CN113968896A - Application of small peptide in preparation of osteoarthritis treatment drug - Google Patents

Abstract

Translated fromChinese

本发明公开了一种小肽在制备环氧化酶‑2抑制剂和治疗骨关节炎药物中的用途，该小肽来源于龟板，其氨基酸序列为N‑Q‑G‑X‑B、K‑N‑G‑P、W‑G‑X‑B中的任一种，该小肽为首次从中药龟板中分离鉴定，为充分利用龟板药材提供了新途径，同时也为相关药物的开发提供了新资源。

The invention discloses the use of a small peptide in the preparation of a cyclooxygenase-2 inhibitor and a drug for treating osteoarthritis. The small peptide is derived from turtle shell, and its amino acid sequence is N-Q-G-X-B, K Any of ‑N‑G‑P, W‑G‑X‑B, this small peptide is the first time to be isolated and identified from the traditional Chinese medicine turtle shell, providing a new way to make full use of turtle shell medicinal materials, and also provide the development of related drugs. new resources.

Description

Application of small peptide in preparation of osteoarthritis treatment drug

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to application of small peptide in treating osteoarthritis.

Background

Osteoarthritis is a chronic degenerative disease that includes articular cartilage loss, subchondral bone sclerosis, osteophytes and low-grade synovitis, meniscal and subgallal fat pad damage. In severe cases, joint deformity and mobility impairment may occur. Modern medicine is mostly treated with anti-inflammatory analgesics such as non-steroidal anti-inflammatory drugs and weak opioids. However, since these drugs can only relieve pain with different symptoms, but cannot effectively prevent articular cartilage degeneration, long-term medication is not good for long-term health, and nonsteroidal anti-inflammatory drugs have potential cardiovascular and gastrointestinal bleeding risks, and patients resist medication, so that the curative effect is poor, and the daily normal activity needs of people are seriously affected. Therefore, the search for safe and effective protective agents or natural preparations for preventing, delaying or even reversing the onset of osteoarthritis is urgently needed.

The tortoise plastron is a traditional Chinese medicine in China, has the functions of nourishing yin, suppressing hyperactive yang, strengthening liver and kidney, resisting and preventing cancers, and has better prevention and treatment effects on yin deficiency, hectic fever, bone steaming, fatigue, dysphoria, hypertension, cerebral apoplexy and other diseases. Some tortoise extracts have good antihypertensive and antibacterial effects. Proteins and peptides extracted from Chinemys reevesii have been shown to have antioxidant and immune system enhancing effects in animal experiments. However, the tortoise plastron peptide has been less studied for anti-inflammatory and its mechanism of action, and its effect on osteoarthritis is unclear. However, researches show that the tortoise-shell glue can delay the degeneration of articular chondrocytes and delay the pathological process of osteoarthritis. In addition, the testudinate peptide molecule is an effective component for treating bone diseases and can treat osteoporosis. However, it has not been confirmed whether the tortoise plastron peptide is effective for arthritis.

The invention extracts the tortoise plastron peptide, and then makes a large amount of analysis and verification on the composition and function of peptide segments in the tortoise plastron peptide, so as to finally obtain the small peptide with better therapeutic action on osteoarthritis.

Disclosure of Invention

The invention aims to provide application of small peptide in preparing a medicament for treating osteoarthritis.

The above purpose is realized by the following technical scheme:

the applicant firstly extracts and separates biological active peptide from traditional Chinese medicine tortoise plastron, and then screens out small peptide with stronger COX-2 enzyme inhibitory activity from the large amount of tests such as liquid chromatography-mass spectrometry, sequence comparison, molecular docking and in vitro enzyme activity inhibition experiments, wherein the amino acid sequence of the small peptide is N-Q-G-X-B (SEQ ID No.1) or K-N-G-P (SEQ ID No.2) or W-G-X-B (SEQ ID No. 3). The small peptide is separated and identified from the traditional Chinese medicine tortoise plastron for the first time, provides a new way for fully utilizing tortoise plastron medicinal materials, and simultaneously provides a new resource for the development of related medicines.

The small peptide can be extracted and separated from tortoise plastron or can be synthesized by using an artificial method well known in the field. When the preparation is carried out by using an extraction separation method, it is necessary to separate a target peptide fragment from the polypeptide obtained by extraction, and the separation of the target peptide fragment can be carried out by using a chromatography method and the like well known in the art, for example, a semi-preparative or preparative liquid chromatography, and the skilled person does not need to overcome technical obstacles in using these separation methods based on the molecular structure of the target peptide fragment being clarified.

When the small peptide is prepared by an artificial synthesis method, the small peptide also has the advantages of simple synthesis process, short period, low cost and the like due to small molecular weight.

The results of in vivo animal experiments show that: the small peptide provided by the invention has no obvious influence on the body weight of the rat, which indicates that the small peptide does not damage the normal growth of the rat. However, the small peptide can obviously improve the cartilage defect and the surface state of the knee joint, which shows that the small peptide has a treatment effect on the bone joint injury; by analyzing the content of CTX-II, COMP and joint fluid TNF-alpha in serum, the small peptide is found to be capable of relieving osteoarthritis symptoms, delaying the degradation and degeneration of joint cartilage, and has mild effect and small side effect.

In conclusion, the small peptide provided by the invention can not only exert the inhibitory activity on COX-2 enzyme in vitro, but also exert the therapeutic action on osteoarthritis in vivo, so that the small peptide can be applied to the preparation of COX-2 inhibitors or osteoarthritis treatment drugs, and has wide application prospects.

Drawings

FIG. 1: effect of small peptides on the trend of body weight change in rats.

FIG. 2: effect of small peptides on the CTX-II content in rat serum.

FIG. 3: effect of small peptides on COMP content in rat serum.

FIG. 4: effect of small peptides on TNF- α content in rat synovial fluid.

Detailed Description

The present invention is further illustrated by the following specific examples.

EXAMPLE 1 preparation of the polypeptide

The total protein in the tortoise plastron is extracted by means of decoction, extraction, concentration, filtration and the like, the contents of fat-soluble and other small molecule water-soluble components such as minerals and the like in the extract are reduced, and then the polypeptide is obtained by carrying out enzymolysis on the total protein, and the specific method is as follows:

1. extraction of Total protein

(1) Killing the small water turtle in hot water, removing viscera, blood and scales, cleaning and crushing tortoise plastron;

(2) soaking the crushed tortoise plastron in 4% acetic acid for 1 hr, cleaning, decocting in a pot for 3 times, each time for 3 hr;

(3) mixing the decoctions obtained by decocting for 3 times, filtering, rotary evaporating at 55 deg.C, and concentrating until no condensate flows out;

(4) extracting the concentrate with petroleum ether for three times to remove fat-soluble impurities, and filtering with dialysis bag to remove part of water-soluble micromolecules;

(5) freeze drying in vacuum freeze drying oven, pulverizing, adding into 0.6moL/L EDTA solution at 6% addition, stirring for 30min to remove calcium bonded in protein, filtering, and freeze drying the filtrate to obtain crude protein extract.

2. Preparation of polypeptides

(1) Weighing the crude protein extract, and adding NaOH solution with pH of 7.5 at a solid-to-liquid ratio of 1:25 (g/mL);

(2) adding alkaline protease to make the concentration of enzyme in the solution be 1.2%, and carrying out water bath enzymolysis for 6h at the constant temperature of 55 ℃;

(3) then putting the mixture into a hot water bath at 95 ℃, and continuously stirring for 15 minutes to inactivate the enzyme;

(4) centrifuging at 4 ℃ at 10000r/min for 10 minutes, and collecting centrifugate;

(5) filtering the centrifugate with ultrafiltration membrane with cut-off molecular weight less than 6000D to remove inactivated enzyme and unhydrolyzed macromolecular protein, rotary evaporating for concentration, freeze-drying in vacuum freeze drying oven, and pulverizing to obtain polypeptide powder.

Example 2 identification and functional verification of peptide fragments

(1) Identification of main peptide segment of tortoise plastron peptide

The tortoise plastron peptide prepared in example 1 was analyzed by high performance liquid chromatography-electrospray mass spectrometry-mass spectrometer (HPLC-ESI-QqTOF-MS/MS), and the peptide solution was separated on an Inter sustatin AQ-C18(250 mm. times.4.6 mm, 5 μm) chromatographic column at a flow rate of 0.8 mL/min; mobile phase: acetonitrile + 0.1% trifluoroacetic acid (phase B), water + 0.1% trifluoroacetic acid (phase A), concentration 10mg/mL,sample volume 8. mu.L,column temperature 30 ℃, detection wavelength 215 nm. Gradient elution conditions: 0-9 min, 0% -9% B; 9-12 min, 9% -13% B; 12-20 min, 13% -26% of B; 20-60 min, 26-70% of B.

The mass spectrum conditions are as follows: ESI + ion source; atomizer pressure was 40.00 psi; capillary voltage, 3500 v; dry gas flow, 10.0L/min; the temperature of dry gas is 325 ℃; m/z is 100-1500, and the spectrum is scanned. The mass spectrum scanning range is set to be between m/z100 and 1500. ESI-MS and MS/MS analysis are carried out by adopting PeakView 2.2 analysis software, the structure of the Chinemys reevesii tortoiseshell peptide is determined, and 5 peaks and 9 peptide segments are identified in total. Specific peptide fragment information is shown in the table below.

Mass spectrometry of small water turtle tortoise plastron peptide

(2) Molecular docking screening functional peptide fragment

Cyclooxygenase-2 (COX-2) has very low activity in normal synovial fluid, and is almost expressed or weakly expressed, and studies such as Jinrongzhi show that COX-2 is expressed in KOA cartilage tissue. After COX-2 is induced to generate, a large amount of prostate E can be generated, and the prostate E and the COX-2 jointly participate in inflammatory reaction, so that not only can the synthesis of type II collagen in extracellular matrix be inhibited, but also the proliferation of chondrocytes be inhibited, the apoptosis of the chondrocytes be promoted, the damage of cartilage matrix is accelerated, and finally the degenerative change of joint cartilage is caused.

Reference documents: study on papain-induced cartilage repair mechanism of rat knee osteoarthritis model by Zhangsheng, dampness resolving and kidney tonifying method [ D ]. Fujian university of traditional Chinese medicine, 2019.

Experimental study of jojun jun yang harming decoction on papain-induced synovial inflammation in rabbit knee osteoarthritis model [ D ]. department of fujian university of traditional Chinese medicine, 2019.)

Preparing target protein molecules.

The receptor was first selected by searching the COX-2 crystal structure from the protein database (http:// www.rcsb.org/pdb). The active site of the COX-2 enzyme with its own ligand serves as the docking site. Removing water molecules carried by the Protein, repairing the problems of side chain deletion, nonstandard amino acid residue naming, inconsistent Protein conformation, unrepaired amino acid residues and protonation state thereof, main chain end defect and the like in COX-2 enzyme by using a Clean Protein tool under Macromolecules module in Discovery Studio 2018, and storing the target Protein in a mol2 format.

Preparing ligand small molecules.

Molecular structure name of docking ligand: J-H, K-N-G-P, N-Q-G-X-B, R-G, W-G-P-G-B, W-G-X-B, W-W-Y-W-R, W-R-W-X-H-T-H-N-W, W-N-A-R-W-P-G-J, and chemdraw is used for drawing A structure.

Preparing the molecular surface file of the target protein molecule after preparing the two basic docking molecules, simultaneously preparing the PDB file of the target protein molecule, and then generating the surface file of the target protein molecule by utilizing dms.

And fourthly, adopting a semi-flexible CDOCKER docking method that the receptor in the Discovery Studio 2018 is rigid and the ligand is flexible. First, the pretreated protein structure was defined as the Receptor molecule using the Define and edition Binding Site tool under the Receptor-Ligand Interactions module. The binding sites of the self-contained ligands in the protein crystal structure were defined as active spheres and adjusted to some extent using the Define Site tool.The size and the position of the active sphere are adjusted, so that the situation that the active sphere is too small and cannot wrap ligand molecules to cause the failure of optimization of the active sphere at an active site is avoided, and the situation that the docking result is inaccurate due to the deviation of the docking position caused by the overlarge active sphere is also avoided. Finally determining the diameter of the active sphere to be

The activity sphere coordinates of COX-2 enzyme are (22.00, 35.76, 13).

Utilizing a Docking Optimization tool under a Receptor-Ligand Interactions module to open the CDOCKER Docking module to run the Docking process.

Molecular docking results:

generally, lower energy indicates lower energy requirements for total energy and interaction between the protein and the small molecule ligand, more stable docking systems and tighter binding states, generally based on total energy. The results show that the three peptide fragments N-Q-G-X-B, K-N-G-P, W-G-X-B have better effect on COX-2 enzyme.

(2) In vitro enzyme activity inhibition assay

(ii) test method

COX-2 enzyme was prepared in 500U/mL solutions using 0.1mol/L Tris-HCl buffer at pH 8.0. mu.L of a sample of the K-N-G-P, N-Q-G-X-B, W-G-X-B peptide fragment was mixed with 110. mu.L of COX-2 enzyme solutions of different concentrations and incubated for 5min at 25 ℃. To the mixture was added 20. mu.L of tetramethylp-phenylenediamine (TMPD) and 20mL of Arachidonic Acid (AA) solution to start the reaction. After 20 minutes of reaction at 25 ℃, the absorbance values were recorded at 610nm using a microplate reader. All experiments were repeated three times. The inhibition rate was calculated using the following formula:

in the formula, A_controlIs the absorbance of Tris-HCL without sample; a. the_sampleIs the absorbance of the sample; a. the_controlblankIs the absorbance of Tris-HCL without sample and enzyme; a. the_sampleBankIs the absorbance of the sample without enzyme. Finally, the half maximal Inhibitory Concentration (IC) of the inhibitor was calculated by SPSS software₅₀) The value is obtained.

Results of the test

IC₅₀Smaller values indicate stronger inhibition. N-Q-G-X-B showed the strongest inhibitory effect (IC) on COX-2 enzyme among 3 peptide fragments₅₀0.045mg/mL), followed by K-N-G-P (IC)₅₀＝0.26mg/mL)、W-G-X-B(IC₅₀0.39mg/mL) consistent with the results of the molecular docking studies.

Example 3 in vivo assay to analyze the effects of Chinemys reevesii tortoise plastron peptide on osteoarthritis of rat knee

Test method 1

(1) Design of experiments

The experimental animal is from SPF SD male rat in research center of experimental animals in Hubei province, and the body weight is 200 +/-10 g. During the experiment, the temperature of the rat breeding environment is controlled to be 25 +/-10 ℃, the humidity is controlled to be 40 +/-10%, the light and shade period is 12h, the rat breeding environment is standard in a pathogen-free environment, and water and food are freely drunk. After 1 week of acclimatization, 50 rats were randomized into 5 groups of 10 rats each: normal (Normal) group (intra-articular injection of 0.15mL of Normal saline, intragastric administration of Normal saline on days 14, 17 and 20); knee arthritis (KOA) group (4%, 0.09mL of papain and 0.3mol/L, 0.06mL of cysteine mixed solution was injected intra-articularly 0.15mL, on days 14, 17 and 20, gastric lavage with physiological saline 45); N-Q-G-X-B group (on days 14, 17 and 20, 4%, 0.09mL of papain, 0.3mol/L and 0.06mL of cysteine mixed solution 0.15mL are injected into joints, and 45mg/kg/d of gastric lavage N-Q-G-X-B is injected); W-G-X-B group (on days 14, 17 and 20, 4%, 0.09mL of papain, 0.3mol/L and 0.06mL of cysteine mixed solution 0.15mL are injected in joints, and 45mg/kg/d of intragastric W-G-X-B is perfused); in the K-N-G-P group, on the 14 th, 17 th and 20 th days, 4 percent, 0.09mL of papain, 0.3mol/L of 0.06mL of cysteine mixed solution, 0.15mL are injected into joints, and the K-N-G-P is perfused at 45 mg/kg/d).

All rats were fasted 1h before the beginning of gavage, and the gavage volume of the rats was calculated from the body weight of the mice on the day on an empty stomach 1h before the experiment, and the body weight was recorded at 10 am each day. The model is made on the 14 th, 17 th and 20 th days, and all groups are anesthetized by 10% chloral hydrate in the abdominal cavity before the model is made. One day before the experiment, the rats were fasted for more than 12h without water treatment. On day 38 of the experiment, 10% chloral hydrate was used for anesthesia, the eyeball was bled, and the neck was cut off to be sacrificed.

(2) Body weight index

Weigh and record body weight daily before gavage.

(3) Blood and tissue extraction

Firstly, taking serum: naturally coagulating the rat blood sample for 1h at room temperature, centrifuging at 3000r/min for 10min at 4 deg.C, and collecting the supernatant to obtain the experimental serum sample. Subpackaging and storing at-80 deg.C for testing. Before measurement, the sample is naturally thawed at 4 ℃ and then used, so that repeated freeze thawing is avoided.

Collecting joint fluid: separating the knee joint layer by layer to the muscle, taking the upper edge of the patella as a positioning, making a transverse incision on the upper edge of the patella, observing the condition in the joint by naked eyes, including the color and the amount of joint fluid, clamping the patella by using a toothed forceps, slightly lifting and pulling the patella upwards, making a longitudinal incision to the upper part of the femoral condyle along the inner side and the outer side of the knee joint respectively, flexing the knee joint of a rat, extracting physiological saline by using an injector to clean the joint, and storing the obtained joint fluid in an EP (EP) tube.

(4) Macroscopic observation of cartilage tissue

Observing color, tissue state, and cartilage morphology change

(5) ELISA detects the content of the carboxyl-terminal peptide (CTX-II) of the type II collagen and the Cartilage Oligomeric Matrix Protein (COMP) in serum and the content of inflammatory cytokine (TNF-alpha) in joint fluid.

First, the standard is diluted, 5 centrifuge tubes of 1.5mL are prepared, and marked. Adding 150 mu L of standard substance diluent into each centrifuge tube, adding 150 mu L of original-time standard substance into the first test tube, uniformly mixing, sucking 150 mu L of standard substance out by using a sample injector, transferring to the second test tube, and repeating the steps to obtain standard solutions with different dilution times;

sample adding: and blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged. The standard sample is accurately loaded by 50 mu L on the enzyme-labeled coating plate, 40 mu L of sample diluent is loaded in the sample hole to be detected, and then 10 mu L of sample to be detected is loaded (the final dilution of the sample is 5 times). And adding the sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and mixing the sample and the enzyme label plate.

And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.

Fourthly, preparing the liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.

Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

Sixthly, adding enzyme: 50 μ L of enzyme-labeled reagent was added to each well, except for blank wells.

And seventhly, incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.

And (b) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.

Ninthly, color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, gently shaking, mixing, and developing at 37 deg.C in dark for 10-20 min.

R terminates: the reaction was stopped by adding 50. mu.L of a stop solution to each well (blue color turned to yellow color), and the absorbance (OD value) of each well was measured in order at a wavelength of 450nm by zeroing the blank well.

2. Test results

(1) Body weight

The test result shows that the weight of rats in each group has obvious weight increment and shows a normal growth trend. Results analysis that the rats belong to normal development increase, and the weight of the peptide group of the small water turtle is higher than that of the other groups, which indicates that the tortoise plastron peptide contributes to the growth and development of the rats. The results are shown in FIG. 1.

(2) Macroscopic observation of cartilage tissue

Normal (Normal) group: the cartilage of the medial condyle of the femur joint is bright red in color and smooth in surface, and no cartilage destruction or osteophyte formation exists.

Group of gonarthritis (KOA): the medial femoral condyle cartilage was dull, partially uneven, and cartilage defects and osteophyte formation were seen.

③ W-G-X-B group: the cartilage surface of the medial condyle of the femur is not glossy and has unevenness in the local area, and cartilage defect and osteophyte formation are observed.

Group K-N-G-P: the medial femoral condyle cartilage surface was less smooth, with mild cartilage loss and osteophyte formation seen, to a significantly lesser extent than the model group.

N-Q-G-X-B group: the medial femoral condyle cartilage surface is less smooth with no macroscopic cartilage defects and osteophyte formation.

The results show that the Chinemys reevesii tortoise plastron peptide is beneficial to restoring rat cartilage tissue damage, and the peptide segment N-Q-G-X-B has the best effect.

(3) Content of CTX-II and COMP in serum

CTX-II and COMP levels can be used as useful indicators for early diagnosis and prediction of osteoarthritis. The content of CTX-II and COMP in the N-Q-G-X-B, K-N-G-P, W-G-X-B group is obviously lower than that in the model group, which shows that the peptide fragments are helpful for reducing the content of CTX-II and COMP, thereby relieving the symptoms of arthritis. The experimental results are shown in FIGS. 2 and 3.

(4) Content of synovial fluid TNF-alpha

TNF-alpha is directly involved in the onset of knee osteoarthritis, selectively inhibits the production of cartilage collagen, reduces proteoglycan synthesis, and destroys articular cartilage. The TNF- α levels in the model group were higher than those in the blank control group and other groups, confirming the presence of abnormal secretion of the above cytokines in rats. The small water turtle tortoise plastron peptide can achieve the effect of relieving knee osteoarthritis symptoms by inhibiting the release of inflammatory factor TNF-alpha, and delay the degradation and degeneration of joint cartilage. The results of the experiment are shown in FIG. 4.

<110> university of agriculture in Huazhong

Application of small peptide in preparation of osteoarthritis treatment drug

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Claims (4)

Translated fromChinese

1.一种小肽在制备环氧化酶-2(COX-2)抑制剂中的用途，其特征在于：所述小肽来源于龟板，其氨基酸序列为N-Q-G-X-B、K-N-G-P、W-G-X-B中的任一种。1. the purposes of a small peptide in the preparation of cyclooxygenase-2 (COX-2) inhibitor, it is characterized in that: described small peptide is derived from turtle shell, and its amino acid sequence is any in N-Q-G-X-B, K-N-G-P, W-G-X-B kind.2.权利要求1中所述的小肽在制备治疗骨关节炎药物中的用途。2. Use of the small peptide described in claim 1 in the preparation of a medicament for the treatment of osteoarthritis.3.如权利要求1或2所述的用途，其特征在于：所述龟板为石金钱龟龟板。3. purposes as claimed in claim 1 or 2, is characterized in that: described tortoise plate is stone money turtle tortoise plate.4.一种环氧化酶-2抑制剂或治疗骨关节炎药物，其活性成分是权利要求1中所述的小肽。4. A cyclooxygenase-2 inhibitor or a drug for treating osteoarthritis, the active ingredient of which is the small peptide described in claim 1.

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