CN114478742A - Anti-helicobacter pylori active polypeptide and application thereof - Google Patents
Anti-helicobacter pylori active polypeptide and application thereof Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of medicines for treating bacterial infection diseases, and particularly relates to an anti-helicobacter pylori active polypeptide and application thereof. The amino acid sequence of the helicobacter pylori active polypeptide is shown in sequence NO: 1. The minimum inhibitory concentration of the polypeptide to helicobacter pylori in gastric acid or gastric protein is as low as 8 mug/mL; the polypeptide can not induce helicobacter pylori to generate drug resistance, and can resist helicobacter pylori strains with antibiotic resistance; the polypeptide shows good selective toxicity to prokaryotic cells and has little toxicity to eukaryotic cells.
Description
Technical Field
The invention belongs to the technical field of medicines for treating bacterial infection diseases, and particularly relates to an anti-helicobacter pylori active polypeptide and application thereof.
Background
Helicobacter pylori infection can lead to clinical symptoms of chronic gastritis, peptic and duodenal ulcers, gastric cancer, and mucosa-associated lymphomas. The world health organization has listed these bacteria as a class of carcinogens. Persons with a high infection rate of H.pylori are generally at greater risk of developing gastric cancer than persons without infection. In china, gastric cancer has become the cause of death after the second largest cancer of lung cancer. The above-mentioned cases of H.pylori infection are associated with the long-term colonization of H.pylori in the stomach. Currently, triple therapy with proton pumps in combination with two antibiotics or quadruple therapy with bismuth is the standard therapy for H.pylori infection. However, H.pylori develops resistance to various antibiotics, and the cure rate of H.pylori is decreasing. Therefore, there is a need to develop new antibacterial compounds capable of killing helicobacter pylori strains resistant to antibiotics.
Among the known compounds, antimicrobial peptides (AMPs) may be suitable candidates, for example LL-37 (a cationic antimicrobial peptide) is an antimicrobial protein derived from neutrophils and various epithelial cells, with a molecular weight of 18 kDa. LL-37 is a member of the human Cathelicidin family and is a polypeptide cleaved from the carboxy terminus of hCAP 18. LL-37 has strong antibacterial effect against both gram-positive and gram-negative bacteria. It has been found that LL-37 has a stronger bactericidal effect not only against wild H.pylori strains than against antibiotics, but also against antibiotic-resistant H.pylori strains. Unfortunately, the acidic environment of the stomach and pepsin have a large effect on its anti-helicobacter pylori activity. Mass spectrometry analysis showed LL-37 to become a short peptide after bathing in simulated gastric fluid. At the same time, its initial anti-H.pylori activity is almost lost.
There is no literature disclosing antimicrobial peptides or other antibiotics that have little or no effect on the activity against helicobacter pylori in the acidic environment of the stomach and in the presence of pepsin.
Disclosure of Invention
In view of the above, the present invention aims to provide an anti-helicobacter pylori active polypeptide or a functional derivative thereof for treating helicobacter pylori infection, wherein the anti-helicobacter pylori active polypeptide has antibacterial activity against helicobacter pylori in gastric acid or pepsin, and the minimum concentration is as low as 8 mug/mL. The amino acid sequence of the anti-helicobacter pylori active polypeptide is shown in sequence NO. 1.
Specifically, the helicobacter pylori active polypeptide is designed based on LL-37.
Specifically, a short peptide KRIVQRIKDFLR-NH2 (named KR-12) of LL-37 at 18 th to 29 th amino acid residues showed stronger antimicrobial activity than LL-37, but this short peptide was degraded in the presence of gastric acid and pepsin, and a shorter peptide KRIVQRIKD-NH2 (named KR-9) of 9 amino acids among the degraded components was determined not to be degraded by pepsin under the conditions of pH <1.3 and pH > 2. Based on the online research data, https:// web. expasy. org/peptide _ cutter/and mass spectrometry analysis, which shows that KR-12 is the smallest LL-37 derived short peptide with antibacterial activity and KR-9 has no antibacterial activity, including anti-helicobacter pylori activity, according to the structure and activity relationship study. Based on this, the invention creatively adds 3 amino acid residues at the C-terminal of KR-9 to form peptides each containing 12 amino acid residues, and 28 peptides are designed in the invention process.
Further, the present invention used on-line work https:// web.expasy.org/peptide _ cutter/and mass spectrometry technique for analysis and mass spectrometry to ensure that 28 designed peptides were stable at low pH and in the presence of pepsin. Then, the polypeptide with the strongest antibacterial activity is selected from the 28 designed polypeptides and named SAMP-12 aa. The bactericidal activity of SAMP-12aa and the safety of clinical application of the same are further verified, and the results show that SAMP-12aa short peptide designed based on LL-37 keeps bactericidal activity and bactericidal kinetic activity in simulated gastric juice; SAMP-12aa exerts an antibacterial effect, does not induce the generation of drug-resistant strains, and has antibacterial activity on antibiotic-resistant helicobacter pylori strains; and its bactericidal mechanism was found to be based on enhancing the permeability of the outer membrane and destroying the integrity of the inner membrane; SAMP-12aa shows good selective toxicity to prokaryotic cells and little toxicity to eukaryotic cells.
The invention also aims to provide SAMP-12aa with good helicobacter pylori killing kinetics in simulated gastric fluid.
Further, the effective dose is more than or equal to the minimum concentration of the anti-helicobacter pylori activity of the anti-helicobacter pylori active polypeptide, namely the drug amount of the minimum inhibitory concentration or bactericidal concentration for generating the antibacterial activity to the helicobacter pylori is selected according to actual needs.
Further, the 8 mug/mL is the minimum inhibitory concentration of the helicobacter pylori resisting polypeptide on helicobacter pylori, and the minimum bactericidal concentration of the helicobacter pylori resisting polypeptide on helicobacter pylori is 32 mug/mL.
Further, the medicament can be prepared into a dosage form suitable for oral administration.
The invention also provides the application of the polypeptide with the anti-helicobacter pylori activity in preparing preparations for inhibiting or killing helicobacter pylori.
Further, the helicobacter pylori has antibiotic resistance. The polypeptide having anti-helicobacter pylori activity also has a killing activity against helicobacter pylori having antibiotic resistance.
Further, the helicobacter pylori is resistant to antibiotics. Helicobacter pylori resistant to existing first-line treatment antibiotics of helicobacter pylori (such as metronidazole, amoxicillin and clarithromycin), and the polypeptide with helicobacter pylori resistance activity also has bactericidal activity.
The invention also provides the use of a polypeptide having an anti-H.pylori activity according to any one of the preceding claims as or in the preparation of an agent for increasing the permeability of the outer membrane of a bacterial cell, in particular H.pylori.
The invention also provides the use of a polypeptide having an anti-helicobacter pylori activity according to any one of the preceding claims as or in the preparation of an agent for disrupting the integrity of the inner membrane of a bacterial cell, in particular helicobacter pylori.
The invention aims to provide application of the anti-helicobacter pylori active polypeptide in preparing a medicament for treating diseases caused by helicobacter pylori infection, wherein the diseases comprise but are not limited to chronic gastritis, peptic ulcer, duodenal ulcer, gastric cancer and mucosa-associated lymphomas.
In the present invention, the term "functional derivative" refers to a modified polypeptide in which the amino acid of the polypeptide having an anti-helicobacter pylori activity is modified but has the same function as the polypeptide having an anti-helicobacter pylori activity.
The invention has the beneficial effects that: the inhibitory concentration of the anti-helicobacter pylori active polypeptide provided by the invention on helicobacter pylori in gastric acid or gastric protein is as low as 8 mu g/mL; but also can not induce drug-resistant helicobacter pylori per se, and has activity of resisting antibiotic helicobacter pylori; it has good selective toxicity to prokaryotic cells and almost no toxicity to eukaryotic cells.
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FIG. 1 shows the bactericidal kinetics of the strain helicobacter pylori ATCC43504 with SAMP-12aa prepared in physiological saline.
FIG. 2 shows the kinetics of sterilization of the strain helicobacter pylori ATCC43504 by SAMP-12aa prepared using artificial gastric juice.
FIG. 3 is a graph showing the effect of SAMP-12aa on permeability of the outer membrane of H.pylori.
FIG. 4 is a graph showing the effect of SAMP-12aa on the permeability of the helicobacter pylori membrane.
FIG. 5 is an in vitro antibiotic induced production of H.pylori resistant strains without the SAMP-12aa peptide itself inducing the production of resistant strains.
FIG. 6 shows that SAMP-12aa has little cytotoxicity to human gastric adenocarcinoma cells and little cytotoxicity indicates that the oral administration is safe.
Wherein, in FIG. 4, A is the cell condition observed by fluorescence confocal microscopy after 1mL of helicobacter pylori suspension, 1mL of PBS (control) and 2mL of 100. mu.M Propidium Iodide (PI) are incubated for 90 minutes at room temperature under microaerobic conditions; b is 1mL of helicobacter pylori suspension, incubated with 2mL of 100. mu.M Propidium Iodide (PI) and 1mL of 16. mu.g/mL SAMP-12aa under microaerobic conditions at room temperature for 90 minutes, and the cells were observed using a fluorescence confocal microscope.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
In the examples of the present invention, artificial gastric juice was prepared according to the following formulation: each liter of the artificial gastric juice contained 2.05 g of sodium chloride, 8.3 g of peptone, 0.6 g of potassium dihydrogen phosphate, 0.37 g of KCl, 3.5 g of D-glucose, 0.11 g of calcium chloride, 0.05 g of bile, 13.3 mg of pepsin and 0.1 g of lysozyme. In addition, 5.5g of urea was supplemented in artificial gastric juice to evaluate the helicobacter pylori urease activity effect.
In the examples of the present invention, the helicobacter pylori strains used were: ATCC43504(NCTC11637), helicobacter pylori ATCC700392(26659), helicobacter pylori ATCC 63629(NCTC 11639), helicobacter pylori strain SS1, clinical strains of helicobacter pylori gastric ulcer and clinical strains of helicobacter pylori gastric cancer were stored in the laboratory of the applicant.
In the present example, the liquid culture medium used was helicobacter pylori (3.85 g, 93 ml of distilled water, sterilized at 121 ℃ for 15 minutes, and 7 ml of fetal bovine serum or sterile defibrinated sheep blood was added at 50-55 ℃) (Qingdao Gaokoubo Biotech Co., Ltd.).
In the practice of the present invention, the helicobacter pylori solution is prepared by: the helicobacter pylori ATCC43504 was revived in the liquid medium prepared above; then, helicobacter pylori was inoculated into colombian blood agar supplemented with 7% defibrinated sheep blood, cultured at 37 ℃ for 36 hours under microaerophilic conditions, and the bacterial preparation in the logarithmic growth phase was collected for use.
In the examples of the present invention, the method for determining the number of H.pylori cells is: viable bacterial cells were counted by plating appropriately diluted H.pylori cells on Columbia blood agar plates and grown into single colonies, and then colony counting was performed on solid medium, and the number of viable H.pylori cells was determined by cell colony counting.
Example 1 polypeptide design and Synthesis
In the practice of the present invention, the acidic and pepsin hydrolyzed fragments and sequencing and on-line tools are based on the human host defensin (antimicrobial peptide) cathelicidin LL-37https://web.expasy.org/peptide_cutter/It was predicted and established by mass spectrometry that 18 to 26 amino acid residues KRIVQRIKD to NH2 of antibacterial peptide LL-37 are stable in the presence of acidic pepsin, but they have no antibacterial activity, and a peptide of 12 amino acid residues was found to be the peptide having the shortest antibacterial activity according to a number of previous experiments of the present invention. In the present example, 28 polypeptides were designed by supplementing the carboxyl terminal (C-terminal) of a peptide with 3 amino acid residues using 9 amino acid residues to a final polypeptide having 12 amino acid residues, as shown in Table 1, and by using an on-line toolhttps://web.expasy.org/peptide_cutter/The polypeptides were analyzed and the short peptides were not degraded by pepsin in the simulated fluid by mass spectrometry.
TABLE 128 polypeptides of the invention
In the examples of the present invention, all polypeptides were synthesized by Biotechnology Inc. or Qiangyao Biotechnology Inc. and the purity was 95% by HPLC.
EXAMPLE 2 screening of the polypeptide having the strongest anti-helicobacter pylori Activity and designated SAMP-12aa
In the embodiment of the invention, the specific steps for detecting the antimicrobial polypeptide with the strongest activity against helicobacter pylori ATCC43504 are as follows: (1) logarithmic growth phase bacteria were obtained by culturing helicobacter pylori under microaerophilic conditions as described above at 37 ℃ for 36 hours, and then a bacterial solution (1X 10)8CFU/ml); (2) the polypeptide was diluted from 256. mu.g/mL to 1. mu.g/mL in liquid medium using the double dilution method (9 serial dilutions in 9 tubes, no polypeptide in 10 th tube as control), and then 10. mu.L of the bacterial solution was added to each tube to give a tube bacterial concentration of about 106CFU/ml; (3) then under microaerophilic conditions at 37 ℃ at a rate of 200 revolutions per minuteCell cultures were shaken for 36 hours, the optical clarity of the tubes was observed, and the Minimum Inhibitory Concentration (MIC) was determined from the absence of visible turbidity or bacterial growth
In the present example, the MIC of the 28 polypeptides designed in example 1 against H.pylori ATCC43504 was determined by the double dilution method to establish antibacterial peptides having the strongest activity against H.pylori ATCC43504, and as a result, as shown in Table 2, there were great differences among the polypeptides, which were KRIVQRIKDVIR having the strongest activity against H.pylori ATCC43504 and an MIC of 8. mu.g/mL, and KRIVQRIKDVIR was designated as SAMP-12 aa.
Anti-helicobacter pylori ATCC43504 strain activity of polypeptides designed in Table 2
Example 3 survival assay of helicobacter pylori in Artificial gastric juice
In the examples of the present invention, the method for the survival analysis of H.pylori ATCC43504 in artificial gastric juice was as follows: (1) culturing 24-hour log phase helicobacter pylori ATCC43504 in a liquid medium at 37 ℃ in a microaerophilic atmosphere at 3000 rpm, centrifuging for 10 minutes to collect bacterial cell pellets; (2) the bacterial cell pellet was then divided into two equal parts for survival experiments and as controls, and then the cells were suspended in physiological saline (PBS) as a control group and artificial gastric juice as survival experiments, respectively, and their concentrations were adjusted to 10, respectively8CFU/ml; (3) then, after 0 minute, 30 minutes and 60 minutes, the number of cells of H.pylori ATCC43504 was measured at various time points of exposure to PBS or artificial gastric juice, and as a result, as shown in Table 3, the survival rate of H.pylori, which is a microaerophilic bacterium to which a certain toxic effect is exerted by high concentration of oxygen, decreased with time, and the survival of H.pylori with little influence of artificial gastric juice on the presence of artificial gastric juiceSounding indicates that H.pylori is highly adapted to artificial gastric juice.
TABLE 3 survival results of the helicobacter pylori ATCC43504 strain in artificial gastric juice
Example 4 minimal inhibitory and bactericidal concentration and kinetics of SAMP-12aa in Artificial gastric juice against helicobacter pylori ATCC43504
In the present examples, in order to demonstrate the bactericidal activity of SAMP-12aa in artificial gastric juice, the Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC) and the bactericidal kinetics of SAMP-12aa were measured in liquid and solid media prepared using physiological saline and artificial gastric juice (prepared by the method of example 3).
(1) Minimum inhibitory concentration test of SAMP-12aa in simulated gastric fluid
In the present example, the minimum inhibitory concentration of SAMP-12aa in artificial gastric juice is 8. mu.g/mL as shown in example 2; the minimum inhibitory concentration in physiological saline was also 8. mu.g/mL.
(2) SAMP-12aa minimum Sterilization concentration test in Artificial gastric fluid
In the present example, 5. mu.l of the mixed solution was taken out from the clarified test tube of SAMP-12aa group in example 2 and inoculated on solid agar medium, respectively; then, they were cultured under microaerophilic conditions at 37 ℃ for 72 hours. The minimum peptide concentration at which no colonies grow is the Minimum Bactericidal Concentration (MBC) of the polypeptide SAMP-12 aa.
As a result, SAMP-12aa was found to exhibit the same antibacterial activity in liquid media prepared with physiological saline or artificial gastric juice, with MIC and MBC of 8. mu.g/mL and 32. mu.g/mL, respectively, indicating that the antibacterial activity of SAMP-12aa was not affected by artificial gastric juice, as shown in Table 4 below.
TABLE 4 minimum inhibitory and bactericidal concentrations of the H.pylori ATCC43504 strain in different environments
| Media preparation | Minimum inhibitory concentration (mug/mL) | Minimum bactericidal concentration (μ g/mL) |
| Physiological saline | 8 | 32 |
| Artificial gastric juice | 8 | 32 |
(3) Sterilization kinetics of SAMP-12aa in simulated gastric fluid
In the practice of the present invention, the method for determining the number of viable cells of helicobacter pylori following exposure to a SAMP-12aa solution is: handle 106The number of viable bacteria (CFU) is measured after the helicobacter pylori of CFU/mL is subjected to warm bath for a certain time with solutions of SAMP-12aa with different concentrations, 100 mu L of helicobacter pylori liquid with different exposure time points is subjected to 10-fold serial dilution, and the diluted helicobacter pylori liquid is coated on a solid agar culture medium for CFU counting, wherein the number of colonies is between 30 and 300, and the number of effective colonies is calculated.
In the examples of the present invention, in order to further determine whether the artificial gastric juice has an effect on the sterilization kinetics of SAMP-12aa, SAMP-12aa was prepared using physiological saline and artificial gastric juice at concentrations of 2 XMIC, 4 XMIC, 8 XMIC and 16 XMIC, respectively, and 10 XMIC was used6CFU/mL H.pylori incubation. The number of viable cells after exposure to SAMP-12aa solutions prepared with physiological saline and artificial gastric juice at 37 ℃ for 0 min, 5 min, 15 min, 30 min, 45 min and 60 min, respectively, was determined. The results are shown in FIGS. 1 and 2:
at 16. mu.g/mL (2MIC) and 5 min, 15 min, 30 min, 45 minAnd an exposure time of 60 minutes, a survival Count (CFU) of helicobacter pylori of 10 in SAMP-12aa solution prepared with physiological saline5.8±0.36,10 5.4±0.31,103.5±0.32,10 3.1±0.28(ii) a SAMP-12aa solution prepared with artificial gastric juice and having helicobacter pylori survival number (CFU) of 105.8±0.45,105.5±0.36,10 3.6±0.35,103.2±0.34(P>0.05 no significant difference).
Survival number of helicobacter pylori (CFU)10 in SAMP-12aa solution formulated with physiological saline at 32. mu.g/mL (4MIC) and exposure times of 5 min, 15 min, 30 min, 45 min and 60 min5.2±0.37，103.5±0.33，101.7±0.31，100.3 ±0.37Preparing SAMP-12aa solution with artificial gastric juice, and survival number of helicobacter pylori (CFU)105.2±0.39，103.7±0.36，101.7 ±0.38，100.4±0.34(P>0.05 no significant difference).
In SAMP-12aa solutions formulated in physiological saline at 64. mu.g/mL (8 MIC. times.2 MBC) and exposure times of 5 min, 15 min, 30 min, 45 min and 60 min, the number of surviving helicobacter pylori bacteria (CFU) was detected only at 5 min and 15 min and 10 min for the number of surviving bacteria4.6±0.35，102.4±0.39No viable bacteria were detected after 30 minutes; SAMP-12aa solution was prepared with artificial gastric juice, and the number of surviving helicobacter pylori bacteria (CFU) was detected only at 5 minutes and 15 minutes and 104.7±0.37，104.5±0.31No viable bacteria were detected after 30 minutes, (P)>0.05 no significant difference).
In SAMP-12aa solutions prepared in physiological saline at 128. mu.g/mL (16MIC ═ 4MBC) and exposure times of 5, 15, 30, 45 and 60 minutes, the survival Count (CFU) of helicobacter pylori was only 10 when the survival count was detected at 5 minutes3.3±0.37No viable bacteria were detected after 15 minutes; SAMP-12aa solutions in artificial gastric juice, the number of viable helicobacter pylori (CFU) was only 10 when the number of viable bacteria was detected at 5 minutes3.4±0.38No viable bacteria were detected after 15 minutes, (P)>0.05 no significant difference).
The above results indicate that the influence of artificial gastric juice on the sterilization kinetics of SAMP-12aa is insignificant, and that SAMP-12aa shows good anti-helicobacter pylori activity in gastric juice.
Example 5SAMP-12aa permeability test for increasing the outer Membrane
In the implementation of the invention, according to the principle that the antibacterial mechanism of the polypeptide is to enhance the permeability of an outer membrane and damage the integrity of a cell membrane, bacteria are not easy to generate drug resistance to the polypeptide; and according to the fact that "helicobacter pylori is a gram-negative bacterium having an outer membrane structure, the outer membrane of the bacterium has a certain inhibitory effect on the entry of a drug into cells, and if the antibacterial agent has the ability to penetrate the outer membrane, it has a better bactericidal effect than the case where it cannot penetrate the outer membrane; 1-N-phenylnaphthylamine (NPN) shows weaker fluorescence in aqueous solution than in hydrophobic environment, and if SAMP-12aa has the ability to enhance the permeability of helicobacter pylori outer membrane, NPN fluorescence intensity will increase when NPN probe enters the hydrophobic environment of bacterial cell wall (outer membrane) from solution. "principle design experiment.
In the embodiment of the invention, the preparation method of the helicobacter pylori ATCC43504 cell suspension comprises the following steps: (1) collecting cell pellets by centrifuging 50 ml of H.pylori cells in logarithmic growth phase at 5000 Xg for 10 minutes; (2) then after three washes with buffer, the cell pellet was suspended in HEPES sodium buffer (pH 7.2); (3) the optical density of the suspended cells was then adjusted to OD600Adjust to 0.5 for use.
In the practice of the present invention, SAMP-12aa solutions were prepared at concentrations of 4, 8, 16, 32 and 64 μ g/mL; 4ml of each of the different concentrations of the test sample contained: 1ml of SAMP-12aa solution, 2ml of H.pylori ATCC43504 cell suspension and 1ml of 40mM NPN; while 4ml of the control sample contained: 1ml of peptide-free HEPES buffer, 2ml of H.pylori ATCC43504 cell suspension and 1ml of 40mM NPN.
In the present example, the effect of SAMP-12aa on the permeability of the outer membrane of helicobacter pylori ATCC43504 was tested using a fluorescence spectrophotometer (F4600, Hitachi, Tokyo, Japan) using a hydrophobic 1-N-phenylnaphthylamine (NPN) as a fluorescent probe, using a method in which the excitation wavelength of 350nm and the emission wavelength of 420nm, the fluorescence density did not increase any more from the start to the time of measurement of the fluorescence intensity at intervals of 30 seconds. As shown in FIG. 3, when the suspension of H.pylori contains SAMP-12aa, the fluorescence intensity of NPN is significantly enhanced, and the enhancement of fluorescence shows dose dependence, and different concentrations of SAMP-12aa can increase membrane permeability to different degrees, as shown by the change of fluorescence intensity with time (when NPN penetrates the hydrophobic environment of H.pylori outer membrane, the fluorescence intensity is increased), i.e., the ability of SAMP-12aa to enhance permeability of H.pylori outer membrane due to the effect of SAMP-12aa on outer membrane is demonstrated.
Example 6 SAMP-12aa integrity test for disrupting cell membranes
In the present example, according to the expression "Propidium Iodide (PI) can enter dead cells, but cannot enter live cells; after the PI enters the cell, the PI is combined with DNA to emit fluorescence; i.e., if SAMP-12aa can destroy the integrity of the bacterial cell membrane and cause cell death, then fluorescence is generated ", the test results can be visually confirmed by using visual means to prove whether SAMP-12aa destroys the integrity of the inner membrane of the helicobacter pylori cell and causes bacterial death, and when the cell is dead, PI absorption is promoted and fluorescence is generated.
In the examples of the present invention, in order to determine whether SAMP-12aa disrupts the integrity of the cell membrane of helicobacter pylori ATCC43504, after thorough mixing, the following method was used for testing: (1) 4mL of the test sample containing 1mL of the helicobacter pylori cell suspension (prepared according to the preparation method of example 5), 1mL of 16. mu.g/mL of the SAMP-12aa polypeptide and 2mL of 100. mu.M Propidium Iodide (PI), and 4mL of the control sample containing 1mL of the helicobacter pylori cell suspension (prepared according to the preparation method of example 5), 1mL of PBS buffer (pH 7.2) and 2mL of 100. mu.M Propidium Iodide (PI) were each stored in the dark at room temperature for 90 minutes; (2) fluorescence was observed using a fluorescent confocal microscope (LSM 710Meta, Zeiss, Jene, Gemany) at an excitation wavelength of 535nm and an emission wavelength of 615 nm. The results are shown in FIG. 4, in which the results of incubation of H.pylori with PBS as a control are shown in A, and no fluorescence spot was observed, indicating that PI could not be taken up by the cells; however, when H.pylori was incubated with SAMP-12aa, the results are shown in B, which has a distinct fluorescence spot, indicating that PI can enter the cells and bind to DNA. That is, SAMP-12aa has the ability to disrupt the integrity of bacterial cell membranes and thus has bactericidal efficacy. This bactericidal mechanism of SAMP-12aa does not readily induce resistance.
Example 7 SAMP-12aa test for resistance to Induction of resistance by itself
In the present example, the helicobacter pylori strain ATCC43504 was used to explore in vitro the SAMP-12aa peptide for antimicrobial use without inducing resistance to the drug. Metronidazole, amoxicillin and clarithromycin were used as positive controls as conventional antibiotics clinically used for the treatment of H.pylori infection. SAMP-12aa and the Minimum Inhibitory Concentration (MIC) of the antibiotic were determined according to the method of example 2.
In the practice of the invention, the drug resistance analysis is performed as follows: (1) incubating helicobacter pylori in exponential growth phase under microaerophilic conditions for 36 hours in a medium containing a sub-inhibitory concentration of a different antibacterial substance, and then centrifuging to collect cells exhibiting approximately 50% growth inhibitory activity; (2) cells were further diluted with fresh medium and adjusted to 105CFU/ml concentration, and again culture, until reaching 15 similar continuous culture; medium without antimicrobial agent was used as negative control; (3) the change in MIC of H.pylori following successive exposure to an antibiotic at sub-inhibitory concentrations was assessed and if the relative MIC for each generation increased, the results indicated that the drug induced the development of resistant strains.
In the present example, the relative MIC for each passage was determined by calculating the ratio of the MIC to the measured value for a given passage culture, which ratio was compared to the value obtained from the first exposure; that is, after 15 serial passages, the relative MIC is the ratio of the MIC obtained for a given subculture to the MIC obtained for the first exposure.
In the examples of the present invention, in order to determine whether or not the antibiotic-resistant H.pylori strain is sensitive to SAMP-12aa, the MIC of SAMP-12aa to the resistant strain was examined according to the method of the above-described resistance analysis, and statistical data was processed from three analyses. As shown in FIG. 5, SAMP-12aa did not induce resistance to H.pylori; the relative MIC of SAMP-12aa for H.pylori remained stable over 15 successive subcultures; however, first-line antibiotics for the treatment of H.pylori infections induce resistance to them to H.pylori to varying degrees. The drug resistance of helicobacter pylori to metronidazole is particularly evident after repeated administration. Metronidazole increased the MIC for H.pylori by 35-fold, followed by clarithromycin, which increased its MIC for H.pylori by 16-fold. Helicobacter pylori induced resistance to amoxicillin, which increased its MIC 6-fold for helicobacter pylori. Moreover, H.pylori strains resistant to antibiotics are still sensitive to SAMP-12 aa. The results indicate that SAMP-12aa can be used to treat drug-resistant H.pylori infections.
Example 8 therapeutic index assay for SAMP-12aa
In the present example, the peptide drug would have good therapeutic potential and relative clinical safety based on the "if the antimicrobial polypeptide drug has prokaryotic selectivity, exhibits optimal antimicrobial activity and kills bacterial cells but not mammalian cells. The Therapeutic Index (TI) reflects the prokaryotic selectivity of the polypeptide. "design test.
In the examples of the present invention, the Therapeutic Index (TI) of the polypeptide was determined by calculating the ratio of the Minimum Haemolytic Concentration (MHC) value to the geometric mean value (GM) of the MIC values obtained for six different strains of helicobacter pylori (helicobacter pylori 43504(NCTC11637), helicobacter pylori 700392(26659), helicobacter pylori SS1ATCC 63629(NCTC 11639), helicobacter pylori SS1, clinical strains of helicobacter pylori gastric ulcer, clinical strains of helicobacter pylori gastric carcinoma); the Minimum Hemolytic Concentration (MHC) was determined as the concentration at which the polypeptide could induce 10% hemolysis; the therapeutic index (PI) was calculated as the ratio of MHC (. mu.g/mL) to GM (. mu.g/mL).
In the examples of the present invention, the hemolytic activity of SAMP-12aa was determined by the following procedure: (1) fresh human blood was collected using a 1000 Xg centrifugation for 7 minutes to obtain human red blood cells (hRBCs) which were then treated with PBS (43mM Na)2HPO4、11mM KH2PO4And 80mM NaCl) were carefully and thoroughly washed 3 times; (2) preparing a 4% (w/v) hRBC suspension using the above-described hRBCs resuspended in PBS; (3) with two-fold serial dilutions from 512 μ g/mL to 32 μ g/mLPreparing five SAMP-12aa with different concentrations, adding 250 mu L of SAMP-12aa with different concentrations and 250 mu L of 4% w/v hRBC suspension into the same 0.5mL centrifuge tube, and fully mixing; in the same manner, 250. mu.L of PBS without SAMP-12aa and 250. mu.L of 4% w/v hRBC suspension were added to the same 0.5mL centrifuge tube as a negative control, and then 250. mu.L of 0.1% Triton X-100 and 250. mu.L of 4% w/v hRBC suspension were added to the same 0.5mL centrifuge tube as the positive control. (4) All mixtures were incubated in a 37 ℃ water bath for 1 hour, then centrifuged at 1000 Xg for 5 minutes, 200 microliters of supernatant was collected from each centrifuge tube and then added to the wells of a 96-well plate; (5) the absorbance of the supernatant was measured at 414nm using a microplate reader.
In the examples of the present invention, the absorbance value (A) of PBSPBS) Identified as zero hemolysis, the absorbance value (A) of the supernatant of 0.1% (v/v) Triton X-100-lysed hRBCsTriton) 100% hemolysis was determined; the hemolytic activity of SAMP-12aa was calculated as a percentage of hemolysis according to the following formula SAMP-12aa hemolysis (%) - (A)sample-APBS)/(ATriton-APBS) X 100. The minimum hemolytic concentration value (minor hemolytic concentration) is expressed in MHC.
In the present examples, for accurate determination of MIC of peptides for six different H.pylori strains, SAMP-12aa was diluted from 4. mu.g/mL to 12. mu.g/mL in 1. mu.g/mL increments, and MIC was determined according to the method of example 2, with the Geometric Mean (GM) of MIC values for the MIC of SAMP-12aa for the different H.pylori strains being indicated as GM.
In the examples of the present invention, the values of GM, MHC and TI of SAMP-12aa calculated according to the above-mentioned method are shown in Table 4. The results show that SAMP-12aa has stronger antibacterial specificity than the evaluated antibiotics; the hemolytic concentration of SAMP-12aa reached 216. mu.g/mL, being low hemolytic activity (high MHC). MIC was 8.5. mu.g/mL, indicating high antibacterial activity (low GM). Thus, the antimicrobial peptide SAMP-12aa is an ideal antimicrobial for the treatment of H.pylori infections.
TABLE 5 Therapeutic Index (TI) of SAMP-12aa
Example 9 cytotoxicity Studies of SAMP-12aa
In the practice of the present invention, human gastric adenocarcinoma cells (ATCC; CRL-1739) were used to determine the cytotoxicity of SAMP-12 aa.
In the present example, SAMP-12aa was used to determine cytotoxicity of CRL-1739 cells using a standard MTT proliferation assay, i.e. assessed by measuring insoluble purple-blue crystalline formazan, a substrate that can only be produced in living cells, in which succinate dehydrogenase in the mitochondria reduces exogenous MTT, whereas dead cells do not; the method comprises the following specific steps:
(1) CRL-1739 cells were cultured in RPMI-1640 medium supplemented with 5% CO for 3-5 days 210% of fetal bovine serum.
(2) Adherent cells were treated by trypsinization to form dispersed single cells, and cell pellets were collected after centrifugation at 1000 × g for 4 minutes. Preparation of 1X 10 Using Medium containing 10% fetal bovine serum5cells/mL of cell suspension.
(3) Then 200 μ L of the cell suspension was added to each well of the 96-well plate, and the plate was incubated at 37 ℃ in 5% CO2And cultured for 24 hours to form adherent cells.
(4) 40 microliters of SAMP-12aa solution was added to the wells, which was serially diluted 2-fold from 256 μ g/mL to 16 μ g/mL with RPMI-1640 medium. Wells containing only 40 μ L RPMI-1640 medium and no SAMP-12aa were used as controls. The plates were further incubated for 48 hours.
(5) Then 20. mu.L of 5mg/mL MTT was added to each well and mixed thoroughly and carefully. The plates were incubated at 37 ℃ for 4 hours. The supernatant from each well was carefully removed.
(6) Then, 150 μ L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the insoluble blue-violet crystalline formazan produced by MTT. A microplate ELISA reader was used to measure absorbance at 550nm of the dissolved perfect formazan crystals.
In the present example, the survival rate of CRL-1739 cells was calculated using the following formula (a550 of SAMP-12aa-treated cells)/a 550 of SAMP-12aa-untreated cells) x 100.
In the present example, SAMP-12aa was evaluated for cytotoxicity against human gastric adenocarcinoma cells (ATCC; CRL-1739) as described above, and the results are shown in FIG. 6, which shows that SAMP-12aa is not toxic to CRL-1739 cells at 128. mu.g/mL, and has more than 90% of viable cells at this concentration. The results indicate that SAMP-12aa is a good candidate for development and use in the treatment of H.pylori infection due to its low toxicity to eukaryotic cells.
Example 10 SAMP-12aa test for the clearance of helicobacter pylori in the mouse stomach
In the implementation of the invention, SAMP-12aa nanoparticles are prepared according to the technical scheme disclosed in patent 201410485607.3 (the patent name is the preparation method of the gastric mucosa nanoparticle drug delivery system of the anti-helicobacter pylori active antibacterial peptide).
In the examples of the present invention, an animal model of H.pylori infection was established using the strain helicobacter pylori SS 1. Mice were randomized into 10 groups (8 in each group) and fasted for 24 hours, then gavaged at 0.3 ml containing 108BIH broth of CFU H.pylori. 14 days after inoculation (2 per group, successful model establishment verified by microscopy, oxidase test, catalase test and urease test), SAMP-12aa and SAMP-12aa nanoparticles were administered orally at a dose of 1, 3, 10 or 30mg/kg body weight, once a day for three consecutive days. Placebo mucoadhesive nanoparticles were administered orally in the same manner as a control. One day after the last dose, mice were euthanized under anesthesia and the stomach was homogenized in 1.5mL BHI broth using a tissue homogenizer. Eradication of helicobacter pylori was determined using microbial culture methods. The remaining homogenate was serially diluted 10-fold and spread evenly on BHI agar plates supplemented with 7% sheep blood and the above antibiotics. The plates were incubated at 37 ℃ for 4 days under microaerophilic conditions. The number of H.pylori bacteria was determined by counting bacterial colonies on agar plates.
The bacteria were then further identified by microscopy, oxidase test, catalase test and urease test. The number of bacterial colonies in each mouse stomach was calculated by counting the colonies on each plate and expressed as log CFU per stomach sample. The results are shown in Table 6.
TABLE 6 comparison of the Effect of mucosal delivery systems taking SAMP-12aa and SAMP-12aa nanoparticles on the eradication of helicobacter pylori from the stomach of mice
Note: ND: not detected (Not detected)
As shown in Table 6, the average bacterial count in the stomach of the mice, the mice of the control group which did not receive the drug, and the average of about 10 in the stomach of each mouse7.57(CFU/stomach) bacterial colonies. The average bacterial count in the stomach of mice treated with oral SAMP-12aa decreased with increasing dose, but complete clearance was not observed, even at the highest dose of 30 mg/kg. However, the gastric mucosa nanoparticle prepared by taking SAMP-12aa obviously enhances the elimination of helicobacter pylori, and the helicobacter pylori in the stomach is completely eliminated when the dosage of 10mg/kg is orally taken.
Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.
Sequence listing
<110> applicant Anhui scientific and technological college
<120> polypeptide with anti-helicobacter pylori activity and application thereof
<130> 2022-3-3
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys Arg Ile Val Gln Arg Ile Lys Asp Val Ile Arg
1 5 10
Claims (10)
1. An anti-helicobacter pylori polypeptide or a functional derivative thereof, wherein the amino acid sequence of the helicobacter pylori polypeptide is shown in sequence NO: 1.
2. A pharmaceutical composition comprising, as an active ingredient, an effective amount of the polypeptide having an anti-helicobacter pylori activity according to claim 1 and a pharmaceutically acceptable carrier or diluent.
3. The pharmaceutical composition of claim 2, wherein the effective dose is not less than the minimum concentration of the anti-helicobacter pylori activity of the anti-helicobacter pylori active polypeptide.
4. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable carrier or diluent is suitable for oral administration.
5. Use of the polypeptide having an anti-helicobacter pylori activity according to claim 1 for the preparation of an agent for suppressing or killing helicobacter pylori.
6. The use according to claim 5, wherein the polypeptide having anti-H.pylori activity does not itself have the effect of inducing resistance to H.pylori.
7. The use according to claim 6, wherein the polypeptide having anti-H.pylori activity has bacteriostatic and bactericidal activity against H.pylori strains having antibiotic resistance.
8. Use of the polypeptide having an anti-helicobacter pylori activity according to claim 1 as or in the preparation of an agent for increasing permeability of the outer membrane of bacterial cells.
9. Use of the polypeptide having an anti-helicobacter pylori activity according to claim 1 as or in the preparation of an agent for disrupting the integrity of the inner membrane of a bacterial cell.
10. Use of the anti-helicobacter pylori active polypeptide according to claim 1, for the preparation of a medicament for the treatment of a disease caused by helicobacter pylori infection, including, but not limited to, chronic gastritis, peptic ulcer, duodenal ulcer, gastric cancer, mucosa-associated lymphomas.
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| CN115960167A (en) * | 2022-08-18 | 2023-04-14 | 齐齐哈尔大学 | Corn anti-adhesion peptide and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115960167A (en) * | 2022-08-18 | 2023-04-14 | 齐齐哈尔大学 | Corn anti-adhesion peptide and preparation method and application thereof |
| CN115960167B (en) * | 2022-08-18 | 2023-11-07 | 齐齐哈尔大学 | Corn anti-adhesion peptide and preparation method and application thereof |
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