloproteinase inhibitor TIMP-1; d. Inhibit the secretion of ET-1 and ICAM-1 in vascular endothelial cells.

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