Application of VMO1 protein in toxicity resistance, oxidation resistance, thrombolysis and sperm motility regulation

Abstract

The invention belongs to the field of natural compounds, and particularly relates to application of VMO1 protein in toxicity resistance, oxidation resistance, thrombolysis and sperm motility regulation. According to the specific technical scheme, the application of the VMO1 protein in preparation of drugs for inhibiting bacteria, resisting toxicity, resisting oxidation and improving or reducing sperm motility is provided, and the amino acid sequence of the VMO1 protein is shown as SEQ NO ID: 1. It is found for the first time that the VMO1 protein extracted from frog follicles not only can inhibit the activity of pseudomonas aeruginosa, but also can resist toxicity, and meanwhile, the VMO1 protein has a good antioxidant effect, a thrombus dissolving effect and a bidirectional sperm motility adjusting effect. Therefore, the VMO1 protein has huge pharmaceutical potential.

Description

Technical Field

[0001] This invention belongs to the field of natural compounds, specifically relating to the application of VMO1 protein in antitoxicity, antioxidation, thrombolysis and regulation of sperm motility. Background Technology

[0002] Vitelline outer layer protein 1 (VMO1), first discovered in chickens, is a basic protein found in the outer layer of the vitelline membrane. It is a crucial component of this outer layer, primarily functioning structurally to separate the yolk from the egg white, preventing their mixing. Subsequent studies have shown that VMO1 can bind to lysozyme C (LYSC), reducing tear surface tension and maintaining tear film stability; this effect is dose-dependent. However, antibacterial experiments against two Gram-negative bacteria have shown that VMO1 has no significant antibacterial activity.

[0003] Antibiotic resistance has become a major threat to global public health, severely impacting the treatment outcomes of infectious diseases. However, current effective treatment strategies against drug-resistant bacteria remain incomplete, making the development of antibiotic alternatives urgent. In recent years, antiviral strategies have received increasing attention due to their mechanism of "disarming" pathogens, becoming an important direction in anti-infective research. Antiviral strategies refer to drugs exerting their antiviral effect by inhibiting the release of virulence factors from microorganisms; antiviral activity does not necessarily inhibit the activity of the microorganism itself. Extracellular proteases and pyocyanin are important virulence factors of *Pseudomonas aeruginosa*. However, due to difficulties in extraction and purification, low bioavailability, and unclear mechanisms of action, the clinical translation of antiviral drugs still faces many challenges, and currently, the market is dominated by synthetic drugs.

[0004] Antioxidants are substances that help capture and neutralize free radicals, thereby eliminating the damage they cause to the human body. Natural antioxidants have advantages such as high activity and low toxicity, and can be used in food processing and storage. They can also be used in areas such as anti-cancer, anti-inflammatory, anti-aging, and prevention and treatment of some chronic diseases such as diabetes and cardiovascular diseases.

[0005] Cardiovascular disease (CVD) seriously threatens people's lives and health. Arterial thrombosis is one of the key factors in CVD, and its formation mechanism is closely related to the abnormal activation, adhesion, and aggregation of platelets. Existing antithrombotic drugs mainly work by inhibiting platelet adhesion and aggregation, while VOM1 mainly dissolves existing thrombi, making it a more suitable candidate for thrombolytic therapy.

[0006] The reproductive process in mammals begins with the successful union of sperm and egg, and its smooth execution is highly dependent on sperm motility. Low sperm motility not only reduces the success rate of natural conception but also affects the success rate of assisted reproductive technologies. Therefore, the development of novel, highly effective, and targeted sperm motility enhancers has become an urgent need in the field of reproductive medicine. Summary of the Invention

[0007] The purpose of this invention is to provide new applications for VMO1 protein in antitoxicity, antioxidation, thrombolysis, and regulation of sperm motility.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: the application of VMO1 protein in the preparation of drugs with antibacterial, antiviral, antioxidant, and sperm motility-enhancing or sperm motility-reducing properties, wherein the amino acid sequence of the VMO1 protein is shown in SEQ NO ID:1.

[0009] Antibacterial, antiviral, antioxidant, sperm motility-enhancing or sperm motility-reducing drugs prepared using VMO1 protein, or antibacterial, antiviral, antioxidant, sperm motility-enhancing or sperm motility-reducing drugs containing VMO1 protein, wherein the amino acid sequence of the VMO1 protein is shown in SEQ NO ID:1.

[0010] The antiviral drugs prepared from the VMO1 protein include resistance to Pseudomonas aeruginosa, specifically by reducing the amount of pyocyanin secreted by Pseudomonas aeruginosa or inhibiting the activity of pyocyanin.

[0011] This invention offers the following beneficial effects: For the first time, this invention discovers that the VMO1 protein extracted from frog follicles not only inhibits the activity of *Pseudomonas aeruginosa* but also exhibits antiviral activity, along with good antioxidant, thrombolytic, and bidirectional regulatory effects on sperm motility. Therefore, the VMO1 protein possesses significant pharmaceutical potential. Attached Figure Description

[0012] Figure 1 Schematic diagram of VMO1's inhibitory activity and antiviral activity against Pseudomonas aeruginosa;

[0013] Figure 2 This is a schematic diagram of the ABTS method for testing the antioxidant activity of VMO1.

[0014] Figure 3 This is a schematic diagram of the FRAP method for testing the antioxidant activity of VMO1.

[0015] Figure 4 This is a schematic diagram of the thrombolysis rate of VMO1 (1 mg / mL). Detailed Implementation

[0016] In their prior research, the inventors' research group extracted a novel VMO1 protein from frog follicles (disclosed in patent CN 116999375 B), the amino acid sequence of which is shown in SEQ NO ID: 1. This invention is the first to discover that the VMO1 protein possesses antitoxic, antioxidant, thrombolytic, and sperm motility-regulating effects.

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data. The preparation method of VMO1 protein used in the embodiments is in accordance with CN 116999375 B, and VMO1 protein can also be artificially synthesized according to its amino acid composition.

[0018] Example 1: Demonstration of the antibacterial and antiviral effects of VMO1 protein

[0019] A single Pseudomonas aeruginosa PAO1 bacterium was picked and inoculated into 5 mL of LB broth medium. After overnight culture, the bacterial cells were collected and adjusted to OD600 = 1 with sterile PBS to obtain the bacterial seed culture.

[0020] 1. Protein hydrolysis zone experiment

[0021] The plates used in this experiment were skim milk powder plates, i.e., M9 solid medium containing 0.5% (w / v) skim milk powder. Three spots of bacterial seed culture were evenly inoculated onto the skim milk powder plates, with each spot containing 2 μL of bacterial seed culture. Different concentrations of VMO1 (2.5, 5, 10, and 20 μg / mL, diluted with 50 mM Tris, 300 mM NaCl buffer) were added to the medium before pouring the plates. A blank control group (without VMO1, all other conditions were identical) was also included. Each treatment was performed in triplicate. After the bacterial culture was dried, the plates were inverted and incubated at 37°C for 24 h. The diameter of the protein hydrolysis zone was then measured.

[0022] 2. Quantitative detection experiment of pyocyanin

[0023] Add 2 mL of LB broth medium to a 15 mL sterile centrifuge tube, and inoculate the bacterial seed culture at a 1% inoculum. Add 20 μg / mL VMO1 (experimental group) or an equal volume of 50 mM Tris, 300 mM NaCl buffer (blank control group). Set up 3 biological replicates. Incubate at 37℃ with shaking for 24 h. Centrifuge and collect the supernatant to a centrifuge tube. Add chloroform at a supernatant:chloroform ratio of 5:3. After repeated inversion, collect the lower chloroform extract. Add HCl to the chloroform extract at a chloroform extract:0.2N HCl ratio of 3:1. After vigorous shaking, collect the upper HCl solution. Measure the absorbance of the solution at 525 nm using a microplate reader. Resuspend the bacterial cells in PBS and measure the OD. 600 Absorbance value. The absorbance value of the HCl solution for each sample divided by the bacterial cell OD is used as the final quantitative detection result for Pseudomonas aeruginosa.

[0024] 3. Quantitative detection experiment of biomembrane

[0025] Using 96-well plates as the biofilm attachment substrate, 200 μL of LB broth medium containing bacterial cells was added to each well (1% inoculum of bacterial seed culture, followed by 20 μg / mL VMO1 or an equal volume of 50 mM Tris, 300 mM NaCl buffer). Seven biological replicates were set up, and the 96-well plates were incubated at 37°C for 24 h. The bacterial culture was removed from the 96-well plates, and the plates were gently washed with PBS buffer and then air-dried. 220 μL of 0.1% crystal violet staining solution was added to each well, and the plates were allowed to stand for 20 mins. The staining solution was then aspirated, and the plates were gently washed with PBS buffer and allowed to air-dry. 230 μL of 95% ethanol solution was added to each well, and the plates were allowed to stand for 10 mins. The liquid was then gently pipetted, and 200 μL of the above mixture was measured at 595 nm. The results are shown in Table 1 (average values). Figure 1 As shown in Table 1, with the data of the blank control group as the baseline (100%), VMO1 data = VMO1 experimental group measured value / blank control group measured value × 100%.

[0026] Table 1. Effects of VMO1 on Pseudomonas aeruginosa pyophytin and biofilm.

[0027]

[0028] The results showed that VMO1 protein could significantly inhibit the growth of biofilms and the activity or production of extracellular proteases, which are important virulence factors of Pseudomonas aeruginosa, and could also inhibit the secretion of pyocyanin by Pseudomonas aeruginosa.

[0029] Example 2: Demonstration of the antioxidant effect of VMO1 protein

[0030] The activity of crude extract from the tree frog's foam nest was tested using a total antioxidant capacity kit. Measurements were performed using the FRAP method (Suzhou Keming Biotechnology Co., Ltd., catalog number: FRAP-1-G) and the ABTS method (Suzhou Keming Biotechnology Co., Ltd., catalog number: ABTS-1-D), with three replicates per group, and the average value was taken. VMO1 samples of different concentration gradients were prepared according to the kit requirements. The wavelength of the microplate reader was adjusted to 734 nm. The working solution was prepared according to the kit instructions. The extract / sample was mixed with the working solution according to the kit operation instructions, and the absorbance value at 734 nm was measured within 10 minutes. ΔA = Ablank - Ameasured.

[0031] The standard curve provided by the kit is: y = 0.7021x - 0.0012R² = 0.9985. Here, x represents the Trolox concentration (μmol / mL), and y represents the absorbance difference ΔA.

[0032] The formula for calculating total antioxidant capacity is: Total antioxidant capacity (μmol Trolox / mg prot) = (△A + 0.0012) ÷ 0.7021 × V sample ÷ (V sample ÷ V sample total × Cpr) = 1.424 × (△A + 0.0012) ÷ Cpr.

[0033] The results are shown in Tables 2 and 3. Figure 2 , 3 As shown.

[0034] Table 2. Comparison table of antioxidant activity of VMO1 tested by ABTS method

[0035] VMO1 VMO1 activity △A Free radical scavenging rate 1000 μg / mL 0.15 1.28 0.90 500 μg / mL 0.16 1.27 0.89 250 μg / mL 0.19 1.25 0.87 100 μg / mL 0.96 0.47 0.33 20 μg / mL 1.26 0.16 0.12 10 μg / mL 1.28 0.15 0.10 5μg / mL 1.32 0.10 0.07

[0036] Table 3. Comparison table of VMO1 antioxidant activity tested by FRAP method

[0037] VMO1 VMO1 activity △A 200 μg / mL 1.76 1.68 100 μg / mL 0.95 0.87 50 μg / mL 0.62 0.54 20 μg / mL 0.17 0.09 10 μg / mL 0.16 0.08 5μg / mL 0.12 0.04 2.5 μg / mL 0.11 0.03

[0038] The results showed that VMO1 exhibited significant antioxidant activity, which was dose-dependent.

[0039] Example 3: Demonstration of the thrombolytic effect of VMO1 protein

[0040] Circular thrombosis: Blood samples were collected from mice in test tubes containing sodium citrate (3.2%) as an anticoagulant. A coagulation inducer of 15% (v / v) was prepared. 67 mM calcium chloride, 25 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), and 137 mM NaCl were used. 5 μL of the coagulation inducer was added dropwise to the bottom of a 96-well plate. Simultaneously, 25 μL of blood was added, and the pipette tip was moved in a circular motion around the well wall to form a blood clot. The plate was incubated at 37°C for 1 hour to form a stable blood clot. A well without the coagulation inducer was set up as a blank control group (1).

[0041] VMO1 thrombolysis: VMO1 was redissolved in 70 μL PBS to obtain a 1 mg / mL VMO1 solution, which served as the experimental group. A control group (2) containing only 70 μL PBS under the same conditions was used. Simultaneously, 70 μL of sample was added to the wells containing the ring thrombus using a microplate reader. The plate was then shaken for 5 seconds (200 rpm) on a microplate shaker before detection. Degradation of the ring thrombus was measured at 37°C using a microplate reader at absorbance changes per minute at 510 nm. x (t), measured for 2 hours to evaluate the fibrinolysis rate.

[0042] Data from a blank control group without coagulation inducers were used as the basis for complete degradation (A). total The negative control (containing a coagulation mixture + PBS) represents the absence of thrombolysis (A). zero ). Specific parameter calculation formula: Degradation percentage: D x (t)=100(A x (t)-A zero (t)) / (A tota (t) l -A zero (t)).

[0043] The results are as follows Figure 4 (Different colors represent different replicates). The results showed that thrombi gradually dissolved over time under the action of VMO1, with the thrombolysis rate exhibiting a typical S-shaped upward trend. The curves among the replicate groups showed good consistency, indicating high experimental reproducibility. Combined with its significant thrombolytic effect and good experimental reproducibility, VMO1 shows promise as a novel thrombolytic candidate molecule for the treatment of thrombosis-related diseases.

[0044] Example 4: Demonstration of the effect of VMO1 protein on regulating sperm motility

[0045] 200 μL of fresh boar semen was centrifuged at 12000 rpm for 5 min, the supernatant was removed, and 190 μL of BTS buffer was added. Then, 10 μL of VMO1 samples diluted with different concentrations of 50 mM Tris and 300 mM NaCl buffer were added, and a blank control group was set up: boar semen treated with the same buffer (50 mM Tris, 300 mM NaCl) was added only. Each sample was mixed with 190 μL of semen and incubated at 37 °C for 10 min. The semen suspension was then maintained at 37 °C and injected into the analysis chamber. The regulatory effects of different protein concentrations on sperm motility parameters (curved velocity VCL, linear velocity VSL, and head lateral sway amplitude ALH) were quantitatively evaluated using a CASA system to screen candidate components that significantly improve sperm motility. At least 40 motile sperm were analyzed in each experiment, and each concentration of VMO1 solution was measured in triplicate and the average value was taken. A 20% increase in sperm curved motility (VCL) was used as the inclusion threshold for active components. The results are shown in Table 4.

[0046] Table 4. VMO1 Sperm Activity Comparison Table

[0047] Group Sperm motility (μm / s) Blank control group 82.47 0.25 μg / mL VMO1 85.24 25 μg / mL VMO1 87.55 50 μg / mL VMO1 93.39 100 μg / mL VMO1 101.57 125 μg / mL VMO1 79.34

[0048] The results showed that low concentrations of VMO1 had a concentration-dependent protective effect, with a concentration of 100 μg / mL increasing sperm VCL by more than 20%. This indicates that low concentrations of VMO1 can act as a sperm function enhancer, improving sperm motility in patients with asthenospermia and increasing the natural conception rate. It is particularly suitable for male reproductive health products and assisted reproductive technology optimization programs, providing a non-invasive intervention option for infertile populations worldwide. High concentrations (125 μg / mL) of VMO1, however, have an inhibitory effect on sperm motility and hold promise for development as a non-hormonal contraceptive. VMO1 possesses the potential for bidirectional regulation—low concentration promoting health and high concentration inhibiting contraception—providing its application potential in male reproductive health and contraceptive products.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of VMO1 protein in the preparation of antibacterial drugs, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

2. An antibacterial drug prepared using VMO1 protein, or an antibacterial drug containing VMO1 protein, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

3. The application of VMO1 protein in the preparation of antiviral drugs, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

4. An antiviral drug prepared using VMO1 protein, or an antiviral drug containing VMO1 protein, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

5. The application of VMO1 protein in the preparation of antioxidant drugs, characterized by: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

6. An antioxidant drug prepared using VMO1 protein, or an antioxidant drug containing VMO1 protein, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

7. The application of VMO1 protein in the preparation of drugs to improve sperm motility, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

8. A sperm motility-enhancing drug prepared using VMO1 protein, or a sperm motility-enhancing drug containing VMO1 protein, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NOID:

1.

9. The application of VMO1 protein in the preparation of drugs that inhibit sperm motility, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NO ID:

1.

10. A sperm motility inhibitor prepared using VMO1 protein, or a sperm motility inhibitor containing VMO1 protein, characterized in that: The amino acid sequence of the VMO1 protein is shown in SEQ NOID:1.

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