CN116327883A - Application of antibacterial peptide in preparation of arthritis treatment medicine - Google Patents
Application of antibacterial peptide in preparation of arthritis treatment medicine Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the field of medicines, in particular to application of antibacterial peptide in preparation of medicines for treating arthritis. The invention separates the strain GBacillus-9 from the intestinal flora of the striped bamboo shark, and carries out induction expression and purification on the main antibacterial peptide component to prepare the antibacterial peptide preparation. By constructing an arthritis model of a rat, regularly feeding an antibacterial peptide preparation to animals, sampling joint tissues of the animals, analyzing the relief condition of joint inflammation, the antibacterial peptide preparation is found to be capable of effectively treating the joint inflammation and has a certain repairing function on joint cartilage, and the antibacterial peptide preparation is a novel medicament for treating the joint inflammation.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of antibacterial peptide in preparation of medicines for treating arthritis.
Background
Currently, more than 1.5 hundred million people worldwide are suffering from cartilage defects, and with the aging of the world population, the population suffering from cartilage defects is more and more. Cartilage defects cause patients to suffer joint pain for a long period of time, and joint movement functions are also very remarkable, so that the influence on the life quality of patients is very great due to the reduction of the ability of the patients to participate in social work and acquire income. The prior method for treating the cartilage tissue mainly comprises oral non-steroidal anti-inflammatory drugs, joint cavity injection sodium hyaluronate preparations, acupuncture treatment, massage treatment and the like. However, these treatments only temporarily relieve pain and improve joint function, but do not prevent the progression of the cartilage defect.
Therefore, the novel arthritis therapeutic drug has important practical significance.
Disclosure of Invention
In view of the above, the invention separates the strain GBacillus-9 from the intestinal flora of the striped bamboo shark, and carries out induced expression and purification on the main antibacterial peptide component to prepare the antibacterial peptide preparation. By constructing an arthritis model of a rat, regularly feeding an antibacterial peptide preparation to animals, sampling joint tissues of the animals, analyzing the relief condition of joint inflammation, the antibacterial peptide preparation is found to be capable of effectively treating the joint inflammation and has a certain repairing function on joint cartilage, and the antibacterial peptide preparation is a novel medicament for treating the joint inflammation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of antibacterial peptide in preparing a medicament for preventing and/or treating arthritis or repairing articular cartilage;
the preparation method of the antibacterial peptide comprises the following steps: the strain with the preservation number of CGMCC No.18080 is used as zymophyte, and fermentation is carried out, and fermentation liquor is collected and purified.
In some embodiments of the invention, the antimicrobial peptide reduces inflammatory infiltration.
In some embodiments of the invention, the antimicrobial peptides significantly increase load-displacement, stress-strain, and elastic modulus.
In some embodiments of the invention, the antimicrobial peptides significantly increase the amount of type II collagen and proteoglycan mRNA expressed.
In some embodiments of the invention, the antimicrobial peptides significantly increase the relative expression levels of type II collagen and a polyprotein polysaccharide protein.
In some embodiments of the invention, the medicament comprises the antimicrobial peptide and a pharmaceutically acceptable adjuvant.
In some embodiments of the invention, the medicament comprises the antimicrobial peptide, as well as any other active ingredient.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient.
The invention separates the strain GBacillus-9 from the intestinal flora of the striped bamboo shark, and carries out induction expression and purification on the main antibacterial peptide component to prepare the antibacterial peptide preparation. By constructing an arthritis model of a rat, regularly feeding an antibacterial peptide preparation to animals, sampling joint tissues of the animals, analyzing the relief condition of joint inflammation, the antibacterial peptide preparation is found to be capable of effectively treating the joint inflammation and has a certain repairing function on joint cartilage, and the antibacterial peptide preparation is a novel medicament for treating the joint inflammation.
Description of biological preservation
Biological material name HD-1; the classification is named: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) has a preservation date of 2019, 07 and 08, a preservation number of CGMCC No.18080 and a preservation unit name of: china general microbiological culture Collection center, with the preservation center address: the institute of microorganisms of national academy of sciences of China, no.1, no. 3, north Chen West Lu, the Korean region of Beijing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the result of electrophoresis; wherein lane (2) shows no induced expression; lane (3) shows induced expression; lane (4) shows recombinant protease after cleavage; lane (5) shows a control;
FIG. 2 shows HE staining to observe the morphological changes of rat cartilage tissue (x 400), respectively; wherein, A1 is the blank group 4 weeks result; a2 is blank group 8 week results; b1 is the result of the GB9 antimicrobial peptide low dose group for 4 weeks, and B2 is the result of the GB9 antimicrobial peptide low dose group for 8 weeks; c1 is the result of 4 weeks in the dose group of GB9 antimicrobial peptides; c2 is the 8 week result for the dose group in GB9 antimicrobial peptide; d1 is the result of GB9 antimicrobial peptide high dose group for 4 weeks; d2 is the result of GB9 antimicrobial peptide high dose group for 8 weeks;
FIG. 3 shows a Western blot of collagen II and polyprotein polysaccharides; wherein group a shows a control; group B shows the low dose group; group C shows the medium dose group; group D high dose group.
Detailed Description
The invention discloses application of antibacterial peptide in preparing an arthritis therapeutic drug, and a person skilled in the art can properly improve process parameters by referring to the content of the antibacterial peptide. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The antibacterial peptide provided by the invention can be used for preparing the arthritis therapeutic drug, and the raw materials and the reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 preparation of antibacterial peptides
The activated p-His-sumo-GP9 (antibacterial peptide) bacterial liquid is induced to be expressed by IPTG (1/1000), a sample tube containing 20mL of bacterial liquid is firstly inserted into crushed ice, and then an ultrasonic breaker probe is immersed into the bacterial liquid for ultrasonic breaking in a pulse mode: stopping for 3s after exceeding 300W for 3s, and performing ultrasonic treatment for 1.5h until bacterial liquid is changed from turbidity to clarification; after the completion of the sonication, a bacterial liquid sample was centrifuged at 12000g for 15min at 4℃to collect the supernatant, and 100. Mu.L of the supernatant was used as a sample 3 for lysis. Filtering the supernatant with a 0.45 μm filter membrane; the filtered supernatant was about 20mL. 1.5mL of column packing (Ni Sepharose particles) was aspirated and packed into an empty column of an affinity chromatography column, and after washing with sterile ddH2O once, buffer A (binding Buffer, 20mM imidazole) was continuously added to equilibrate the column packing, allowing the Buffer to flow out naturally, and balancing 15mL. After the chromatographic column is prepared, continuously adding the filtered supernatant into the column until the addition is finished, and naturally flowing out the solution after incubation for 20min by light shaking; collecting 0.5mL of effluent (sample 4) for subsequent protein electrophoresis analysis, taking 10mL of buffer A, washing the column, and repeating the washing of the column for 5 times, wherein the total amount of the effluent is 50mL; 5mL buffer B (50 mM imidazole) was used to elute the protein of interest (twice); the total eluate was collected and 0.1mL of the sample (sample 5) was taken for subsequent protein electrophoresis analysis.
EXAMPLE 2 purification (dialysis) and concentration of recombinant proteins
The dialysis bag was placed in a solution of 250mL of 2% NaHCO3 (w/v) and 1mM EDTA at pH 8.0 and boiled for 10min; after cooling, the dialysis bag is thoroughly washed by ddH2O, and then can be used; or storing in 25% ethanol at 4deg.C, and adding ddH before use 2 Thoroughly cleaning O; or directly decocting in boiling water for 5-10min, and washing with distilled water. And clamping one end of the pretreated dialysis bag by a dialysis clamp, transferring the sample into the dialysis bag, removing air in the bag, and clamping the other end of the dialysis bag. The two ends are clamped and placed into a ddH2O big beaker with 2L precooling. The beaker was transferred to a magnetic stirrer in a freezer, continuously stirred by a magnetic stirrer, replaced with ddH2O every 30min 6 times, and dialyzed for 3-4h. Collecting the sample in the dialysis bag, transferring the protein liquid collected after dialysis into a 50mL ultrafilter tube, and centrifuging at 4 ℃ and 4000rpm for 10min; taking out the ultrafiltration tube, pouring out the filtered liquid in the ultrafiltration sleeve, supplementing a protein sample in the ultrafiltration tube, and uniformly mixing; repeating centrifugation until ultrafiltration is completedAll protein samples; about 1.5-2mL of sample was retained in the final ultrafiltration concentrate tube.
EXAMPLE 3 cleavage and Activity detection of recombinant proteins
Measuring the concentration of the concentrated protein sample, and calculating the protein content; adding ULP1 enzyme according to the product specification; after being evenly mixed, the mixture is subjected to enzyme digestion at room temperature for overnight at 4 ℃; and taking 0.1mL of the sample after enzyme digestion for protein electrophoresis analysis. Taking 0.1mL of sample bacterial liquid before cracking, centrifuging at 12000rpm for 1min, and discarding the supernatant; adding 100mL of PBS to resuspend the bacterial precipitate; taking 100 mu L of the sample after the lysis as a lysed analysis sample; adding 1/4 volume of 5 XSDS gel sample-carrying buffer into each of the six samples, uniformly mixing, and boiling for 10min at the constant temperature of 95-100 ℃ in a metal bath. After cooling, the supernatant was centrifuged at 12000rpm and used as a protein electrophoresis sample.
EXAMPLE 4 SDS-PAGE analysis of protein expression and freeze drying
Preparing PAGE gel, respectively taking 20 mu L of boiled samples in two gels in the same electrophoresis tank, loading the samples into 120V electrophoresis, and adjusting the voltage to 180V when the samples enter the separation gel. After bromophenol blue had approached about 0.5cm at the bottom of the gel, the electrophoresis was stopped, the glass plate was removed, and the gel was removed for coomassie brilliant blue R250 staining (0.1%). The freshly prepared staining solution was used to stain for 1h on a shaker. The staining solution was discarded, and after washing the gel with tap water several times, the gel was decolorized with a decolorization solution (30% methanol+10% glacial acetic acid in water) until the background disappeared. Freeze-drying the protein after induced expression, preparing powder, diluting with normal saline to prepare an antibacterial peptide preparation, and analyzing the effect of treating arthritis.
As shown in FIG. 1, it can be seen from the result of electrophoresis that the target protein has been expressed, and its size is 26kDa.
EXAMPLE 5 analysis of the Effect of antibacterial peptides on arthritis treatment
Animals: SPF class, 3 week old SD rats, 40 purchased from Hangzhou university laboratory animal center; all rats were housed in the Hangzhou university laboratory animal center and the laboratory was approved for consent by the animal ethics committee.
Drug and reagent: the GB9 antimicrobial peptide preparation used in the research is provided by Zhejiang university of Armillariella; hematoxylin, eosin was purchased from the katsumadai doctor bio-limited company; neutral formalin, alcohol, xylene from the company mikumi, the family Tianjin; TRAIL is available from Pepro Tech company, uk; secondary antibodies such as HRP goat anti-rabbit IgG, HRP goat anti-mouse IgG, and the like were purchased from Thermo corporation of the united states.
Grouping and modeling: adaptively feeding 40 SD rats for 1 week, and dividing the SD rats into 4 groups by adopting a random number table method, wherein each group comprises 10 SD rats, and the group A is a blank group; group B is GB9 antimicrobial peptide low dose group (1 mg/ml); group C is the dose group (5 mg/ml) of GB9 antimicrobial peptides; group D is GB9 high dose group (10 mg/ml) of antibacterial peptide. All rats were placed in a sterile super clean bench using 3% pentobarbital sodium (1 m1.kg -1 ) The rat is anesthetized and fixed on an experimental animal table, the conventional skin preparation disinfection towel of bilateral knee joints is adopted, the medial longitudinal incision of the knee is taken, the patellar ligament is moved to the outside, the intercondylar notch of the femur is exposed, the articular surface point of the pulley of the femur is drilled under the aseptic condition, the diameter is 3.5mm and the depth is 3.0mm, the full-layer cartilage defect is caused, and fresh blood seeps.
Detecting items
HE staining separately observed the morphological changes of rat cartilage tissue: the rats of each group are sacrificed in batches by air embolism of 4 and 8 weeks after operation, 5 rats are sacrificed in each group at 4 weeks after operation, and 5 rats are sacrificed in each group at 8 weeks after operation; the cartilage repair area of the rat is prepared into a specimen, after the specimen is fixed for 3 days by 10 percent neutral formaldehyde solution (0.1 mol/L, pH=7.4), the specimen is placed in 100 g/LETTA for decalcification for one month (the decalcification liquid is changed every three days in the first half month and the decalcification liquid is changed every day in the second half month), and the decalcification liquid is changed for 1 time every week until the pin can pierce into cortical bone to complete decalcification standard. After decalcification is completed, the mixture is dehydrated step by step, xylene is transparent, the defect area is divided by longitudinal lines, paraffin embedded slices are cut, the thickness of the slices is 4 mu m, and HE staining is observed under 400 times of light mirrors.
Pathology Mankin score: sections prepared at week 8 above were stained with toluidine blue, visualized with light and scored using a pathological Mankin [7] The total score was 14 points, with higher scores indicating more cartilage damage and the Mankin score was recorded for each group of rats.
Biomechanical testing machine determines young's modulus of elasticity of cartilage tissue: taking the prepared specimen, melting at room temperature, and then using a sharp knife to make the articular cartilageResecting, exposing subchondral bone, reference to Todd et al [3] Is a method of (2). And a universal mechanical testing machine is adopted, and a miniature sensor is used. The load-displacement curve, stress-strain curve and compressive modulus were measured.
Western blot detection of type II collagen and expression level of the polyprotein polysaccharide protein: taking rat cartilage repair area tissues, shearing the tissues by using scissors, then digesting the tissues by using trypsin, extracting total proteins by using 10% SDS-polyacrylamide gel electrophoresis, transferring the proteins to a PVDF film by a semi-dry method, placing the PVDF film in 5% skimmed milk powder, sealing the milk powder at room temperature for 2 hours, adding primary antibodies and secondary antibodies of the proteins to be detected, incubating the milk powder for 2 hours, taking GADPH as an internal reference protein, and adopting a chromogenic liquid for developing and then performing absorbance analysis to calculate the relative expression quantity of the proteins.
RT-PCR (reverse transcription-polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) expression level of type II collagen and polyprotein polysaccharide eggs: after the rat cartilage repair area tissue is pretreated by trypsin, total RNA of cells is extracted by Trizol, the purity and concentration of the RNA are determined by a spectrophotometer, and a cDNA chain is synthesized by reverse transcription. The chain is used as a template for amplification by a quantitative PCR instrument, and actin is used as an internal reference gene, and the method comprises the following steps: pre-denaturation at 95℃for 2min; the temperature was maintained at 95℃for 8s, 58℃for 35s, 72℃for 40s,40 cycles. Relative value of target gene expression rq=2 -ΔΔCt 。
Statistical methods: statistical analysis was performed using the SPSS 17.0 statistical software package. Experimental data are all expressed in (x±s), and single-factor analysis of variance is used for comparison among multiple groups, and independent sample t-test is used for comparison among two groups. The alpha value of the checking level is taken as two sides 0.05.
Effect example
HE staining for observing the morphological change of rat cartilage tissue
As can be seen from the HE staining results, the cartilage of the group A rats is damaged to a significant extent, the cell arrangement is disordered, and the cartilage tissue is cracked and inflammatory infiltrated; the arrangement of the cartilage tissue cells of the rats in the group B and the group C is more orderly than that of the rats in the group A, and the occurrence of cracks and inflammatory infiltration are obviously reduced; the cartilage tissue cells of the group D rats are orderly arranged, a small amount of inflammatory infiltration is realized, and no tissue crack exists. See fig. 2.
Comparison of the Mankin scores of the rat pathology of each group
Comparing the rats in each group by Mankin score, the Mankin score of the rats in the B group and the C group is obviously reduced (P < 0.05) compared with the rats in the A group; group D rats had significantly reduced Mankin scores (P < 0.05) compared to both group B and group C; within each group, the Mankin score was significantly reduced (P < 0.05) for the 8 week rats compared to 4 weeks, see table 1.
Table 1 group of rats pathology Mankin score comparisons
Note that: compared with group A * P<0.05; compared with group B or group C # P<0.05; within each group compared to 4 weeks & P<0.05
Young's elastic modulus change of cartilage tissue of rats of each group
The change of the elastic modulus of the cartilage of each group of rats is detected, and compared with the group A, the load-displacement, the stress-strain and the elastic modulus of the group B and the group C are obviously increased (P < 0.05); group D rats had significantly higher load-displacement, stress-strain and elastic modulus (P < 0.05) than group B and group C, see table 2.
TABLE 2 Young's elastic modulus changes in cartilage tissue of rats of each group
Note that: compared with group A * P<0.05; compared with group B or group C # P<0.05
mRNA and protein expression of rat cartilage tissue type II collagen and polyprotein polysaccharide
The detection of mRNA and protein expression of type II collagen and polyproteins in rat cartilage tissues of each group shows that compared with the group A, the relative expression level of type II collagen and polyproteins and the relative expression level of type II collagen and polyproteins in rats of group B are obviously increased (P < 0.05); compared with the B group and the C group, the expression amount of the type II collagen and the polyproteins mRNA and the relative expression amount of the type II collagen and the polyproteins protein are obviously increased (P < 0.05), and the expression amounts are shown in figure 3 and table 3.
TABLE 3 mRNA and protein expression of rat cartilage tissue type II collagen and polyprotein polysaccharide
Note that: compared with group A * P<0.05; compared with group B or group C # P<0.05
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Use of an antibacterial peptide in the preparation of a medicament for preventing and/or treating arthritis, or repairing articular cartilage;
the preparation method of the antibacterial peptide comprises the following steps: the strain with the preservation number of CGMCC No.18080 is used as zymophyte, and fermentation is carried out, and fermentation liquor is collected and purified.
2. The use of claim 1, wherein the antimicrobial peptide reduces inflammatory infiltration.
3. The use of claim 1, wherein the antimicrobial peptide significantly increases load-displacement, stress-strain, and elastic modulus.
4. The use of claim 1, wherein the antimicrobial peptide significantly increases type ii collagen and proteoglycan mRNA expression levels.
5. The use according to claim 1, wherein the antimicrobial peptide significantly increases the relative expression of type ii collagen and a polyprotein polysaccharide protein.
6. The use according to any one of claims 1 to 5, wherein the medicament comprises the antibacterial peptide and a pharmaceutically acceptable adjuvant.
7. The use according to any one of claims 1 to 5, wherein the medicament comprises the antimicrobial peptide, as well as any other active ingredient.
8. The use according to any one of claims 1 to 7, wherein the medicament further comprises a pharmaceutically acceptable adjuvant.
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