Application of a polypeptide in the preparation of drugs to inhibit inflammatory pain

Abstract

This invention discloses the application of a polypeptide in inhibiting inflammatory pain, belonging to the field of biomedicine. The polypeptide is Mexneurin-3 (Mx-3), one of three Mexneurin polypeptides formed by the cleavage of ProMexneurin, a protein precursor found in the mammalian central nervous system, by proprotein convertase. This invention reveals that Mx-3, as an endogenous polypeptide, has significant analgesic effects, inhibiting both acute and chronic inflammatory pain with fewer side effects and greater safety. This invention also elucidates the analgesic mechanism of Mx-3, finding that it upregulates diacylglycerol (DAG) levels in the spinal cord and activates protein kinase C (PKC). The application of Mx-3 in inhibiting inflammatory pain provided by this invention can be used to develop safer and more effective novel analgesics, providing new ideas and references for the treatment of inflammatory pain.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a polypeptide in the preparation of drugs for inhibiting inflammatory pain. Background Technology

[0002] Pain, often referred to as the fifth vital sign, is one of the most common public health problems worldwide. Inflammatory pain, in particular, is one of the most common and challenging human diseases. Prolonged pain not only causes anxiety and depression but also imposes a significant socioeconomic burden. Inflammation is a biochemical response of the immune and nervous systems to tissue damage and infection, and pain is a symptom of inflammation. If acute inflammatory pain is not promptly relieved, the continued activation of primary afferent fibers can lead to central sensitization, eventually developing into intractable chronic inflammatory pain. Currently, common analgesics have significant side effects. For example, nonsteroidal anti-inflammatory drugs (NSAIDs) can cause nephrotoxicity and gastrointestinal bleeding; tricyclic antidepressants, serotonin reuptake inhibitors (STIs), and GABA analogs have short-lasting analgesic effects; and acetaminophen has hepatotoxicity. Opioids, the most widely used analgesics in clinical practice, can lead to addiction and respiratory depression with long-term use. Current treatment methods do not meet the needs of patients, and there is an urgent need to develop safer and more effective new analgesics.

[0003] Endogenous opioid peptides are naturally occurring in the human body and possess natural anti-inflammatory and analgesic effects. They can not only reduce the production of inflammatory mediators and improve inflammation-related symptoms, but also alleviate pain through multiple pathways. Furthermore, they can promote homeostasis and reduce the long-term effects of inflammation on the body. Developing endogenous opioid peptides as novel analgesics could reduce the side effects of traditional analgesics, such as addiction and respiratory depression, and also lower development costs. Based on these considerations, this patent application has been filed. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects and problems existing in the prior art and to provide an application of a polypeptide in the preparation of drugs for inhibiting inflammatory pain. This polypeptide has a naturally occurring basis in the human body, reduces the side effects of traditional analgesics such as addiction and respiratory depression, and exhibits a significant analgesic effect.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the application of a polypeptide in the preparation of drugs for inhibiting inflammatory pain, wherein the polypeptide is Mexneurin-3 (Mx-3). The polypeptide can be used to treat various types of inflammatory pain.

[0007] Specifically, the polypeptide described herein has an amino acid sequence (see...). Figure 1 )for:

[0008] NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

[0009] Furthermore, in the above applications, the peptide can inhibit acute inflammatory pain. The acute inflammatory pain that the peptide can inhibit includes infectious inflammatory pain, chemical inflammatory pain, or mechanical inflammatory pain, etc.

[0010] Furthermore, in the above applications, the peptide can inhibit chronic inflammatory pain. The chronic inflammatory pain that the peptide can inhibit includes neurogenic inflammatory pain, degenerative inflammatory pain, or metabolic inflammatory pain, etc.

[0011] In this invention, the mechanism of action of the polypeptide in inhibiting inflammatory pain is related to the upregulation of DAG at the spinal cord level and PKC phosphorylation. The polypeptide exerts its analgesic effect by upregulating DAG levels in the spinal cord and activating PKC.

[0012] Furthermore, the present invention can process, modify, or alter the amino acid sequence of the polypeptide.

[0013] The present invention also provides a pharmaceutical composition for treating inflammatory pain, wherein the pharmaceutical composition comprises the aforementioned polypeptide Mexneurin-3.

[0014] Furthermore, the composition can be prepared into injectable, oral, or topical dosage forms. For example, conventional excipients in the art can be added to prepare the corresponding dosage forms for injection, oral administration, or topical application to patients. In other words, a method for treating inflammatory pain is provided.

[0015] ProMexneurin is a protein precursor found in the mammalian central nervous system, discovered during research on endorphin precursor proteins. ProMexneurin mRNA is widely expressed in pain-related regions such as the mouse cortex, brainstem, spinal cord, thalamus, hippocampus, and striatum. ProMexneurin is cleaved by proprotein convertase to form three active polypeptide fragments: Mexneurin-1 (Mx-1), Mexneurin-2 (Mx-2), and Mexneurin-3 (Mx-3). This invention has found that Mx-3 is a G protein-coupled receptor ligand with potential analgesic activity.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1) The polypeptide Mx-3 provided by this invention is an endogenous bioactive peptide that has a natural basis in the human body, reducing the side effects of traditional analgesics, such as addiction and respiratory depression.

[0018] 2) The peptides provided by this invention exhibited significant analgesic effects in both acute and chronic inflammatory pain models. When exerting their inhibitory effect on inflammatory pain, the peptides provided by this invention had no significant impact on the opioid system, cannabinoid system, NMDA / MAPK / NF-κB / CREB pathway, or glial cells.

[0019] 3) The mechanism of action of the polypeptide provided by this invention in inhibiting inflammatory pain is related to the upregulation of DAG at the spinal cord level and PKC phosphorylation. The polypeptide can exert an analgesic effect by upregulating DAG levels in the spinal cord and activating PKC. Attached Figure Description

[0020] Figure 1 The amino acid sequence is Mx-3.

[0021] Figure 2 The effect of intraventricular injection (ICV) of Mexneurin on pain perception in formalin-induced mouse models. (a) Effect of ICV injection of three Mexneurin peptides at the same dose on pain perception in formalin-induced mouse models. (b, c) Effect of ICV injection of different concentrations of Mx-1 or Mx-3 on formalin-induced mouse models 5 min before formalin model establishment, with morphine as a positive control. (d, e) Effect of ICV injection of different concentrations of Mx-1 or Mx-3 on formalin-induced mouse models 5 min after formalin model establishment; 6-12 mice per group. All data are expressed as Mean ± SEM. Statistical differences compared with the control group are expressed as * P < 0.05, ** P < 0.01, *** P < 0.001.

[0022] Figure 3 This study investigated the effect of intraventricular injection (ICV) of Mexneurin on pain perception in an acetate-induced writhing mouse model. Five minutes before establishing the acetate-induced writhing mouse model, different concentrations of Mexneurin were injected via ICV to assess its effect on pain perception in the mice, with morphine serving as a positive control. Each group consisted of 6–8 mice. All data are expressed as mean ± SEM. Statistical differences compared to the control group are expressed as *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4The effect of intraventricular injection (ICV) of Mexneurin on pain perception in a fully Freund's adjuvanted mouse model. The effects of ICV doses of Mx-1, Mx-2, and Mx-3 (1–30 nmol / mouse) at 20 nmol / mouse on mechanical withdrawal threshold (PWT) (A, B) and thermal withdrawal latency (PWL) (C, D) were investigated, with normal saline (NS) serving as the control group. Note: *P<0.05, **P<0.01, ***P<0.001 represent comparisons with the control group (id NS + ICV NS); # P<0.05 ## P<0.01 ### P<0.001 indicates a comparison with the CFA model group (id CFA + icv NS); the upward arrow indicates the time of subcutaneous injection of CFA or NS in the plantar surface; the downward arrow indicates the time of icv Mexneurin or NS; N=4-10.

[0024] Figure 5 To investigate the effect of Mx-3 on G protein-coupled receptor-related kinases, (a) Western blot was used to detect the effects of icv Mx-3 on the total protein levels and protein phosphorylation levels of PKA, PKC, and CAMKII in the spinal cord of formalin-induced mouse models, with β-tubulin as an internal control. (b) Image J was used to perform grayscale analysis on protein bands (p-PKC, p-PKA, and p-CAMKII); six mice were in each group, and all data are expressed as mean ± SEM. Statistical differences compared with the control group are expressed as * P < 0.05, ** P < 0.01, and *** P < 0.001.

[0025] Figure 6 The effect of icv Mx-3 on the levels of IP3, DAG, and GABA in the spinal cord of formalin-induced mouse model mice was detected by ELISA. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] This invention provides the application of a polypeptide in the preparation of drugs for inhibiting inflammatory pain, wherein the polypeptide is Mexneurin-3 (Mx-3), synthesized by Shanghai Jier Biochemical Co., Ltd., and its amino acid sequence is shown in (see...). Figure 1 )for:

[0028] NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

[0029] The following examples provide relevant experiments for three peptides: Mexneurin-1 (Mx-1), Mexneurin-2 (Mx-2), and Mexneurin-3 (Mx-3).

[0030] The innovation of this invention lies in the discovery that peptides have the effect of inhibiting inflammatory pain. In the following examples, the inflammatory pain test involved can be performed using conventional techniques in the art or by referring to the kit instructions, unless detailed steps or operation instructions are given.

[0031] Example 1: Effects of Mexneurin on Acute Inflammatory Pain in Mice

[0032] 1.1 Preparation of experimental reagents

[0033] Remove Mx-1, Mx-2, and Mx-3 from the -20 ℃ freezer. After 5 minutes, use a spatula to take an appropriate amount of the drug and place it into a sterile 1.5 mL centrifuge tube without enzymes. Weigh the drug and dilute it with pre-cooled physiological saline to the required concentration. Place the prepared drug solution on ice immediately for use.

[0034] Table 1 Preparation of experimental reagents

[0035]

[0036] 1.2 Construction of a formalin-induced acute inflammatory pain mouse model

[0037] Before the experiment, the sole of the mouse's right paw was disinfected with a cotton ball soaked in 75% alcohol. 20 μL of 2.5% formalin solution was injected subcutaneously into the sole of the mouse's right paw using a microsyringe. The injection was held for 5 seconds, then the needle was rotated and withdrawn to prevent leakage of the formalin solution. Before the experiment, the mouse was placed in a 500 mL transparent glass beaker 20 cm above the ground for 30 minutes to acclimatize. Five minutes before modeling, 4 μL of peptide solution was administered to the lateral ventricle. After modeling, the mouse was immediately returned to the suspended glass beaker, and the experiment was started. A stopwatch was used to record the total time (in seconds) of licking / biting the paw in the first phase (0-5 min after modeling) and the second phase (15-45 min after modeling). A mirror was placed below the beaker at a 45° angle to the table to observe the mouse's behavior. The experiment was conducted using a double-blind method. The ambient temperature was maintained at 22 ± 1 ℃ during the experiment to prevent low temperatures from affecting the inflammatory response in the second phase.

[0038] 1.3 Construction of the Acetic Acid Writhing Mouse Model

[0039] Before modeling, the injection area on the right side of the mouse abdomen was disinfected with an alcohol swab. 0.3 mL of 1% glacial acetic acid solution was injected into the mouse's peritoneal cavity using a disposable syringe. Successful modeling was indicated by the mouse's ability to perform a complete "writhing movement." 4 μL of the peptide solution was administered into the lateral ventricle 5 minutes before modeling. Immediately after modeling, the mouse was placed in a transparent container (294×185×125 mm), and the number of writhing movements was recorded over the next 30 minutes. The experiment was conducted using a double-blind method. During the experiment, the ambient temperature was maintained at approximately 22±1 °C.

[0040] 1.4 Administration method

[0041] Lateral ventricle injection (ICV): Attach a 1 mL syringe tip to the needle of a microsyringe, ensuring the needle protrudes 3 mm. The injection target is located at the intersection of the anterior fontanelle (0.5 mm horizontally) and the midline of the skull (1.5 mm vertically) to the left or right, with a depth of 3 mm. During injection, keep the needle vertical and advance at a constant speed. The injection volume per mouse is 4 μL. After injection, hold the needle in place for 10 seconds to prevent leakage. After the experiment, confirm the accuracy of the injection site by injecting methylene blue dye into the lateral ventricle. The procedure should be rapid and gentle to minimize human interference with the experimental results.

[0042] Intraperitoneal injection: Hold the mouse still, ensuring its head is tilted downwards to avoid abdominal organs being in the injection area. The injection point is located 1-2 cm from the midline of the mouse's abdomen. Using a sterile syringe, insert the needle into the mouse's abdomen at a 45° angle, tilting the needle downwards. Then, aspirate the syringe; if no blood or urine flows back, the injection can proceed. After injection, hold the needle in place for 5 seconds, then gently withdraw it by rotating the needle to prevent leakage of the injection site.

[0043] 1.5 Data Analysis

[0044] All experimental data are expressed as mean ± standard error (SEM). Data analysis was performed using Graphpad Prism 8.0 software. The t-test was used to compare significant differences between groups, and one-way ANOVA was used to compare significant differences among multiple groups. P < 0.05 was indicated by *, P < 0.01 by **, and P < 0.001 by ***.

[0045] 1.6 Effects of Mexneurin on Pain Sensation in Formalin-Based Mice

[0046] Five minutes before modeling, icv Mx-1, Mx-2, and Mx-3 (20 nmol) were administered to mice to compare their effects. The results are shown in […]. Figure 2 . Figure 2 (a) shows that the licking / biting time was shortened in both the Mx-1 and Mx-3 groups in the first and second phases, while there was no difference in the Mx-2 group. The effects of Mx-1 and Mx-3 at different doses (1 nmol, 10 nmol, 15 nmol and 20 nmol) were further compared, with morphine as a control. Figure 2 (b) and (c) show that at 20 nmol, the licking / biting time of both the first and second phases of Mx-1 was shortened; at 10 nmol, the licking / biting time of the first phase of Mx-3 was significantly shortened; and at 15 nmol and 20 nmol, the licking / biting time of both the first and second phases of Mx-3 was shortened.

[0047] Five minutes after modeling, Mx-1 and Mx-3 were injected via ICV at different doses (1 nmol, 10 nmol, 15 nmol and 20 nmol). Figure 2 (d) and (e) show that Mx-1 showed no difference at the four doses (1 nmol, 10 nmol, 15 nmol and 20 nmol), while Mx-3 showed a shortened licking / biting paw time in the second phase at 10 nmol, 15 nmol and 20 nmol.

[0048] 1.7 Effects of Mexneurin on Pain Sensation in Acetic Acid-Induced Writhing Model Mice

[0049] Five minutes before modeling, different doses (1 nmol, 10 nmol, 15 nmol, and 20 nmol) of Mx-3 were injected via ICV, with morphine as a positive control, to investigate the effects of different doses of Mx-3 on the model mice. The results are shown in […]. Figure 3 . Figure 3 The results showed that Mx-3 significantly reduced the number of writhing movements in mice at doses of 1 nmol, 10 nmol, 15 nmol, and 20 nmol.

[0050] Example 2: Effects of Mexneurin on Chronic Inflammatory Pain in Mice

[0051] 2.1 Establishment of a mouse model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA)

[0052] Before modeling, the mouse paws were rubbed with alcohol swabs, and 20 μL of CFA (provided by Sigma-Aldrich, USA, model: F5881) was injected using a sterile syringe. Modeling was performed using a hot plate test to assess the heat withdrawal latency (PWL) of all mice to evaluate their thermal pain sensitivity. Before testing, each mouse was placed in a hot plate device without heating for 30 min to allow it to acclimatize. It was ensured that both hind paws of the mouse gently contacted the surface of the plate without applying any force, and the hot plate temperature was set at 55 °C with a cutoff time of 30 s. Pain response was defined by the reaction time of the mouse shaking, withdrawing, or licking its paw, and PWL was recorded. Mechanical hyperalgesia was assessed by analyzing the 50% withdrawal response threshold (PWT) of the Von Frey fiber stimulation response. After the mice had acclimatized to the metal mesh for 30 min, the hind paws were stimulated with Von Frey fibers, and PWT was measured using Dixon's up-and-down method. The experiment was conducted in a double-blind manner.

[0053] 2.2 Administration method

[0054] Intradermal injection (id): The injection site is the surface of the left hind paw of the mouse. Before injection, the injection site is disinfected with 75% alcohol. A 25 μL microsyringe is used, and 20 μL of CFA is given to each mouse.

[0055] Intraventricular injection (ICV): Same as Example 1 "1.4".

[0056] 2.3 Data Analysis

[0057] Same as Example 1, "1.5".

[0058] 2.4 Effects of Mexneurin on Pain Sensation in Full Freund's Adjuvant Model Mice

[0059] like Figure 4 As shown in Figure A, compared with the blank control group (id NS + icv NS), the PWT of the CFA model group (idCFA + icv NS) decreased significantly at 0, 15, 30, and 45 min, indicating successful modeling. Compared with the CFA model group, Mx-3 (20 nmol / animal) significantly increased PWT at 15 min; Mx-1 (20 nmol / animal) and Mx-2 (20 nmol / animal) had no effect on PWT at 15, 30, and 45 min. Figure 4B shows the effect of icv Mx-3 (1-30 nmol / mouse) on PWT in CFA model mice. Compared with the CFA model group, Mx-3 (20 nmol / mouse) significantly increased PWT at 15 min; Mx-3 (30 nmol / mouse) significantly increased PWT at both 15 and 30 min; Mx-3 (1, 10 nmol / mouse) had no effect on PWT. The results indicate that Mx-3 (20, 30 nmol / mouse) can increase the mechanical pain threshold in CFA model mice, i.e., exert an analgesic effect.

[0060] like Figure 4 As shown in Figure C, the PWL of the CFA model group (id CFA + icv NS) decreased significantly at 0, 15, 30, and 45 min, indicating successful modeling. Mx-3 (20 nmol / animal) significantly improved PWL at 15 and 30 min; Mx-1 (20 nmol / animal) and Mx-2 (20 nmol / animal) had no effect on PWL. Figure 4 As shown in Figure D, compared with the CFA model group, Mx-3 (1 and 10 nmol / mouse) had no effect on PWL, while Mx-3 (20 nmol / mouse) significantly increased PWL at 15 and 30 min; Mx-3 (30 nmol / mouse) significantly increased PWL at 15, 30, and 45 min. These results indicate that Mx-3 (20 and 30 nmol / mouse) can increase the thermal pain threshold in CFA model mice, thus exerting an analgesic effect.

[0061] Example 3: Study on the analgesic mechanism of Mexneurin-3

[0062] 3.1 Preparation of experimental reagents

[0063] Same as Example 1 "1.1".

[0064] 3.2 Construction of a formalin-induced acute inflammatory pain mouse model

[0065] Same as Example 1 "1.2".

[0066] 3.3 Administration method

[0067] Same as Example 1, "1.4".

[0068] 3.4 Obtaining mouse spinal cord tissue

[0069] Before the experiment, instruments were sterilized at high temperature, and all sample collection was performed on ice, with rapid and gentle movements. Five minutes before modeling, mice were administered 20 nmol Mx-3 via ICV, and euthanized 30 minutes later. The spinal cord was flushed with a 200 μL pipette tip. The spinal cord was first removed and placed in PBS, then 3 mL of PBS was drawn up using a syringe and flushed out of the spinal canal from the neck. The L4-L6 lumbar enlargements were collected, placed in cryovials, and flash-frozen in liquid nitrogen. The final samples were stored at -80 °C.

[0070] 3.5 Protein Immunoblotting

[0071] (1) Extraction of total protein from tissues

[0072] Proteins are easily degraded, so the entire process should be carried out on ice.

[0073] (a) Remove the spinal cord tissue from the -80 ℃ freezer into a 1.5 mL centrifuge tube, add pre-cooled tissue protein lysis buffer, and mix according to the ratio of RIPA:protease inhibitor:phosphatase inhibitor = 100:1:1, and add 2 grinding beads. Grind in a -10 ℃ cryogenic grinder until the tissue fragments disappear.

[0074] (b) After removing the grinding beads, pass the sample through liquid nitrogen. Place the sample on ice, thaw it, and then lyse it on ice for 30 min.

[0075] (c) Centrifuge at 12,000 rpm for 10 min at 4 °C, transfer the supernatant to a new tube, and store at -80 °C if not to be used immediately.

[0076] (2) Determination of total protein concentration by BCA method

[0077] (a) Preparation of standard protein solution.

[0078] Table 2. Required system for preparing standard protein solution

[0079]

[0080] Based on the required amount of BSA standard, dilute the 2 mg / mL BSA stock solution to a 0.5 mg / mL working solution, and prepare 8 standard protein solutions of different concentrations according to the table above.

[0081] (b) Calculate the required amounts of the test protein and sample diluent (PBS), and dilute the test sample accordingly. For example, for a 5-fold dilution, the test sample is: 4 μL test sample + 16 μL PBS; the sample control is: 4 μL RIPA + 16 μL PBS.

[0082] (c) Calculate the required amount of BCA working solution, prepare it according to the ratio of solution A to solution B = 50:1, and store it away from light.

[0083] (d) Determination of tissue protein concentration using the microplate method. Standard protein solutions of different concentrations (20 μL), test protein solutions (20 μL), and sample controls (20 μL) were added sequentially to a 96-well plate, with each sample requiring replicates.

[0084] (e) Add 200 μL of BCA working solution to each well, seal the microplate, and incubate at 37 °C for 30 min in the dark.

[0085] (f) After removing the 96-well plate and allowing it to return to room temperature, measure the absorbance (OD value) of the sample at a wavelength of 526 nm using an ELISA reader.

[0086] (g) Plot a standard protein curve with standard protein concentration and OD value on the x and y axes, and calculate the concentration of the sample to be tested based on the standard protein curve.

[0087] (3) Protein denaturation

[0088] Dilute the protein to a uniform concentration with PBS, add 5 × loading buffer, then heat in a 100 °C metal bath for 10 min, and store at -20 °C after cooling to room temperature.

[0089] (4) Protein electrophoresis

[0090] (a) Clean the gel casting plate and electrophoresis comb with a thickness of 1.5 mm, and then rinse 3 times with ddH2O.

[0091] (b) Leak test. Place the glue-making plate on the glue-making frame, fill the glue-making plate with ddH2O, and let it stand for 30 minutes.

[0092] (c) Pour ddH2O out of the gel plate, absorb the moisture with filter paper, let stand for 10 minutes, and let it dry.

[0093] (d) Prepare two 1.5 mm lower gels. Bring the kit to room temperature. Then, take a 50 mL centrifuge tube and mix 7 mL of gel solution, 7 mL of buffer solution, and 140 μL of coagulant. Pour the mixture into the gel plate to 2 / 3 full, level it, and let it solidify at room temperature for 30 min.

[0094] (e) Pour ddH2O out of the gel plate and blot dry with filter paper.

[0095] (f) Prepare two 1.5 mm upper gels. Bring the kit to room temperature, then take a 50 mL centrifuge tube and mix 2.5 mL of gel solution + 2.5 mL of buffer + 50 μL of coagulant. Pour the mixed upper gel solution onto the already solidified lower gel, then immediately insert the electrophoresis comb to ensure no air bubbles are generated. Let it solidify at room temperature for 30 minutes.

[0096] (g) Place the gel casting frame into the electrophoresis tank, fill it with electrophoresis solution, and remove the electrophoresis comb.

[0097] (h) Sample loading. The denatured protein sample was taken out from -20 °C, thawed on ice, centrifuged, and then 10 μL of protein sample was added sequentially. 1 μL and 3 μL of marker were added to both sides of the protein sample, respectively.

[0098] (i) Electrophoresis. Connect the electrophoresis tank lid to the electrodes, run the stacking gel at 80 V for 30 min, run the separating gel at 120 V for 1 h, and stop electrophoresis when the bromophenol blue reaches the bottom.

[0099] (j) Transfer. Cut the PVDF membrane and activate it with methanol. Prepare the transfer solution, soaking the sponge and filter paper. Place the gel block into the solution and remove the top layer of gel. Assemble the "sandwich" transfer structure, place it in the tank, ensuring that the black side of the transfer clamp faces the black side of the transfer tank and the white side faces the red side of the transfer tank. Add the transfer solution and connect the electrodes. The transfer time depends on the molecular weight; perform a constant current of 300 mA for transfer.

[0100] (k) Blocking. Remove the PVDF membrane and place it in an incubator with 5% skim milk, back to back. Block at room temperature with gentle shaking for 1 hour.

[0101] (l) Primary antibody incubation. Place the PVDF membrane containing the target protein into an incubation bag, add the antibody, seal the bag with a sealing machine, fix the incubation bag on a 3D shaker, and incubate overnight at 4 °C.

[0102] (m) Antibody recovery. The next day, the incubation bag was cut open to recover the antibody. The PVDF membrane was placed in an incubation box containing TBST washing buffer and washed on a shaker at room temperature for 10 min per wash, for a total of 3 washes.

[0103] (n) Secondary antibody incubation. After washing the membrane, discard TBST, add secondary antibody of the same species as the primary antibody, incubate on a slow shaker at room temperature for 1 hour, and then wash the membrane 3 times at a frequency of 10 min / time at room temperature.

[0104] (o) Development. Prepare the luminescent solution (A:B=1:1), taking care to avoid light. After blotting off the TBST on the PVDF film, place the exposure plate and drop the luminescent solution on it, remove the excess solution, and place it in the developing apparatus for exposure.

[0105] 3.6 Enzyme-linked immunosorbent assay

[0106] (1) Sample preparation. The extraction of tissue protein and the determination of total tissue protein concentration by BCA method are the same as above.

[0107] (2) Dilution of the standard. Following the concentration guidelines provided in the instructions, dilute the standard to six different concentrations using the standard diluent.

[0108] (3) Sample dilution. Dilute serum and tissue proteins to an appropriate factor using sample diluent.

[0109] (4) Sample loading. Add standard solutions of different concentrations (replicas required) and sample solutions sequentially to the microplate, 50 μL per well. Also set up sample blank wells and standard blank wells (replicas required). After loading, incubate the microplate in a 37 ℃ incubator for 30 min.

[0110] (5) Washing the plate. Calculate the required amount of washing solution and dilute the 30× concentrated washing solution to 1× washing solution. Add 300 μL to each well, let stand for 30 seconds, then discard the solution and pat dry on filter paper. Repeat five times.

[0111] (6) Add enzyme-labeled reagent. Add 50 μL of enzyme-labeled reagent to each well, except for the sample blank well and the standard blank well. Then put the plate back into the 37 ℃ incubator and continue to incubate for 30 min.

[0112] (7) Wash the plate.

[0113] (8) Add chromogenic solution. Calculate and prepare the required amount of chromogenic solution. Mix solution A and solution B in a 1:1 ratio, and add 100 μL of the mixed chromogenic solution to each well. Place the microplate in a 37 ℃ incubator in the dark for 20 min. Adjust the reaction time according to the color change.

[0114] (9) Add stop solution. Add 50 μL of stop solution to each well to terminate the reaction.

[0115] (10) Measure the OD value with an ELISA reader. Within 15 minutes after adding the stop solution, measure the absorbance (OD value) of the sample at a wavelength of 450 nm using an ELISA reader.

[0116] (11) Prepare a standard protein curve. Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis, and calculate the concentration of the protein to be tested based on the curve.

[0117] 3.7 Data Analysis

[0118] Same as Example 1, "1.5".

[0119] 3.8 Effects of Mexneurin-3 on G protein-coupled receptor-related kinases

[0120] G protein-coupled receptors are key targets for analgesics. Western blot experiments showed that icv 20 nmolMx-3 was administered to formalin-induced mice to investigate whether its analgesic effect was related to downstream kinases of G protein-coupled receptors, such as PKA, PKC, and CAMKII. Figure 5The results showed that the phosphorylation level of PKC in the spinal cord of mice in the Mx-3 group was significantly increased, while the phosphorylation levels of PKA and CAMKII remained unchanged, suggesting that the analgesic effect of Mx-3 may be related to PKC phosphorylation.

[0121] 3.9 Effect of Mexneurin-3 on DAG content

[0122] The ELISA assay was used to detect changes in the levels of related second messengers (IP3 and DAG) and GABA in the 5-HT2A / PLC / PKC / KCC2 / GABAA pathway. Figure 6 As shown, the DAG content in the spinal cord of mice in the Mx-3 group was increased.

[0123] In summary, Mx-3, as an endogenous polypeptide, exhibits significant analgesic effects, inhibiting both acute and chronic inflammatory pain with fewer side effects and greater safety. This invention also elucidates the analgesic mechanism of Mx-3, discovering its upregulation of diacylglycerol (DAG) levels in the spinal cord and activation of protein kinase C (PKC). This invention provides the application of Mx-3 in inhibiting inflammatory pain, which can be used to develop safer and more effective novel analgesics, offering new ideas and references for the treatment of inflammatory pain.

[0124] Matters not covered in this invention are common knowledge.

[0125] The above embodiments are merely illustrative of the features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of a polypeptide in the preparation of drugs for inhibiting inflammatory pain, characterized in that, The polypeptide is Mexneurin-3; Its amino acid sequence is: NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

2. The application according to claim 1, characterized in that, The polypeptide can inhibit acute inflammatory pain.

3. The application according to claim 1, characterized in that, The polypeptide can inhibit chronic inflammatory pain.

4. The application according to claim 1, characterized in that, The polypeptide can exert analgesic effects by upregulating DAG levels in the spinal cord and activating PKC.

5. A pharmaceutical composition for treating inflammatory pain, characterized in that, The pharmaceutical composition comprises the polypeptide Mexneurin-3 as described in any one of claims 1-4.

6. The pharmaceutical composition according to claim 5, characterized in that, The composition is prepared into an injectable, oral, or topical dosage form.

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