Anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity as well as preparation method and application of anti-inflammatory peptide

Abstract

The invention discloses an anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity as well as a preparation method and application of the anti-inflammatory peptide. A series of MLIF anti-inflammatory peptide derivatives are obtained through chemical structure modification based on a straight-chain MLIF anti-inflammatory peptide template (MQCNS-NH2), and the MLIF anti-inflammatory peptide derivatives comprise the steps that 1-site amino acid M is replaced into a D type, 2-site amino acid Q is replaced into N-methylation modified Q, 3-site amino acid C is replaced into methyl-heptapolyethylene glycol modified C, or a combination of two groups of the three modifications; the anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity can be prepared through a solid-phase synthesis method, the preparation method is simple and easy to implement, and the obtained product is high in yield and purity. In-vivo and in-vitro experiments prove that the anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity can remarkably inhibit generation of NO, is good in biocompatibility and high in enzyme stability and anti-inflammatory activity, can remarkably relieve osteoarthritis inflammation of mice and improve cartilage injury, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to an anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Osteoarthritis is the most common chronic disease today. Osteoarthritis can cause joint pain and motor dysfunction in patients, resulting in limited physical activity, difficulties in personal life and work, and even difficulty in maintaining family expenses, which has placed a heavy burden on patients, families and society. According to literature reports, the number of people affected by osteoarthritis is increasing worldwide, with more than 300 million osteoarthritis patients, and it is one of the main causes of disability among the elderly. Moreover, with the increasing degree of aging, the prevalence of osteoarthritis has a gradual upward trend, and it is expected to become the largest cause of disability in the general population by 2030. At present, due to the unclear pathogenesis of osteoarthritis and the poor self-healing ability of articular cartilage, the treatment options for this disease are limited. Traditional drug therapy is still the most commonly used treatment option for osteoarthritis, which can only control the symptoms of osteoarthritis and cannot really delay the progression of the disease. In addition, traditional drug treatment is often accompanied by adverse reactions such as liver and kidney damage, gastrointestinal discomfort, and skin bleeding. Therefore, the development of more effective new osteoarthritis treatment drugs with fewer side effects is urgent.

[0003] Peptide drugs, with their advantages of high affinity, high selectivity, and high safety, occupy an important position in drug development and are highly favored by developers. Given their attractive pharmacological characteristics and intrinsic properties, peptides are a good starting point for designing new treatment methods. However, the drugability defects of peptide molecules, such as poor stability, short half-life, and high plasma clearance, seriously restrict their clinical application. It is crucial to obtain peptide analogs with better drugability through a variety of peptide modification technologies. Monocyte locomotion inhibitory factor (MLIF) is a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory activity. It is reported that the natural peptide MLIF has good selective anti-inflammatory effects and can downregulate the expression of pro-inflammatory chemokines (MIP-1α, MIP-1β, and I-309). However, like other natural peptides, the MLIF anti-inflammatory peptide still faces the problems of poor enzyme stability and low anti-inflammatory activity in clinical translation applications. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity and a preparation method and application thereof in view of the shortcomings of MLIF anti-inflammatory peptide such as poor enzyme stability and low anti-inflammatory activity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity, wherein the anti-inflammatory peptide is selected from any one of the following a)-e):

[0007] a) MQCNS-NH2 is used as a peptide chain template, in which amino acid M at position 1 is replaced with D-type, and its chemical structure is as follows:

[0008]

[0009] b) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid Q at position 2 is replaced by an N-methylated Q, and its chemical structure is shown below:

[0010]

[0011] c) MQCNS-NH2 was used as a peptide chain template, wherein the amino acid C at position 3 was replaced by C modified with methyl-heptapeglycol, and its chemical structure is shown below:

[0012]

[0013] d) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid M at position 1 is replaced with a D-type, and the amino acid C at position 3 is replaced with a C modified with methyl-heptapeglycol, and its chemical structure is shown below:

[0014]

[0015] e) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid Q at position 2 is replaced by an N-methylated Q, and the amino acid C at position 3 is replaced by a C modified by methyl-heptapeglyene glycol, and its chemical structure is shown below:

[0016]

[0017] In a second aspect, the present invention provides a method for preparing the anti-inflammatory peptide as described above, comprising the following steps:

[0018] Step 1): using a deprotection reagent to remove the Fmoc protecting group on the amino resin to expose the amino group;

[0019] Step 2): using a condensation reagent to couple the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the amino resin obtained after deprotection in step 1);

[0020] Step 3): using a deprotection reagent to remove the Fmoc protecting group on the amino acid;

[0021] Step 4): Repeat the coupling-deprotection operations described in step 2) and step 3) to synthesize a peptide chain according to the amino acid sequence; wherein, when the amino acid C at position 3 in the anti-inflammatory peptide is a C modified with methyl-heptapeglycol, a methyl-heptapeglycol-bromo-modified Fmoc-Cys-O t Bu compounds, removing O t Bu, to obtain Fmoc-Cys-mPEG7, was condensed to the amino group after asparagine deprotection;

[0022] Step 5): Use a cleavage reagent to cut the peptide chain from the resin, and use ice ether to precipitate to obtain a crude polypeptide;

[0023] Step 6): Separate and purify the crude polypeptide to obtain a pure anti-inflammatory peptide, which is a chemically modified MLIF anti-inflammatory peptide.

[0024] Preferably, the amino resin in step 1) is Rink Amide MBHA resin, and the sample loading amount of the resin is 0.2-0.4 mmol / g;

[0025] And / or, the deprotection reagent in step 1) is a mixed solution of piperidine and DMF, with a volume ratio of 1:3 to 5;

[0026] And / or, in the step 1), the deprotection is carried out by using a deprotection reagent for 5 minutes and then for another 5 minutes; the reaction temperature for removing the Fmoc protecting group is 20-30°C, more preferably 25°C.

[0027] Preferably, the condensing agent used in step 2) is a DIC-Oxyme condensation system, the activating agent is DIC, and DMF is used as the solvent;

[0028] And / or, the temperature of the coupling reaction in step 2) is 50-60° C., more preferably 55° C.; the time of the coupling reaction is 20-30 min, more preferably 20 min.

[0029] More preferably, the ratio of amino acid, oxygen, DIC and DMF in step 2) is 1 mol: 1 mol: 1 mol: 5-7 mL.

[0030] Preferably, the methyl-heptapeglycol-bromo-modified Fmoc-Cys-O t Bu is composed of Fmoc-Cys-O t It is prepared by coupling reaction of Bu and methyl-heptapeptide-bromide under the action of phase transfer catalyst.

[0031] More preferably, the synthesis of the methyl-heptapeglycol-bromo-modified Fmoc-Cys-O tThe reagents used in Bu are tetrabutylammonium iodide (TBAI), methyl-heptapeptide-bromide, Fmoc-Cys-O t Bu, NaHCO3 aqueous solution / EA, the ratio is 1 mol: 0.8~1.2 mol: 3~5 mL: 3~5 mL; then, remove O t The reagents used by Bu are TFA and DCM with a volume ratio of 1:2-4.

[0032] Preferably, the cutting reagent in step 5) is a mixed solution of TIPS, H2O and TFA, and the volume ratio thereof is 2-3:2-3:95; the volume mass ratio of the cutting reagent to the resin is 1 mL:40-60 mg.

[0033] And / or, in said step 5), the cutting temperature is 20-30° C., more preferably 25° C.; the cutting time is 3-5 h, more preferably 4 h.

[0034] Preferably, in step 6), reverse phase high performance liquid chromatography is used for purification and separation, the instrument used for purification and separation is SHIMADZU (LC-6A) reverse phase high performance liquid chromatography (RP-HPLC), the chromatographic column is a C18 column (Daisogel, 20×250 mm), the flow rate is 10 mL / min; the mobile phase buffer A is acetonitrile plus 0.1% TFA, and the mobile phase buffer B is water plus 0.1% TFA; starting from 3% buffer A, linear gradient elution is performed to 0% buffer A within 40 minutes to obtain the target polypeptide.

[0035] In a third aspect, the present invention provides use of the anti-inflammatory peptide as described above in the preparation of anti-inflammatory products.

[0036] Preferably, the anti-inflammatory products include anti-inflammatory drug molecules, anti-inflammatory drug preparations, and anti-inflammatory device applications.

[0037] Preferably, the anti-inflammatory product comprises an anti-osteoarthritis product.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity provided by the present invention is a chemically modified MLIF anti-inflammatory peptide, which can more significantly inhibit the production of NO and has a better anti-inflammatory effect; and the anti-inflammatory peptide after chemical structure modification has high safety for RAW264.7 cells; higher enzyme stability; and after chemical structure modification, it can more significantly reduce osteoarthritis inflammation and improve cartilage damage; therefore, the chemically modified MLIF anti-inflammatory peptide has good clinical application prospects and market value.

[0040] (2) The anti-inflammatory peptide of the present invention is prepared by solid phase synthesis, which is simple and easy to prepare, and the product yield is high and the purity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the sequence of the anti-inflammatory peptide prepared in Example 1 (lowercase letters represent D-type amino acids);

[0042] Figure 2 This is the small molecule synthesis route of Example 1;

[0043] Figure 3 The synthetic route of the anti-inflammatory peptide of Example 1 is shown;

[0044] Figure 4 The H-NMR spectrum (A) and C-NMR spectrum (B) of compound a;

[0045] Figure 5 The H-NMR spectrum (A) and C-NMR spectrum (B) of compound b;

[0046] Figure 6 The high performance liquid chromatography and mass spectrum of the anti-inflammatory peptide MILF;

[0047] Figure 7 The high performance liquid chromatography and mass spectrum of the chemical structure modified MILF anti-inflammatory peptide 8 (MILF derivative 8);

[0048] Figure 8 The high performance liquid chromatography and mass spectrometry of the chemical structure modified MILF anti-inflammatory peptide 14 (MILF derivative 14);

[0049] Fig. 9 The high performance liquid chromatography and mass spectrometry of the chemical structure modified MILF anti-inflammatory peptide 20 (MILF derivative 20);

[0050] Fig.10 The high performance liquid chromatography and mass spectrometry of the chemically modified MILF anti-inflammatory peptide 29 (MILF derivative 29);

[0051] Fig.11 The high performance liquid chromatography and mass spectrum of the chemical structure modified MILF anti-inflammatory peptide 30 (MILF derivative 30);

[0052] Fig.12 This is a graph showing the inhibition of NO production by the anti-inflammatory peptide prepared in Example 1;

[0053] Fig.13 The cell activity diagram of the anti-inflammatory peptide prepared in Example 1;

[0054] Fig.14 This is a serum stability diagram of the anti-inflammatory peptide prepared in Example 1;

[0055] Fig.15 This is a Mankin score chart for the severity of articular cartilage treated with the anti-inflammatory peptide prepared in Example 1;

[0056] Fig.16 This is a graph showing the effect of the anti-inflammatory peptide prepared in Example 1 on the relative expression level of Il-1b mRNA in articular cartilage. DETAILED DESCRIPTION

[0057] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0058] In the following embodiments, the abbreviations involved are explained as follows:

[0059] Fmoc: fluorenylmethoxycarbonyl

[0060] DCM: dichloromethane

[0061] DCE: 1,2-dichloroethane

[0062] DMF: N,N-dimethylformamide

[0063] Oxyme: Ethyl 2-oxime cyanoacetate

[0064] DIC: N,N-diisopropylcarbodiimide

[0065] Cys: Cysteine

[0066] PEG: polyethylene glycol

[0067] TBAI: Tetrabutylammonium iodide

[0068] NaHCO3: Sodium bicarbonate

[0069] TFA: trifluoroacetic acid

[0070] TIPS: Triisopropylsilane

[0071] RAW 264.7: Mouse mononuclear macrophage leukemia cells

[0072] %: Unless otherwise specified in the present invention, all refer to mass percentage.

[0073] The sources of experimental materials involved are as follows:

[0074] Amino acids, Rink Amide MBHA resin, and ethyl 2-oximecyanoacetate (Oxyme) were purchased from Shanghai Jier Biochemical Co., Ltd.; N,N-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), and acetonitrile (chromatographic grade) were purchased from Beijing Bailingwei Technology Co., Ltd.; N,N-dimethylformamide (DMF), isopropyl ether, dichloromethane (DCM), and piperidine were all analytically pure and purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.

[0075] Example 1

[0076] This example provides an anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity, and the preparation method thereof is as follows:

[0077] 1. Synthesis of small molecules (such as Figure 2 (shown)

[0078] Preparation of compound a

[0079] (Fmoc-Cys-O t Bu)2 (10 g, 12.5 mmol) was dissolved in DCM (200 mL), and then dithiothreitol (DTT, 2.89 g, 18.75 mmol) and triethylamine (NEt3, 2.6 mL, 18.75 mmol) were added. The reaction was stirred at room temperature for 3 hours to completely break the disulfide bond. The residue was dissolved in DCM, washed with water three times, collected and dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by PE / EA (2 / 1) silica gel column chromatography to obtain compound a, and its characterization results are shown as follows: Figure 4 shown.

[0080] (2) Preparation of compound b

[0081] Compound a (2.0 g, 2.5 mmol) was dissolved in ethyl acetate (EA, 10 mL), and an equal volume of saturated sodium bicarbonate solution (10 mL) was added to mix. Then tetrabutylammonium iodide (TBAI, 3.69 g, 10 mmol) and methyl-heptapyrrolidone (1.0 g, 2.5 mmol) were added and stirred gently at room temperature overnight. The residue was dissolved in EA, washed three times with water, dried over anhydrous Na2SO4 after collection, filtered and concentrated under reduced pressure. The crude product was purified by PE / EA (2 / 1) silica gel column chromatography to obtain compound b, and its characterization results are shown as follows: Figure 5 shown.

[0082] 2. Synthesis of peptides (such as Figure 3 (shown)

[0083] (1) Preparation of Compound 1

[0084] Take 500 mg of amino resin (loading amount is 0.30 mmol / g) and add it to the solid phase synthesis reaction tube, soak it in DCM for 20 min to make the resin fully swell, and then drain it for use.

[0085] Add 20% (v / v) piperidine-DMF solution until the resin is completely submerged, shake at 25°C for 5 min×2 to remove Fmoc on the resin, and wash the resin with DCM and DMF for 3 times each.

[0086] (2) Preparation of Compound 2

[0087] The first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol) and DIC (155 μL, 1 mmol) were mixed and dissolved in 6 mL DMF, added to the resin and shaken at 60° C. for 20 min. The resin was washed with DCM and DMF three times each.

[0088] (3) Preparation of Compound 3

[0089] Repeat steps (1) and (2), deprotection → condensation → deprotection, until all amino acids are connected. After the last amino acid is deprotected, wash the resin with DCM and DMF three times each.

[0090] (4) Preparation of target compound

[0091] Wash the resin and drain it, add 10 mL of TIPS:H2O:TFA=2.5:2.5:95 (V / V / V), shake at room temperature for 4 hours, filter, wash the resin with a little TFA, and collect the filtrate. Use argon bubbling to blow away the excess TFA, pour into ice isopropyl ether for precipitation and centrifugation, discard the supernatant, continue to wash repeatedly with ice ether and centrifuge for 3 times, and blow dry with argon to obtain the crude polypeptide.

[0092] 3. Purification of target anti-inflammatory peptides

[0093] The crude peptide was dissolved in acetonitrile and water and purified by preparative RP-HPLC. The separation conditions were as follows:

[0094] Instrument: SHIMADZU (LC-6A) reverse phase high performance liquid chromatography (RP-HPLC)

[0095] Chromatographic column: C18 column (Daisogel, 20×250mm)

[0096] Mobile phase: Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA;

[0097] Steps and parameters: starting from 10% buffer A, linear gradient elution to 75% buffer A within 50 minutes, flow rate of 10 mL / min, injection volume of 5 mL, detection wavelength of 214 nm.

[0098] Through the above steps, this embodiment synthesized Figure 1 The polypeptide molecules shown.

[0099] Example 2: Identification and purity analysis of products

[0100] The product obtained in Example 1 was subjected to purity analysis by HPLC and molecular weight identification by ESI-MS. ESI-MS was measured on a Shimadzu LCMS-8040 mass spectrometer. Scanning mode: positive ion; measurement time: 0-2 min; measurement range: 400-2000; data acquisition time: 2 min; total flow rate: 0.2 mL / min; buffer A is water plus 0.1% formic acid, and buffer B is acetonitrile plus 0.1% formic acid. 80% of buffer B was maintained for 2 min for compound analysis. The peak time was consistent with that of the main peak of the crude product, and the purity of the polypeptide prepared by this method was >98%. The analysis results of HPLC and ESI-MS mass spectrometers are as follows: Figure 6-11 shown.

[0101] Example 3: Cultivation of RAW264.7 cells

[0102] RAW 264.7 cells were obtained from Shanghai Institute of Biological Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 100 μg / mL penicillin-streptomycin (Thermo Scientific, Waltham, MA, USA). Cells were cultured in a humidified incubator at 37 °C with 5% CO2. The medium was changed every other day until the cells grew confluent. Cells were then digested with 0.25% trypsin-EDTA for cell splitting or experiments.

[0103] Example 4: Determination of anti-inflammatory activity of anti-inflammatory peptides

[0104] The density is 5×10 5RAW 264.7 cells at 1 μg / mL were seeded in a 96-well plate and cultured with DMEM for 24 h. MLIF and its derivatives were dissolved in PBS. Then, 1 μg / mL MLIF or MLIF derivatives were used to act on the cells with DMEM at equimolar concentrations for 24 h. The supernatant was taken, washed with PBS three times, and 1 μg / mL DMEM without LPS serum was added for 6 h. The cells in the control group were treated with PBS. The NO level was detected using the NO fluorescent probe DAF-FM Diacetate (AAT Bioquest, USA). GraphPadPrism 8.4.2 was used for statistical evaluation, using one-way analysis of variance and Bonferroni's post hoc test. The statistical significance was *p<0.05, **p<0.01, ***p<0.001. It can be seen from the results that the chemically structure-modified MLIF anti-inflammatory peptide of the present invention has a stronger effect in inhibiting the production of NO than MLIF. The results are as shown in Fig.12 shown.

[0105] Example 5: Determination of the cellular activity of the anti-inflammatory peptide prepared in Example 1

[0106] The density is 5×10 5 RAW 264.7 cells at 1 μg / mL were seeded in 96-well plates and cultured for 24 h. The MLIF and MLIF derivative groups were pretreated with 1 μg / mL equimolar concentration of MLIF or MLIF derivatives for 24 h. The control group treated cells with PBS. Then CCK-8 reagent was added to each well and incubated with the cells for 30 min. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, USA), and the cell viability percentage was calculated as follows: Cell viability (%) = (Abspeptide-Absblank) / (Abscontrol-Absblank) × 100%, where Abscontrol is the absorbance of cells treated with PBS and Absblank is the absorbance of cell-free culture medium to evaluate cell viability. The determination was performed in three independent experiments, and the standard error (SD) is shown in the histogram. Statistical evaluation was performed using GraphPad Prism 8.4.2, using one-way analysis of variance and Bonferroni's post hoc test. The statistical significance is *p<0.05, **p<0.01, ***p<0.001. The results show that the chemical structure modified MLIF anti-inflammatory peptide of the present invention has good biocompatibility. Fig.13 shown.

[0107] Example 6: Determination of the stability of the anti-inflammatory peptidase prepared in Example 1

[0108] The serum stability of the anti-inflammatory peptides was detected by reverse phase high performance liquid chromatography (RP-HPLC). A 2 mg / mL peptide solution was prepared in water and incubated with human serum at a final volume ratio of 1:4 at 37°C. Sampling was performed at different time intervals, and 50 μL of the mixture was added to 50 μL of acetonitrile to terminate the enzymatic hydrolysis. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant was collected and analyzed by HPLC at a wavelength of 214 nm. The integrated peak area of ​​the intact peptide was recorded, and the percentage of the remaining peptide was calculated based on the integrated area, as shown below: Remaining amount of polypeptide (%) = peak area of ​​remaining polypeptide / peak area of ​​intact polypeptide × 100%. As can be seen from the results, the MLIF derivatives 14, 20, 29, and 30 described in the present invention have higher serum stability than MLIF, as shown in the following. Fig.14 shown.

[0109] Example 8: In vivo efficacy experiment of the anti-inflammatory peptide prepared in Example 1 on cartilage inflammation and damage

[0110] All animal experiments and surgical procedures were approved by the Animal Care and Use Committee of Shanghai University (ECSHU2024-003) and followed the international ethical guidelines for the use of experimental animals. C57BL / 6j male mice, weighing 20-25 g, were purchased from Cavens Laboratory Animal Company, Changzhou, China. After anesthesia, the medial skin of the right knee joint was incised longitudinally to expose the medial edge of the patellar tendon. The knee joint was flexed to 45°, and 10 μL PBS solution containing 1 μL type II collagenase was vertically injected using a microbore tube. The incision was sutured. Collagenase-induced osteoarthritis (CIOA) mice were obtained by continuous administration of collagenase on days 0, 3, and 9, and the sham operation group was injected with PBS. The obtained CIOA mice were randomly divided into 8 groups, with 5 mice in each group. Mice in each group were injected with MLIF or MLIF derivatives at a dose of 1 mg / kg, and the sham operation group and control group were injected with PBS. The treatment was repeated 3 times on days 0, 3, and 9. All mice were killed on day 14, and the articular cartilage was collected. The mice were killed with an overdose of 4% pentobarbital (Merck KGaA, Darmstadt, Germany). The results show that the polypeptides 29 and 30 of the present invention significantly reduce osteoarthritis inflammation in mice and significantly improve cartilage damage compared to MLIF. Fig.15 , 16 shown.

[0111] The above examples show that the present invention successfully prepared a derivative based on the straight-chain template peptide MQCNS-NH2 (chemically modified MLIF anti-inflammatory peptide), and proved that the chemically modified MLIF anti-inflammatory peptide of the present invention can significantly inhibit the production of NO, has high biocompatibility, high serum stability, and good anti-inflammatory activity, can significantly reduce osteoarthritis inflammation in mice, improve cartilage damage, and has good application prospects.

[0112] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. An anti-inflammatory peptide with high enzyme stability and anti-inflammatory activity, characterized in that: The anti-inflammatory peptide is selected from any one of the following a)-e): a) MQCNS-NH2 is used as a peptide chain template, in which amino acid M at position 1 is replaced with D-type, and its chemical structure is as follows: b) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid Q at position 2 is replaced by an N-methylated Q, and its chemical structure is shown below: c) MQCNS-NH2 was used as a peptide chain template, wherein the amino acid C at position 3 was replaced by C modified with methyl-heptapeglycol, and its chemical structure is shown below: d) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid M at position 1 is replaced with a D-type, and the amino acid C at position 3 is replaced with a C modified with methyl-heptapeglycol, and its chemical structure is shown below: e) MQCNS-NH2 is used as a peptide chain template, wherein the amino acid Q at position 2 is replaced by an N-methylated Q, and the amino acid C at position 3 is replaced by a C modified by methyl-heptapeglyene glycol, and its chemical structure is shown below:

2. The method for preparing the anti-inflammatory peptide according to claim 1, characterized in that: The following steps are involved: Step 1): using a deprotection reagent to remove the Fmoc protecting group on the amino resin to expose the amino group; Step 2): using a condensation reagent to couple the carboxyl group of the Fmoc-protected amino acid with the exposed amino group on the amino resin obtained after deprotection in step 1); Step 3): using a deprotection reagent to remove the Fmoc protecting group on the amino acid; Step 4): Repeat the coupling-deprotection operations described in step 2) and step 3) to synthesize a peptide chain according to the amino acid sequence; wherein, when the amino acid C at position 3 in the anti-inflammatory peptide is a C modified with methyl-heptapeglycol, a methyl-heptapeglycol-bromo-modified Fmoc-Cys-O t Bu compounds, removing O t Bu, to obtain Fmoc-Cys-mPEG7, was condensed to the amino group after asparagine deprotection; Step 5): Use a cleavage reagent to cut the peptide chain from the resin, and use ice ether to precipitate to obtain a crude polypeptide; Step 6): Separate and purify the crude polypeptide to obtain a pure anti-inflammatory peptide.

3. The preparation method according to claim 2, characterized in that: The amino resin in step 1) is Rink Amide MBHA resin, and the sample loading amount of the resin is 0.2-0.4 mmol / g; And / or, the deprotection reagent in step 1) is a mixed solution of piperidine and DMF, with a volume ratio of 1:3 to 5; And / or, in the step 1), the deprotection is carried out by using a deprotection reagent for 5 minutes and then for another 5 minutes; the reaction temperature for removing the Fmoc protecting group is 20-30°C, more preferably 25°C.

4. The preparation method according to claim 2, characterized in that: The condensing agent used in step 2) is a DIC-Oxyme condensation system, the activating agent is DIC, and DMF is used as the solvent; And / or, the temperature of the coupling reaction in step 2) is 50-60° C., more preferably 55° C.; the time of the coupling reaction is 20-30 min, more preferably 20 min.

5. The preparation method according to claim 4, characterized in that: The ratio of amino acid, oxygen, DIC and DMF in the step 2) is 1 mol: 1 mol: 1 mol: 5-7 mL.

6. The preparation method according to claim 2, characterized in that: The methyl-heptapeptide-bromo-modified Fmoc-Cys-O t Bu is composed of Fmoc-Cys-O t It is prepared by coupling reaction of Bu and methyl-heptapeptide-bromide under the action of phase transfer catalyst.

7. The preparation method according to claim 2, characterized in that: The cutting reagent in step 5) is a mixed solution of TIPS, H2O and TFA, and the volume ratio thereof is 2-3:2-3:95; the volume mass ratio of the cutting reagent to the resin is 1 mL:40-60 mg. And / or, in step 5), the cutting temperature is 20-30° C., and the cutting time is 3-5 hours.

8. The preparation method according to claim 2, characterized in that: In the step 6), reverse phase high performance liquid chromatography is used for purification and separation.

9. Use of the anti-inflammatory peptide according to claim 1 in the preparation of anti-inflammatory products.

10. The use according to claim 9, characterized in that The anti-inflammatory products include anti-inflammatory drug molecules, anti-inflammatory drug preparations and anti-inflammatory devices; And / or, the anti-inflammatory effect includes anti-osteoarthritis.

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