Application of nicotinamide adenine dinucleotide in the preparation of anti-Burkholderia cepacia products

Abstract

This invention belongs to the field of biomedical technology, and provides the application of nicotinamide adenine dinucleotide in the preparation of products against Burkholderia cepacia. This invention studies and discovers that nicotinamide adenine dinucleotide (NAD...) + It has pharmacological activity, NAD + It is non-toxic to human cells and can inhibit the motility and biofilm formation of Burkholderia cepacia, thereby suppressing its pathogenicity, at a low concentration. Furthermore, due to NAD... + The antibacterial effect against Burkholderia cepacia does not depend on direct inhibition of Burkholderia cepacia cell growth, therefore NAD+... + It does not exert selective pressure on Burkholderia cepacia, thus preventing the emergence of drug-resistant pathogens. NAD + It has great application prospects and value in the development of new antibacterial drugs, especially in the development of drugs against Burkholderia cepacia infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to nicotinamide adenine dinucleotide (NAD). + Application in the preparation of products against Burkholderia cepacia. Background Technology

[0002] Burkholderia cenocepacia is a common and serious opportunistic pathogen that is abundant in the natural environment and hospitals. It typically infects immunocompromised individuals, such as patients with cystic fibrosis (CF), and the resulting clinical symptoms are known as "Burkholderia cenocepacia syndrome," which can lead to lung failure and sepsis.

[0003] Due to the complex and diverse resistance genes and mechanisms of Burkholderia cepacia, as well as the irregular use of antibiotics, it has developed strong resistance to many commonly used antibiotics, making the treatment of infections caused by Burkholderia cepacia very difficult.

[0004] Currently, there is an urgent need for an antibacterial drug that can effectively inhibit the pathogenicity of Burkholderia cepacia, has high safety, and does not cause Burkholderia cepacia to develop drug resistance. Summary of the Invention

[0005] The present invention aims to solve the above problems and provides the application of nicotinamide adenine dinucleotide in the preparation of anti-Burkholderia cepacia products. Nicotinamide adenine dinucleotide can inhibit the pathogenicity of Burkholderia cepacia without inhibiting its growth, thus solving the problem of difficulty in inhibiting the bacteria due to the strong drug resistance of Burkholderia cepacia.

[0006] The purpose of this invention is to provide the application of nicotinamide adenine dinucleotide in the treatment of Burkholderia cepacia.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention has discovered nicotinamide adenine dinucleotide (NAD) + It has antibacterial activity against Burkholderia cepacia, NAD+ + Without inhibiting the growth of *Burkholderia cepacia*, NAD+ can inhibit its motility and biofilm formation, thereby suppressing its pathogenicity, and at a low concentration is required. In human cytotoxicity experiments, NAD+... + It is non-toxic to human cells and can significantly inhibit the killing effect of Burkholderia cepacia on human cells. Therefore, the present invention provides the following applications:

[0009] Application of nicotinamide adenine dinucleotide in the preparation of products against Burkholderia cepacia.

[0010] Application of nicotinamide adenine dinucleotide in antibacterial activity against Burkholderia cepacia.

[0011] Application of nicotinamide adenine dinucleotide in inhibiting the pathogenicity of Burkholderia cepacia.

[0012] Application of nicotinamide adenine dinucleotide in inhibiting biofilm formation in Burkholderia cepacia.

[0013] Application of nicotinamide adenine dinucleotide in the preparation of products that inhibit biofilm formation by Burkholderia cepacia.

[0014] Application of nicotinamide adenine dinucleotide in inhibiting the motility of Burkholderia cepacia.

[0015] Application of nicotinamide adenine dinucleotide in the preparation of products that inhibit the motility of Burkholderia cepacia.

[0016] Application of nicotinamide adenine dinucleotide in the preparation of drugs for the treatment or prevention of diseases caused by Burkholderia cepacia.

[0017] Application of nicotinamide adenine dinucleotide in the preparation of drugs for the treatment or prevention of symptoms caused by Burkholderia cepacia.

[0018] Application of nicotinamide adenine dinucleotide in the preparation of drugs for the prevention or treatment of Burkholderia cepacia infection.

[0019] Application of nicotinamide adenine dinucleotide in the preparation of drugs for the prevention or treatment of infectious diseases caused by Burkholderia cepacia.

[0020] In addition, this invention also tested NAD+. + NAD + The capsule's antibacterial effect against Burkholderia cepacia. The NAD... + The capsule's ingredients include NAD. + Niacin, lipoic acid, chromium, bioflavonoids, choline, leucine, piperine.

[0021] Therefore, the NAD + The application of the capsule in inhibiting Burkholderia cepacia, in inhibiting the pathogenicity of Burkholderia cepacia, in inhibiting the biofilm formation of Burkholderia cepacia, in inhibiting the motility of Burkholderia cepacia, and in the preparation of related products should all be within the scope of protection of this invention.

[0022] The present invention has the following beneficial effects:

[0023] This invention has discovered nicotinamide adenine dinucleotide (NAD) + It possesses pharmacological activity, inhibiting the motility and biofilm formation of *Burkholderia cepacia* without inhibiting its growth, and at a low concentration. In human cytotoxicity experiments, NAD+... + It is non-toxic to human cells and can significantly inhibit the killing effect of Burkholderia cepacia on human cells. Furthermore, due to NAD... + The antibacterial effect against Burkholderia cepacia does not depend on direct inhibition of Burkholderia cepacia cell growth, therefore NAD+... + It does not exert selective pressure on Burkholderia cepacia, thus preventing the emergence of drug-resistant pathogens. NAD + It has great application prospects and value in the development of new antibacterial drugs, especially in the development of drugs against Burkholderia cepacia infection. Attached Figure Description

[0024] Figure 1 For NAD + and NAD + Figure 1 shows the results of capsule inhibition of biofilm formation by Burkholderia cepacia (Figure A shows NAD). + Figure B shows the results of inhibiting biofilm formation in Burkholderia cepacia. + The results of the capsule inhibiting biofilm formation of Burkholderia cepacia are shown in the figure. The data shows the average results of 5 biological replicates. Error bars reflect the standard deviation, where * indicates a significant difference compared with the control group (p < 0.05), ** indicates a significant difference compared with the control group (p < 0.01), *** indicates an extremely significant difference compared with the control group (p < 0.001), and **** indicates an extremely significant difference compared with the control group (p < 0.0001).

[0025] Figure 2 For NAD + and NAD + Figure 1 shows the results of capsule inhibition of Burkholderia cepacia motility (Figure A shows NAD). + The results of inhibiting the motility of Burkholderia cepacia are shown in Figure B, where NAD is represented. + The results of capsule inhibition of Burkholderia cepacia motility are shown in the figure; the data are the average results of three biological replicates, and the error bars reflect the standard deviation, where ** indicates a significant difference compared with the control group (p < 0.01), and *** indicates an extremely significant difference compared with the control group (p < 0.001).

[0026] Figure 3 For NAD + and NAD +The effect of capsules on the growth rate of Burkholderia cepacia (Figures A, B, and C represent NAD). + The results of the effect on the growth rate of Burkholderia cepacia are shown in Figures D, E, and F, which represent NAD. + The effects of capsules on the growth rate of Burkholderia cepacia are shown in the following figures: Figures A and D show the growth rate of Burkholderia cepacia cultured in LB liquid medium; Figures B and E show the growth rate of Burkholderia cepacia cultured in NYG liquid medium; Figures C and F show the growth rate of Burkholderia cepacia cultured in MM liquid medium.

[0027] Figure 4 For NAD + and NAD + The results of the capsule inhibiting the cytotoxicity of Burkholderia cepacia against A549 cells (where Figure A represents different concentrations of NAD). + The results of detecting the inhibition of Burkholderia cepacia infection in A549 cells are shown in Figure B. Figure B shows the results of 100 μM NAD. + Figure C shows the results of cytotoxicity detection of A549 cells; Figure C shows different concentrations of NAD. + The results of the detection of the capsule's inhibition of Burkholderia cepacia infection in A549 cells are shown in Figure D, where 100 μM NAD is used. + The figure shows the results of the cytotoxicity test of the capsules on A549 cells. The data is the average result of four biological replicates. The error bars reflect the standard deviation. * indicates a significant difference compared with the control group (p < 0.05), ** indicates a significant difference compared with the control group (p < 0.01), *** indicates an extremely significant difference compared with the control group (p < 0.001), and **** indicates an extremely significant difference compared with the control group (p < 0.0001). Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The Burkholderia cenocepacia strain used in the following examples is Burkholderia cenocepacia strain B. cenocepacia H111, which has been disclosed in the literature "Carlier A., ​​et al. Genome Sequence of Burkholderiacenocepacia H111, a Cystic Fibrosis Airway Isolate. Genome Announcements, 2014, 2(2): 1-2".

[0031] LB solid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L.

[0032] LB liquid medium culture: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, solvent is water.

[0033] MM liquid culture medium formula: K2HPO4 10.5g / L, KH2PO4 4.5g / L, (NH4)2HPO4 2g / L, MgSO4·7H2O 0.2g / L, FeSO4 0.005g / L, CaCl2 0.01g / L, MnCl2 0.002g / L, mannitol 2g / L, glycerol 2g / L.

[0034] The formula for the motility-enhancing culture medium is: 8 g / L tryptone, 5 g / L glucose, and 3 g / L agar powder.

[0035] NYG liquid culture medium formula: yeast extract 3g / L, peptone 5g / L, glycerol 20g / L.

[0036] CytoTox The Non-Radioactive Cytotoxicity Assay kit was purchased from Promega, catalog number G1780.

[0037] The fetal bovine serum is branded as DIB, and its product number is DIB-12B-50ml.

[0038] The DMEM culture medium is from Pronosai, and its product number is PM150210-500.

[0039] NAD + It is a biological substance, also known as NAD, with the Chinese name nicotinamide adenine dinucleotide, abbreviated as oxidized coenzyme I, CAS number 53-84-9, and its structural formula is:

[0040]

[0041] NAD+ The capsule brand is NaturElan (Germany), product number DNE7150, and its main ingredient is NAD+. + Niacin, lipoic acid, chromium, bioflavonoids, choline, leucine, piperine, with each two tablets containing NAD+ + The capsule contains 150mg of NAD.

[0042] Example 1 NAD + and NAD + Effects of capsules on Burkholderia cepacia biofilm formation

[0043] I. Experimental Methods

[0044] Activation method of Burkholderia cepacia: Burkholderia cepacia strain H111 was used as the test strain. Burkholderia cepacia was streaked on LB plates and incubated overnight at 37°C for activation.

[0045] Preparation method of bacterial suspension: Activated Burkholderia cepacia were picked and cultured overnight in LB liquid medium, and the OD of the bacterial suspension was measured. 600 The bacterial culture was diluted to OD using MM liquid medium. 600 =0.01.

[0046] NAD + Solution preparation: Use sterile water as the solvent, NAD + Using NAD as the solute, NAD+ solutions with concentrations of 100 mM, 50 mM, 25 mM, 12.5 mM, and 6.25 mM were prepared. + The solution was then filtered and sterilized using a 0.22 μm sterile filter membrane.

[0047] NAD + Capsule solution preparation: Dissolve NAD in sterile water. + Capsules, formulated with sterile water as a solvent, to produce NAD. + NAD+ concentrations of 100 mM, 50 mM, 25 mM, 12.5 mM, and 6.25 mM were used. + The capsule solution was then filtered and sterilized using a 0.22 μm sterile filter membrane.

[0048] Set up treatment and control groups:

[0049] Processing Group 1: Press NAD + The volume ratio of solution to bacterial culture was 1:999, and different concentrations of NAD were added. + The solution was added to the bacterial culture.

[0050] Processing Group 2: Press NAD + The volume ratio of capsule solution to bacterial solution was 1:999, and different concentrations of NAD were added. +The capsule solution was added to the bacterial culture.

[0051] Control group: Sterile water was added to the bacterial solution at a volume ratio of sterile water to bacterial solution of 1:999.

[0052] 150 μL of the mixture was added to each treatment group and control group in a 96-well plate, with five replicates per group. The plates were incubated at 37°C and 200 rpm with shaking. After 12 hours, the culture medium was discarded, and 200 μL of 0.1% (w / v) crystal violet was added. The plates were incubated at room temperature for 30 minutes. The crystal violet was then discarded, and the plates were washed three times with ddH₂O. The plates were then dried in a 60°C oven, and 200 μL of 95% ethanol was added. The plates were then allowed to stand at room temperature for 20 minutes before measuring the OD. 570 The values ​​are processed using GraphPad Prism 8 software.

[0053] II. Experimental Results

[0054] NAD + and NAD + The results of the capsule inhibiting the formation of Burkholderia cepacia biofilm are as follows: Figure 1 As shown, the results indicate that, compared with the control group, the final concentration of NAD+ at 6.25 μM was significantly higher. + and NAD + Encapsulation significantly reduced the formation of Burkholderia cepacia biofilm, by approximately 20%. This was further reduced by NAD+ concentration of 50 μM. + and NAD + Encapsulation treatment of Burkholderia cepacia reduced biofilm formation by more than 50%. This result indicates that NAD+ + and NAD + The capsule has an inhibitory effect on biofilm formation of Burkholderia cepacia.

[0055] Example 2 NAD + and NAD + Effects of capsules on the motility of Burkholderia cepacia

[0056] I. Experimental Methods

[0057] Activation method of Burkholderia cepacia and different concentrations of NAD + Solution and NAD + The preparation method of the capsule solution is the same as in Example 1.

[0058] Set up treatment and control groups:

[0059] Processing Group 1: Press NAD + The volume ratio of solution to motile medium was 1:999, and different concentrations of NAD were added. +Add the solution to the motile culture medium, pour into plates, 15 mL per plate, and use a toothpick to pick up the activated fresh Burkholderia cepacia cells and inoculate them in the center of the plate.

[0060] Processing Group 2: Press NAD + The volume ratio of NAD+ capsule solution to motility medium is 1:999. + Add the capsule solution to the motility medium, pour into plates, 15 mL per plate, and use a toothpick to pick up the activated fresh Burkholderia cepacia cells and inoculate them in the center of the plate.

[0061] Control group: Sterile water was added to the motility medium at a volume ratio of 1:999. The medium was poured into plates, 15 mL per plate. Fresh, activated Burkholderia cepacia cells were picked up with a toothpick and inoculated into the center of the plate.

[0062] Each treatment group and control group was set up in 3 replicates and incubated in an incubator at 37°C. After 18 hours, the swimming distance of Burkholderia cepacia on the petri dish was measured and the experimental data were recorded.

[0063] II. Experimental Results

[0064] NAD + and NAD + The results of the capsule inhibiting the motility of Burkholderia cepacia were as follows: Figure 2 As shown, the results indicate that, compared to the control group, the levels of NAD+ at a final concentration of 12.5 μM were significantly lower. + and NAD + In the culture medium containing capsules, the motility of Burkholderia cepacia was significantly reduced. This was observed in the presence of NAD+ at a final concentration of 50 μM. + and NAD + In the culture medium containing the capsules, the motility of Burkholderia cepacia was reduced by more than 50%. This result indicates that NAD... + and NAD + The capsule medication has a good inhibitory effect on the motility of Burkholderia cepacia.

[0065] Example 3 NAD + and NAD + Determination of the effect of capsules on the growth of Burkholderia cepacia

[0066] I. Experimental Methods

[0067] Activation method of Burkholderia cepacia and different concentrations of NAD + Solution and NAD + The preparation method of the capsule solution is the same as in Example 1.

[0068] Activated Burkholderia cepacia were picked and cultured overnight on LB broth. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were then resuspended in LB broth, NYG broth, and MM broth to OD200. 600 =0.01, to obtain a bacterial suspension.

[0069] Set up treatment and control groups:

[0070] Processing Group 1: Press NAD + The volume ratio of solution to bacterial suspension was 1:999, and different concentrations of NAD were added. + The solution was added to bacterial suspensions resuspended in different liquid culture media.

[0071] Processing Group 2: Press NAD + The volume ratio of capsule solution to bacterial suspension was 1:999, and different concentrations of NAD were used. + The capsule solution was added to bacterial suspensions resuspended in different liquid culture media.

[0072] Control group: Sterile water was added to bacterial suspensions resuspended in different liquid culture media at a volume ratio of sterile water to bacterial suspension = 1:999.

[0073] 200 μL of the mixture was added to each treatment group and control group into a 96-well plate, with 5 replicates for each treatment group and control group. The plates were incubated at 37°C and 200 rpm with shaking. OD was measured every 4 hours. 600 Values ​​were set, and experimental results were observed after 2 days. Data was processed using GraphPadPrism 8.

[0074] II. Experimental Results

[0075] NAD + and NAD + The effect of capsules on the growth rate of Burkholderia cepacia is as follows: Figure 3 As shown, the results indicate that, compared with the control group, in LB liquid medium ( Figure 3 Figures A and D), NYG liquid culture medium ( Figure 3 Figures B and E), MM liquid culture medium ( Figure 3 (Figures C and F) show NAD+ concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. + and NAD + The growth rate of Burkholderia cepacia was not affected after capsule treatment. This result indicates that NAD+... + and NAD + The capsules do not primarily inhibit Burkholderia cepacia by killing bacterial cells, thus reducing the likelihood of drug resistance.

[0076] Example 4 NAD + and NAD + Effects of capsules on the virulence of Burkholderia cepacia

[0077] I. Experimental Methods

[0078] (1) Resuscitation and culture of human non-small cell lung cancer cell line A549

[0079] Frozen-thawed A549 cells were transferred to DMEM medium containing 10% fetal bovine serum and cultured overnight at 37°C and 5% CO2 for resuscitation.

[0080] (2) Preparation of A549 cells

[0081] The revived A549 cells were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1.5 × 10⁶ cells / mL. 4 Cells were cultured overnight in 96-well plates at a concentration of 1 cell / well. When the cells covered 80% of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed three times with 1×PBS (pH=7.4, 0.1M).

[0082] (3) Prepare cell culture medium containing Burkholderia cepacia.

[0083] The activation method of Burkholderia cepacia is the same as in Example 1.

[0084] Freshly activated Burkholderia cepacia were inoculated into LB broth and cultured overnight at 37°C and 200 rpm with shaking. The cells were collected by centrifugation at 5000 rpm for 5 min, washed three times with 1×PBS, and then... 9 A concentration of cfu / mL was dispersed in DMEM cell culture medium containing 1% fetal bovine serum (v / v) to obtain a cell culture medium containing bacteria.

[0085] (4) Cytotoxicity assay

[0086] Different concentrations of NAD + Solution and NAD + The preparation method of the capsule solution is the same as in Example 1.

[0087] Treatment group, control group and negative control group were set up:

[0088] Processing Group 1: Press NAD + The volume ratio of solution to bacterial cell culture medium was 1:999, and different concentrations of NAD+ were added. + The solution was added to the bacterial cell culture medium.

[0089] Processing Group 2: Press NAD+ The volume ratio of capsule solution to bacterial cell culture medium is 1:999. Different concentrations of NAD+ are then added. + The capsule solution was added to the bacterial cell culture medium.

[0090] Treatment group 3: 100 μM NAD + Solution (prepared with sterile water as solvent).

[0091] Treatment group 4: 100 μM NAD + Capsule solution (NAD dissolved in sterile water) + (Capsules, prepared with sterile water as a solvent).

[0092] Control group: Sterile water was added to the bacterial cell culture medium at a volume ratio of 1:999.

[0093] Negative control group (control): sterile water.

[0094] 100 μL of liquid was taken from each treatment group, control group, and negative control group and added to each well of a 96-well plate containing pre-prepared A549 cells at a rate of 100 μL / well. The plates were then incubated at 37°C in a 5% CO2 incubator for 8 hours. Four replicates were set up for each treatment group and control group. The CytoTox reagent kit was used. The Non-Radioactive Cytotoxicity Assay was used to measure the LDH (lactate dehydrogenase) activity of cells. The specific measurement method was described in the instruction manual, followed by data analysis.

[0095] II. Experimental Results

[0096] The cytotoxicity of Burkholderia cepacia to A549 cells was determined by detecting LDH release levels, and NAD+ was also measured. + and NAD + The effect of capsules on the virulence of Burkholderia cepacia was investigated by using the LDH release of the control group as 100% and calculating the effects of different concentrations of NAD added. + and NAD + The LDH release ratio of the capsule.

[0097] NAD + and NAD + The results of the capsule inhibiting the cytotoxicity of Burkholderia cepacia against A549 cells are shown in the figure below. Figure 4 As shown, the results indicate that, under the condition of adding Burkholderia cepacia, NAD+ levels were lower than those in the control group. + At 6.25 μM, the virulence of Burkholderia cepacia significantly decreased, and NAD... +At 12.5 μM, the virulence of Burkholderia cepacia was significantly reduced. NAD + and NAD + At 100 μM, the virulence of Burkholderia cepacia was reduced to below 50%. Figure 4 (Figures A and C).

[0098] In the absence of Burkholderia cepacia, compared with the negative control group, 100 μM NAD+... + and NAD + There was no significant difference in LDH release between the capsule-treated group and the negative control group, indicating that 100 μM NAD+ release is effective. + and NAD + The capsule is non-toxic to cells. Figure 4 (Figures B and D).

[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of nicotinamide adenine dinucleotide in the preparation of anti-Burkholderia cepacia products.

2. Application of nicotinamide adenine dinucleotide in the preparation of drugs for the prevention or treatment of Burkholderia cepacia infection.

3. NAD + Application of capsules in the preparation of anti-Burkholderia cepacia products, wherein the NAD + The capsule's ingredients include NAD. + Niacin, lipoic acid, chromium, bioflavonoids, choline, leucine, piperine.

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