A polypeptide and uses thereof

Abstract

The application provides a polypeptide and application thereof. The amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2; the 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; the 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; the 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; and the 29th amino acid in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine and tryptophan. The polypeptide provided by the application has good analgesic function, can selectively inhibit the activation of Nav1.7 channel, thereby inhibiting pain, and has extremely important clinical significance for pain diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide and its applications. Background Technology

[0002] Voltage-gated sodium channels (VGSCs) are transmembrane glycoproteins expressed on the cell membrane with multiple subunits. They consist of an α subunit (functional unit) and a β subunit. The α subunit is composed of four homologous transmembrane domains (I-IV), each of which contains six transmembrane hydrophobic α helices (S1-S6). The positively charged S4 segment functions as a voltage sensor, regulating the hydrophilic pore between S5 and S6 that allows sodium ions to pass through, causing cell depolarization or hyperpolarization, and completing the transmembrane signal transmission.

[0003] Compared to other sodium ion channel subtypes, Nav1.7 channels exhibit rapid activation, rapid inactivation, and slow inactivation kinetics. They can be activated by minor stimuli, promoting further depolarization of the cell membrane. Nav1.7 is the predominant sodium ion channel expressed in peripheral neurons, and its expression is more pronounced in nociceptive sensory neurons than in non-nociceptive sensory neurons.

[0004] Nav1.7 is a sodium ion channel that was previously found to be related to the sensation of pain (i.e., pain perception) through genetic studies of patients with rare pain disorders. Neurons use electrical currents to send signals to the brain and throughout the body, and sodium ion channels are crucial for the cell's ability to generate these currents. When neurons are stimulated, Nav1.7 channels open, allowing positively charged sodium ions to cross the cell membrane and enter a previously negatively charged cell. The change in charge on the cell membrane generates an electrical current, increases the excitability of the neuron, and initiates a cascade of events that lead to pain. Therefore, developing a peptide that can selectively inhibit Nav1.7 channel activation has extremely important clinical significance for pain disorders. Summary of the Invention

[0005] This invention provides a polypeptide and its application. The polypeptide has good analgesic function and can selectively inhibit the activation of Nav1.7 channels, thereby inhibiting pain. It has extremely important clinical significance for pain diseases.

[0006] According to a first aspect of the present invention, a polypeptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2; the 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; the 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; the 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; and the 29th amino acid in the amino acid sequence of the polypeptide is selected from isoleucine, leucine, and tryptophan.

[0007] The polypeptide provided by this invention has good analgesic function and can selectively inhibit the activation of Nav1.7 channel, thereby suppressing pain, which has extremely important clinical significance for pain diseases.

[0008] Preferably, the amino acid sequence of the above-mentioned polypeptide is selected from one of SEQ ID NO: 3 to 18.

[0009] According to a second aspect of the present invention, a nucleic acid molecule is provided, the nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned polypeptide.

[0010] According to a third aspect of the present invention, a recombinant expression vector is provided, the recombinant expression vector comprising the above-described nucleic acid molecule.

[0011] According to a fourth aspect of the present invention, a recombinant host cell is provided, the recombinant host cell containing the above-described recombinant expression vector.

[0012] According to a fifth aspect of the invention, the above-described polypeptide is provided for use in the preparation of a medicament with analgesic function.

[0013] Preferably, the above-mentioned drug can inhibit the activation of voltage-gated sodium ion channels.

[0014] Preferably, the voltage-gated sodium ion channel mentioned above includes the Nav1.7 channel.

[0015] Preferably, the formulation type of the above-mentioned analgesic drug includes at least one of colloidal solution, emulsion or suspension, oral instantaneous film, oral liquid, capsule, injection or transdermal absorption formulation.

[0016] According to a sixth aspect of the present invention, a pharmaceutical composition is provided, wherein the active ingredient in the pharmaceutical composition comprises the aforementioned polypeptide.

[0017] The polypeptide provided by this invention can selectively inhibit the activation of voltage-gated Nav1.7 sodium ion channels, thereby suppressing pain. When the polypeptide provided by this invention is applied to the preparation of analgesic drugs, the resulting drugs have good analgesic effects and are of great clinical significance for pain diseases. Attached Figure Description

[0018] Figure 1 The figure shows the effect of the peptide provided in this invention on the current trajectory of HEK293 cells overexpressing Nav1.7.

[0019] Figure 2 The figure shows the effect of peptide PPN-01 on the current trajectory of HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7.

[0020] Figure 3 Nav1.7 current trajectory diagrams under different concentrations of peptide PPN-01 dosing.

[0021] Figure 4 The figure shows the inhibition results of different concentrations of peptide PPN-01 on the peak current of Nav1.7.

[0022] Figure 5 The figure shows the current-voltage dependent inhibition results of the peptide PPN-01 on the Nav1.7 channel.

[0023] Figure 6 The figure shows the effect of the polypeptide provided by this invention on the time of first writhing and the number of writhing movements in mice.

[0024] Figure 7 The figure shows the effect of different doses of the polypeptide PPN-01 provided by this invention on the number of writhing movements in mice.

[0025] Figure 8 The figure shows the effect of different concentrations of the polypeptide PPN-01 provided by this invention on the electrical signal of the sciatic nerve in rats. Detailed Implementation

[0026] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] This embodiment provides a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1. The 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; the 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; the 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; and the 29th amino acid in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine, and tryptophan.

[0029] The amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 represent the general formula of the polypeptide provided by the present invention. In SEQ ID NO: 1 and SEQ ID NO: 2, the 12th, 19th, 28th and 29th amino acids are all represented by X.

[0030] The difference between SEQ ID NO:1 and SEQ ID NO:2 is that SEQ ID NO:1 contains a total of 29 amino acids, while SEQ ID NO:2 has an extra amino acid (W) added to the N-terminus of the amino acid sequence of SEQ ID NO:1.

[0031] Table 1. Amino acid sequences of the peptides

[0032]

[0033]

[0034] This embodiment lists several polypeptides that meet the above requirements and their corresponding amino acid sequences in Table 1. All polypeptides involved in this invention are prepared using the following steps:

[0035] 1. Using 4-toluenehydroamine resin (MBHA Resin, 1.0 mmol / g) at S = 0.3 mmol / g, the Fmoc synthesis process was employed to sequentially condense amino acids from the C-terminus to the N-terminus of the peptide until the linear peptide was fully condensed, yielding a resin peptide. Acetic anhydride and pyridine were then added to acetylate the N-terminus of the peptide. The peptide was then cleaved from the resin using a cleavage solution (calculated by volume ratio: trifluoroacetic acid TFA: anisole thioether: phenol: EDT: water = 87.5: 5: 2.5: 2.5: 2.5) to obtain a linear peptide.

[0036] 2. Add the linear peptide to an acetonitrile solution (acetonitrile:water = 1:1 by volume), mix thoroughly to obtain a peptide solution with a concentration of 5 mg / mL; mix 60 mg of oxidized glutathione, 30 mg of reduced glutathione and 2.58 g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) thoroughly, and adjust the pH of the mixture to 7.5 with 5 M NaOH to obtain the folded solution;

[0037] 3. Take 30 mL of the above polypeptide solution, add 90 mL of folding solution, mix well, and place in a 4℃ refrigerator for 48 h. Take a sample and detect it using high performance liquid chromatography. After the reaction is basically completed, purify it using preparative liquid chromatography under the conditions shown in Table 2.

[0038] Table 2. Preparation and Liquid Phase Purification Conditions

[0039]

[0040] Example 2: Inhibitory effects of different peptides on Nav1.7 channels

[0041] The purpose of this embodiment is to study the inhibitory effects of different peptides on the Nav1.7 channel using patch-clamp technology.

[0042] The recording electrode used in this embodiment is a glass microelectrode, which is prepared as follows: a glass electrode blank (outer diameter 1.50 mm, inner diameter 0.84 mm) is drawn into a single tube glass microelectrode with a tip diameter of 1-2 μm in four steps using a P-1000 microelectrode horizontal drawing instrument for subsequent experiments. When drawing the electrode, care should be taken not to touch the platinum sheet heated in the middle, and care should also be taken not to touch the drawn electrode tip to prevent the tip from breaking. In addition, the tip of the glass microelectrode is very easy to attract dust, so it is generally used immediately after drawing and should not be left overnight.

[0043] The method of using glass microelectrodes is as follows: Before use, the glass microelectrodes should be cleaned. Then, place all the glass microelectrodes in a large beaker, add anhydrous ethanol to completely immerse the glass microelectrodes in the anhydrous ethanol, and place the beaker on an ultrasonic cleaner for ultrasonic vibration for 30 minutes. After that, pour out the anhydrous ethanol, rinse the glass microelectrodes with ultrapure water 3-4 times, cover the microelectrodes with another small beaker to prevent dust from contaminating the electrodes, and put them in an oven to dry the glass microelectrodes. After drying, place the glass microelectrodes in a clean box for later use.

[0044] In patch-clamp experiments, the measured sodium currents were corrected for leakage current. Raw data were recorded using Clampfit 10.6, and data acquisition was performed using pCLAMP software. Three to five sweeps (a term in patch-clamp software referring to a series of sampling points) where the current was stable before peptide addition were analyzed. The average peak current was calculated from these sweeps and used as the control current amplitude. Then, three to five sweeps where the current was stable after peptide addition were analyzed, and the average of the remaining peak currents was calculated as the residual current amplitude.

[0045] The effects of the glass microelectrodes prepared above on the electrostimulation of HEK293 cells overexpressing Nav1.7 were studied using whole-cell patch-clamp technique (n=1). In voltage clamp mode, the clamping voltage was maintained at -90mV, and a square wave stimulation was applied from -90mV to +40mV for 30ms. The concentration of each drug (peptide) was 300nM. The six peptides with significant effects were replicated (n=2).

[0046] In voltage clamp mode, the clamping voltage was maintained at -90mV, and a square wave stimulus with a depolarization from -90mV to +40mV and a duration of 30ms was applied. The experimental results are as follows: Figure 1 As shown, compared to the negative control (without added peptides), all 16 peptides (PPN-01, PPN-02, PPN-03, PPN-04, PPN-05, PPN-06, PPN-07, PPN-08, PPN-09, PPN-10, PPN-11, PPN-12, PPN-13, PPN-14, PPN-15, and PPN-16) provided in Example 1 inhibited the expression of Nav1.7 in HEK293 cells. Among them, PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16 showed significant inhibitory effects on Nav1.7-overexpressing HEK293 cells.

[0047] Example 3: Selectivity of Peptide PPN-01

[0048] The purpose of this embodiment is to study the selectivity of the polypeptide PPN-01 provided in Example 1 for voltage-gated sodium ion channels Nav1.4, Nav1.5, Nav1.6, and Nav1.7.

[0049] The glass microelectrode prepared in Example 2 was used to study the effect of the peptide PPN-01 provided in Example 1 on the voltage-gated sodium ion channel using whole-cell patch-clamp technique. The specific operation is as follows:

[0050] 1. First, thaw the electrode fluid on ice. After thawing, use a 1mL syringe to draw an appropriate amount of electrode fluid. Attach a homemade injector to the syringe tip and purge the air from the tubing, filling the injector with electrode fluid. Then, use the tail-end injection method to slowly inject the electrode fluid from the tail of the glass microelectrode. The injection volume is generally 1 / 3 to 1 / 2 of the length of the glass microelectrode (ensuring sufficient contact between the silver electrode wire and the electrode fluid while avoiding excessive electrode fluid contamination). After the glass microelectrode is filled with electrode fluid, gently tap the wall of the glass microelectrode to expel air bubbles, ensuring the tip is fully filled with electrode fluid. The resistance of the filled glass microelectrode is 4.0–6.0 MΩ. Note that the force used to tap the air bubbles should not be too great, and the tube wall should be supported with the middle finger of the left hand; otherwise, the glass microelectrode may break.

[0051] 2. The electrostimulation effect of the peptide PPN-01 provided in Example 1 on HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 was studied using the glass microelectrode prepared above and the whole-cell patch-clamp technique (n=5). In voltage clamp mode, the clamping voltage was maintained at -90mV, and a square wave stimulation was applied from -90mV to +40mV for 30ms. The concentration of the peptide was 300nM each time it was administered.

[0052] 3. HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 were added to glass microelectrodes perfused with electrode fluid, respectively. The polypeptide provided in Example 1 was also added to each microelectrode. Under voltage-clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulation was applied, depolarizing from -90 mV to +40 mV for 30 ms. The experimental results showed that the polypeptide PPN-01 provided in Example 1 had a significant inhibitory effect on HEK293 cells overexpressing Nav1.7, but no significant inhibitory effect on HEK293 cells overexpressing Nav1.4, Nav1.5, and Nav1.6. The amino acid sequence provided in Example 1 (SEQ ID) was added as shown in Example 1. The current trajectory after applying the peptide PPN-01 shown in NO:3 to a glass microelectrode overexpressing HEK293 cells containing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 in voltage-clamp mode, followed by square wave stimulation from -90mV to +40mV for 30ms, is shown in the figure. Figure 2 As shown.

[0053] Depend on Figure 2 It can be seen that when HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 (referred to as Nav1.4 group, Nav1.5 group, Nav1.6 group, and Nav1.7 group, respectively) were added to glass microelectrodes perfused with electrode fluid, and the polypeptide PPN-01 with the amino acid sequence shown in Example 1 as SEQ ID NO: 3 was added to each microelectrode, the clamping voltage was maintained at -90mV, and a square wave stimulation was applied from -90mV to +40mV for a duration of 30ms. The current trajectory of the Nav1.7 group showed obvious fluctuations, while the current trajectory fluctuations of the Nav1.4, Nav1.5, and Nav1.6 groups were not obvious.

[0054] The above results demonstrate that the peptide PPN-01 provided in Example 1 can selectively inhibit the activation of the voltage-gated sodium ion channel Nav1.7.

[0055] Example 4: Inhibitory effect of different concentrations of peptide PPN-01 on Nav1.7 channel

[0056] The purpose of this embodiment is to use patch-clamp technique to study the inhibitory effect of different concentrations of peptides (the amino acid sequence of which is shown in SEQ ID NO: 3, hereinafter referred to as "peptide PPN-01" for ease of description) on the Nav1.7 channel.

[0057] In the patch-clamp experiment (refer to Example 2), the measured sodium currents were corrected for leakage current. Raw experimental data were recorded using Clampfit 10.6, and data acquisition was performed using pCLAMP software. Three to five sweeps (a term in patch-clamp software referring to a series of sampling points) before the addition of peptide PPN-01, where the current was stable, were selected for analysis. The average peak current was calculated as the control current amplitude. Then, three to five sweeps after the addition of peptide PPN-01, where the current was stable, were selected for analysis, and the average remaining peak current was calculated as the residual current amplitude. The inhibition rate of peptide PPN-01 on Nav1.7 current was calculated using the following equation: Inhibition rate (%) = [1 - (Residual current amplitude) / (Control current amplitude)] × 100%.

[0058] The dose-response curve was fitted by the Hill equation: I drug / I control =1 / [1+(C / IC)] 50 ) H ];

[0059] Among them, I control I represents the average current amplitude of the control group. drug IC represents the average current amplitude at different drug concentrations C (i.e., different concentrations of peptide-17). 50 The drug concentration (i.e. peptide concentration) required to block 50% of the Nav1.7 channel, where H is the Hill coefficient.

[0060] The inward sodium current curve is obtained by fitting 90% to 10% of the data between the peak and the baseline using a single-exponential or double-exponential equation during the rising and decaying phases.

[0061] Among them, A i and τ i , where are the initial current amplitude and the activation / deactivation time constant, respectively, and C is the time-independent component.

[0062] The activation and deactivation curves of the Nav1.7 channel were both fitted using the Boltzmann equation: G = G max / [1+exp((V 1 / 2 -Vm ) / k)];

[0063] Where G is the sodium conductivity, G max V represents the maximum sodium conductivity. 1 / 2 It is the voltage when half of the channels are activated, V m It is the clamping voltage, and k is the slope factor.

[0064] The sodium conductivity G is calculated using the following formula: G = I / (V) m -Vrev);

[0065] Where G is sodium conductivity, I is current, and V is... m It is the clamping voltage, V rev This is the reversal potential of the sodium current, which is calculated using the Nernst equation. rev It is +66mV.

[0066] In this invention, all statistics were analyzed using one-way ANOVA between two groups, with P < 0.05 considered statistically significant (* indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001). All statistical data were plotted using Graphpad Prism 9.0 software.

[0067] The current was extracted from a 30ms square wave stimulation with a clamp voltage of -90mV depolarized to +40mV. Representative Nav1.7 current trajectory graphs (n=7) recorded before and after administration of peptide PPN-01 (low concentration: 30nM; medium concentration: 100nM; high concentration: 300nM) at different concentrations are shown below. Figure 3 As shown, the control group represents those who did not receive the medication.

[0068] Dose-response curves were plotted based on the percentage inhibition of Nav1.7 peak current by each peptide concentration. Data fitting was performed according to the steps outlined above, and the results are as follows: Figure 4 As shown.

[0069] Depend on Figure 3 and Figure 4 It can be seen that the peptide PPN-01 suppresses the peak current of Nav1.7 using an IC. 50 The value was 52.7 ± 7.8 nM, and the Hill coefficient was 1.18. The above results indicate that there is no intermolecular synergy when the peptide PPN-01 interacts with the Nav1.7 channel.

[0070] Example 5: Voltage dependence of peptide PPN-01 on Nav1.7 channel blocking

[0071] The purpose of this embodiment is to investigate the voltage dependence of peptide PPN-01 (whose amino acid sequence is shown in SEQ ID NO: 3) on Nav1.7 channel blockade using patch-clamp technique.

[0072] In voltage-clamp mode, with a clamping voltage of -90 mV, HEK293 cells overexpressing Nav1.7 were stimulated with a square wave train, depolarized from -90 mV to +80 mV, with a step voltage of 10 mV and a duration of 30 ms, to elicit current trajectories at different voltages. The experimental group included 300 nM PPN-01, and the control group did not include 300 nM PPN-01. The peak amplitude of the Nav1.7 current was plotted against the membrane potential, constructing an IV curve (n = 6, P < 0.05) related to the Nav1.7 current. Simultaneously, a graph showing the inhibition percentage against the membrane potential was also plotted. The results are as follows: Figure 5 As shown, where, Figure 5 Figure A shows the Nav1.7 current trajectory induced by a square wave train stimulus with a duration of 30 ms, which depolarizes from a clamping voltage of -90 mV to +80 mV (step voltage of 10 mV). Figure 5 Figure B shows the IV (current-voltage) relationship curves of the Nav1.7 channel before and after administration of peptide PPN-01. Figure 5 Inner graph C shows the relationship between the percentage of current suppression caused by 300 nM peptide PPN-01 and membrane potential.

[0073] Depend on Figure 5 As shown in inner diagram A, current inhibition was observed at all test potentials that activated the Nav1.7 channel, indicating that the current blocking induced by the peptide PPN-01 occurred across the entire potential activation range; Figure 5 As shown in insets B and C, peptide PPN-01 exhibits voltage-dependent inhibition between -40 mV and +40 mV. The current inhibition rate increases from 43.57% ± 6.95% at -40 mV to 73.57% ± 6.26% at -30 mV (n = 6, P < 0.01), and further from 73.57% ± 6.26% at -30 mV to 89.59% ± 2.46% at +40 mV (n = 6, P < 0.05). These results indicate that peptide PPN-01 has a stronger inhibitory effect on the open Nav1.7 channel.

[0074] Example 6: Effects of different peptides on a mouse acetic acid-induced writhing model

[0075] The purpose of this embodiment is to study the effects of different peptides on a mouse acetic acid writhing model using patch-clamp technology.

[0076] Seventy healthy SPF-grade C57BL / 6 mice (half male and half female), weighing 22–25 g and aged 8–10 weeks, were randomly selected. The 70 C57BL / 6 mice were weighed and numbered from lowest to highest weight (S1 to S70). They were then randomly divided into seven groups using a random number table: a saline group and an experimental group. The experimental group included PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16. Each group received a drug concentration (i.e., the concentration of the added peptides PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16) of 10 mg / kg, with 10 mice in each group.

[0077] Saline group: Ten healthy mice were routinely fed for one week and then injected intraperitoneally with saline at a dose of 0.1 mL / 10 g for 7 consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0078] Experimental group: Ten healthy mice were routinely fed for one week and then injected intraperitoneally once with different polypeptides at a dose of 0.1 mL / 10 g for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0079] During the above experiment, immediately after the injection of glacial acetic acid solution, the time when the mice in each group began to writhe (writhing latency) and the number of writhing movements per mouse within 15 minutes were recorded. A complete writhing response was defined as the appearance of abdominal retraction, trunk and hind limb extension, and raised buttocks. The results are shown in Table 3 and... Figure 6 As shown.

[0080] Table 3 Effects of different peptides on the mouse acetic acid writhing model

[0081]

[0082] From Table 3 and Figure 6 The results showed that the experimental group mice had fewer writhing movements compared to the saline group. This indicates that peptides PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16 all have a certain analgesic effect on the acetic acid writhing model in mice, which in turn significantly reduces the number of writhing movements in mice.

[0083] Example 7: Effects of different concentrations of peptide PPN-01 on a mouse acetic acid-induced writhing model

[0084] The purpose of this embodiment is to investigate the effects of different concentrations of the peptide PPN-01 (whose amino acid sequence is shown in SEQ ID NO: 3) on a mouse acetic acid writhing model using patch-clamp technology.

[0085] Forty SPF-grade healthy C57BL / 6 mice, half male and half female, weighing 22–25 g and aged 8–10 weeks, were randomly selected. The 40 C57BL / 6 mice were weighed and numbered from lowest to highest weight (number 1–40). They were then randomly divided into 5 groups using a random number table: saline group, low-dose group (2.5 mg / kg), medium-dose group (10 mg / kg), high-dose group (40 mg / kg), and diclofenac sodium group (30 mg / kg), with 8 mice in each group.

[0086] Saline group: Eight healthy mice were routinely fed for one week and then injected intraperitoneally with saline at a dose of 0.1 ml / 10 g once for seven consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0087] Low-dose group (2.5 mg / kg): Eight healthy mice were routinely fed for one week and injected intraperitoneally once daily with a 0.5 μM polypeptide PPN-01 solution at a dose of 10 mL / kg. The dosage of the mice was 2.5 mg / kg for 7 consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0088] Medium-dose group (10 mg / kg): Eight healthy mice were routinely fed for one week and injected intraperitoneally once daily with a 2 μM polypeptide PPN-01 solution at a dose of 10 mL / kg for 7 consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0089] High-dose group (40 mg / kg): Eight healthy mice were routinely fed for one week and injected intraperitoneally once daily with 8 μM PPN-01 solution at a dose of 10 mL / kg. The dosage of the mice was 40 mg / kg for 7 consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0090] Diclofenac sodium group (10 mL / kg): Eight healthy mice were routinely fed for one week and were injected intraperitoneally once a day with a 3 mg / mL diclofenac sodium solution at a dose of 10 mL / kg. The dosage of the mice was 30 mg / kg for 7 consecutive days. Fifteen minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was injected intraperitoneally, and the writhing behavior of each mouse was observed.

[0091] During the above experiment, the time when the mice in each group began to writhe (writhing latency) and the number of writhing episodes per mouse within 15 minutes were recorded immediately after the injection of glacial acetic acid solution. A complete writhing response was defined as the appearance of a concave abdomen, extension of the trunk and hind limbs, and raised buttocks. The analgesic effect of the peptide PPN-01 was evaluated by the inhibition rate (%) and the analgesic rate (%).

[0092] Inhibition rate (%) = [(mean latency of the drug treatment group - mean latency of the saline group) / mean latency of the saline group] × 100%

[0093] Analgesia rate (%) = [(Mean number of writhing movements in the saline group - Mean number of writhing movements in the drug treatment group) / Mean number of writhing movements in the saline group] × 100%

[0094] All statistics were analyzed using one-way ANOVA, with P < 0.05 considered statistically significant (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001). All statistical data were plotted using GraphpadPrism 9.0 software.

[0095] The writhing inhibition rates of different concentrations of peptide PPN-01 on mice in each group are shown in Table 4. The number of writhing episodes in each group after injection of different concentrations of peptide PPN-01 are shown in Table 4. Figure 7 As shown.

[0096] Table 4. Results of writhing inhibition rate in each group of mice.

[0097] Group Inhibition rate (%) saline group —— Diclofenac sodium group 77.6 low-dose group 43.5 medium dose group 72.5 High-dose group 75.7

[0098] From Table 4 and Figure 7 It can be seen that after intervention with diclofenac sodium or different concentrations of peptide PPN-01, the writhing response of mice caused by inflammatory pain decreased. With the increase of the dose of peptide PPN-01, the inhibition rate of writhing in mice became higher and higher, reaching a maximum of 75.7%, which was slightly lower than that of the diclofenac sodium group.

[0099] Example 8: Effect of peptide PPN-01 on the electrical signal of the sciatic nerve in rats

[0100] The purpose of this embodiment is to study the effect of peptide PPN-01 (whose amino acid sequence is shown in SEQ ID NO: 3) on the electrical signal of the sciatic nerve in rats.

[0101] Four healthy SPF-grade Sprague-Dawley (SD) rats (half male and half female), weighing 180–200 g and aged 6–8 weeks, were randomly selected. The rats were acclimatized for one week and fasted for 12 hours before the experiment, but allowed free access to water. Rats weighing 180–200 g were anesthetized with 7% chloral hydrate solution at a dose of 0.4 mL / 100 g. Complete anesthesia was indicated when the rats did not exhibit a rolling reflex and showed no response when their tails were squeezed. The fully anesthetized rats were then placed on the operating table with their backs facing upwards and their paws secured with ropes. The skin and connective tissue of the legs were sequentially cut open. The muscles between the biceps femoris and the superficial gluteal muscles were bluntly dissected, and the sciatic nerve was extracted between the two muscles using a glass needle. Two sutures were passed through the two ends of the sciatic nerve to facilitate subsequent experiments.

[0102] Electrodes specifically designed to record nerve impulses are placed on the sciatic nerve. The electrical signals generated by the nerve impulses are converted into digital signals by a multi-channel physiological signal acquisition and processing system. These signals are displayed as compound action potentials (CAPs) on a computer, which are then input into the computer for storage and subsequent analysis. Each experiment is performed alternately between the left and right legs. During the experiment, the sciatic nerve is moistened with saline solution from time to time to avoid prolonged direct exposure of the nerve to air.

[0103] PPN-01 group: The sciatic nerve of one side of the rat was isolated, and a special electrode for recording nerve impulses was placed on the sciatic nerve to record CAP (capillary action). The nerve was stabilized for 5 minutes, during which time it was moistened with 0.9% saline. After 5 minutes, recording began by adding 20 μL of 0.9% saline to the sciatic nerve and recording for 5 minutes. Then, the rat's foot was stimulated with a syringe needle, and recording was repeated for 5 minutes. Next, 20 μL of low-concentration (30 nM), medium-concentration (100 nM), and high-concentration (300 nM) PPN-01 were sequentially added to the sciatic nerve. Before adding the next reagent, the previous reagent was blotted clean with a paper towel. The recording method was the same as with saline. The other sciatic nerve was treated with PPN-01 first, rinsed, and then saline was added as a control. Different rats alternated the order of drug administration and saline.

[0104] Negative control group (lidocaine): The sciatic nerve of one side of the rat was isolated and an electrode specifically designed to record nerve impulses was placed on the sciatic nerve to record CAP. The nerve was stabilized for 5 minutes, during which time the sciatic nerve was moistened with 0.9% saline. After 5 minutes, recording began. 20 μL of 0.9% saline was added to the sciatic nerve and recorded for 5 minutes. Then, the rat's foot was stimulated with a syringe needle and recorded for another 5 minutes. Next, 20 μL of lidocaine was added to the sciatic nerve and recorded for 5 minutes. The rat's foot was stimulated with a syringe needle and recorded for another 5 minutes. The bilateral control group of different rats followed the same procedure.

[0105] Rats in the PPN-01 group and the lidocaine negative control group were observed, and the effect of each concentration of PPN-01 on the peak amplitude of the positive and negative phases of the sciatic nerve CAP was evaluated by the inhibition rate. The inhibition rate was calculated according to the following formula: Inhibition rate (%) = [(peak amplitude of the drug group - peak amplitude of the negative control group) / peak amplitude of the negative control group] × 100%.

[0106] Four to six CAPs within 2 minutes after each recording time period were selected for analysis. The average peak amplitude was calculated as the representative CAP peak amplitude and time course for each group. The peak amplitude of CAP was the difference between the baseline and the peak CAP level.

[0107] The effects of peptide PPN-01 on the electrical signals of the sciatic nerve in rats are as follows: Figure 8 As shown, where, Figure 8 Figure A shows the representative trajectory of the complex action potential of the sciatic nerve in rats before and after acupuncture, before and after lidocaine administration. Figure 8 Figure B shows the representative trajectories of the complex action potential of the sciatic nerve in rats of different concentrations of peptide PPN-01 before and after acupuncture. Figure 8 Inner plot C represents the statistical analysis results comparing the positive phase amplitude of CAP before and after acupuncture in rats with different concentrations of peptide PPN-01 and lidocaine. Figure 8 Inner plot D represents the statistical analysis results of the comparison of the negative phase amplitude of CAP in rats before and after acupuncture in the PPN-01 group and lidocaine group with different concentrations of polypeptide. All data are expressed as mean ± standard deviation (x ± s); "*", "**", and "***" indicate that compared with the saline group before acupuncture, P < 0.05, P < 0.01, and P < 0.001, respectively; "###" indicates that compared with the saline group after acupuncture, P < 0.001; "$" indicates that compared with the lidocaine group after acupuncture, P < 0.05.

[0108] Depend on Figure 8It was found that the peak amplitude of the saline group was the largest before and after acupuncture. After drug intervention, the peak amplitude of each group was reduced to varying degrees. Compared with the negative control group, all concentrations of PPN-01 groups significantly reduced the peak amplitude of CAP before and after acupuncture (P<0.05). Among them, the high-dose PPN-01 group had the greatest inhibition rate on the peak amplitude of both positive and negative phases, while the low-dose PPN-01 group had the smallest inhibition rate. Compared with the positive control drug lidocaine, the high-dose PPN-01 group had the greatest inhibition rate before acupuncture, while the lidocaine group had the greatest inhibition rate after acupuncture. Compared with the high-dose PPN-01 group, the low-dose PPN-01 group and the medium-dose PPN-01 group had smaller inhibition rates before and after acupuncture, but there was no statistically significant difference between the two groups and the high-dose PPN-01 group (P>0.05). The above results indicate that the polypeptide PPN-01 can effectively reduce the amplitude of the positive and negative phase peaks of the sciatic nerve CAP in rats. This effect is similar to that of lidocaine (P>0.05), and the higher the drug dose, the more obvious the effect.

[0109] In summary, the polypeptide provided by this invention can selectively inhibit the activation of voltage-gated Nav1.7 sodium ion channels, thereby suppressing pain. When the polypeptide provided by this invention is applied to the preparation of analgesic drugs, the resulting drugs have good analgesic effects and are of great clinical significance for pain-related diseases.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; The 12th amino acid in the amino acid sequence of the polypeptide is either alanine or glutamic acid. The 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine. The 28th amino acid in the amino acid sequence of the polypeptide is isoleucine; The 29th amino acid in the amino acid sequence of the polypeptide is either isoleucine or tryptophan. The amino acid sequence of the polypeptide is selected from one of SEQ ID NO: 6, 13, 14, 18.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule is a nucleotide sequence encoding the polypeptide as described in claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule as described in claim 2.

4. A recombinant host cell, characterized in that: The recombinant host cell contains the recombinant expression vector as described in claim 3.

5. The use of the polypeptide as described in claim 1 in the preparation of a drug with analgesic function, characterized in that: The drug can inhibit the activation of a voltage-gated sodium ion channel, which is a Nav1.7 channel.

6. The use of the polypeptide as described in claim 5 in the preparation of a drug with analgesic function, characterized in that: The formulation types of the analgesic drugs include at least one of colloidal solution, emulsion or suspension, oral instantaneous film, oral liquid, capsule, injection or transdermal absorption formulation.

7. A pharmaceutical composition, characterized in that: The active ingredient in the pharmaceutical composition contains the polypeptide as described in claim 1.

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