Application of polypeptide in aspect of preparing medicine for inhibiting inflammatory pain

Abstract

The invention discloses an application of a polypeptide in the aspect of inhibiting inflammatory pain, and belongs to the field of biological medicines, the polypeptide is Mexneurin-3 (Mx-3), and the Mx-3 is one of three Mexneurin polypeptides formed after a protein precursor ProMexneurin existing in a central nervous system of a mammal is cut by a proprotein convertase. Research finds that Mx-3 serving as endogenous polypeptide has a remarkable analgesic effect, can inhibit acute inflammatory pain and chronic inflammatory pain, and is small in side effect and safer. The invention also discloses an analgesic action mechanism of the Mx-3, and discovers that the Mx-3 up-regulates the diacylglycerol (DAG) level in spinal cord and activates protein kinase C (PKC). The application of Mx-3 in the aspect of inhibiting inflammatory pain can be used for developing safer and more effective novel analgesic drugs, and a new idea and reference are provided for treating inflammatory pain.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a polypeptide in the preparation of a drug for inhibiting inflammatory pain. Background Art

[0002] Pain is known as the fifth vital sign and is the most common public health problem in the world. Among them, inflammatory pain is one of the most common and difficult human diseases. Long-term pain not only causes anxiety and depression, but also creates a huge socioeconomic burden. Inflammation is a biochemical response of the immune system and nervous system to tissue damage and infection, and pain is a symptom of inflammation. If acute inflammatory pain cannot be eliminated in time, the continuous activation of primary afferent fibers will lead to central sensitization, which will then develop into intractable chronic inflammatory pain. At present, common analgesics have obvious side effects. For example, non-steroidal analgesics can cause nephrotoxicity and gastrointestinal bleeding, tricyclic antidepressants, serotonin reuptake inhibitors and GABA analogs have a short analgesic time, and acetaminophen is hepatotoxic. Opioids are the most widely used analgesics in clinical practice. Long-term use can lead to addiction, respiratory depression, etc. The current treatment methods cannot meet the needs of patients, and there is an urgent need to develop new, safer and more effective analgesics.

[0003] Endogenous opioid peptides exist naturally in the human body and have natural anti-inflammatory and analgesic effects. They can not only reduce the production of inflammatory mediators and improve inflammation-related symptoms, but also relieve pain through various pathways. At the same time, they can promote the stability of the internal environment and reduce the long-term effects of inflammation on the body. If endogenous opioid peptides are developed as new analgesics, on the one hand, the side effects of traditional analgesics, such as addiction and respiratory depression, can be reduced, and on the other hand, the development cost can be reduced. Based on this, this patent application is proposed. Summary of the invention

[0004] The present invention aims to overcome the defects and problems of the above-mentioned prior art and provides an application of a polypeptide in the preparation of a drug for inhibiting inflammatory pain. The polypeptide has a natural basis in the human body, reduces the side effects of traditional analgesics, such as addiction, respiratory depression, etc., and exhibits a significant analgesic effect.

[0005] To achieve the above object, the present invention adopts the following technical solution: The present invention provides an application of a polypeptide in the preparation of a drug for inhibiting inflammatory pain, wherein the polypeptide is Mexneurin-3 (Mx-3). The polypeptide can be used to treat various types of inflammatory pain.

[0006] Specifically, the polypeptide, its amino acid sequence (see Figure 1 )for: NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

[0007] Furthermore, in the above application, the polypeptide can inhibit acute inflammatory pain. The acute inflammatory pain that the polypeptide can inhibit includes infectious inflammatory pain, chemical inflammatory pain or mechanical inflammatory pain.

[0008] Furthermore, in the above application, the polypeptide can inhibit chronic inflammatory pain. The chronic inflammatory pain that the polypeptide can inhibit includes neuropathic inflammatory pain, degenerative inflammatory pain or metabolic inflammatory pain.

[0009] In the present invention, the mechanism of action of the polypeptide in inhibiting inflammatory pain is related to the upregulation of DAG and PKC phosphorylation at the spinal cord level. The polypeptide can exert an analgesic effect by upregulating the DAG level in the spinal cord and activating PKC.

[0010] Furthermore, the present invention can process, modify or transform the amino acid sequence of the polypeptide.

[0011] The present invention also provides a pharmaceutical composition for treating inflammatory pain, wherein the pharmaceutical composition comprises the polypeptide Mexneurin-3.

[0012] Furthermore, the composition can be prepared into an injection, oral or external dosage form, etc. For example, conventional adjuvants in the art can be added to prepare a corresponding dosage form, and then injected, orally or externally administered to the patient, etc. That is, a method for treating inflammatory pain is provided.

[0013] ProMexneurin is a protein precursor found in the central nervous system of mammals when people were exploring endorphin precursor proteins. ProMexneurin mRNA is widely expressed in pain-related areas 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). The present invention found that Mx-3 is a G protein-coupled receptor ligand with potential analgesic effects.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1) The polypeptide Mx-3 provided by the present invention is an endogenous bioactive peptide that exists naturally in the human body, reducing the side effects of traditional analgesics, such as addiction and respiratory depression.

[0015] 2) The polypeptide provided by the present invention exhibits significant analgesic effects in both acute inflammatory pain models and chronic inflammatory pain models. When the polypeptide provided by the present invention exerts its effect of inhibiting inflammatory pain, it has no significant effect on the opioid system, cannabinoid system, NMDA / MAPK / NF-κB / CREB pathway and glial cells.

[0016] 3) The mechanism of action of the polypeptide provided by the present invention in inhibiting inflammatory pain is related to the upregulation of DAG and PKC phosphorylation at the spinal cord level. The polypeptide can exert analgesic effects by upregulating DAG levels in the spinal cord and activating PKC. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the amino acid sequence of Mx-3.

[0018] Figure 2 The effect of intracerebroventricular (icv) injection of Mexneurin on pain perception in formalin model mice. (a) Effects of icv injection of three Mexneurin peptides at the same dose on pain perception in formalin model mice. (b, c) Effects of icv injection of different concentrations of Mx-1 or Mx-3 on formalin model mice 5 min before formalin model establishment, with morphine as a positive control. (d, e) Effects of icv injection of different concentrations of Mx-1 or Mx-3 on formalin model mice 5 min after formalin model establishment; 6-12 mice in each group, all data are expressed as Mean ± SEM. Statistical differences compared with the control group are indicated by * P<0.05, ** P<0.01, and *** P<0.001.

[0019] Figure 3 The effect of intracerebroventricular injection (icv) of Mexneurin on pain perception in mice with acetic acid writhing model. The effect of icv injection of different concentrations of Mx-3 on acetic acid writhing model mice 5 min before the establishment of acetic acid writhing model, with morphine as positive control; 6-8 mice in each group, all data are expressed as Mean ± SEM. Statistical differences compared with the control group are represented by * P < 0.05, ** P < 0.01, *** P < 0.001.

[0020] Figure 4The effect of intracerebroventricular (icv) injection of Mexneurin on pain perception in mice with complete Freund's adjuvant model. The effect of icv injection of 20 nmol / mouse Mx-1, Mx-2, Mx-3 and Mx-3 (1-30 nmol / mouse) on mechanical paw withdrawal threshold (PWT) (A, B) and thermal paw withdrawal latency (PWL) (C, D) of mice, with normal saline (NS) as the control group. Note: *P<0.05, **P<0.01, ***P<0.001, compared with the control group (id NS + icv NS); # P<0.05, ## P<0.01, ### P<0.001, compared with the CFA model group (id CFA + icv NS); the upward arrow indicates the time of subcutaneous injection of CFA or NS; the downward arrow indicates the time of icv Mexneurin or NS; N=4-10.

[0021] Figure 5 Effects 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 model mice, with β-tubulin as an internal reference. (b) Image J was used to perform grayscale analysis of protein bands (p-PKC, p-PKA and p-CAMKII); 6 mice in each group, all data are expressed as Mean ± SEM. Statistical differences compared with the control group are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001.

[0022] Figure 6 ELISA was used to detect the effect of icv Mx-3 on the contents of IP3, DAG and GABA in the spinal cord of formalin model mice. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] The present invention provides an application of a polypeptide in the preparation of a drug for inhibiting inflammatory pain, wherein the polypeptide is Mexneurin-3 (Mx-3), which is synthesized by Shanghai Jier Biochemical Co., Ltd., and its amino acid sequence (see Figure 1 )for: NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

[0025] In the following examples, related experiments on three polypeptides, Mexneurin-1 (Mx-1), Mexneurin-2 (Mx-2) and Mexneurin-3 (Mx-3), are given.

[0026] The innovation of the present invention is that the polypeptide has the effect of inhibiting inflammatory pain. In the following embodiments, if there are no detailed steps or operating instructions for the inflammatory pain test involved, it can be carried out using conventional techniques in the art or referring to the kit instructions.

[0027] Example 1: Effect of Mexneurin on Acute Inflammatory Pain in Mice 1.1 Preparation of experimental drugs Take out Mx-1, Mx-2, and Mx-3 from the -20 ℃ refrigerator. After 5 minutes, use a medicine spoon to take an appropriate amount of medicine and put it into a sterile, enzyme-free 1.5 mL centrifuge tube. After weighing the medicine, dilute it with pre-cooled physiological saline to the required concentration. Put the prepared medicine solution on ice immediately for use.

[0028] Table 1 Preparation of experimental drugs 1.2 Construction of formalin acute inflammatory pain mouse model Before the experiment, the sole of the right foot of the mouse was disinfected with a cotton ball containing 75% alcohol. A microsyringe was used to inject 20 μL of 2.5% formalin solution into the subcutaneous part of the sole of the right foot of the mouse. Keep it for 5 s, then rotate the needle and pull it out to prevent the formalin solution from leaking out from the injection point. Before the experiment, the mouse should be placed in a 500 mL transparent glass beaker 20 cm above the ground for 30 minutes. 5 minutes before modeling, 4 μL of peptide solution was given to the lateral ventricle. After the modeling was completed, the mouse was immediately placed back in the hanging glass beaker, the experiment was started, and the total time (s) of the mouse licking / biting its paw in the first phase (0-5 min after modeling) and the second phase (15-45 min after modeling) was recorded with a stopwatch. A mirror was placed at a 45° angle under the beaker and at the tabletop to observe the behavior of the mouse. The experiment was conducted in a double-blind manner. During the experiment, the ambient temperature was maintained at 22±1 ℃ to prevent low temperature from affecting the inflammatory response in the second phase.

[0029] 1.3 Construction of acetic acid writhing mouse model Before modeling, the injection area on the right side of the mouse's abdomen was disinfected with an alcohol cotton ball. 0.3 mL of 1% glacial acetic acid solution was injected into the mouse's peritoneal cavity with a disposable syringe. The sign of successful modeling is that the mouse can perform a complete "twisting movement". 4 μL of peptide solution was given to the lateral ventricle 5 minutes before modeling. After modeling, the mouse was immediately placed in a transparent container (294×185×125 mm) and the number of twists of the mouse was recorded in the next 30 minutes. The experiment was conducted in a double-blind manner. During the experiment, the ambient temperature should be maintained at approximately 22±1 °C.

[0030] 1.4 Administration Intracerebroventricular injection (icv): Install a 1 mL gun tip onto the needle part of the microsyringe, ensuring that the needle is exposed 3 mm. The injection target point is located at the intersection of 0.5 mm behind the mouse's anterior bregma (horizontally) and 1.5 mm to the left or right of the midline of the skull (vertically), and the injection depth is 3 mm. During the injection process, the needle should be kept vertical and pushed forward at a constant speed. The injection volume for each mouse is 4 μL. After the injection, keep the needle in place for 10 s to prevent leakage of the drug solution. After the experiment is completed, confirm whether the injection position is accurate by injecting methylene blue dye into the lateral ventricle. The operation should be quick and gentle to reduce human interference with the experimental results.

[0031] Intraperitoneal injection: The mouse is fixed by hand, making sure that its head is tilted downward to avoid abdominal organs in the injection area. The injection point is located 1-2 cm from the midline of the mouse's abdomen. Use a sterile syringe to pierce the mouse's abdomen at a 45° angle with the needle tilted downward. Then, withdraw the needle, and if there is no blood or urine reflux, the injection can be performed. After the injection, keep the needle in place for 5 seconds and then rotate the needle and gently pull it out to prevent the injection solution from leaking out from the injection point.

[0032] 1.5 Data Analysis All experimental data were expressed as mean ± standard error (SEM), and data were analyzed using Graphpad Prism 8.0 software. The t-test was used to compare the significant differences between groups, and one-way ANOVA was used to compare the significant differences between multiple groups. P < 0.05 was indicated by *, P < 0.01 was indicated by **, and P < 0.001 was indicated by ***.

[0033] 1.6 Effect of Mexneurin on Pain Sensation in Formalin Model Mice 5 min before modeling, icv Mx-1, Mx-2, and Mx-3 (20 nmol) were administered to mice, and the effects on mice were compared. The results are shown in Figure 2 . Figure 2(a) shows that the licking / biting time in the first and second phases of the Mx-1 and Mx-3 groups were shortened, 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 the control. Figure 2 (b) and (c) show that when Mx-1 was 20 nmol, the licking / biting time of the first and second phases were shortened; when Mx-3 was 10 nmol, the licking / biting time of the first phase was significantly shortened; when Mx-3 was 15 nmol and 20 nmol, the licking / biting time of the first and second phases were shortened.

[0034] Five minutes after modeling, different doses (1 nmol, 10 nmol, 15 nmol, and 20 nmol) of Mx-1 and Mx-3 were injected icv. Figure 2 (d) and (e) show that there was no difference in the four doses of Mx-1 (1 nmol, 10 nmol, 15 nmol and 20 nmol), while the licking / biting time of the second phase was shortened at 10 nmol, 15 nmol and 20 nmol of Mx-3.

[0035] 1.7 Effect of Mexneurin on Pain Sensation in Mice with Acetic Acid Writhing Model 5 min before modeling, different doses (1 nmol, 10 nmol, 15 nmol and 20 nmol) of Mx-3 were injected icv, and morphine was used as a positive control to explore the effects of Mx-3 at different doses on model mice. The results are shown in Figure 3 . Figure 3 The results showed that Mx-3 at doses of 1 nmol, 10 nmol, 15 nmol and 20 nmol significantly reduced the number of writhing times in mice.

[0036] Example 2: Effect of Mexneurin on Chronic Inflammatory Pain in Mice 2.1 Establishment of the complete Freund’s adjuvant (CFA) chronic inflammatory pain mouse model Before modeling, the plantar of the mouse was wiped with alcohol cotton balls, and 20 μL of CFA (provided by Sigma-Aldrich, USA, model: F5881) was injected with a sterile syringe. Modeling used a hot plate to test the thermal withdrawal latency (PWL) of all mice to evaluate the thermal pain sensitivity of mice. Before the test, each mouse was placed in a hot plate device without heating for 30 min to adapt to it. It should be ensured that the two hind paws of the mouse gently touch the surface of the plate without applying any force, and the temperature of the hot plate was set at 55 ℃ with a cut-off time of 30 s. The pain response was defined by the reaction time of the mouse shaking, retracting or licking the paw, and the PWL was recorded. Mechanical hyperalgesia was evaluated by analyzing the 50% withdrawal threshold (PWT) of the Von Frey fiber stimulation response. After the mouse adapted to the metal mesh for 30 min, the Von Frey fiber stimulated the hind paw, and the PWT was measured by Dixon's up-and-down method. The experiment was conducted in a double-blind manner.

[0037] 2.2 Administration Intradermal injection (id): The injection site was the plantar surface of the left hind paw of the mouse. The injection site was disinfected with 75% alcohol before injection. A 25 μL microsyringe was used and the volume of CFA was 20 μL for each mouse.

[0038] Intracerebroventricular injection (icv): same as Example 1 "1.4".

[0039] 2.3 Data Analysis Same as Example 1 “1.5”.

[0040] 2.4 Effect of Mexneurin on Pain Sensation in Complete Freund's Adjuvant Model Mice like Figure 4 As shown in 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 that the modeling was successful. Compared with the CFA model group, Mx-3 (20 nmol / mouse) significantly increased PWT at 15 min; Mx-1 (20 nmol / mouse) and Mx-2 (20 nmol / mouse) had no effect on PWT at 15, 30, and 45 min. Figure 4B shows the effect of icv Mx-3 (1-30nmol / mouse) on PWT of 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 15 and 30 min; Mx-3 (1, 10 nmol / mouse) had no effect on PWT. The results showed that Mx-3 (20, 30 nmol / mouse) can increase the mechanical pain threshold of CFA model mice, that is, play an analgesic effect.

[0041] like Figure 4 As shown in C, the PWL of the CFA model group (id CFA + icv NS) decreased significantly at 0, 15, 30, and 45 min, indicating that the modeling was successful. Mx-3 (20 nmol / mouse) significantly increased PWL at 15 and 30 min; Mx-1 (20 nmol / mouse) and Mx-2 (20 nmol / mouse) had no effect on PWL. Figure 4 As shown in D, compared with the CFA model group, Mx-3 (1, 10 nmol / mouse) had no effect on PWL, 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. The results showed that Mx-3 (20, 30 nmol / mouse) could increase the thermal pain threshold of CFA model mice, that is, play an analgesic role.

[0042] Example 3: Study on the analgesic mechanism of Mexneurin-3 3.1 Preparation of experimental drugs Same as Example 1 “1.1”.

[0043] 3.2 Construction of formalin acute inflammatory pain mouse model Same as Example 1 “1.2”.

[0044] 3.3 Administration Same as Example 1 “1.4”.

[0045] 3.4 Obtaining mouse spinal cord tissue Before the experiment, the instruments were sterilized at high temperature, and the whole process of sampling was carried out on ice, with quick and gentle movements. 5 min before modeling, icv20 nmol Mx-3, and the mice were killed 30 min later. The spinal cord was flushed with a 200 μL pipette tip. The spinal cord was first removed and placed in PBS, and then 3 mL of PBS was aspirated with a syringe to flush the spinal cord out of the neck from the caudal vertebral canal of the mouse. The L4-L6 lumbar enlargement was taken, stored in a cryopreservation tube and quickly frozen in liquid nitrogen, and the final sample was stored in a -80 ℃ refrigerator.

[0046] 3.5 Western Blot (1) Extraction of total protein from tissues Proteins are easily degraded and should be operated on ice throughout the process.

[0047] (a) Take out the spinal cord tissue from the -80 ℃ refrigerator and put it into a 1.5 mL centrifuge tube. Add pre-cooled tissue protein lysis buffer, mix according to the ratio of RIPA: protease inhibitor: phosphatase inhibitor = 100:1:1, and add 2 grinding beads. Grind in a -10 ℃ cryo-grinder until tissue fragments disappear.

[0048] (b) After removing the grinding beads, the samples were lysed on ice for 30 min after thawing.

[0049] (c) Centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new tube or store at -80°C if not used immediately.

[0050] (2) BCA method for total protein concentration (a) Preparation of standard protein solution.

[0051] Table 2 System required for preparation of standard protein solution According to the required amount of BSA standard, dilute the 2 mg / mL BSA stock solution to 0.5 mg / mL working solution, and prepare 8 concentrations of standard protein solutions according to the table above.

[0052] (b) Calculate the amount of protein to be tested and sample diluent (PBS) required to dilute the sample to be tested. Taking 5-fold dilution as an example, the sample to be tested is: 4 μL of the sample to be tested + 16 μL of PBS; the sample control is: 4 μL of RIPA + 16 μL of PBS.

[0053] (c) Calculate the required volume of BCA working solution, prepare it according to the ratio of solution A: solution B = 50:1, and store it away from light.

[0054] (d) Microplate method for measuring tissue protein concentration: Standard protein solutions (20 μL), test protein solutions (20 μL) and sample controls (20 μL) of different concentrations were added to a 96-well plate in sequence, and each sample was plated in duplicate.

[0055] (e) Add 200 μL of BCA working solution to each well, seal the microplate, and incubate at 37 °C for 30 min in a dark incubator.

[0056] (f) After the 96-well plate is taken out and returned to room temperature, the absorbance (OD value) of the samples is measured at a wavelength of 526 nm using an ELISA reader.

[0057] (g) Draw a standard protein curve with the standard protein concentration and OD value as the horizontal and vertical axes, and calculate the concentration of the sample to be tested based on the standard protein curve.

[0058] (3) Protein denaturation The protein was diluted to a uniform concentration with PBS, 5 × loading buffer was added, and then heated in a 100 °C metal bath for 10 min and stored at room temperature and -20 °C.

[0059] (4) Protein electrophoresis (a) Clean the 1.5 mm thick gel sheet and electrophoresis comb and rinse them three times with ddH2O.

[0060] (b) Leak test: Place the rubber sheet on the rubber rack, fill the rubber sheet with ddH2O, and let it stand for 30 minutes.

[0061] (c) Pour out the ddH2O from the gelatin plate, absorb the moisture with filter paper, and let it stand for 10 min to dry.

[0062] (d) Prepare two 1.5 mm lower layer gels. Bring the kit to room temperature. Next, take a 50 mL centrifuge tube, mix 7 mL of gel solution + 7 mL of buffer + 140 μL of coagulant, pour into the gel plate to 2 / 3 of the surface, add water to level, and place at room temperature for 30 min to solidify.

[0063] (e) Pour the ddH2O from the gelatin plate and absorb the water with filter paper.

[0064] (f) Prepare two 1.5 mm upper gels. Bring the reagent kit to room temperature, then take a 50 mL centrifuge tube, mix 2.5 mL gel solution + 2.5 mL buffer + 50 μL coagulant, pour the mixed upper gel solution onto the solidified lower gel, then immediately insert the electrophoresis comb to ensure that no bubbles are generated, and leave at room temperature for 30 minutes to solidify.

[0065] (g) Place the gel rack into the electrophoresis tank, fill it with electrophoresis solution, and remove the electrophoresis comb.

[0066] (h) Sample loading. The denatured protein sample was taken out from -20 ℃, thawed on ice, centrifuged, and then 10 μL of protein sample was added in sequence. 1 μL and 3 μL of marker were added on both sides of the protein sample, respectively.

[0067] (i) Electrophoresis. Connect the electrophoresis tank cover to the electrodes, run the stacking gel at 80 V for 30 min, and the separation gel at 120 V for 1 h. Stop the electrophoresis when the bromophenol blue reaches the bottom.

[0068] (j) Transfer. Cut the PVDF membrane and activate it with methanol. Prepare the transfer solution and soak the sponge and filter paper. Put the gel block into the solution and remove the upper gel. Assemble the "sandwich" transfer structure and put it into the tank, making sure the black side of the transfer clip faces the black side of the transfer tank and the white side faces the red side of the transfer tank. After adding the transfer solution, connect the electrodes. The transfer time depends on the molecular weight, and the constant current is 300 mA for transfer.

[0069] (k) Blocking. Take out the PVDF membrane and put it into a 5% skim milk incubation box with the two membranes back to back. Block at room temperature with slow shaking for 1 h.

[0070] (l) Primary antibody incubation. Place the PVDF membrane containing the target protein in an incubation bag, add the antibody, seal it with a film sealer, fix the incubation bag on a 3D shaker, and incubate it at 4°C overnight.

[0071] (m) Recover antibodies. The next day, cut the incubation bag to recover antibodies, put the PVDF membrane into an incubation box containing TBST membrane washing solution, and wash the membrane on a fast shaker at room temperature for 10 min / time, for 3 times.

[0072] (n) Secondary antibody incubation. After washing the membrane, discard TBST, add a secondary antibody of the same species as the primary antibody, incubate at room temperature for 1 h on a slow shaker, and then wash the membrane three times at room temperature at a frequency of 10 min / time.

[0073] (o) Development. Prepare the luminescent solution (A:B=1:1) and avoid light. After absorbing the TBST on the PVDF membrane, place the exposure plate and drip the luminescent solution, remove the excess solution, and put it into the developer for exposure.

[0074] 3.6 ELISA (1) Sample preparation. The extraction of tissue protein and the determination of total tissue protein concentration by BCA method were the same as above.

[0075] (2) Dilution of the standard. According to the concentration instructions provided in the instructions, use the standard diluent to dilute the standard into 6 different concentrations.

[0076] (3) Sample dilution: Dilute serum and tissue proteins to an appropriate multiple using sample diluent.

[0077] (4) Sample loading. Add different concentrations of standard solution (repeated wells are required) and sample solution to the ELISA plate in sequence, 50 μL per well. Set up sample blank wells and standard blank wells (repeated wells are required). After sample loading, place the ELISA plate in a 37 ℃ incubator and incubate for 30 min.

[0078] (5) Wash the plate. Calculate the amount of wash solution required and dilute the 30× concentrated wash solution to 1× wash solution. Add 300 μL to each well, let stand for 30 seconds, discard, and pat dry on filter paper. Repeat five times.

[0079] (6) Add enzyme labeling reagent. Add 50 μL of enzyme labeling reagent to each well except the sample blank well and the standard blank well. Then return the ELISA plate to the 37 ℃ incubator and continue incubating for 30 min.

[0080] (7) Wash the board.

[0081] (8) Add colorimetric solution. Calculate and prepare the required amount of colorimetric solution. Mix solution A and solution B in a ratio of 1:1 and add 100 μL of the mixed colorimetric solution to each well. Place the ELISA plate in a 37°C incubator in the dark for 20 min. Adjust the reaction time according to the color change.

[0082] (9) Add stop solution. Add 50 μL of stop solution to each well to terminate the reaction.

[0083] (10) Measure the OD value using an ELISA reader. Within 15 minutes after adding the stop solution, measure the sample absorbance (OD value) at a wavelength of 450 nm using an ELISA reader.

[0084] (11) Prepare a standard protein curve. Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis, and calculate the concentration of the protein to be tested based on the curve.

[0085] 3.7 Data Analysis Same as Example 1 “1.5”.

[0086] 3.8 Effects of Mexneurin-3 on G protein-coupled receptor-related kinases G protein-coupled receptors are the key targets of analgesics. Western blot experiments showed that icv 20 nmolMx-3 was used in formalin model mice to explore whether its analgesic effect was related to the downstream kinases of G protein-coupled receptors, PKA, PKC, and CAMKII. Figure 5 The 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 did not change, suggesting that the analgesic effect of Mx-3 may be related to PKC phosphorylation.

[0087] 3.9 Effect of Mexneurin-3 on DAG Content ELISA experiments were used to detect changes in the content 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.

[0088] In summary, Mx-3, as an endogenous polypeptide, has a significant analgesic effect, can inhibit acute inflammatory pain and chronic inflammatory pain, and has fewer side effects and is safer. The present invention also reveals the analgesic mechanism of Mx-3, and finds that it upregulates diacylglycerol (DAG) levels in the spinal cord and activates protein kinase C (PKC). The present invention provides an application of Mx-3 in inhibiting inflammatory pain, which can be used to develop safer and more effective new analgesic drugs, providing new ideas and references for the treatment of inflammatory pain.

[0089] Matters not covered by the present invention are known technologies.

[0090] The above embodiments are only for illustrating the features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a drug for inhibiting inflammatory pain, characterized in that: The polypeptide is Mexneurin-3.

2. The use according to claim 1, characterized in that: Its amino acid sequence is: NH2-PPPRSPYSGKGLGPGNLLHSALLPIHCICLC-COOH.

3. The use according to claim 2, characterized in that: The polypeptide can inhibit acute inflammatory pain.

4. The use according to claim 3, characterized in that: The acute inflammatory pain that can be inhibited by the polypeptide includes infectious inflammatory pain, chemical inflammatory pain, or mechanical inflammatory pain.

5. The use according to claim 2, characterized in that: The polypeptide can inhibit chronic inflammatory pain.

6. The use according to claim 5, characterized in that: The chronic inflammatory pain that the polypeptide can inhibit includes neuropathic inflammatory pain, degenerative inflammatory pain, or metabolic inflammatory pain.

7. The use according to claim 1, characterized in that: The polypeptide can exert analgesic effect by upregulating DAG level in the spinal cord and activating PKC.

8. The use according to claim 1, characterized in that: The amino acid sequence of the polypeptide is processed, modified or altered.

9. A pharmaceutical composition for treating inflammatory pain, characterized in that: The pharmaceutical composition comprises the polypeptide Mexneurin-3 described in any one of claims 1 to 7.

10. The pharmaceutical composition according to claim 9, characterized in that The composition is prepared into an injection, oral or external dosage form.

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