Polypeptide AAMP and Its Application in Analgesia and Anti-Inflammation

Abstract

The present invention relates to a polypeptide AAMP and its application in analgesia and anti - inflammation. The amino acid sequence of the polypeptide AAMP is shown as SEQ ID NO.1. This polypeptide AAMP has good anti - inflammatory and analgesic activities; it can relieve the pain reaction caused by PNI and has good therapeutic effects on diseases such as fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, and nerve injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and provides a polypeptide AAMP and its application in analgesia and anti - inflammation. Background Art

[0002] Inflammatory pain is mediated by inflammatory factors, and central or peripheral sensitization is achieved through receptors, ion channels, neurotransmitters, and regulatory proteins, triggering the body to produce spontaneous pain. These inflammatory factors are mainly pro - inflammatory cytokines, including TNF - α, TNF - β, IL - 1β, IL - 6, etc. They can bind to different receptors on the surface of surrounding tissue cells, and then activate signal molecules such as MAPK, PKA, PKC, PI3K, Src, etc., thereby increasing the excitability of neurons and the release of neurotransmitters, leading to chronic and persistent pain perception. Therefore, inhibiting the production of pro - inflammatory cytokines is crucial for reducing inflammatory pain.

[0003] Inflammatory factors are mainly secreted by immune cells, including monocytes, macrophages, mast cells, neutrophils, dendritic cells, and NK cells, etc. These immune cells will differentiate into different phenotypes under different environments, and typical subtypes include M1 type and M2 type. Among them, the M1 type mainly secretes pro - inflammatory cytokines, while the M2 type mainly secretes anti - inflammatory cytokines, including IL - 4, IL - 10, TGF - β, etc. The former mainly participates in the inflammatory response and causes hyperalgesia, while the latter can inhibit the excessive activation of inflammation and promote tissue repair.

[0004] In the related art, there is no effective clinical means related to reducing inflammatory pain, etc., so there is an urgent need for improvement. Summary of the Invention

[0005] The present invention provides a polypeptide AAMP and its application in analgesia and anti - inflammation. The polypeptide AAMP has good anti - inflammatory and analgesic activities; it can relieve the pain reaction caused by PNI, and has good therapeutic effects on diseases such as fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, and nerve injury.

[0006] In the first aspect, the present application provides a polypeptide AAMP, and its amino acid sequence is as shown in SEQ ID NO.1.

[0007] In some optional embodiments, the polypeptide AAMP is prepared by solid - phase synthesis.

[0008] In some optional embodiments, the polypeptide AAMP is obtained by modifying the painless short peptide NPL7, and the amino acid sequence of the painless short peptide NPL7 is as shown in SEQ ID NO.2.

[0009] Specifically, the painless short peptide NPL7: T-D-I-K-C-K-E-K-M urgently needs to be modified; that is, the 5th cysteine (C) in its sequence is replaced with methionine (M). The painless short peptide NPL7 is obtained by modifying the first hairpin loop region Loop I (T-T-A-T-D-I-K-G-K-E-V-M-V-L) in the nerve growth factor sequence.

[0010] In some optional embodiments, the molar concentration of the polypeptide AAMP is 40 to 60 μmol / L.

[0011] In some optional embodiments, the molar concentration of the polypeptide AAMP is 50 μmol / L.

[0012] In a second aspect, the present application provides a drug for analgesia and anti-inflammation, comprising the polypeptide AAMP of the first aspect.

[0013] In a third aspect, the present application provides an application of the polypeptide AAM in the preparation of a drug for analgesia and anti-inflammation.

[0014] In a fourth aspect, the present application provides an application of the polypeptide AAM in the preparation of a drug for treating any one of the diseases of fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, and nerve injury.

[0015] This application has at least the following beneficial effects:

[0016] The polypeptide AAMP provided by the present invention can effectively promote the transformation of the macrophage cell line Raw264.7 cells from the M1 / M0 type to the M2 type, and inhibit the expression of anti-inflammatory related genes, such as TNF-α, TNF-β, IL-1β, and the secretion of anti-inflammatory cytokines. At the same time, it enhances the production of pro-inflammatory related genes, such as IL-4, IL-10, TGF-β, and pro-inflammatory cytokines. At the same time, the polypeptide AAMP has good anti-inflammatory and analgesic activities; it can relieve the pain response caused by peripheral nerve injury (PNI). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Next, the features, advantages, and technical effects of the exemplary embodiments of the present application will be described with reference to the drawings.

[0018] Figure 1 Shows the results of immunofluorescence experiments after different treatments of cells in the embodiments of the present application;

[0019] Figure 2 Shows the effect diagram of the expression of pro-inflammatory related genes after LPS stimulation of Raw264.7 cells in the embodiments of the present application;

[0020] Figure 3The figure shows the effect diagram of the content of pro-inflammatory cytokines in the supernatant of different groups of cells in the embodiments of the present application;

[0021] Figure 4 The figure shows the comparison diagram of the thickness of the right hind toes of mice after treatment with different groups in the embodiments of the present application;

[0022] Figure 5 The figure shows the effect diagram of the pain degree caused by acetic acid in mice after treatment with different groups in the embodiments of the present application;

[0023] Figure 6 The figure shows the comparison diagram of the foot licking time of mice in the early and late stages after treatment with different groups in the embodiments of the present application;

[0024] Figure 7 The figure shows the comparison diagram of the mechanical withdrawal threshold and the hyperalgesia time at different times after treatment with different groups on the operative side of rats in the embodiments of the present application. Detailed implementation manners

[0025] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0026] With the increasing maturity of polypeptide chemical synthesis technology and biotechnology, more and more polypeptide drugs have received extensive attention and applications in the fields of prevention, treatment and diagnosis of diabetes, tumors, AIDS, hepatitis and cardiovascular diseases. Such drugs are synthesized by chemically connecting different amino acids in a certain sequence, and have the advantages of easy synthesis, high biological activity, wide indications, low immunogenicity and significant curative effects.

[0027] To relieve acute and chronic pain caused by inflammation, through research, a polypeptide AAMP containing a 9-amino acid sequence: T-D-I-K-M-K-E-K-M, has good anti-inflammatory and analgesic effects. This polypeptide is obtained by modifying the painless short peptide NPL7: T-D-I-K-C-K-E-K-M, that is, replacing the 5th cysteine (C) in its sequence with methionine (M), and the painless short peptide NPL7 is obtained by modifying the first hairpin loop region Loop I (T-T-A-T-D-I-K-G-K-E-V-M-V-L) in the nerve growth factor sequence.

[0028] In vitro and in vivo experimental results have confirmed that polypeptide AAMP can effectively promote the transformation of macrophage line Raw264.7 cells from the M1 / M0 type to the M2 type, inhibit the expression of anti-inflammatory related genes (TNF-α, TNF-β, IL-1β) and the secretion of anti-inflammatory cytokines, and at the same time, enhance the production of pro-inflammatory related genes (IL-4, IL-10, TGF-β) and pro-inflammatory cytokines. At the same time, the acetic acid writhing animal pain model, formalin animal pain model and carrageenan animal pain model were used to confirm that polypeptide AAMP has good anti-inflammatory and analgesic activities. In addition, the PNI model was used to find that AAMP can relieve the pain response caused by PNI.

[0029] In summary, polypeptide AAMP has good anti-inflammatory and analgesic effects and has good therapeutic effects on diseases such as fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, and nerve injury.

[0030] Example 1

[0031] Synthesis and purification of polypeptide AAMP

[0032] Using the solid-phase synthesis method, the Rink-Amide-MBHA resin was swollen in dichloromethane (DCM) and N,N-dimethylformamide (DMF), and the Fmoc protecting group was removed using 50% morpholine; subsequently, after washing alternately with DCM and DMF three times, the amino acid to be incorporated was mixed with 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in an equivalent ratio of 1:1:1 and reacted for 1.5 h under nitrogen bubbling.

[0033] According to the above steps, different Fmoc-protected amino acids were sequentially incorporated according to the polypeptide sequence of AAMP. After the synthesis of the linear peptide resin was completed, a cleavage solution (V 三氟乙酸 :V 三异丙基硅烷 :V 纯水 = 95:2.5:2.5) was added and the mixture was lysed on a shaker for 1 - 1.5 h; after removing the resin, 50% acetonitrile solvent was added, and the polypeptide was separated and purified by high performance liquid chromatography (HPLC). Samples with different peak patterns in HPLC at different time points were collected, and the molecular weight was identified using a mass spectrometer. The target polypeptide with a molecular weight of 1148 Da was obtained, and the pure peptide liquid showing this peak pattern was collected and placed in a freeze dryer for lyophilization to obtain solid AAMP.

[0034] Example 2

[0035] Polypeptide AAMP induces the polarization of Raw264.7 cells into the M2 type

[0036] To investigate whether AAMP can promote the transformation of macrophages into the M2 type, linear peptide AAMP was used to treat RAW264.7 cells stimulated with lipopolysaccharide (LPS), and the polarization of cells in each group was observed by immunofluorescence staining.

[0037] First, RAW264.7 cells of more than three passages were seeded on 24-well plates at a density of 2.5×10 4 / mL. After 24 h of culture, the cells were divided into 4 groups, namely the control group (90% 1640 medium, which can be from Gibco, +10% fetal bovine serum, and the control group only has these components), the group with LPS (100 ng / mL lipopolysaccharide was added to the control group), the group with only AAMP (50 μM polypeptide AAMP was added to the control group), or the group with AAMP (50 μM) and LPS (100 ng / mL), that is, they were divided into the control group, the LPS group, the AAMP group, and the LPS+AAMP group in sequence. The 4 groups were then placed in an incubator for 24 h, and then, the cells in each group were subjected to immunofluorescence experiments. In this experiment, the M0 type of RAW264.7 cells was labeled with CD68, the M1 type was labeled with CD86, the M2 type was labeled with CD206, and the cell nuclei in each group were labeled with DAPI. The results are as Figure 1 shown.

[0038] As Figure 1 shown, A and C are parallel experimental groups labeled with different antibodies. A and C are the fluorescence pictures taken. B is the quantitative and statistical difference analysis of the fluorescence intensity values of each group in A using Image J. D is the quantitative and statistical difference analysis of the fluorescence intensity values of each group in C using Image J. After Raw264.7 cells were stimulated with LPS, the fluorescence intensity of CD86 increased sharply, while the simultaneous addition of LPS and AAMP could weaken the fluorescence intensity of CD86 and enhance the fluorescence intensity of CD206 at the same time, while the simple addition of AAMP did not change the fluorescence intensity of the CD86 and CD206 markers ( Figure 1 in A and C). The statistical results of the immunofluorescence intensity of CD68 and CD206 in each group also showed that compared with the LPS group, the relative fluorescence intensity value of CD68 in the LPS+AAMP group was significantly smaller ( *** P < 0.001), while the relative fluorescence intensity value of CD206 increased sharply (***P <0.001).

[0039] M2 macrophages can produce anti-inflammatory cytokines, which aim to reduce the level of inflammation and thus relieve pain, while M1 macrophages have the opposite effect. This example shows that AAMP can inhibit the polarization of macrophage line RAW264.7 cells into M1 and promote their polarization into M2, thereby reducing the production of pro-inflammatory cytokines and promoting the production of anti-inflammatory factors.

[0040] Example 3

[0041] Polypeptide AAMP downregulates the transcription of pro-inflammatory related genes and upregulates the expression of anti-inflammatory related genes

[0042] To explore whether AAMP can inhibit inflammation, linear peptide AAMP treatment was given to LPS-stimulated RAW264.7 cells, and the expressions of pro-inflammatory related genes (including TNF-α, IFN-γ and IL-6) and anti-inflammatory related genes (including IL-4, IL-10 and TGF-β) in each group were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).

[0043] First, RAW264.7 cells above passage 3 were seeded on 6-well plates at a density of 2.5×10 4 / mL and cultured for 24 h. Then they were divided into 4 groups, namely the control group (adding nothing, only containing 90% 1640 medium + 10% fetal bovine serum), LPS-containing (100 ng / mL), simply adding AAMP (50 μM), or LPS (100 ng / mL) plus AAMP (50 μM) medium, that is, they were divided into the control group, LPS group, AAMP group and LPS+AAMP group in turn. The 4 groups were then placed in an incubator and cultured for 24 h. Then, the medium was aspirated and Trizol reagent was added to lyse for 5 min, followed by total RNA extraction, reverse transcription, amplification and relative quantification of target genes. In this experiment, β-actin was used as an internal reference, and the primer sequences for target gene amplification are shown in Table 1: Table 1. Primer sequences for qRT-PCR.

[0044] As Figure 2 shown, after LPS stimulation of Raw264.7 cells, the expression results of pro-inflammatory related genes (including TNF-α, IFN-γ and IL-6) are shown in Figure A; the expression results of anti-inflammatory related genes (including IL-4, IL-10 and TGF-β) are shown in Figure B; significant upregulation and downregulation occurred in the two groups of results respectively, and the transcriptional levels of these genes were reversed after adding linear polypeptide AAMP. Therefore, linear polypeptide AAMP can significantly promote the transcription of anti-inflammatory related genes and inhibit the expression of pro-inflammatory related genes, thereby preventing the secretion of downstream inflammatory factors.

[0045] Example 4

[0046] Multiple AAMP inhibits the expression of pro-inflammatory cytokines and increases the secretion of anti-inflammatory cytokines

[0047] To investigate the effect of AAMP on inhibiting inflammation, RAW264.7 cells stimulated with lipopolysaccharide (LPS) were treated with the linear peptide AAMP, and the expressions of pro-inflammatory cytokines (including TNF-α and IFN-γ) and anti-inflammatory cytokines (including TGF-β and IL-10) in each group were detected by enzyme-linked immunosorbent assay (ELISA).

[0048] First, RAW264.7 cells of more than three generations were seeded on a 12-well plate at a density of 2.5×10 4 / mL. After culturing for 24 h, the medium was replaced with a medium containing LPS (100 ng / mL) or LPS (100 ng / mL) plus AAMP (50 μM), and then placed in an incubator for 24 h. Then, the supernatant was aspirated and the content of related inflammatory factors was detected by ELISA.

[0049] As Figure 3 shown, compared with the Control group, the contents of pro-inflammatory cytokines (including TNF-α and IFN-γ) in the cell supernatant of the LPS group were significantly increased, while the levels of anti-inflammatory cytokines (including TGF-β and IL-10) were down-regulated. In addition, compared with the LPS group, the levels of pro-inflammatory cytokines (including TNF-α and IFN-γ) in the cell supernatant of the LPS+AAMP group were significantly down-regulated, while the levels of anti-inflammatory cytokines (including TGF-β and IL-10) were significantly up-regulated; meanwhile, AAMP itself had no effect on the expression changes of these cytokines. Therefore, under LPS stimulation, AAMP can significantly down-regulate the expression of pro-inflammatory cytokines and, at the same time, increase the secretion of anti-inflammatory cytokines, indicating that AAMP has a significant effect on inhibiting the inflammatory response.

[0050] Example 5

[0051] Carrageenan-induced paw swelling experiment in mice

[0052] The anti-inflammatory effect of linear peptide AAMP was evaluated by the carrageenan-induced paw swelling experiment. SPF-grade 4-week-old C57BL / 6 mice (weighing 18 - 22 g) were randomly divided into a normal group, a model group (carrageenan), an AAMP group (30 mg / kg linear peptide AAMP), and a positive control group (30 mg / kg indomethacin), with 10 mice in each group. Linear peptide AAMP and indomethacin were dissolved in PBS solution and diluted to a concentration of 3 mg / mL. Each mouse in the AAMP group and the positive control group was intraperitoneally injected with 200 μL of linear peptide AAMP and indomethacin, respectively; each mouse in the normal group and the model group was injected with an equal volume of PBS. 30 minutes later, 30 μL of 1% carrageenan was injected into the right hind paw of the mice in the model group, the AAMP group, and the positive control group, while the mice in the normal group were injected with an equal volume of normal saline into the right hind paw. Subsequently, the thickness of the right hind toes of the mice was measured every 1 h.

[0053] As Figure 4 shown, compared with the control group, the thickness of the mouse toes in the model group increased significantly 1 h after injecting carrageenan, and the thickness of the toes did not decrease significantly over time, indicating that the mouse toes showed significant swelling and maintained for a long time, and the degree of swelling reached the peak at 2 - 3 h. Compared with the model group, the AAMP group inhibited the carrageenan-induced swelling of the right hind paw of mice at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after administration to varying degrees, and the inhibitory effect was better than that of the indomethacin group, indicating that the linear peptide AAMP group had a good effect on inhibiting inflammation and was better than indomethacin.

[0054] Example 6

[0055] Antipyretic analgesic experiment of the mouse acetic acid writhing pain model

[0056] To verify the analgesic effect of AAMP, SPF-grade 4-week-old C57BL / 6 mice were randomly divided into a control group (PBS solution), an AAMP group (30 mg / kg linear peptide AAMP), and a positive control group (30 mg / kg aspirin), with 10 mice in each group. Linear peptide AAMP was dissolved in PBS solution and diluted to a concentration of 3 mg / mL, and then 200 μL was injected into the left abdominal cavity of the mice using a 1 mL syringe; the mice in the positive drug group and the control group were intraperitoneally injected with an equal volume of aspirin solution and PBS solution, respectively. 30 minutes later, 250 μL of 0.6% acetic acid solution was injected into the right abdominal cavity of the three groups of mice, and the time of the first writhing after injecting acetic acid and the number of writhings within 20 minutes were recorded in sequence.

[0057] As Figure 5As shown, compared with the control group, the linear peptide AAMP can significantly prolong the time of the first writhing in mice and reduce the number of acetic acid-induced writhing in mice, and the effect is better than that of the positive control group. These results show that the linear peptide AAMP has an obvious analgesic effect on acetic acid-induced animal pain.

[0058] Example 7

[0059] Formalin-induced pain experiment in mice

[0060] SPF-grade 4-week-old C57BL / 6 mice were randomly divided into a control group (PBS solution), an AAMP group (30 mg / kg linear peptide AAMP), and a positive control group (30 mg / kg aspirin), with 10 mice in each group. The linear peptide AAMP was dissolved in PBS solution and diluted to a concentration of 3 mg / mL. Then, 200 μL of the drug was injected into the mice intraperitoneally using a 1 mL syringe; mice in the positive drug group and the control group were intraperitoneally injected with equal volumes of aspirin (3 mg / mL) and PBS solution, respectively. 30 minutes later, 25 μL of 5% formalin solution was injected into the right hind paw of the mice, and the licking foot time in the early stage (5 minutes) and the late stage (30 minutes) was recorded.

[0061] As Figure 6 shown, the experimental results show that the linear peptide AAMP can significantly reduce the licking foot time of mice in the early and late stages, and the time is less than that of the positive control group, indicating that the linear peptide AAMP has a significant analgesic effect on both acute pain and tonic phase pain.

[0062] Example 8

[0063] AAMP can relieve the chronic pain response caused by peripheral nerve injury (PNI)

[0064] To further explore whether AAMP has the effect of relieving the pain response caused by nerve injury, we exposed the sciatic nerve of 8-week-old SD rats and cut the nerve trunk along the sciatic notch to make a sciatic nerve transection injury model. After surgery, 80 ng of AAMP (100 μL) or an equal dose of NPL7 was injected in situ at the injury site and continuously administered for 1 month, and they were divided into the AAMP group and the NPL7 group. In the PNI group, after transecting the sciatic nerve, the same volume of normal saline was injected continuously for 1 month, while in the sham group, only the sciatic nerve was exposed and no surgical section was performed. The pain sensation changes of rats in each group were tested by Paw pressure test and Hargreaves test at the 1st, 2nd, and 3rd months after surgery.

[0065] By Figure 7The detection results show that in Figure A. During mechanical stimulation, compared with the sham group, the pressure required to withdraw the ipsilateral hindlimb of rats in the stimulated PNI group was significantly reduced, indicating that the pain threshold of mechanical dull pain in rats in the PNI group was decreased, showing hyperalgesia, and it was most severe in the first month of the test; administering AAMP increased the pressure required to withdraw the ipsilateral hindlimb of PNI rats, indicating that the pain threshold of mechanical dull pain in rats in this group was increased, thus significantly weakening the hyperalgesic behavior caused by PNI, and the mechanical withdrawal thresholds measured in the AAMP group at each time period were higher than those in the PNI group. However, the mechanical threshold values measured in the NPL7 group at each time period were similar to those in the PNI group. As shown in Figure B, during infrared thermal stimulation, the time for the ipsilateral toes of rats after PNI or treated with NPL7 to withdraw was significantly reduced, indicating that the sensitivity of these two groups of rats to thermal stimulation increased, and it was inferred that severe hyperalgesia occurred in their ipsilateral toes. However, the treatment with AAMP significantly blunted this hyperalgesia. Therefore, AAMP can relieve neuropathic pain caused by PNI.

[0066] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A polypeptide AAMP, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The polypeptide AAMP according to claim 1, characterized in that The polypeptide AAMP is prepared by solid phase synthesis.

3. The polypeptide AAMP according to claim 1, characterized in that The polypeptide AAMP is obtained by modifying the painless short peptide NPL7, and the amino acid sequence of the painless short peptide NPL7 is shown in SEQ ID NO.

2.

4. The polypeptide AAMP according to claim 1, characterized in that The molar concentration of the polypeptide AAMP is 40 to 60 μmol / L.

5. The polypeptide AAMP according to claim 4, characterized in that The molar concentration of the polypeptide AAMP is 50 μmol / L.

6. A drug for analgesia and anti-inflammatory, characterized in that: Comprising the polypeptide AAMP according to any one of claims 1 to 5.

7. Use of the polypeptide AAM according to any one of claims 1 to 5 in the preparation of analgesic and anti-inflammatory drugs.

Images