Lipopeptide and application thereof in treatment of mouse mastitis

Abstract

The invention provides a lipopeptide, and the sequence of the lipopeptide is C18-Lys (Lymphocyte-Gly-Gly-Lys-NH2). The invention further provides a preparation method of the lipopeptide. The invention also provides application of the lipopeptide in treatment of mouse mastitis. The lipopeptide is simple in design and low in preparation cost, compared with traditional antibiotics, the unique antibacterial mode of the lipopeptide enables bacteria to hardly generate drug resistance through conventional mutation, the drug resistance risk is low after long-term use, and the treatment effect can be kept more enduringly. The C18KGGK lipopeptide disclosed by the invention can be used for remarkably inhibiting the malignant progression of an inflammation part, is small in harm to an organism, is used for inhibiting the generation of inflammatory factors and is used for promoting the healing and recovery of inflammatory cells. Therefore, the lipopeptide provided by the invention has a good application prospect in the aspect of preparing clinical antibacterial drugs, and is expected to become a candidate drug of novel antibiotics.

Description

Technical Field

[0001] The present invention relates to lipopeptide and application thereof in treating mouse mastitis. Background Art

[0002] Mastitis is primarily caused by pathogens such as Streptococcus agalactiae invading the mammary ducts. Clinical manifestations include mammary gland redness and swelling, milk abnormalities, and a sudden drop in milk production. In severe cases, it can lead to mammary fibrosis and even systemic sepsis. In mouse research models, mastitis not only affects the health and reproductive performance of mice but also severely hinders mouse-related biomedical research. The prevention and treatment of mouse mastitis is crucial for breeding mice for scientific research and biopharmaceutical production. Currently, antibiotics are the standard treatment for mouse mastitis. However, recurrent mastitis caused by persistent pathogens can lead to the development of bacterial resistance. This not only complicates further medication use by veterinarians but also poses a potential threat to public health. Therefore, reducing or eliminating antibiotic use is an inevitable trend in the prevention and treatment of mastitis. Traditional Chinese herbal medicine treatments are still in the exploratory stage and face challenges such as long medication cycles, inconsistent efficacy, and unclear active ingredients. Therefore, developing new, safe, and effective methods or products for the treatment of mastitis is of great practical significance.

[0003] In recent years, lipopeptides have garnered significant attention due to their novel mode of action and broad-spectrum antimicrobial activity against a wide range of pathogens. Traditional antibiotics primarily kill bacteria by inhibiting bacterial cell wall synthesis or interfering with bacterial protein or nucleic acid synthesis, leading to the potential for bacteria to selectively develop resistance. However, due to their lipophilic nature, lipopeptides can kill bacteria by disrupting their cell membranes, causing irreparable damage. Therefore, compared to traditional antibiotics, bacterial resistance to lipopeptides is less likely. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides lipopeptides and their use in treating mouse mastitis.

[0005] The first object of the present invention is to provide a lipopeptide having a sequence of C18-Lys-Gly-Gly-Lys-NH2; the structural formula of the lipopeptide is shown in the following formula (I):

[0006]

[0007] (Ⅰ).

[0008] The second object of the present invention is to provide the use of the above-mentioned lipopeptide in the preparation of a drug for treating mouse mastitis.

[0009] Preferably, the mastitis is caused by infection with Streptococcus agalactiae.

[0010] Preferably, the medicine is an injection.

[0011] Preferably, the concentration of the lipopeptide in the drug is 1 mg / mL-10 mg / mL.

[0012] The third object of the present invention is to provide a medicine, the active ingredient of which includes the above-mentioned lipopeptide.

[0013] Preferably, the medicine is an injection.

[0014] Preferably, the concentration of the lipopeptide in the drug is 1 mg / mL-10 mg / mL.

[0015] The lipopeptide of the present invention has a simple design and low preparation cost. Compared with traditional antibiotics, the unique antibacterial mode of lipopeptides makes it difficult for bacteria to develop drug resistance through conventional mutations. The risk of drug resistance after long-term use is low, and the therapeutic effect can be maintained more sustainably. The C18KGGK lipopeptide of the present invention can significantly inhibit the malignant progression of inflammatory sites, causes little damage to the body, inhibits the production of inflammatory factors, and promotes the healing and recovery of inflammatory cells. Therefore, the lipopeptide of the present invention has a good application prospect in the preparation of clinical antibacterial drugs and is expected to become a candidate drug for new antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 This is a graph showing the hemolytic activity of the lipopeptide of the present invention.

[0018] Figure 2 This is a diagram showing the therapeutic effect of the lipopeptide of the present invention on mouse mastitis.

[0019] Figure 3 This is a comparison of the therapeutic effects of different lipopeptides on mouse mastitis.

[0020] Figure 4 These are the results of HE staining of mammary gland pathological sections of mice in the C18KGGK group.

[0021] Figure 5 Comparison results of HE staining of mammary gland pathological sections of mice in different lipopeptide treatment groups.

[0022] Figure 6 Statistical analysis results of the bacterial load detection test in mouse mammary tissue.

[0023] Figure 7 These are the statistical analysis results of the IL-6 and TNF-α factors in the mouse mammary tissue inflammatory factor detection test. DETAILED DESCRIPTION

[0024] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0025] Example 1 Preparation of lipopeptides

[0026] The three lipopeptides of this embodiment are fatty acid-modified polypeptides containing two or four amino acids, which are obtained by combining lysine (Lys, K) with each other or with glycine (Gly, G) to obtain ultra-short sequence polypeptides containing two or four amino acids; the C-terminus of the ultra-short sequence polypeptide is then amidated, and the N-terminus is modified with a C16 or C18 saturated straight-chain fatty acid, and its structural formula is as follows: C16-Lys-Gly-Gly-Lys-NH2, marked as C16KGGK; C18-Lys-Lys-NH2, marked as C18KK; C18-Lys-Gly-Gly-Lys-NH2, marked as C18KGGK. The above-mentioned lipopeptides were prepared by the classic solid-phase synthesis method and synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China). The product is in the form of a lyophilized powder. The lipopeptide was prepared into a storage solution with a final concentration of 10 mg / mL using sterile PBS and stored at -80°C for use.

[0027] Among them, the structural formula of C18KGGK is as follows:

[0028]

[0029] Example 2 Determination of Minimum Inhibitory Concentration (MIC)

[0030] The minimum inhibitory concentration of the three lipopeptides was determined by the microbroth dilution method to evaluate the antibacterial activity of the novel derivative peptides. The test Streptococcus agalactiae GS032 strain was cultured in tryptone soy broth (TSB) medium at 37°C to mid-logarithmic phase and then diluted to 5×10 5 CFU / mL. The peptide solution was diluted twofold to a final concentration of 0.05-400 μg / mL. 10 μL of peptide solution and 90 μL of bacterial suspension in the logarithmic growth phase were added to a 96-well microtiter plate and incubated at 37°C for 18-24 hours. All assays were repeated three times. The minimum inhibitory concentration was defined as the lowest concentration at which no visible bacterial growth occurred after overnight incubation. The results are shown in Table 1. By measuring the minimum inhibitory concentration (MIC), it was found that the three lipopeptides all exhibited certain antibacterial activity against Streptococcus agalactiae.

[0031] Table 1 Minimum inhibitory concentration of synthetic lipopeptides

[0032] MIC (μg / mL) C16KGGK C18KK C18KGGK GS032 3.125 3.125-6.25 3.125-6.25

[0033] Example 3 Red blood cell hemolysis experiment

[0034] Mouse blood cells were collected and washed three times with 0.9% NaCl solution. 100 μL of 2% (v / v) red blood cell solution was mixed with 100 μL of peptide (C16KGGK, C18KK, and C18KGGK) to a final peptide concentration of 0.2-400 μg / mL. After incubation at 37°C for 1 hour, the mixture was centrifuged at 1000 rpm / min for 10 minutes at 4°C. 100 μL of the supernatant was transferred to a new microcentrifuge tube, and the absorbance (ODS) was measured at 540 nm. The values ​​of 0.9% NaCl solution (ODB) and 0.1% TritonX-100 solution (ODP) were used as controls. The percentage of peptide hemolysis was calculated using the following formula: Hemolysis rate (%) = [(ODS–ODB) / (ODP–ODB)] × 100%.

[0035] The results are as follows Figure 1 As shown, the hemolytic test results showed that all three lipopeptides exhibited hemolytic activity at high concentrations. When the C16KGGK concentration was below 12.5 μg / mL, over 93% of red blood cells remained intact. C18KK at 12.5 μg / mL caused hemolysis in approximately 15% of red blood cells, and C18KGGK at 100 μg / mL caused hemolysis in approximately 15% of red blood cells. C18KGGK exhibited extremely low hemolytic activity. Taking the lipopeptide concentration that maintained 80% of red blood cell integrity as its maximum concentration, the safe red blood cell concentrations for C16KGGK, C18KK, and C18KGGK were 12.5 μg / mL, 12.5 μg / mL, and 100 μg / mL, respectively. The hemolytic activity of lipopeptides is the most commonly used method for preliminary evaluation of the safety of lipopeptide drugs. Hemolytic activity can be used as an indicator of drug safety. By comparison, the hemolytic activity of C18KGGK is significantly lower than that of C16KGGK and C18KK, and it is safer.

[0036] Figure 1 This is a graph showing the hemolytic activity of the lipopeptide of the present invention.

[0037] Example 4 Establishment of Mouse Mastitis Model

[0038] 1. For primiparous and lactating rats 4-6 days after delivery, place them in a supine position after inhalation anesthesia. Disinfect the abdominal mammary glands and surrounding skin with 75% alcohol. Use a microsyringe to infuse Streptococcus agalactiae GS032 through the milk ducts, injecting 5×10 5 CFU / mL Streptococcus agalactiae resuspension was used to construct an infection model of strain GS032.

[0039] 2. Start administering lipopeptides 24 hours after the S. agalactiae injection. Separate the mother mouse from the pups for 2 hours before lipopeptide treatment. After the mammary glands are filled with milk, anesthetize the mother mouse and inject 10 μg of the lipopeptide solution prepared in Example 1 through the mammary duct at a concentration of 1 mg / mL. Two hours after the drug injection, house the mother mouse and the pups in the same cage. Administer the drug once a day for 7 days, and keep the mice for an additional 2 days after the treatment trial. Observe the status of the mice daily.

[0040] 3. The experiment was divided into 5 groups, with 3 mice in each group. The experimental groups were as follows:

[0041] (1) Group N (control group): mice were not treated with any treatment.

[0042] (2) Group P (mastitis group): Mastitis model of mice infected with Streptococcus agalactiae, without any treatment.

[0043] (3) Lipopeptide treatment group: A mouse mastitis model infected with Streptococcus agalactiae was treated with C16KGGK lipopeptide prepared in Example 1 injected into the mammary ducts.

[0044] (4) Lipopeptide treatment group 2: The mouse mastitis model was infected with Streptococcus agalactiae and treated with the C18KK lipopeptide prepared in Example 1 by injection into the mammary duct.

[0045] (5) Lipopeptide treatment in group three: The mastitis model of mice infected with Streptococcus agalactiae was treated by injection of the C18KGGK lipopeptide prepared in Example 1 into the mammary ducts.

[0046] 4. The results are as follows Figure 2 、 Figure 3 As shown, the mammary glands of mice in group N were normal and did not suffer from mastitis; mice in group P suffered from mastitis; and mice in group C18KGGK suffered from mastitis and, after treatment, their symptoms were alleviated. This indicates that the C18KGGK lipopeptide of the present invention has a certain inhibitory effect on mastitis during injection therapy and can accelerate the healing time of a mouse mastitis model infected with Streptococcus agalactiae. Comparing the therapeutic effects of the three lipopeptides, the symptoms of mastitis in all treatment groups were alleviated, while some areas of the mammary gland in the C18KK treatment group were still accompanied by inflammation, congestion, or bleeding.

[0047] Figure 2 This is a diagram showing the therapeutic effect of the lipopeptide of the present invention on mouse mastitis.

[0048] Figure 3 This is a comparison of the therapeutic effects of different lipopeptides on mouse mastitis.

[0049] Example 5 Breast Tissue Pathology Section Analysis

[0050] Pathological analysis was performed on the mouse mastitis model constructed in Example 4. The specific experimental steps are as follows:

[0051] 1. Dewax the paraffin sections to water: sequentially place the sections in xylene I (20 min), xylene II (20 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% alcohol (5 min), 90% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min), and wash with distilled water (10 s).

[0052] 2. Staining: Place the sections in hematoxylin solution for 5 minutes, rinse with tap water, and rinse with 1% hydrochloric acid alcohol for 5 seconds until the sections turn light red. Place them under running water to restore the blue color, and then stain with eosin for 15 minutes.

[0053] 3. Dehydration and sealing: Place the slices in 95% alcohol I (5 min), 95% alcohol II (5 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), xylene I (5 min), and xylene II (5 min) in sequence for dehydration and transparency. Take the slices out of xylene and let them dry slightly. Seal the slices with neutral gum and observe and photograph under a microscope.

[0054] The results are as follows Figure 4 、 Figure 5 As shown, mammary gland sections from mice in group N showed no obvious signs of inflammation. Mammary gland sections from mice in group P showed obvious signs of inflammation, with a large amount of serous fluid and inflammatory cells within the mammary alveoli, and an incomplete alveolar structure. Mammary alveoli in mice treated with C18KGGK showed very few inflammatory cells, clear boundaries, relatively intact structure, and no interstitial fibrosis. Mammary alveoli in mice treated with C16KGGK and C18KK showed fewer inflammatory cells and relatively intact alveolar structure.

[0055] Figure 4 These are the results of HE staining of mammary gland pathological sections of mice in the C18KGGK group.

[0056] Figure 5 Comparison results of HE staining of mammary gland pathological sections of mice in different lipopeptide treatment groups.

[0057] Example 6 Determination of bacterial load in breast tissue

[0058] The bacterial load of the mouse mastitis model constructed in Example 4 was measured. The specific experimental steps are as follows:

[0059] Weigh 0.1 g of mammary gland tissue from experimental mice and homogenize it with 0.9 mL of sterile PBS. Serially dilute the homogenate, and plate 100 μL of each dilution onto a TSB plate. Invert the plate and incubate overnight at 37°C. Count the colonies growing on the plate and analyze them statistically.

[0060] The results are as follows Figure 6As shown, the number of bacteria in the mammary gland tissue of mice in the C18KGGK group was significantly lower than that in the untreated mastitis group.

[0061] Figure 6 Statistical analysis results of the bacterial load detection test in mouse mammary tissue.

[0062] Example 7 Determination of inflammatory factors in breast tissue

[0063] Inflammatory factors were measured in the mouse mastitis model constructed in Example 4. The specific experimental steps are as follows:

[0064] Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of inflammatory factors in mammary gland tissue. Total RNA was extracted from mouse mammary gland tissue using TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized by reverse transcription using PrimeScriptTM reverse transcriptase mixed reagent according to the manufacturer's instructions. GreenMaster Mix (High ROX Premixed) was used to detect the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in breast tissue by real-time quantitative PCR (qRT-PCR) to determine the degree of inflammation. The primer sequences are shown in Table 2. The reaction system is: qPCR Green Master Mix 10 μL; reverse primer 0.4 μL; forward primer 0.4 μL; cDNA 5 μL; double-distilled water 4.2 μL. Reaction conditions were as follows: initial denaturation at 95°C for 30 seconds; 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Results were calculated using the comparative Ct method (2-ΔΔct) and normalized to the internal control level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0065] Table 2 Primer sequences for real-time quantitative PCR

[0066]

[0067] The results are as follows Figure 7 As shown in the figure, compared with the mammary gland tissue of mice in the P group, the expression levels of IL-6 and TNF-α in the mammary gland tissue of mice in the C18KGGK group were significantly decreased.

[0068] Figure 7 These are the statistical analysis results of the IL-6 and TNF-α factors in the mouse mammary tissue inflammatory factor detection test.

[0069] Figure 1-Figure 7 In the table, * indicates p < 0.01, **** indicates <p<0.0001。

[0070] The above examples confirm the C18KGGK lipopeptide prepared by the present invention; through the minimum inhibitory concentration determination experiment and the red blood cell hemolysis experiment, it was found that C18KGGK exhibited a certain antibacterial ability against Streptococcus agalactiae, and the hemolytic activity was significantly lower than that of C16KGGK and C18KK, and it was safer. Through the construction of a mouse mastitis model, lipopeptide injection treatment and pathological section observation, it was found that C18KGGK lipopeptide can promote the healing of inflammatory tissue, and the therapeutic effect is better than C16KGGK and C18KK; through bacterial load determination and inflammatory cytokine determination experiments, it was confirmed that C18KGGK lipopeptide has an inhibitory effect on inflammatory factors, thereby promoting the recovery of inflammatory tissue.

[0071] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A lipopeptide, characterized in that: The sequence of the lipopeptide is: C18-Lys-Gly-Gly-Lys-NH2; the structural formula of the lipopeptide is shown in the following formula (I):

2. Use of the lipopeptide according to claim 1 in the preparation of a medicament for treating mouse mastitis.

3. The use according to claim 1, characterized in that: The mastitis is caused by infection with Streptococcus agalactiae.

4. The use according to claim 1, characterized in that: The medicine is an injection.

5. The use according to claim 4, characterized in that: The concentration of the lipopeptide in the drug is 1 mg / mL-10 mg / mL.

6. A drug, characterized in that: The active ingredient of the medicine includes the lipopeptide according to claim 1.

7. The drug according to claim 6, characterized in that: The medicine is an injection.

8. The drug according to claim 7, characterized in that: The concentration of the lipopeptide in the drug is 1 mg / mL-10 mg / mL.

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