Puffer fish TRPV1 (transient receptor potential vanilloid 1) inhibitory peptide as well as preparation method and application thereof

Abstract

The invention discloses a puffer fish TRPV1 inhibitory peptide, a preparation method and application, and belongs to the field of polypeptides, and the amino acid sequence of the puffer fish TRPV1 inhibitory peptide is RLF. According to the puffer fish skin TRPV1 inhibitory peptide, the preparation method and the application, the puffer fish skin is used as a starting raw material, and the amino acid sequence of the puffer fish skin TRPV1 inhibitory peptide is successfully obtained through a specific process. Experiments prove that the prepared puffer fish TRPV1 inhibitory peptide shows an excellent inhibitory effect on TRPV1. The inhibitory peptide can be used for preparing a TRPV1 antagonist, and the antagonist can play an anti-inflammatory regulation role through multiple pathways and effectively lower the high reactivity of skin microvessels.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, and particularly relates to a pufferfish TRPV1 inhibitory peptide, a preparation method and an application thereof. Background Art

[0002] The skin is the largest defense organ of the human body, and the imbalance between its barrier function and neuroimmunity is the pathogenesis of sensitive skin. The transient receptor potential vanilloid 1 (TRPV1), as a multimodal-activated, calcium-permeable, non-selective cation channel, is widely distributed in skin keratinocytes and sensory nerve endings, and mediates the influx of Ca 2 + by stimuli such as capsaicin, high temperature (>43°C), and low pH (<5.9), triggering a neurogenic inflammatory response. Research has shown that TRPV1 plays an important role in sensitive skin, and its abnormal activation can lead to the release of pro-inflammatory factors (such as IL-6, TNF-α), the secretion of neuropeptides (substance P, CGRP), and hyperreactivity of vascular endothelium, thereby resulting in skin erythema, burning, and barrier function damage. However, existing TRPV1 antagonists have problems such as low transdermal absorption efficiency and skin desensitization caused by long-term use. Therefore, it is necessary to develop a new type of inhibitor with high efficiency, safety, and multi-target regulation characteristics.

[0003] Due to the advantages of small molecular weight, strong targeting, and high biocompatibility, polypeptide compounds have become a hot spot for the development of active substances for alleviation in recent years. Currently, the APHC peptide from sea anemones has been found to be the first reported peptide antagonist that binds to the TRPV1 channel and shows strong inhibitory effects on capsaicin-induced TRPV1 activation. In vivo studies have shown that the APHC peptide has significant analgesic effects in different pain models. There is also a new TRPV1-targeted peptide (TIP). TIP can effectively inhibit capsaicin-induced calcium influx and TRPV1 activation. TIP reduces UV-induced erythema and the expression of inflammatory factors in human skin in vivo, providing a treatment method for UV-induced inflammation and photoaging. However, there is very little research on TRPV1 inhibitors from fish at present. Summary of the Invention

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

[0005] To achieve this purpose, the present invention adopts the following technical solutions:

[0006] A pufferfish TRPV1 inhibitory peptide provided by the present invention has an amino acid sequence of: 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 polypeptide from pufferfish skin

[0009] Use alkaline protease to enzymatically hydrolyze the skin of pufferfish. After the enzymatic hydrolysis is completed, inactivate the enzyme, and then screen out polypeptides with a molecular weight not greater than 1 kDa from the enzymatic hydrolysate, and freeze-dry to obtain the enzymatically hydrolyzed polypeptide from pufferfish skin;

[0010] S2. Sequence identification and preliminary screening

[0011] Perform mass spectrometry on the enzymatically hydrolyzed polypeptide from pufferfish skin. Analyze the results of the mass spectrometry using mass spectrometry analysis software, and use the screening conditions of confidence -10lgP > 20 and the number of amino acids < 10 to screen out multiple non-repeating polypeptide sequences;

[0012] S3. Virtual screening

[0013] Use software to perform molecular docking of the polypeptide sequences screened in step S2 with the TRPV1 receptor, and screen out polypeptide sequences with strong binding ability to the TRPV1 receptor;

[0014] S4. Polypeptide synthesis

[0015] Solid-phase synthesize the polypeptide sequences screened in step S3 to obtain the pufferfish TRPV1 inhibitory peptide.

[0016] Preferably, before step S1, it further includes step SO: first cut the skin of pufferfish into small pieces, and then perform freeze-drying treatment on the cut small pieces of skin; then soak the freeze-dried skin in NaCl solution and continuously stir for 12 - 36 h, and change the NaCl solution every 6 - 12 h. The mass concentration of the NaCl solution is 6 - 12%; after the skin soaking is completed, wash with distilled water and filter with nylon yarn, and repeat 3 - 5 times.

[0017] Preferably, in step S1: the conditions for alkaline protease to enzymatically hydrolyze the skin of pufferfish are: solid-liquid ratio 1:10, enzyme addition amount 8000 U / g, enzymatic hydrolysis temperature 50 °C, enzymatic hydrolysis pH value 9.0, enzymatic hydrolysis time 4 h.

[0018] Preferably, in step S1: the enzymatic hydrolysate is first microfiltered through a ceramic membrane, and then separated by an ultrafiltration membrane with a molecular weight cut-off of 1 kDa to obtain polypeptides with a molecular weight not greater than 1 kDa.

[0019] Preferably, the mass spectrometry determination of the puffer fish skin hydrolysate polypeptide in step S2 is specifically as follows: Dissolve the puffer fish skin hydrolysate polypeptide in solvent A to obtain a sample peptide; Load 1 μL of the sample peptide onto a 25 cm analytical column, and separate it starting from 2% buffer B with a 60-minute gradient, gradually increasing to 35% in 47 minutes, increasing to 100% in 1 minute, and maintaining for 12 minutes; The column flow is maintained at 300 nL / min, and the column temperature is 40 °C; The electrospray voltage is set to 2 kV; The mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS modes; The 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; Select precursor ions to enter the collision cell for high-energy collision dissociation; Fragmentation, 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 ACN with a mass concentration of 80% and FA with a mass concentration of 0.1%.

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

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

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

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

[0025] Starting from puffer fish skin as the raw material, the amino acid sequence of the puffer TRPV1 inhibitory peptide was successfully obtained through a specific process. Through experimental verification, the prepared puffer TRPV1 inhibitory peptide exhibits excellent inhibitory effects on TRPV1. This inhibitory peptide can be used to prepare a TRPV1 antagonist, which can exert an anti-inflammatory regulatory effect through multiple pathways and effectively downregulate the high reactivity of skin microvessels. The puffer TRPV1 inhibitory peptide shows significant effects in soothing the skin and nerve-immune regulation. The present invention provides a new strategy for the development of functional ingredients for sensitive skin and is expected to contribute to the upgrading and innovation of sensitive skin care products. Description of the Drawings

[0026] Figure 1It is the 3D docking and complex mode diagram of T21 and TRPV1 of the present invention.

[0027] Figure 2 It is the 2D docking and complex mode diagram of T21 and TRPV1 of the present invention.

[0028] Figure 3 It is the cytokine array diagram (each pair of points represents 2 parallels of each cytokine or chemokine) of RAW 264.7 cells of the present invention.

[0029] Figure 4 It is the heat map of the gene expression of 40 cytokines of the present invention.

[0030] Figure 5 It is the inhibitory effect diagram of T21 on the expression level of ET-1 of the present invention.

[0031] Figure 6 It is the inhibitory effect diagram of T21 on the expression level of ICAM-1 of the present invention.

[0032] Figure 7 It is the expression level diagram of TRPV1 protein detected by Western Blot method of the present invention.

[0033] Figure 8 It is the effect diagram of T21 on the expression of TRPV1 protein in CAP-induced HaCaT cells of the present invention.

[0034] Figure 9 It is the effect diagram of T21 on the mortality rate of zebrafish of the present invention.

[0035] Figure 10 It is the fluorescence diagram of neutrophils in zebrafish treated with T21 of the present invention.

[0036] Figure 11 It is the effect diagram of T21 on the number of neutrophils in zebrafish of the present invention.

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

[0038] Figure 13 It is the effect diagram of T21 on the total movement distance of zebrafish of the present invention.

[0039] Figure 14 It is the effect diagram of T21 on the rotation frequency of zebrafish of the present invention.

[0040] Figure 15 It is the effect diagram of T21 on the activity state of zebrafish of the present invention. Specific embodiments

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

[0042] A kind of pufferfish TRPV1 inhibitory peptide provided in this embodiment has an amino acid sequence of: RLF.

[0043] The present invention also provides a preparation method of the above-mentioned pufferfish TRPV1 inhibitory peptide, which comprises the following steps:

[0044] Step SO: Pretreatment of fish skin

[0045] In this embodiment, the pufferfish used is Takifugu obscurus. First, cut the fish skin of the pufferfish into small pieces with a meat slicer, and then freeze-dry the cut fish skin to remove the moisture in the fish skin, and store it in vacuum packaging. Then soak the freeze-dried fish skin in a NaCl solution with a mass concentration of 9%, and continuously stir for 24 h, and replace the NaCl solution every 12 h. After the fish skin soaking is completed, wash it with distilled water and filter it with nylon yarn, and repeat 3 times to remove non-collagen.

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

[0047] Use alkaline protease (B8360, Solarbio) to enzymatically hydrolyze the fish skin of the pufferfish. The optimal enzymatic hydrolysis conditions are: solid-liquid ratio 1:10, enzyme addition amount 8000 U / g, enzymatic hydrolysis temperature 50 °C, enzymatic hydrolysis pH value 9.0, and enzymatic hydrolysis time 4 h.

[0048] After the enzymatic hydrolysis is completed, inactivate the enzyme for 10 min. The enzymatic hydrolysate is first microfiltered through a ceramic membrane, and then ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 1 kDa to separate polypeptides with a molecular weight not greater than 1 kDa, and then vacuum freeze-dried and stored at -20 °C to obtain enzymatically hydrolyzed polypeptides from pufferfish skin.

[0049] S2. Sequence identification and preliminary screening

[0050] Perform mass spectrometry on the enzymatically hydrolyzed polypeptides from pufferfish skin. The results of the mass spectrometry are analyzed using mass spectrometry analysis software, and multiple non-repetitive polypeptide sequences are screened out with the screening conditions of confidence -10lgP > 20 and the number of amino acids < 10.

[0051] Specifically, the polypeptide sequence was identified by Nano-HPLC-MS / MS. The polypeptide was redissolved in solvent A (A: 0.1% formic acid aqueous solution) to obtain sample peptides, which were analyzed by an Orbitrap Q-Exactive Plus and EASY-nanoLC 1200 system (Thermo Fisher Scientific, MA, USA). 1 μL of the sample peptides was loaded onto a 25-cm analytical column (inner diameter 75 μm, resin 1.9 μm (Dr Maisch)), and separation was started with a 60-min gradient from 2% buffer B (80% ACN (acetonitrile), 0.1% FA (formic acid)), gradually increased to 35% in 47 min, increased to 100% in 1 min, and maintained for 12 min. The column flow was maintained at 300 nL / min, and the column temperature was 40 °C. The electrospray voltage was set at 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 the Orbitrap at 70,000 resolution. The automatic gain control (AGC) target was 3e6, and the maximum injection time was 50 ms. Precursor ions were selected to enter the collision cell for high-energy collision dissociation (HCD) fragmentation, and the normalized collision energy was 28%. The MS / MS resolution was set at 17,500, the automatic gain control (AGC) target was 1e5, the maximum injection time was 45 ms, and the dynamic exclusion time was 30 s.

[0052] The polypeptide sequence was identified by nano-HPLC-MS / MS. In the peptide results, the -10lgP index represents the confidence level of the corresponding spectral identification, and the larger the value, the better the matching result. According to the mass spectrometry analysis results, peptides with a spectral identification confidence of -10lgP > 20 and the number of amino acids < 10 were screened, and 190 polypeptides were preliminarily screened.

[0053] S3. Virtual screening

[0054] The polypeptide sequences screened in step S2 were molecularly docked with the TRPV1 receptor through software to screen out polypeptide sequences with strong binding ability to the TRPV1 receptor.

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

[0056] S4. Peptide synthesis

[0057] The lead peptide sequences screened in step S3 were solid-phase synthesized to screen out lead peptide sequences with good inhibitory effects on TRPV1, namely the TRPV1 inhibitory peptide RLF (T21) of puffer fish.

[0058] Next, the lead peptide RLF (T21) was experimentally verified.

[0059] 1. Experimental materials and methods

[0060] 1.1 Molecular docking

[0061] Using TRPV1 as the receptor protein and T21 as the ligand, the Discovery studio 2019 software was used to dehydrate, protonate, add hydrogen, and charge the TRPV1 protein. The ChemDraw 20.0 was used to draw the 2D structure of the polypeptide, and then the Chem3D was used to draw the 3D structure of the polypeptide and minimize its energy. The TRPV1 pocket position was determined in Discovery studio, and TRPV1 was docked with the polypeptide ligand using Libdock. Finally, the conformation with the best score was output, and the intermolecular interaction forces were visually analyzed in Discovery studio 2019.

[0062] 1.2 Molecular dynamics

[0063] Molecular dynamics simulations were performed on the D chain of the 8GFA protein using Amber 20: After deleting the ABC chains and water molecules and repairing the structure, the protein was processed using the AMBERff19SB force field. The small molecule peptide was hydrogenated and charge-optimized, and then parameterized using the GAFF force field and AM1-BCC charges. The system was constructed with an OPC water model to form a truncated octahedral solvent box ( Buffer), add NaCl to neutralize the charge, and perform stepwise energy minimization (constrain heavy atoms → full atom relaxation until energy convergence). The stepped equilibration includes heating to 300 K in 0.02 ns NVT, 200 ps of NPT equilibration with heavy atom constraints, and 1 ns of free NVT relaxation. Finally, a 100 ns production simulation (300 K, 2 fs time step) is carried out, 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 culture supernatant of Raw264.7 cells was detected using the Proteome ProfilerTM antibody array kit (ARY006, R&D Systems). The experimental procedure was as follows: Collect the cell supernatants of each treatment group and process them according to the kit operation procedure (take 300 μL of supernatant for each sample). First, warm the reagents to room temperature and block the array membrane for 2 h; simultaneously incubate the detection antibodies with the supernatant at room temperature for 1 h, and then transfer them to the membrane and bind overnight at 4 °C. The next day, wash thoroughly 3 times with the washing solution (≥10 min each time), add HRP-streptavidin and incubate at room temperature for 2 h, wash again, and then develop using chemiluminescent reagents. Collect the signals through a chemiluminescent imaging system, and quantitatively analyze the integrated optical density value using ImageJ software.

[0066] 1.4. Stimulatory response of HUVEC cells

[0067] Take cells in the logarithmic growth phase and inoculate them into 24-well plates at 2×10 5 cfu / mL, culture overnight, discard the supernatant, first add different concentrations of T21 (50, 100, 200, 400 μM) to each well in 24-well plates and culture for 6 h, then add LPS (10 μg / mL), and co-culture for 24 h. Collect the cell culture supernatants, and detect the expression levels of ET-1 and ICAM-1 according to the ELISA kit instructions, and calculate the inhibition rate.

[0068] Inhibition rate = (Expression level in LPS group - Expression level in polypeptide group) / (Expression level in LPS group - Expression level in negative control group) × 100%.

[0069] 1.5. Western Blot determination of the expression of TRPV1 protein in HaCaT cells by T21

[0070] T21 was pre-treated for 6 h and then co-cultured with CAP for 24 h. After washing with DPBS, it was lysed with RIPA lysis buffer on ice for 1 h, centrifuged at 4 °C to obtain the supernatant, and the protein concentration was determined by the BCA method. Equal amounts of protein were mixed with Loading Buffer (1:3) and denatured at 95 °C for 5 min. After SDS-PAGE electrophoresis (10 μL / lane), it was transferred to PVDF, and blocked with 5% non-fat milk-TBST for 2 h. Antibodies against TRPV1 (A00128, BOSTER, Wuhan, China) and GAPDH (CL594-6004, Abclonal, Wuhan, China) were prepared according to the instructions and incubated overnight at 4 °C. After washing with TBST, it was incubated with HRP secondary antibody at room temperature for 1 h. The final wash was performed with TBST, and the bands were developed with ECL reagent (K-12045-D50, Advansta, San Jose, CA, USA), and the gray values of the bands were quantitatively analyzed by ImageJ.

[0071] 1.6. Determination of zebrafish embryo survival rate

[0072] Wild-type zebrafish embryos at 24 h post-fertilization (24 hpf) of the AB strain were used, and the toxicity of T21 was evaluated using standard E3 culture medium as the matrix. Six concentration gradients of 0, 100, 200, 300, 400, and 500 μM were set for each compound, with 30 zebrafish embryos in each well. Fresh culture medium containing the corresponding concentration of the compound was replaced twice a day, and the exposure continued until 3 dpf. The embryo survival rate was recorded every 24 h during the observation period to determine the highest non-toxic concentration, and the experiment was repeated three times.

[0073] 1.7. Neutrophil experiment in zebrafish

[0074] Transgenic neutrophil fluorescent zebrafish (Tg mpeg1:EGFP, 2 dpf) obtained by natural mating were used to construct an inflammation model, and the inflammatory response was induced by sodium dodecyl sulfate (SLS). The experiment was divided into a normal control group, a model group (200 μM SLS), and a positive control group (1000 μM dipotassium glycyrrhizinate + 200 μmol / L SLS), and a multi-concentration treatment group of T21 was set: T21 (three concentration gradients of 1, 5, and 25 μM), with 30 zebrafish in each group. Each treatment group was mixed and exposed with 200 μM SLS, and after 24 h of treatment in the dark at 28 °C, it was placed under a fluorescence microscope for photographing and data collection, and the number of neutrophil aggregations in the spinal cord and abdominal regions of zebrafish was quantitatively analyzed.

[0075] 1.8. Zebrafish behavioral experiment

[0076] An inflammation model was constructed by inducing 2dpf AB strain zebrafish with 1000 μM SLS. The experiment was divided into a normal control group (untreated), a model group (1000 μM SLS), and a T21 polypeptide treatment group (1 μM + 1000 μM SLS), and they were continuously exposed for 72 h under light - avoiding conditions at 28 ± 0.5 °C. After the exposure, a zebrafish behavior analysis system was used to detect the total movement distance, rotation frequency, and activity status within 30 min. The data acquisition parameters were set as follows: the activity level was less than 20% in the static state, 20% - 60% in the active state, and higher than 60% in the manic state.

[0077] 2. Results

[0078] 2.1 Interaction analysis of T21 and TRPV1

[0079] T21 binds to TRPV1 through hydrogen - bond interactions, electrostatic interactions, salt - bridge interactions, and hydrophobic interactions ( Figure 1 ); forms 6 hydrogen bonds with ASN438, PHE488, ARG491, TYR554, TYR555, GLU513; forms two electrostatic interactions with TYR554, GLU513; forms one salt - bridge interaction with GLU513; and forms 4 hydrophobic interactions with PHE435, VAL441, TYR554, TYR555. The binding energy ΔG of the docking of T21 and TRPV1 is - 4.86 kcal / mol.

[0080] As Figure 2 shown, in the early stage of the simulation, the amplitude of the complex system is relatively large, which belongs to the normal conformational adjustment stage, and the molecule explores and adapts to the new environment at this stage. As the simulation progresses, different complex systems show different stability characteristics. The TRPV1 - T21 complex enters a stable state after 18 ns. At the end of the 100 - ns simulation process, the amplitude of the TRPV1 - T21 complex is less than This result indicates that the complex system has reached an equilibrium state, meaning that the molecule gradually stabilizes during the simulation process and forms 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 form a relatively tight binding.

[0081] 2.2 Effect of T21 on the cytokine expression level of RAW 264.7 cells

[0082] From Figure 3-4It can be seen that, compared with the normal group, the LPS group significantly up-regulated the expression levels of cytokines such as ICAM-1, IL-1ra, IL-6, CCL3, CCL5, and TNF-α; the expression levels of cytokines such as G-CSF, GM-CSF, IL-23, CXCL10, CXCL2, and TIMP-1 were slightly up-regulated. After incubating with T21, the expression levels of cytokines G-CSF, GM-CSF, ICAM-1, IL-1ra, IL-6, IL-23, CCL3, CXCL2, CCL5, and TNF-α were down-regulated; there was little change in CXCL10 and CXCL2; the expression level of TIMP-1 cytokine was significantly up-regulated.

[0083] 2.3 T21 reduces the stimulatory response of HUVEC

[0084] As Figure 5 shown, when HUVEC cells were stimulated with 10 μg / mL of LPS for 24 h, the expression level of ET-1 increased significantly from 2.14 pg / mL to 3.0 pg / mL. When pretreated with T21 for 6 h and then co-cultured with LPS for 24 h, T21 showed a dose-dependent effect of reducing the ET-1 expression level. When the concentration of T21 was 50 μM, the ET-1 expression level decreased to 2.74 pg / mL, and the inhibition rate reached 29%; when the concentration of T21 was increased to 400 μM, the ET-1 expression level decreased to 2.33 pg / mL, and the inhibition rate was as high as 78%, and the difference was statistically significant.

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

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

[0087] Western blot analysis revealed the regulatory effect of T21 on the expression of TRPV1 channel protein in CAP-induced HaCaT cells. The experimental data showed that after HaCaT cells were stimulated with 20 μM CAP for 24 h, the expression level of TRPV1 protein in HaCaT cells was significantly higher than that in the control group. As Figure 7-8As shown, after treatment with T21 at a concentration gradient of 50 - 400 μM, the TRPV1 expression level showed a significant dose-dependent inhibitory effect. This result indicates that T21 can effectively inhibit the CAP-induced neurogenic inflammatory response by targeting and regulating the TRPV1-mediated calcium signaling pathway.

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

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

[0090] As Figure 10 and 11 shown, the number of neutrophil infiltrations in the epidermis of zebrafish in the blank control group was (16.4 ± 7.0) per field of view (n = 30), and it significantly increased to 50.1 ± 18.1 after stimulation with 1000 μM SLS for 24 h. After adding four lead peptides, it was shown that the number of neutrophils decreased to 20.3 ± 9.4 at 1 μM for T1, with an inhibition rate of 88.2%; it increased to 24.9 ± 10.9 at 5 μM, with an inhibition rate of 74.8%; for T11, it was 26.0 ± 13.5 at 1 μM, with an inhibition rate of 71.6%; it abnormally increased to 36.1 ± 20.5 at 5 μM; for T22, it was 23.7 ± 13.8 at 1 μM, with an inhibition rate of 78.1%, and it increased sharply to 55 ± 21.9 at 5 μM, showing significant concentration toxicity; while T21 stably inhibited the number of neutrophils within the range of 1 - 5 μM, from 23.7 ± 15.9 to 25.6 ± 10.8, with an inhibition rate of 72.7% - 78.3%. The results showed that there were significant differences in the anti-inflammatory potencies of the four groups of peptides at 1 μM. Therefore, 1 μM was selected as the unified concentration for subsequent experiments.

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

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

[0093] In terms of the proportion of zebrafish activity time, as Figure 15 shown, the proportion of static time of T21 reached 97.2 ± 1.8%, which was significantly higher than 94.5 ± 3.1% of the model group; the proportion of active movement time in the T21 group decreased to 2.3 ± 0.9%, which was 52.1% lower than 4.8 ± 1.6% of the model group; the proportion of abnormal activity time in the T21 group was 0.5 ± 0.2%, which was 83.3% lower than 3.0 ± 0.7% of the model group. The results showed that in the experimental group with T21 added, zebrafish were irritable and had a shorter continuous activity time and a longer static time, indicating that after treatment with T21, the stimulation of SLS on zebrafish could be effectively reduced.

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

[0095] Among them, the pufferfish TRPV1 inhibitory peptide is applied to one or more of the following expressions:

[0096] a. Inhibiting TRPV1 protein expression.

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

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

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

[0100] The above examples are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A pufferfish TRPV1 inhibitory peptide, characterized in that: Its amino acid sequence is: RLF.

2. A method for preparing the pufferfish TRPV1 inhibitory peptide as described in claim 1, characterized in that, It includes the following steps: S1. Extraction of enzymatically hydrolyzed polypeptide from pufferfish skin Use alkaline protease to hydrolyze the skin of pufferfish. After the hydrolysis is completed, inactivate the enzyme, and then screen out polypeptides with a molecular weight not greater than 1 kDa from the hydrolysate, and freeze-dry to obtain the enzymatically hydrolyzed polypeptide of pufferfish skin; S2. Sequence identification and preliminary screening Perform mass spectrometry on the enzymatically hydrolyzed polypeptide of pufferfish skin. The results of the mass spectrometry are analyzed using mass spectrometry analysis software, and polypeptides with a confidence level of -10lgP > 20 and the number of amino acids < 10 are used as screening conditions to screen out multiple non-repeating polypeptide sequences; S3. Virtual screening Use software to perform molecular docking of the polypeptide sequences screened in step S2 with the TRPV1 receptor, and screen out polypeptide sequences with strong binding ability to the TRPV1 receptor; S4. Polypeptide synthesis Solid-phase synthesize the polypeptide sequences screened in step S3 to obtain the pufferfish TRPV1 inhibitory peptide.

3. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 2, characterized in that, Before step S1, it also includes step SO: First cut the skin of pufferfish into small pieces, and then perform freeze-drying treatment on the cut skin; then soak the freeze-dried skin in NaCl solution and continuously stir for 12 - 36 h, and change the NaCl solution every 6 - 12 h. The mass concentration of the NaCl solution is 6 - 12%; after the skin soaking is completed, wash with distilled water and filter with nylon yarn, and repeat 3 - 5 times.

4. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 2, characterized in that, In step S1: The conditions for alkaline protease to hydrolyze the skin of pufferfish are: solid-liquid ratio 1:10, enzyme addition amount 8000 U / g, hydrolysis temperature 50 °C, hydrolysis pH value 9.0, hydrolysis time 4 h.

5. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 2, characterized in that, In step S1: The hydrolysate is first microfiltered through a ceramic membrane, and then polypeptides with a molecular weight not greater than 1 kDa are separated by an ultrafiltration membrane with a molecular weight cut-off of 1 kDa.

6. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 2, characterized in that, In step S2, the specific mass spectrometry of the enzymatically hydrolyzed polypeptide of pufferfish skin is as follows: Dissolve the enzymatically hydrolyzed polypeptide of pufferfish skin in solvent A to obtain a sample peptide; load 1 μL of the sample peptide onto a 25 cm analytical column, and separate it from 2% buffer B in a 60 min gradient, gradually increasing to 35% in 47 min, increasing to 100% in 1 min, and maintaining for 12 min; the column flow is maintained at 300 nL / min, and the column temperature is 40 °C; the electrospray voltage is set to 2 kv; the mass spectrometer operates in data-dependent acquisition mode and automatically switches 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; select precursor ions to enter the collision cell for high-energy collision dissociation; fragment, 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.

7. The preparation method of the pufferfish TRPV1 inhibitory peptide according to claim 6, characterized in that, 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 ACN with a mass concentration of 80% and FA with a mass concentration of 0.1%.

8. According to the method for preparing a pufferfish TRPV1 inhibitory peptide according to claim 2, characterized in that: The crystal structure of the TRPV1 receptor is 8GFA.

9. Use of the pufferfish TRPV1 inhibitory peptide according to claim 1 in the preparation of a TRPV1 antagonist.

10. The use according to claim 9, wherein: the pufferfish TRPV1 inhibitory peptide is used for one or more of the following expressions: a. inhibiting the expression of TRPV1 protein; b. down-regulating the release of pro-inflammatory factors; c. up-regulating the level of matrix metalloproteinase inhibitor TIMP-1; d. inhibiting the secretion of ET-1 and ICAM-1 in vascular endothelial cells.

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