This application is related to, and claims priority benefit of, Chinese Patent Application Serial No. 202010418358.1, filed May 18, 2020. The content of the aforementioned priority application is hereby expressly incorporated by reference in its entirety into this disclosure.
The present invention relates to the technical field of medicines, and in particularly to use of melatonin in preparation of a medicament for inhibiting and/or killing a bacterium.
A written Sequence Listing for the sequences described herein is appended hereto and the same Sequence Listing is provided in computer-readable form (CRF) encoded in a file that is submitted concurrently herewith and incorporated herein by reference. Such file, generated on Aug. 18, 2020, is entitled “ST25.txt”. The content of the CRF is the same, and the information recorded in the CRF is identical to, the written Sequence Listing provided herein, pursuant to 37 C.F.R. § 1.821(f).
Among human and animal diseases, infectious diseases caused by pathogenic bacteria are spread most widely, which seriously threatens the public health around the world. So far, the discovery and application of antibiotics undoubtedly play an important role in resisting bacterial infection. However, due to the overuse and abuse of antibiotics in the pharmaceutical industry or animal husbandry, the emergence and prevalence of antibiotic resistance in human beings, animals and ecological environment are caused irreversibly. Alarmingly, plasmid-mediated interspecific and interspecific horizontal transfer of an antibiotic resistance gene accelerates the rapid spread of a resistance genetic element. Furthermore, residual antibiotics in veterinary drugs, animal products and the like products will also lead to serious environmental pollution, all constituting a great threat to human health. Therefore, there is an urgent need for more environmentally friendly, safer and more effective compounds to treat bacterial infectious diseases.
For bacterial infectious diseases, especially diseases caused by gram-negative bacterial infection, therapeutic drugs are very limited. The main reasons include: 1) the outer layer of the cell membrane of the gram-negative bacterium is composed of lipopolysaccharide, and can serve as an osmotic barrier to prevent antibiotics from entering the interior of the bacterium; and 2) the gram-negative bacterium has acquired multidrug resistance. Therefore, finding a safe and effective compound to inhibit the gram-negative bacterium is still an urgent problem to be solved in the current medical field.
Melatonin (N-acetyl-5-methoxytryptamine) is a natural compound, mainly a hormone secreted from the pineal gland of a vertebrate. Current studies have found that melatonin is widely used in sleep promoters by inhibiting orexin neurons in hypothalamus that interact with MT1 receptors. However, so far there has been no report on researches that melatonin inhibits bacteria and bacterial infectious diseases.
To address the aforementioned technical problem, the present invention provides use of safe and nontoxic melatonin in preparation of a medicament for inhibiting and/or killing a bacterium, and in particular melatonin can effectively inhibit and/or kill a gram-negative bacterium.
In order to realize the aforementioned objective of the present invention, the present invention provides the following technical solutions.
In at least one embodiment, a medicament for inhibiting and/or killing a bacterium is provided that comprises melatonin.
Preferably, the aforementioned bacterium is a gram-positive bacterium or a gram-negative bacterium.
Preferably, the gram-positive bacterium includes Bacillus cereus, Streptococcus pneumoniae, Pneumococcus, Listeria monocytogenes, Lactobacillus johnsonii or Staphylococcus aureus.
Preferably, the gram-negative bacterium includes Pasteurella multocida, Klebsiella pneumoniae, avian pathogenic Escherichia coli, Enterobacter sakazakii, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella enteritidis, bovine meningitic Escherichia coli or Mannheimia haemolytica.
In certain embodiments, the present invention provides use of melatonin in preparation of a medicament that damages the cell membrane of a bacterium.
The present invention provides use of melatonin in preparation of a bacterial metabolic blocker composition.
Preferably, the pathways blocked by the aforementioned bacterial metabolic blocking composition may include, for example, pyruvate metabolism, citric acid cycle and tryptophan metabolism.
In other embodiments, the present invention provides use of melatonin in preparation of a medicament for inhibiting a citrate synthase activity in a gram-negative bacterium.
The present invention provides use of melatonin in preparation of a medicament for preventing and/or treating a bacterial infection or related disease.
The present invention provides use of melatonin in preparation of a medicament for inhibiting pulmonary inflammation.
Compared with the prior art, the technical solution of the present invention has the following beneficial effects.
The present invention provides novel uses of melatonin to inhibit and/or kill a bacterium for the first time, and in particular, is capable of inhibiting and/or killing a gram-negative bacterium. The bacteriostatic or bactericidal mechanism of melatonin indicates that melatonin damages the metabolic flux of a bacterium by specifically targeting citrate synthase in a gram-negative bacterium, resulting in membrane damage and release of cell contents. When administered to a host, compositions of the present disclosure comprising melatonin, and the methods of using the same in treatments, can also reduce the pathological changes of tissues and organs of the host, and inhibit the colonization of pathogenic bacteria in the host body, thereby effectively improving the survival of animals (including, without limitation, mammals) infected with pathogenic bacteria. In this manner, the compositions and methods hereof can be used to prevent and/or treat diseases caused by infection with pathogenic bacteria, and provide a basis for the development of new drugs. Furthermore, the natural bacteriostatic substance melatonin is harmless, pollution-free, safe and effective, and, when used as described herein, solves the problems of drug resistance, pollution and the like caused by antibiotic abuse.
In the figures, “CA” represents citric acid, and MT represents melatonin;
The present invention provides a novel method for the use of melatonin in a preparation of a medicament for inhibiting and/or killing a bacterium.
In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes a compound or composition that comprises melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally.
Preferably, the aforementioned bacterium is a gram-positive bacterium or a gram-negative bacterium.
Preferably, the aforementioned gram-positive bacterium includes Bacillus cereus, Streptococcus pneumoniae, Pneumococcus, Listeria monocytogenes, Lactobacillus johnsonii or Staphylococcus aureus.
Preferably, the gram-negative bacterium includes Pasteurella multocida, Klebsiella pneumoniae, avian pathogenic Escherichia coli, Enterobacter sakazakii, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella enteritidis, bovine meningitic Escherichia coli or Mannheimia haemolytica.
The present invention provides use of melatonin in preparation of a medicament that damages the cell membrane of a bacterium.
In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally. The present invention provides use of melatonin in preparation of a bacterial metabolic blocker. In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally. Preferably, the pathways of the aforementioned bacterial metabolism blocking include pyruvate metabolism, citric acid cycle and tryptophan metabolism.
The present invention provides use of melatonin in preparation of a medicament for inhibiting a citrate synthase activity in a gram-negative bacterium.
In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally.
The present invention provides use of melatonin in preparation of a medicament for preventing and/or treating a bacterial infection disease.
In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally. The present invention provides use of melatonin in preparation of a medicament for inhibiting pulmonary inflammation.
In the present invention, the medicament for inhibiting and/or killing a bacterium preferably includes melatonin as an active ingredient and a pharmaceutically acceptable carrier. In the present invention, the content of melatonin in the medicament is preferably 1.5625 mg/mL. In the present invention, the pharmaceutically acceptable carrier for example includes one or more of a diluent, a colorant, a sweetener, a coating agent, an adhesive, an absorbent, a disintegrating agent, a releasing agent, a dispersing agent, a wetting agent, a cosolvent, a buffer and a surfactant. The dosage form of the medicament is preferably a solid, liquid or gas formulation, wherein the solid formulation is preferably powder, tablets, granules, pills, hard capsules, cream, ointment, plaster, gels, pastes, pulvis or patches. The liquid formulation is preferably a solution, a suspension, an injection, syrup, liniment, an emulsion, tincture, pulvis or a patch. The gas formulation is preferably an aerosol or spray. The medicament of the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously and intranasally.
Experimental Materials
1. Strains Mainly Used in Examples
The present invention has no special limitation on the source of the strains, and it will be appreciated that any strain well known to those skilled in the art can be used.
2. Materials Mainly Used in the Examples
For the examples provided herein, melatonin is available from Sangon Biotech under the product code A600605. O-nitrophenyl-β-D-galactopyranoside is available from Sangon Biotech under the product code A602361.
The kit for protein detection is available from Beyotime under the product code P0006C.
The alkaline phosphatase kit is available from Nanjing Jiancheng Bioengineering Institute under the product code A059-1-1.
The reducing sugar kit is available from Solarbio under the product code BCO230.
The LIVE/DEAD BacLight bacterial detection kit is available from Invitrogen under the product code L7012.
The kit used for determining the citrate synthase activity is available from Sigma under the product code MAK193.
The LDH detection kit is available from Beyotime under the product code C0017.
The mice used in the examples of the present disclosure were female C57BL/6 mice purchased from Experimental Animal Center, Third Military Medical University, Chongqing, China. The mice weighed 18-22 g and were 6-8 weeks old. They were raised in individual ventilated cages without pathogens (at a temperature of 20-30° C., relative humidity of 50-60%, and an illumination period of 12 hours every day), and had free access to feedstuff and water.
In the present disclosure, analysis was conducted by a statistical method adopting one-way analysis of variance, and is expressed herein as an average±SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, wherein P<0.05 refers to the difference being statistically significant.
In the present disclosure, the raw constituents mentioned are all commercially available products well known to those skilled in the art, unless otherwise specified.
The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are merely provided as explanatory examples, are not intended to be limiting, and indeed do not comprise all of the embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
1 mL of a fresh suspension of bacteria (1×109 CFU) at the logarithmic growth stage was added into 100 mL containing different concentrations (50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.125 mg/mL, 1.5625 mg/mL, 0.78125 mg/mL, 0.390625 mg/mL) of melatonin. A melatonin (MT)-free solution diluted with DMSO was used as a control group. The samples were shaken at 37° C. and a rotation speed of 120 r/min with protection from light for 12 hours, and then subjected to detection of the minimal inhibitory concentration (MIC) of MT on bacteria. Then, 0.1 mL of the aforementioned transparent bacterial solution was pipetted onto a sterilized agar plate of a specific culture medium and coated evenly, incubated in an incubator at 37° C. for 24 hours, and then subjected to detection of the minimum bactericidal concentration (MBC) of MT on pathogens, wherein the minimum concentration of MT at which no bacterium grew was the MBC of MT on pathogens. Each experiment was carried out for 3 times, and each group was set in triplicate.
The MIC and MBC results of melatonin on gram-negative bacteria and gram-positive bacteria are shown in Table 1:
Pasteurella multocida
Klebsiella pneumoniae
Enterobacter sakazakii
Pseudomonas aeruginosa
Acinetobacter baumannii
Salmonella enteritidis (ATCC85090)
Escherichia coli C83902 (ATCC72864)
Mannheimia haemolytica
Bacillus cereus
Streptococcus pneumoniae
Pneumococcus
Listeria monocytogenes (ATCC53005)
Lactobacillus johnsonii
Staphylococcus aureus (ATCC25923)
It can be seen from Table 1 that melatonin exhibited a bacteriostatic or bactericidal activity against both gram-negative bacteria and gram-positive bacteria, with MIC values ranging from 1.5625 to 50 mg/mL and MBC values ranging from 3.125 to 50 mg/mL. Notably, melatonin showed the most effective antibacterial or bactericidal activity against Pasteurella multocida, with MIC and MBC of 1.5625 mg/mL and 3.125 mg/mL respectively.
In order to further verify the bacteriostatic activity of melatonin against Pasteurella multocida, the bacterial growth curve was determined in view of an increase of melatonin concentration. Here, 1 mL of Pasteurella multocida (1.0×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing a MT concentration lower than MBC (1 mg/mL, 1.5 mg/mL and 2 mg/mL), and melatonin-free DMSO was used as a solvent control group. Then the bacteria were cultured in a shaker at a constant temperature of 37° C. at 220 rpm, and the bacterial culture was determined for OD600 every 2 hours with an ultraviolet spectrophotometer to detect the effect of melatonin on the bacterial growth curve. Each experiment was carried out for three times, and each group was set in triplicate.
The results shown in
Into 100 mL of fresh and sterile Martin broth medium, 1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added. The bacteria were cultured in a constant-temperature shaker under conditions of 37° C. and 220 rpm for 8 hours. Subsequently, MT of different concentrations (4 mg/mL, 8 mg/mL and 10 mg/mL) higher than the MBC were added into the bacterial culture, while the MT-free DMSO was used as the solvent control group. After the addition of MT, the bacterial culture was continuously measured for OD600 every 20 minutes to depict a growth curve under the action of melatonin at a concentration higher than the MBC. At the same time, 100 μL of the bacterial culture was evenly coated on a Martin agar plate, and incubated at 37° C. for 24 h so as to calculate the number of living bacteria for bacterial counting.
The results of these studies are shown in
1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations (1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL and 10 mg/mL) of MT. Melatonin-free DMSO was used as a solvent control group, ampicillin (100 μg/mL) was used as a positive control group, and bacterial cells were cultured in a shaker at 37° C. and 220 rpm for 12 h. Thereafter, 1 mL of the bacterial solution was taken and centrifuged at 3000 r/min for 10 minutes, and the supernatant and precipitated cells were collected, respectively. At the same time, 2.5 mmol/mL of o-nitrophenyl-β-D-galactopyranoside (ONPG) and Pasteurella multocida were added to detect β-galactosidase. The concentrations of protein, alkaline phosphatase (AKP) and reducing sugar in the supernatant were determined sequentially using respective determination kits. The β-galactosidase in the supernatant was determined by measuring the optical density at 420 nm (OD420). Each experiment was carried out for three times, and each group was set in triplicate.
The results of these studies are shown in
The integrity of bacterial cell membrane was evaluated with a LIVE/DEAD BacLight bacterial detection kit. 1 mL of bacterial culture was centrifuged at 3000 rpm for 10 minutes, the supernatant was removed, and the cells were washed with 10 mL of 0.85% NaCl for 3 times. The precipitated cells were resuspended and mixed with 1 mL of 0.85% NaCl. Then a mixture of SYTO9 fluorescent nucleic acid dye and propidium iodide mixed at the equal volume (3 μL each) was added into the resuspended solution. The mixture was thoroughly mixed and incubated in the dark at room temperature for 15 minutes. 5 μL of suspension of stained bacteria was added between a slide glass and a 18 mm square cover glass. (The combination of two nucleic acid dyes in this kit can distinguish living bacteria with intact membranes from dead bacteria with damaged membranes.) The living bacteria were dyed green with SYTO9, while the dead bacteria presented as red because of the interaction between propidium iodide and the double-stranded DNA of the bacteria. The fluorescence micrographs of stained bacteria were taken by OLYMPUS IX51 inverted microscope. Each experiment was carried out in triplicate for three times.
The effect of melatonin on the morphologies of P. multocida cells were evaluated employing SEM and TEM. 1 mL of fresh Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh Martin liquid medium containing different concentrations (1, 2 or 3 mg/mL) of MT. MT-free DMSO was used as a solvent control group and ampicillin (100 μg/mL) was used as a positive control group. All cultures were shaken at 220 rpm in a shaker at 37° C. for 8 hours, 12 hours or 16 hours. The bacterial samples were centrifuged at 3000 r/min for 10 minutes, the supernatant was removed, and cells were collected and washed with PBS for 3 times. Then the cells were fixed with a fixation solution of 2.5% fresh glutaraldehyde for 6 hours, and washed with PBS for 3 times, each time for 10 minutes. Subsequently, the SEM samples were dehydrated with 50%, 70%, 80% and 90% ethanol for 15 minutes respectively, and then dehydrated with 100% ethanol for 3 times, each time for 30 minutes. Ethanol was removed by washing with tert-butyl alcohol for three times, each time for 30 minutes. The samples were dried by a freeze dryer and plated with 10 nm of gold film by an ion sputtering device. The samples were examined using a field scanning electron microscope (FESEM, Hitch SU8010).
The TEM samples were fixed with 1%-2% citric acid for 2-3 hours, and washed with phenyl-5-(4-diphenyl)-1,3,4-oxazole (PBD) 3 times, each time for 10 minutes. The samples were dehydrated with 30%, 50%, 70%, 80% and 90% ethanol for 20 minutes respectively, and then dehydrated with 100% ethanol 3 times, each time for 30 minutes. Ethanol was replaced with acetone three times, each time for 30 minutes. The bacterial samples were placed on a copper grid with a formvar membrane, and negatively stained with phosphotungstic acid (2% v/v, pH=6.7). The surface area of the sample was less than 0.2 mm×0.2 mm, and slice thickness was between 50 and 90 nm (Lycra UC7). The samples were detected by TEM (Hitachi H-7650) at 80 kV, and photographs of the samples were taken by a Gatan832 CCD camera (Gatan). Each experiment was carried out twice, and each group was set in triplicate.
Therefore, the results illustrated in
1 mL of fresh Pasteurella multocida (P. multocida) at the logarithmic growth stage was added into 100 mL of fresh Martin liquid medium containing 1 mg/mL of MT, and MT-free DMSO was used as a solvent control group. All cultures were incubated with shaking at 37° C. and 220 rpm for 12 hours. The bacterial samples were centrifuged at a speed of 3000 rpm/min at 4° C. for 10 minutes, the supernatant was removed, and the bacterial cells were collected and washed with PBS for 3 times. The bacterial cells were rapidly frozen in liquid nitrogen, transferred to dry ice, and sent to Shanghai Baiqu Biomedical Technology Co., Ltd. for metabolomic analysis (Q Exactive Orbitrap, Thermo Fisher Scientific, USA). Through the non-targeted metabonomic study of Pasteurella multocida cells before and after melatonin treatment, it was clarified which metabolic pathway of the bacteria is affected by melatonin in exerting the antibacterial activity of melatonin.
As shown in
Based on the discovery of metabolomics, the enzyme activity of citrate synthase after melatonin treatment was studied. 1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations (1 mg/mL, 2 mg/mL, and 3 mg/mL) of MT. Melatonin-free DMSO was used as a solvent control group, and the bacterial cells were cultured in a constant-temperature shaker at 37° C. and 220 rpm for 12 h. Thereafter, 1 mL of the bacterial solution was centrifuged at 3000 r/min for 10 minutes, and the supernatant and precipitated cells were collected, respectively. The citrate synthase activity was determined using a determination kit according to the protocol of the instructions. Each experiment was repeated for three times, and each group was set in triplicate.
In order to explore whether the decrease of citric acid induced by melatonin is the key link of the bacteriostasis of melatonin, the influence of exogenous citric acid on the bacteriostatic effect of melatonin was also studied. Here, 1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations of citric acid (CA-1 mg/ml, CA-2 mg/mL, and CA-3 mg/mL), and citric acid-free DMSO was used as a solvent control group. The bacterial cells were cultured in a constant-temperature shaker at 37° C. and 220 rpm for 30 minutes. Then, the growth curve of the cells was determined. Each experiment was repeated for three times, and each group was set in triplicate.
Additionally, 1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations of melatonin and citric acid (CA) (MT-1 mg/mL, MT-2 mg/mL, MT-3 mg/mL, CA-1 mg/mL, CA-2 mg/mL, CA-3 mg/mL, MT-1 mg/mL-CA-1 mg/mL, MT-2 mg/mL-CA-2 mg/mL, MT-3 mg/mL-CA-3 mg/mL). The melatonin-free DMSO was used as a solvent control group, and the bacterial cells were cultured in a shaker at 37° C. and 220 rpm for 12 h. Thereafter, 1 mL of the bacterial solution was taken and centrifuged at 3000 r/min for 10 minutes, and the supernatant and precipitated cells were collected, respectively. At the same time, 2.5 mmol/mL of o-nitrophenyl-3-D-galactopyranoside (ONPG) and Pasteurella multocida were added to detect (3-galactosidase. The concentrations of protein, alkaline phosphatase (AKP) and reducing sugar in the supernatant were determined sequentially using respective determination kits. The β-galactosidase in the supernatant was determined by measuring the optical density at 420 nm (OD420). Each experiment was carried out for three times, and each group was set in triplicate.
1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was also added into 100 mL of fresh and sterile Martin broth medium containing different concentrations of melatonin and citric acid (CA) (MT-1 mg/mL, MT-1 mg/mL-CA-1 mg/mL, MT-1 mg/mL-CA-2 mg/mL, MT-1 mg/mL-CA-3 mg/mL; MT-2 mg/mL, MT-2 mg/mL-CA-1 mg/mL, MT-2 mg/mL-CA-2 mg/mL, MT-2 mg/mL-CA-3 mg/mL; MT-3 mg/mL, MT-3mg/mL-CA-1 mg/mL, MT-3 mg/mL-CA-2 mg/mL, and MT-3 mg/mL-CA-3 mg/mL). DMSO not containing melatonin and citric acid was used as a solvent control group, and the bacterial cells were cultured in a constant-temperature shaker at 37° C. and 220 rpm for 12 h. Then the growth curve of the cells were determined. Each experiment was carried out for three times, and each group was set in triplicate.
Further, 1 mL of Pasteurella multocida (1×109 CFU) at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations of melatonin and citric acid (MT-1 mg/mL, MT-2 mg/mL, MT-3 mg/mL, CA-1 mg/mL, CA-2 mg/mL, CA-3 mg/mL, MT-1 mg/mL-CA-1 mg/mL, MT-2 mg/mL-CA-2 mg/mL, MT-3 mg/mL-CA-3 mg/mL). The melatonin-free DMSO was used as a solvent control group, and the bacterial cells were cultured in a constant-temperature shaker at 37° C. and 220 rpm for 12 h. Thereafter, 1 mL of the bacterial solution was centrifuged at 3000 r/min for 10 minutes, and the supernatant and precipitated cells were collected, respectively. The citrate synthase activity was determined using a determination kit according to the protocol of the instructions. Each experiment was repeated for three times, and each group was set in triplicate.
The results shown in
In view of the important role of citrate synthase in mammalian cells, the cytotoxicity of melatonin in mouse peritoneal primary macrophages and mouse lung epithelial cells BNCC341334 was examined.
5×105 cells per well were inoculated in a 24-well cell culture plate, and cultured in a 5% CO2 incubator at 37° C. until full confluence of the cells. The non-adherent cells were washed away with PBS, and the cells were added with medium with the final melatonin concentrations of 1, 2, 3 and 10 mg/mL sequentially. The melatonin-free DMSO solvent was used as the control group. After melatonin treatment for 12 h, the supernatant was collected for LDH test. For specific test steps, please refer to the LDH test kit as is known in the art. Each experiment was repeated three times, and each group was set in hexaplicate.
2×105 mouse lung epithelial cells per well were inoculated into a 48-well microplate, and cultured in a 5% CO2 incubator at 37° C. until full confluence of the cells. The non-adherent cells were washed away with 1×PBS, and the cells were treated with melatonin with the final concentrations of 1, 2, 3, 10, 20 and 40 mg/ml, while the control group was treated with the same volume of DMSO. After melatonin treatment for 30 min, 1MOI Pasteurella multocida was added into the culture medium for continue culture. After 12 h, the bacterial cells were collected, lysed by ultrasound at a low temperature, and then the enzyme activity of citrate synthase was analyzed. For specific test steps, please refer to the kit for detecting the enzyme activity of the citrate synthase. Each experiment was repeated three times, and each group was set in hexaplicate.
In additional studies, 1 mL of Klebsiella pneumoniae, Pseudomonas aeruginosa, Diplococcus pneumoniae and Streptococcus pneumoniae at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth medium containing different concentrations of melatonin and citric acid (CA) (MT-1 mg/mL, MT-1 mg/mL-CA-1 mg/mL, MT-1 mg/mL-CA-2 mg/mL, MT-1 mg/mL-CA-3 mg/mL; MT-2 mg/mL, MT-2 mg/mL-CA-1 mg/mL, MT-2 mg/mL-CA-2 mg/mL, MT-2 mg/mL-CA-3 mg/mL; MT-3 mg/mL, MT-3 mg/mL-CA-1 mg/mL, MT-3 mg/mL-CA-2 mg/mL MT-3 mg/mL-CA-3 mg/mL). DMSO (that did not contain melatonin and citric acid) was used as a solvent control group, and bacterial cells were cultured in a constant-temperature shaker at 220 rpm and 37° C., and then 12 h later the growth curve of the cells was determined.
Further, 1 mL of Klebsiella pneumoniae, Pseudomonas aeruginosa, Diplococcus pneumoniae and Streptococcus pneumoniae at the logarithmic growth stage was added into 100 mL of fresh and sterile Martin broth liquid medium containing different concentrations of melatonin and citric acid (MT-1 mg/mL, MT-2 mg/mL, MT-3 mg/mL, CA-1 mg/mL, CA-2 mg/mL, CA-3 mg/mL, MT-1 mg/mL-CA-1 mg/mL, MT-2 mg/mL-CA-2 mg/mL, MT-3 mg/mL-CA-3 mg/mL) added was. Melatonin-free DMSO was used as the solvent control group, and the bacterial cells were cultured in a constant-temperature shaker at 37° C. and 220 rpm for 12 h. Thereafter, 1 mL of the bacterial solution was pipetted and centrifuged at 3000 r/min for 10 minutes. The bacterial cells were then collected and determined for citrate synthase activity according to the protocol of the instructions for the kit. Each experiment was repeated three times, and each group was set in triplicate.
It can be seen in
It can be seen from
In view of the remarkable in vitro bacteriostatic activity and novel bacteriostatic mechanism of melatonin, the in vivo preventive and therapeutic potential of melatonin was studied.
1. Test of Prevention of Pasteurella multocida Infection by Melatonin:
C57 mice purchased from the Third Military Medical University were randomly divided into four groups (10 mice in each group): a group of Pasteurella multocida+solvent control, a group of Pasteurella multocida+MT (30 mg/kg), a group of Pasteurella multocida+MT (60 mg/kg), and a group of Pasteurella multocida+MT (120 mg/kg). Before Pasteurella multocida infection, MT was injected intraperitoneally to the experimental group (injection once in the morning) for consecutive 7 days (P) or 6 days (P-1), while the control mice received the equal amount of DMSO. Thereafter, mice were infected by intraperitoneal injection of suspension of Pasteurella multocida (2.2∴105 CFU). The survival rate of mice within 7 days was monitored and recorded. Blood samples were collected from mice, and the serum was stored at −20° C. for later ELISA test. Lung samples were collected and homogenized aseptically, and then the bacterial load was quantified by serial dilution, plate inoculation and CFU counting.
2. Test of Treatment of Pasteurella multocida Infection by Melatonin:
Mice were randomly divided into four groups (10 mice in each group) as described in #1, described above. In the experimental group, after infection with 2.2×105 CFU of Pasteurella multocida through intraperitoneal injection, MT was injected intraperitoneally for four times, with the first injection 30 minutes after Pasteurella multocida infection, then the injection was performed once every 6 hours, for three times in total. Control mice were given an equal amount of DMSO at the same four time points. Subsequently, the survival rate of mice within 7 days was recorded. At the same time, mice were euthanized at 12 h, 16 h, 24 h and 32 h after infection to collect tissue and serum samples for later ELISA and bacterial count analysis.
3. Test of Colonization of Mouse Lung Bacteria Before and After Melatonin Treatment
In order to measure the colonization of mouse lung bacteria before and after melatonin treatment, the lung tissues of mice were collected 24 hours and 32 hours after infection. The tissues were homogenized under aseptic conditions, and the bacterial content was determined by continuous dilution of 10 times in saline. These different dilutions were coated on the Martin broth agar in triplicate and incubated in a constant-temperature incubator at 37° C. for 24 hours to count the bacterial CFU.
4. RT-PCR Analysis:
The mouse lung tissue samples were collected, and the total RNA of mouse lung tissues was extracted according to the instructions of the total RNA extraction kit (Invitrogen). According to the instructions of a TaKaRa reverse transcription kit, cDNA was prepared and reverse transcribed with the general process as follows:
1) Genomic DNA Elimination Reaction:
The reaction system was as follows:
The reaction procedure was 42° C. for 2 min, and stored at 4° C.
2) Reverse Transcription Reaction:
The reaction system was as follows:
The reaction procedure was 37° C. for 15 min, 85° C. for 5 s, and stored at 4° C.
3) qRT-PCR
The process of qRT-PCR was carried out with reference to previous studies, and (3-actin was used as a reference gene to normalize the transcription level of the target gene. The specific primer sequences used are shown in Table 2. The relative expression level of the gene was calculated according to the equation 2−(ΔΔCt), wherein ΔΔCt=(CT of the target gene−Ct of the internal reference) of the experimental group−(Ct of the target gene−Ct of the internal reference) of the control group.
5. Enzyme Linked Immunosorbent Assay (ELISA):
ELISA samples were divided into serum samples and lung tissue homogenate samples. Among them, the process of collecting serum was collecting mouse whole blood from the eyeball, storing overnight at 4° C., centrifuging at 5000 rpm for 10 min, collecting the supernatant and storing it at −20° C. for later use. While the preparation process of the mouse lung homogenate was collecting lung tissues, adding 2 mL sterile normal saline to prepare a homogenate, then repeatedly freezing and thawing the homogenate in liquid nitrogen (5 min) and ice-water bath for 3 times, then centrifuging at 4° C. and 12000 rpm for 10 min, and collecting the supernatant for later use. According to the instructions of the kit, the serum and the supernatant of lung tissue homogenate as prepared were detected for IL-10, IL-4, IL-6, IL-10, TNF-α and IFN-γ (eBioscience).
The data illustrated in
Furthermore, before Pasteurella multocida infection, all mice were intraperitoneally injected with melatonin for consecutive 7 days to test the preventive effect of melatonin against bacterial infection. The results shown in
Finally, melatonin was injected intraperitoneally for four times respectively at 30 minutes, 6 hours, 12 hours and 18 hours after the bacterial infection to explore the therapeutic effect of melatonin on Pasteurella multocida infection. The results show that it had obvious protective effects, including improving the survival rate and reducing bacterial colonization in mouse lungs (see
The above description describes preferred embodiments of the present invention and is not intended to be limiting. It should be pointed out that, for those of ordinary skills in the art, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as falling into the claimed scope of the present invention.
Number | Date | Country | Kind |
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202010418358.1 | May 2020 | CN | national |