Polypeptide AAMP and application thereof in analgesia and anti-inflammation

Abstract

The invention relates to a polypeptide AAMP and application thereof in analgesia and anti-inflammation. The amino acid sequence of the polypeptide AAMP is as shown in SEQ ID NO. 1. The polypeptide AAMP has better anti-inflammatory and analgesic activity; the traditional Chinese medicine composition can relieve pain reaction caused by PNI, and has a good treatment effect on fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, nerve injury and other diseases.

Description

Technical Field

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

[0002] Inflammatory pain is a type of pain that is mediated by inflammatory factors and triggers the body to produce spontaneous pain through central or peripheral sensitization of receptors, ion channels, neurotransmitters and regulatory proteins. These inflammatory factors are mainly proinflammatory cytokines, including: TNF-α, TNF-β, IL-1β, IL-6, etc. They can bind to different receptors on the surface of surrounding tissue cells, thereby activating signaling 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. Therefore, inhibiting the production of proinflammatory cytokines is crucial to reducing inflammatory pain.

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

[0004] In the related technologies, there are no effective clinical means to reduce inflammatory pain, etc., and therefore improvement is urgently needed. 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 activity; it can relieve the pain response caused by PNI, and has a good therapeutic effect on diseases such as fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, nerve damage, etc.

[0006] In a first aspect, the present application provides a polypeptide AAMP, whose amino acid sequence is 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 shown in SEQ ID NO.2.

[0009] Specifically, the pain-free short peptide NPL7: TDIKCKEKM is in urgent need of modification; that is, the fifth cysteine ​​(C) in its sequence is replaced with methionine (M). The pain-free short peptide NPL7 is obtained by modifying the first hairpin loop region Loop I (TTATDIKGKEVMVL) in the nerve growth factor sequence.

[0010] In some optional embodiments, the 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 a use of a polypeptide AAM in the preparation of analgesic and anti-inflammatory drugs.

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

[0015] This application has at least the following beneficial effects: The polypeptide AAMP provided by the present invention can effectively promote the transformation of the macrophage cell line Raw264.7 cells from M1 / M0 type to 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, while enhancing 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 activity; it can relieve the pain response caused by peripheral nerve injury (PNI). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The features, advantages and technical effects of exemplary embodiments of the present application will be described below with reference to the accompanying drawings.

[0017] Figure 1 The results of immunofluorescence experiments after different treatments of cells in the examples of the present application are shown; Figure 2 The effect diagram of the expression of pro-inflammatory related genes after LPS stimulation of Raw264.7 cells in the embodiment of the present application is shown; Figure 3 The effect diagram of the content of pro-inflammatory cytokines in the supernatant of cells of different groups in the examples of the present application is shown; Figure 4 A comparison of the thickness of the right hind toes of mice after treatment in different groups in the examples of the present application is shown; Figure 5 The effect diagram of the pain degree caused by acetic acid on mice after different groups of treatment in the examples of the present application is shown; Figure 6 A comparison of the paw licking time of mice in the early and late stages after treatment in different groups in the examples of the present application is shown; Figure 7 A comparison chart of mechanical paw withdrawal thresholds and hyperalgesia time at different times after treatment in different groups of rats on the surgical side of the embodiment of the present application is shown. DETAILED DESCRIPTION

[0018] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0019] With the increasing maturity of peptide chemical synthesis technology and biotechnology, more and more peptide drugs have received extensive attention and application in the fields of prevention, treatment and diagnosis of diabetes, tumors, AIDS, hepatitis and cardiovascular diseases. This type of drug is made by connecting different amino acids in a certain sequence through chemical synthesis, and has the advantages of easy synthesis, high biological activity, wide indications, low immunogenicity and significant efficacy.

[0020] In order to relieve acute and chronic pain caused by inflammation, a 9-amino acid peptide AAMP: TDIKMKEKM was found to have good anti-inflammatory and analgesic effects. The peptide was obtained by modifying the painless short peptide NPL7: TDIKCKEKM, that is, the fifth cysteine ​​(C) in its sequence was replaced with methionine (M), and the painless short peptide NPL7 was modified from the first hairpin loop region Loop I (TTATDIKGKEVMVL) in the nerve growth factor sequence.

[0021] The results of in vitro and in vivo experiments have confirmed that the peptide AAMP can effectively promote the transformation of the macrophage cell line Raw264.7 cells from M1 / M0 to M2, and inhibit the expression of anti-inflammatory related genes (TNF-α, TNF-β, IL-1β) and the secretion of anti-inflammatory cytokines, while enhancing 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 confirmed that the peptide AAMP has good anti-inflammatory and analgesic activity. In addition, the PNI model was used to find that AAMP can relieve the pain response caused by PNI.

[0022] In summary, peptide AAMP has excellent anti-inflammatory and analgesic effects, and has good therapeutic effects on diseases such as fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis, and nerve damage.

[0023] Example 1 Synthesis and purification of peptide AAMP Using the solid phase synthesis method, 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 with DCM and DMF alternately for 3 times, the amino acid to be connected was mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in an equivalent ratio of 1:1:1 and reacted under nitrogen blowing for 1.5 h.

[0024] According to the above steps, different Fmoc protected amino acids were inserted in sequence according to the peptide sequence of AAMP. After the linear peptide resin was synthesized, shearing solution (V 三氟乙酸 :V 三异丙基硅烷 :V 纯水 = 95:2.5:2.5) and shake for lysis for 1-1.5 h; after removing the resin, add 50% acetonitrile solvent, separate and purify the polypeptide by high performance liquid chromatography (HPLC), collect samples with different peaks on HPLC at different time points, identify the molecular weight by mass spectrometer, obtain the target polypeptide with a molecular weight of 1148 Da, collect the pure peptide liquid with the peak and put it into a freeze dryer for freeze drying, so as to obtain solid AAMP.

[0025] Example 2 Peptide AAMP induces Raw264.7 cells to polarize to M2 type To explore whether AAMP can promote the transformation of macrophages to M2 type, RAW264.7 cells stimulated with lipopolysaccharide (LPS) were treated with linear peptide AAMP, and the polarization of cells in each group was observed by immunofluorescence staining.

[0026] First, RAW264.7 cells over three generations were cultured at 2.5×10 4 / mL was plated on a 24-well plate. After culturing for 24 hours, the cells were divided into four groups, namely the control group (90% 1640 culture medium, which can be from Gibco, +10% fetal bovine serum, the control group only has these components), LPS-containing (100 ng / mL added to the control group to add lipopolysaccharide), AAMP alone (50 μM of polypeptide AAMP was added to the control group) or AAMP (50 μM) and LPS (100 ng / mL) culture medium, that is, they were divided into the control group (control group), LPS group, AAMP group and LPS+AAMP group. The four groups were cultured in the incubator for another 24 hours, and then the cells in each group were subjected to immunofluorescence experiments. In this experiment, the M0 type of RAW264.7 cells was marked with CD68, the M1 type was marked with CD86, the M2 type was marked with CD206, and the cell nuclei of each group were marked with DAPI. The results are shown in the following figure. Figure 1 shown.

[0027] like Figure 1 As shown, A and C are parallel experimental groups labeled with different antibodies. A and C are fluorescent images taken, and 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 by 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. However, the simple addition of AAMP did not change the fluorescence intensity of CD86 and CD206 markers ( Figure 1 The statistical results of the immunofluorescence intensity of CD68 and CD206 in each group also showed that the relative fluorescence intensity of CD68 in the LPS+AAMP group was significantly lower than that in the LPS group ( *** P < 0.001), while the relative fluorescence intensity of CD68 increased dramatically (***P < 0.001).

[0028] M2 macrophages can produce anti-inflammatory cytokines, which have the purpose of reducing inflammation levels and thus relieving pain, while M1 macrophages have the opposite effect. This example shows that AAMP can inhibit the polarization of macrophage cell line RAW264.7 cells to M1 and promote their polarization to M2, thereby reducing the production of pro-inflammatory cytokines and promoting the production of anti-inflammatory factors.

[0029] Example 3 Peptide AAMP downregulates the transcription of pro-inflammatory genes and upregulates the expression of anti-inflammatory genes To explore whether AAMP can inhibit inflammation, linear peptide AAMP was given to RAW264.7 cells stimulated with lipopolysaccharide (LPS), and the expressions of pro-inflammatory genes (including TNF-α, IFN-γ and IL-6) and anti-inflammatory genes (including IL-4, IL-10 and TGF-β) in each group were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).

[0030] First, RAW264.7 cells over three generations were cultured at 2.5 × 10 4 / mL density was planted on a 6-well plate. After culturing for 24 hours, it was divided into 4 groups, which were the control group (nothing was added, only 90% 1640 medium + 10% fetal bovine serum), medium containing LPS (100ng / mL), medium with AAMP (50 μM) alone, or medium with LPS (100 ng / mL) plus AAMP (50 μM), that is, the control group, LPS group, AAMP group and LPS+AAMP group. The 4 groups were cultured in an incubator for another 24 hours, then the medium was removed and Trizol reagent was added for lysis for 5 minutes, followed by total RNA extraction, reverse transcription, amplification and relative quantification of the target gene. In this experiment, β-actin was used as the internal reference, and the primer design required for the amplification of the target gene is shown in Table 1: Table 1. Primer sequences for qRT-PCR.

[0031] like Figure 2 As shown, after LPS stimulated Raw264.7 cells, the expression results of pro-inflammatory genes (including TNF-α, IFN-γ and IL-6) are shown in Figure A; the expression results of anti-inflammatory genes (including IL-4, IL-10 and TGF-β) are shown in Figure B; in the two groups of results, significant upregulation and downregulation were respectively observed, and the transcription levels of these genes were reversed after the addition of linear peptide AAMP. Therefore, linear peptide AAMP can significantly promote the transcription of anti-inflammatory genes and inhibit the expression of pro-inflammatory genes, thereby preventing the secretion of downstream inflammatory factors.

[0032] Example 4 Multi-AAMPs inhibit the expression of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines To explore the inhibitory effect of AAMP on inflammation, RAW264.7 cells stimulated with lipopolysaccharide (LPS) were treated with 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).

[0033] First, RAW264.7 cells over three generations were cultured at 2.5×10 4 / mL were seeded on a 12-well plate and cultured for 24 h. The culture medium was replaced with LPS (100 ng / mL) or LPS (100 ng / mL) plus AAMP (50 μM) and cultured in an incubator for another 24 h. Then, the supernatant was aspirated and the content of related inflammatory factors was detected by ELISA.

[0034] like Figure 3 As shown in the figure, compared with the Control group, the levels 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 downregulated. 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 downregulated, while the levels of anti-inflammatory cytokines (including TGF-β and IL-10) were significantly upregulated; at the same time, AAMP itself had no effect on the expression changes of these cytokines. Therefore, under LPS stimulation conditions, AAMP can significantly downregulate the expression of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines, indicating that AAMP has a significant effect in inhibiting inflammatory responses.

[0035] Example 5 Carrageenan-induced foot swelling experiment in mice The anti-inflammatory effect of linear peptide AAMP was evaluated by carrageenan-induced paw swelling test. SPF 4-week-old C57BL / 6 mice (weight 18-22 g) were randomly divided into normal group, model group (carrageenan), AAMP group (30 mg / kg linear peptide AAMP), and 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 positive control group was intraperitoneally injected with 200 μL of linear peptide AAMP and indomethacin, respectively; while each mouse in the normal group and model group was injected with an equal volume of PBS. After 30 min, 30 μL of 1% carrageenan was injected into the right hind paw of mice in the model group, AAMP group, and positive control group, respectively, while the right hind paw of mice in the normal group was injected with an equal volume of normal saline. The thickness of the right hind toe of the mice was then measured every 1 h.

[0036] like Figure 4As shown in the figure, compared with the control group, the thickness of the mouse toes in the model group increased significantly 1 hour after carrageenan injection, and the thickness of the toes did not decrease significantly over time, indicating that the mouse toes were significantly swollen and maintained for a long time, and the swelling reached a peak at 2-3 hours. Compared with the model group, the AAMP group inhibited carrageenan-induced swelling of the right hind paw of mice to varying degrees at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours after administration, and the inhibition degree was better than that of the indomethacin group, indicating that the linear peptide AAMP group has a good effect in inhibiting inflammation, which is better than indomethacin.

[0037] Example 6 Analgesia experiment of acetic acid writhing pain model in mice To verify the analgesic effect of AAMP, we randomly divided SPF-grade 4-week-old C57BL / 6 mice 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 was injected into the left abdominal cavity of the mouse using a 1 mL syringe; the positive drug group and the control group mice were injected with equal volumes of aspirin solution and PBS solution in the left abdominal cavity, respectively. After 30 minutes, 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 of the mice after the injection of acetic acid and the number of writhing within 20 minutes were recorded.

[0038] like Figure 5 As shown, compared with the control group, the linear peptide AAMP can significantly prolong the time before the first writhing of mice and reduce the number of acetic acid writhing times of mice, and the effect is better than that of the positive control group. These results show that the linear peptide AAMP has a significant analgesic effect on animal pain caused by acetic acid.

[0039] Example 7 Formalin-induced pain experiment in mice 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 intraperitoneally injected into the mice using a 1 mL syringe; the positive drug group and the control group mice were intraperitoneally injected with equal volumes of aspirin (3 mg / mL) and PBS solution, respectively. After 30 minutes, 25 μL of 5% formalin solution was injected into the right hind foot of the mice, and the early (5 min) and late (30 min) licking time was recorded.

[0040] like Figure 6 As shown, the experimental results showed that linear peptide AAMP can significantly reduce the paw licking time of mice in the early and late stages, and the time is shorter than that of the positive control group, indicating that linear peptide AAMP has a significant analgesic effect on both acute pain and tonic pain.

[0041] Example 8 AAMPs can alleviate chronic pain responses after peripheral nerve injury (PNI) To further explore whether AAMP can relieve 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 create a sciatic nerve transection injury model. After surgery, 80 ng of AAMP (100 μL) or an equal amount of NPL7 was injected in situ at the injury site for 1 month, and the rats were divided into the AAMP group and the NPL7 group. After sciatic nerve transection, the PNI group was injected with the same volume of saline for 1 month, while the sham group only exposed the sciatic nerve without surgical transection. The pain changes of rats in each group were tested by Paw pressure test and Hargreaves test at 1, 2, and 3 months after surgery.

[0042] Depend on Figure 7 The test results are shown in Figure A. During mechanical stimulation, the pressure required to stimulate the hind limbs of the PNI rats on the surgical side to withdraw their feet was significantly reduced compared with the sham group, indicating that the pain threshold of mechanical dull pain in the PNI rats was reduced, and hyperalgesia occurred, which was most severe in the first month of the test; the administration of AAMP increased the pressure required to withdraw the hind limbs of the PNI rats on the surgical side, indicating that the pain threshold of mechanical dull pain in the rats in this group increased, thereby significantly weakening the hyperalgesia behavior caused by PNI, and the mechanical withdrawal threshold of the AAMP group tested at each time period was higher than that of the PNI group. However, the mechanical threshold values ​​of the NPL7 group tested at each time period were similar to those of the PNI group. As shown in Figure B, during infrared heat stimulation, the time of toe withdrawal on the surgical side of rats after PNI or treated with NPL7 was significantly reduced, indicating that the sensitivity of these two groups of rats to heat stimulation increased, and it was inferred that the toes on the surgical side had severe hyperalgesia, but the treatment of AAMP significantly blunted this hyperalgesia. Therefore, AAMP can relieve neuropathic pain caused by PNI.

[0043] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is determined by the attached 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 substance amount 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: It comprises 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.

8. Use of the polypeptide AAM according to any one of claims 1 to 5 in the preparation of a medicament for treating any one of fibromyalgia syndrome, rheumatoid arthritis, rheumatic arthritis, ankylosing spondylitis and nerve damage.

Images