A pufferfish TRPV1 inhibitory peptide, its preparation method and application

Abstract

This invention relates to a pufferfish TRPV1 inhibitory peptide, its preparation method, and its application. The amino acid sequence of the peptide is LDIF. The preparation method includes: 1. Enzymatically hydrolyzing pufferfish skin, then inactivating the enzyme, and screening for peptides with a molecular weight not greater than 1 kDa from the hydrolysate, followed by freeze-drying to obtain the pufferfish skin enzymatic hydrolysate peptide; 2. Performing mass spectrometry analysis on the pufferfish skin enzymatic hydrolysate peptide, using mass spectrometry analysis software, and selecting multiple non-repeating peptide sequences based on the criteria of confidence level -10lgP > 20 and amino acid count < 10; 3. Molecularly docking the selected peptide sequences with the TRPV1 receptor using software to select peptide sequences with strong binding affinity to the TRPV1 receptor; 4. Solid-phase synthesis of the selected peptide sequences to obtain the peptide LDIF. This invention uses pufferfish skin as raw material to obtain a peptide with good inhibitory effect on TRPV1, which can be used to prepare skin care products for soothing sensitive skin.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a pufferfish TRPV1 inhibitory peptide, its preparation method, and its application. Background Technology

[0002] Sensitive skin refers to a hyperreactive state of the skin under physiological or pathological conditions, primarily affecting the face. Clinically, it manifests as subjective symptoms such as burning, stinging, itching, and tightness when stimulated by physical, chemical, or psychological factors, with or without objective signs such as erythema, scaling, and telangiectasia. The "Chinese Expert Consensus on the Diagnosis and Treatment of Sensitive Skin" indicates that the incidence rate is generally higher in women than in men, approximately 36.1%. The development of sensitive skin is triggered by the interaction of external and internal factors, including environmental and climatic changes, improper use of cosmetics, stress, and dietary habits. The formation of sensitive skin is a complex and subtle process involving multiple factors such as damage to the skin barrier, abnormal excitation of neurovasculature, and activation of immune inflammation.

[0003] When the skin barrier function is impaired, transient vanilla-like receptors (TRPV1) are activated. TRPV1 is a multimodally activated, calcium-permeable, non-selective cation channel involved in the transmission of sensitivity-related sensory symptoms. When TRPV1 receptors are activated, the TRPV1 channel induces an action potential, causing a large amount of calcium to be released. 2+ Influx of TRPV1 leads to symptoms such as burning, stinging, and itching in sensitive skin. Activation of TRPV1 can also promote the release of substance P, vasoactive intestinal peptide, and neurotensin in the local skin. It also induces the release of interleukin-23 (IL-23) and interleukin-31 (IL-31) from keratinocytes (HaCaT) and mast cells near sensory nerve endings, subsequently activating antigen-presenting cells and T cells, triggering skin immune and inflammatory responses. Endothelin (ET-1) is a polypeptide that induces vasoconstriction and vasodilation, mainly synthesized by various cells including endothelial cells, neurons, and macrophages. When vascular endothelial cells are stimulated, ET-1 secretion increases significantly. ET-1 can induce the upregulation of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells, promoting the release of inflammatory mediators and leading to vascular dysfunction. Therefore, finding safe and effective inhibitors of TRPV1 activity is of great significance.

[0004] Currently, artificially synthesized TRPV1 antagonists include trans-4-tert-butylcyclohexanol, acitrepp, capsicum, etc.; in addition, many extracts with TRPV1 antagonistic effects have been obtained from plants and have been shown to accelerate the recovery of skin barrier damage.

[0005] Natural bioactive peptides possess various metabolic and physiological regulatory functions in the human body, exhibiting excellent biocompatibility and extremely high safety. They are currently among the most popular research topics and promising functional factors in the international food and cosmetics industries. Peptide TRPV1 antagonists have become a research hotspot in the development of soothing active substances in recent years. APHC peptides from sea anemones are the first reported peptide antagonists binding to the TRPV1 channel, showing a strong inhibitory effect on capsaicin (CAP)-induced TRPV1 activation. In vivo studies have shown that APHC peptides have significant analgesic effects in different pain models. Kang et al. developed a novel TRPV1-targeting peptide (TIP), finding that TIP effectively inhibits CAP-induced calcium influx and TRPV1 activation. TIP also attenuates 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, current research on TRPV1 inhibitors is extremely limited, primarily focusing on fish. Summary of the Invention

[0006] The purpose of this invention is to provide a pufferfish TRPV1 inhibitory peptide, its preparation method, and its application. The method uses pufferfish skin as raw material to obtain the amino acid sequence of the pufferfish TRPV1 inhibitory peptide, and the prepared pufferfish TRPV1 inhibitory peptide has a good inhibitory effect on TRPV1. It can be used to prepare skin care products that soothe sensitive skin.

[0007] To achieve the above objectives, this invention discloses a pufferfish TRPV1 inhibitory peptide with the amino acid sequence LDIF.

[0008] This invention also discloses a method for preparing the above-mentioned pufferfish TRPV1 inhibitory peptide, which includes the following steps:

[0009] S1. Extraction of enzymatically hydrolyzed polypeptides from pufferfish skin

[0010] 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.

[0011] S2. Sequence Identification and Preliminary Screening

[0012] 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.

[0013] S3, Virtual Filtering

[0014] 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.

[0015] S4, polypeptide synthesis

[0016] The polypeptide sequences screened in step S3 were synthesized in a solid phase to obtain the pufferfish TRPV1 inhibitory peptide.

[0017] Preferably, the process also includes step S0, which is performed before step S1. Step S0 is a pretreatment of the fish skin, specifically: first, the pufferfish skin is cut into small pieces, and then the small 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. The mass concentration of the NaCl solution is 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.

[0018] 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.

[0019] 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.

[0020] Preferably, the mass spectrometry determination of the pufferfish skin enzymatic hydrolysate peptides in step S2 is specifically as follows: the pufferfish skin enzymatic hydrolysate peptides are dissolved in solvent A to obtain sample peptides; 1 μL of sample peptides 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, the column temperature at 40 °C; the electrospray voltage is set to 2 kV; the mass spectrometer is operated 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 minutes. MS; Precursor ions were selected and entered into the collision cell for high-energy collision dissociation; fragmentation was performed, and the normalized collection energy was 28%; MS / MS resolution was set to 17500, automatic gain control target was 1e5, maximum injection time was 45 ms, and dynamic exclusion time was 30 s; Solvent A was a 0.1% formic acid aqueous solution, and buffer B was an aqueous solution containing 80% ACN and 0.1% FA.

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

[0022] Furthermore, this invention also discloses the application of the above-mentioned pufferfish TRPV1 inhibitory peptide in the preparation of skin care products.

[0023] Preferably, the mass concentration of pufferfish TRPV1 inhibitory peptide in the skin care product is not less than 400 μM.

[0024] The present invention has the following beneficial effects:

[0025] This invention uses pufferfish skin as raw material to obtain the amino acid sequence of pufferfish TRPV1 inhibitory peptide, and the prepared pufferfish TRPV1 inhibitory peptide has a good inhibitory effect on TRPV1. It can be used to prepare skin care products to soothe sensitive skin, and provides a good candidate compound for the development of skin care products for sensitive skin. Attached Figure Description

[0026] Figure 1 The effect of different concentrations of T14 on the viability of RAW 264.7 cells.

[0027] Figure 2 The inhibitory effect of different concentrations of T14 on NO content.

[0028] Figure 3 The fluorescence intensity of HaCaT cells loaded with Fluo-4 AM at different concentrations of T14 in response to CAP.

[0029] Figure 4 The effect of T14 treatment time on intracellular calcium induced by CAP 2+ The effect of increased concentration.

[0030] Figure 5 3D view of the optimal configuration for docking T14 and TRPV1.

[0031] Figure 6 for Figure 5 Enlarged schematic diagram of part A in the diagram.

[0032] Figure 7 This is a 2D diagram of the interaction between T14 and TRPV1.

[0033] Figure 8 The inhibitory effect of different concentrations of T14 on ET-1 expression.

[0034] Figure 9 The inhibitory effect of different concentrations of T14 on ICAM-1 expression.

[0035] Figure 10 This is a RAW 264.7 cytokine array atlas.

[0036] Figure 11 Heatmap of gene expression for 40 cytokines.

[0037] Note: Figure 10 Each pair of dots represents two parallel pairs of each cytokine or chemokine. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] This invention discloses a pufferfish TRPV1 inhibitory peptide, the amino acid sequence of which is LDIF, as shown in SEQ ID NO: 1.

[0040] The above-mentioned method for preparing the pufferfish TRPV1 inhibitory peptide includes the following steps:

[0041] S0, Fish skin pretreatment

[0042] The pufferfish used in this experiment was the dark-spotted pufferfish from Zhangpu, Fujian. First, the pufferfish skin was cut into small pieces using a meat slicer. Then, the pieces were freeze-dried to remove moisture and vacuum-packed for preservation. Next, the freeze-dried skin was soaked in a 9% NaCl solution with continuous stirring for 24 hours, with the NaCl solution replaced every 12 hours. After soaking, the skin was washed with distilled water and filtered through nylon mesh, repeated three times to remove non-collagenous proteins.

[0043] S1. Extraction of enzymatically hydrolyzed polypeptides from pufferfish skin

[0044] The optimal hydrolysis conditions for pufferfish skin were as follows: solid-liquid ratio 1:10, enzyme dosage 8000 U / g, hydrolysis temperature 50℃, hydrolysis pH 9.0, and hydrolysis time 4h.

[0045] After enzymatic hydrolysis, the enzyme was inactivated for 10 minutes. The hydrolysate was first passed through a ceramic membrane for microfiltration, and then separated by an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Peptides with a molecular weight not greater than 1 kDa were screened out, and then freeze-dried under vacuum and stored at -20℃ to obtain pufferfish skin enzymatic hydrolysate.

[0046] S2. Sequence Identification and Preliminary Screening

[0047] 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.

[0048] 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.

[0049] The peptide sequences were identified using nano-HPLC-MS / MS. In the peptide results, the -10 lgP 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 -10 lgP > 20 and an amino acid count < 10 were screened, and 190 peptides were initially screened.

[0050] S3, Virtual Filtering

[0051] 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.

[0052] Specifically, based on the mass spectrometry analysis results, peptides were initially screened using the confidence level of the spectral identification (-10 lgP) 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, and hydrogen and charge were added for energy optimization. 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 LDIF (T14) was selected based on the Grid Score and binding mode.

[0053] S4, polypeptide synthesis

[0054] The polypeptide sequence (LDIF(T14)) selected in step S3 was synthesized in a solid phase to obtain the pufferfish TRPV1 inhibitory peptide.

[0055] The following analysis examines the effects on cell viability, inhibition of NO levels, and the impact of CAP-induced Ca2+ levels in HaCaT cells. 2+ The inhibitory effect of influx and the reduction of the stimulatory response of HUVEC cells were experimentally verified by the selected lead peptide LDIF.

[0056] I. Experimental Cell Culture

[0057] RAW 264.7 and HUVEC cells were purchased from the Peking Union Medical College Cell Bank, and HaCaT cells were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences Cell Bank. RAW 264.7 and HaCaT cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 10% FBS (Sigma, F8687) and 1% penicillin-streptomycin (Beyotime, C0222). HUVEC cells were cultured in DMEM (Gibco, C11995500BT) complete medium containing 1% glutamine, 1% non-essential amino acids (NEAA), 2% sodium pyruvate, 10% FBS (Sigma, F8687), and 1% penicillin-streptomycin (Beyotime, C0222).

[0058] II. Measurement of cell viability

[0059] The effect of peptides on the proliferation of Raw 264.7 cells was determined using the MTT assay (MTS, Promega; PMS, Sigma). Specifically, the activity of T14 in RAW 264.7 cells at concentrations ranging from 100 to 800 μM was measured using the MTT assay, and a blank control group (CK) was included. The results are as follows: Figure 1 As shown. From Figure 1 It can be seen that the cell survival rate of T14 is the lowest at 200 μM, which is 86%. When the cell survival rate is above 80%, it is considered to be non-toxic to cells. Therefore, T14 is non-toxic to RAW 264.7 cells at a concentration of 800 μM.

[0060] III. Determination of NO content

[0061] RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. RAW 264.7 cells in the logarithmic growth phase were collected, suspended, and the cell density was adjusted to 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at 500 µL per well in 24-well plates with cfu / mL concentrations and incubated for 8-12 h. The supernatant was discarded, and cells were treated with different concentrations (50, 100, 200, 400 μM) of T14 solution for 2 h, followed by incubation with LPS (final concentration 1 μg / mL) (L8880, Solarbio) for 24 h. NO expression levels were detected according to the NO kit instructions. Results are shown below. Figure 2 shown (Note: Figure 2 Groups marked with "0" on the horizontal axis represent blank control, while those marked "LPS" only have LPS added to the group representation. Figure 3 The coordinate identification method is similar to Figure 2 (This will not be elaborated further).

[0062] from Figure 2 It can be seen that, compared with the LPS group, T14 has an inhibition rate of 14.6% at 200 μM and an inhibition rate of 46.1% at 400 μM, which can significantly inhibit the production of NO.

[0063] IV. Ca 2+ Inflow Experiment

[0064] HaCaT cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 CFU / mL was seeded into 96-well black-walled clear plates and cultured overnight. The supernatant was discarded, and 200 μM T14 was added to each well for 2 h, 6 h, 12 h, and 24 h. Then, 100 μL CAP was added to each well for 30 min of stimulation. A blank control group was set up. Images were taken using an inverted fluorescence microscope and processed using imaje.j. The average fluorescence intensity was calculated.

[0065] An in vitro model was established using CAP-induced HaCaT cells to induce Ca2+. 2+ Using influx as an indicator, this study investigates the skin anti-allergy mechanism based on TRPV1. For example... Figure 3-4 As shown, CAP can induce intracellular Ca2+ in HaCaT cells. 2+ Increased concentration, after incubation of HaCaT cells with 200 μM T14, reduced CAP-induced intracellular Ca2+ levels. 2+ The inhibitory effect on the content was significant and time-dependent; when the incubation time was extended to 12 and 24 hours, the inhibitory effect on Ca was reduced. 2+ The inhibitory effect was more significant at higher concentrations. After 24 hours of incubation, T14 showed a greater inhibitory effect on Ca. 2+ The fluorescence intensity suppression rate was 73.1% ± 7.55%.

[0066] V. Interaction Analysis between T14 and TRPV1

[0067] The docking results of T14 and TRPV1 are as follows: Figure 5-7 As shown, T14 binds within the cavity of TRPV1, forming hydrogen bonds with residue THR550 (2.24 Å), hydrophobic interactions with MET572 (4.26 Å), LEU553 (5.27 Å), TYR511 (4.59 Å), LEU515 (5.32 Å), LEU553 (5.27 Å), LEU577 (4.83 Å, 4.96 Å), and ILE569 (4.78 Å), and sulfur bonds with MET568 (5.86 Å) and MET683 (5.95 Å); these three forces contribute to the structural stability of the complex.

[0068] VI. HUVEC cell stimulation response

[0069] HUVEC cells in logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5 CFU / mL was seeded into 24-well plates and cultured overnight. The supernatant was discarded. Different concentrations of T14 (50, 100, 200, 400 μM) were added to each well and cultured for 6 h. Then, LPS (10 μg / mL) was added, and the plates were cultured 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 using the following formula:

[0070] Inhibition rate = (Expression level of LPS group - Expression level of peptide group) / (Expression level of LPS group - Expression level of negative control group) × 100%

[0071] When vascular endothelial cells are stimulated by endogenous or exogenous factors, ET-1 secretion increases. This increase in ET-1 stimulates neutrophils and macrophages to release various pro-inflammatory factors, leading to a sharp rise in ICAM-1 expression. This affects vascular permeability, causing skin redness and swelling, and resulting in vascular irritation. Figure 8 As shown, stimulation of HUVEC cells with 10 μg / mL LPS for 24 h increased ET-1 expression from 2.05 pg / mL to 2.7 pg / mL. Pretreatment with T14 for 6 h followed by co-culturing with LPS for 24 h resulted in a dose-dependent reduction in ET-1 expression. At 100 μM T14, ET-1 expression decreased to 0.54 pg / mL (inhibition rate 24.47%), and at 400 μM T14, it decreased to 2.2 pg / mL (inhibition rate 75%), demonstrating a significant difference.

[0072] like Figure 9 As shown, the expression level of ICAM-1 increased from 0.43 pg / mL to 0.57 pg / mL under LPS stimulation. Pretreatment with T14 dose-dependently reduced ICAM-1 expression. At 50 μM T14, ICAM-1 expression decreased to 0.54 pg / mL, with an inhibition rate of 20.65%. At 400 μM T14, ICAM-1 expression decreased to 0.50 pg / mL, with an inhibition rate of 48.96%, showing a significant difference.

[0073] VII. Mouse Cytokine Array Analysis

[0074] The protein expression of 40 cytokines and chemokines in the supernatant of Raw 264.7 cell culture was measured using the Proteome Profiler™ Antibody Arrays Mouse Cytokine Array Panel A kit. Cell culture supernatant was collected from each treatment group, with 300 μL of supernatant used for each array. At the start of the experiment, reagents were equilibrated to room temperature, and the membrane was blocked for 2 hours. Simultaneously, the antibody mixture was added to the cell supernatant and incubated at room temperature for 1 hour, followed by overnight incubation at 4°C. After incubation, the membrane was washed three times with washing buffer for at least 10 minutes each time. Following washing, HRP-conjugated streptavidin was added and incubated at room temperature for 2 hours to allow for complete binding. The membrane was then washed again and exposed with a chemiluminescent reagent. Array images displaying chemiluminescent signals were obtained using a chemiluminescence imaging system, and the accumulated light density was analyzed using an image.j software densitometer.

[0075] from Figure 10-11As can be seen, compared with the normal group, the LPS group significantly upregulated the expression levels of G-CSF, GM-CSF, ICAM-1, IL-3, IL-6, TNF-α, CCL2, CCL3, CCL4, CXCL2, CCL5, and TIMP-1 cytokines. After incubation with T14, the expression levels of G-CSF, GM-CSF, ICAM-1, IL-3, IL-6, IL-23, and TNF-α cytokines were significantly downregulated, while the expression level of TIMP-1 was upregulated. This indicates that T14 can inhibit the expression of pro-inflammatory factors produced by LPS-stimulated RAW 264.7 cells while increasing the expression level of TIMP-1.

[0076] Based on the above experiments, this invention also discloses the application of the aforementioned pufferfish TRPV1 inhibitory peptide (LDIF) in the preparation of skincare products, wherein the mass concentration of the pufferfish TRPV1 inhibitory peptide in the skincare product is not less than 400 μM. This skincare product is particularly suitable for sensitive skin.

[0077] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A scorpion TRPV1 inhibitory peptide, characterized in that, The amino acid sequence of which is: LDIF.

2. The puffer fish TRPV1 inhibitory peptide for use in the preparation of a skin care product according to claim 1.

3. Use according to claim 2, wherein: The mass concentration of the puffer fish TRPV1 inhibitory peptide in the skin care product is not less than 400 μM.

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