CN115040551A - Application of lactobacillus reuteri LN0214 for regulating and controlling level of host melatonin - Google Patents
Application of lactobacillus reuteri LN0214 for regulating and controlling level of host melatonin Download PDFInfo
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Abstract
The invention relates to an application of lactobacillus reuteri LN0214, and belongs to the technical field of melatonin regulation. The invention provides application of lactobacillus reuteri (Lactobacillus reuteri) LN0214 in preparation of a medicament for regulating and controlling melatonin, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24542. The lactobacillus reuteri LN0214 has a regulation effect on the level of host melatonin, can specifically regulate the level of host serum and colon melatonin, and provides a theoretical basis for development of a new drug for regulating and controlling synthesis and secretion of the melatonin.
Description
Technical Field
The invention relates to the technical field of melatonin regulation and control, and particularly relates to application of a Lactobacillus reuteri LN0214 strain.
Background
Melatonin (N-acetyl-5-methoxytryptamine) is a natural compound, primarily a hormone secreted in the pineal gland of vertebrates. However, there is experimental evidence that melatonin synthesis is not restricted to the pineal gland, and other organs and cells, even non-vertebrates, are able to synthesize melatonin, especially in the mitochondria. In recent decades, melatonin has been widely used as a sleep promoter, and in addition to it, exhibits various other physiological and pharmacological biological functions, such as regulation of circadian rhythm, immunoregulation, scavenging of free radicals, promotion of normal cell proliferation and inhibition of apoptosis, and several of them have been explored to treat various human diseases.
The structure of the intestinal microorganisms of mammals is complex and the quantity is huge. Generally, the number of the microorganisms is 500-1000, and the total number is more than 10 13 The main components of the medicine include bacteria, fungi, saccharomycetes, viruses, protozoa, archaea and the like. Wherein the bacteria are the main part of the intestinal symbiosis. The gut microbiota is a complex ecosystem that is critical to the host's resistance to infection, nutrient metabolism and tissue repair. In view of the important roles of intestinal microorganisms in nutrient absorption, metabolism and immune regulation, it is suggested that intestinal microorganisms are regarded as a special and essential component of the animal body, and that the amino acid metabolism of the host is also affected by intestinal microorganisms. Melatonin is a metabolite of tryptophan, and its metabolic regulation in the host may be affected by intestinal microorganisms.
It is generally considered that melatonin is synthesized from the pineal gland, but the melatonin content in the intestinal tract is the largest. The enterochromaffin cells in the intestinal tract synthesize melatonin which is secreted to the digestive tract, and the microorganisms colonized in the intestinal tract as part of the intestinal system play a crucial role in the amino acid metabolism of the intestinal tract. In addition, since the intestinal tract is an important nerve-regulating organ, intestinal microorganisms may influence the melatonin synthesis and secretion of the pineal gland through the brain-intestinal axis, thereby affecting the melatonin content in blood. Research has shown that the expression level of a key rate-limiting enzyme AANAT in a melatonin synthesis pathway is related to the change of the melatonin level, and the expression level has a positive regulation effect on the melatonin synthesis. Intestinal microorganisms have been partially reported to regulate host amino acid metabolism, however, how intestinal microorganisms regulate host melatonin levels is still poorly understood.
Disclosure of Invention
The invention aims to provide application of a Lactobacillus reuteri LN 0214. The lactobacillus reuteri LN0214 has a regulation effect on the level of host melatonin, can specifically regulate the level of host serum and colon melatonin, and provides a theoretical basis for development of a new drug for regulating and controlling synthesis and secretion of the melatonin.
The invention provides application of lactobacillus reuteri LN0214 in preparation of a medicament for regulating and controlling melatonin, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention also provides application of lactobacillus reuteri LN0214 in preparation of a medicine for reducing serum melatonin level, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention also provides application of lactobacillus reuteri LN0214 in preparing a medicine for improving the level of the colonic melatonin, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention also provides application of the lactobacillus reuteri LN0214 in preparation of a medicine for improving the expression level of the colon AANAT protein, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention provides application of lactobacillus reuteri LN0214 in preparation of a medicament for regulating and controlling melatonin. The lactobacillus reuteri LN0214 has the effect of remarkably regulating the level of host melatonin, and particularly has the effect of regulating the level of host melatonin in serum and colon tissues; the invention provides a novel melatonin regulation and control mode. Lactobacillus reuteri LN0214 affects melatonin levels in mouse serum and colon. These findings highlight the great potential of Lactobacillus reuteri LN0214 in regulating melatonin levels in the host. The method determines the content difference of the melatonin of the serum and the colon of mice treated by different antibiotics through a metabonomics technology. Protein expression difference of colon AANAT of mice in a control group and a lactobacillus reuteri LN0214 group is detected by a Western blot technology, and melatonin content difference of serum of a sterile mouse is determined by a metabonomics technology. In the in vivo colonization model of the strain, lactobacillus reuteri LN0214 can reduce the melatonin level in the host serum and can increase the melatonin level in the host colon tissue. These findings indicate that intestinal bacteria (lactobacillus reuteri) have great potential in regulating host melatonin levels and can be used as important therapeutic and prophylactic means for regulating biorhythm and intestinal health.
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FIG. 1 is a graph showing the result of the metabolic change of melatonin in mice treated with mixed antibiotics colonized by Lactobacillus reuteri LN0214 according to the present invention; wherein, A: a flow chart of a field planting experiment of mixed antibiotic treatment mouse lactobacillus reuteri LN 0214; b: a result graph of melatonin level change in serum of mice colonized by lactobacillus reuteri LN 0214; c: a graph of the change result of melatonin level in the colon of a mouse colonized by lactobacillus reuteri LN 0214; d: a result graph of mouse colon AANAT protein Westernblot fixedly planted by lactobacillus reuteri LN 0214; b and C data were analyzed by t-test and expressed as mean. + -. SD; p < 0.05; abx is mixed antibiotic; L.R LN 214: lactobacillus reuteri LN 0214;
FIG. 2 is a graph showing the result of the metabolic change of melatonin of a sterile mouse colonized by Lactobacillus reuteri LN0214 provided by the present invention; a: a flow chart of an experiment for permanent planting of sterile mouse lactobacillus reuteri LN 0214; b: sequencing result chart of genus level relative abundance of 16S rRNA gene amplicon of sterile mouse lactobacillus reuteri LN0214 colonized colon content; c: a result graph of melatonin level change in serum of mice colonized by lactobacillus reuteri LN 0214; c data were analyzed by t-test and expressed as mean ± SD; p < 0.05; GF: and (5) sterilizing.
Biological preservation information
Lactobacillus reuteri LN0214, which is preserved in the China general microbiological culture Collection center, with a preservation time of 2022 years, 03 months and 24 days. The address is No. 3 of West Lu No. 1 of Beijing, Chaoyang, Beijing, and the microbial research institute of Chinese academy of sciences, and the preservation number is CGMCC No. 24586.
Detailed Description
The preservation number of the Lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention provides application of Lactobacillus reuteri LN0214 in preparation of a medicament for regulating and controlling melatonin, wherein the preservation number of the Lactobacillus reuteri LN0214 is CGMCC No. 24586. In order to explore the role of lactobacillus reuteri LN0214 in regulating the level of host melatonin, lactobacillus reuteri LN0214 is implanted in vivo in mice treated with mixed antibiotics to explore the influence of the strain on the level of host melatonin.
The invention also provides application of lactobacillus reuteri LN0214 in preparation of a medicine for reducing serum melatonin level, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention also provides application of lactobacillus reuteri LN0214 in preparing a medicine for improving the level of the colonic melatonin, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
The invention also provides application of the lactobacillus reuteri LN0214 in preparation of a medicine for improving the expression level of the colon AANAT protein, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
In an in-vivo colonization model of lactobacillus reuteri LN0214, a targeted metabonomics detection result shows that the strain of bacteria can obviously reduce the melatonin level in the serum of a mouse and obviously increase the melatonin level in the colon tissue of the mouse. Meanwhile, the expression of melatonin synthesis rate-limiting enzyme 5-hydroxytryptamine-N-acetyltransferase (AANAT) in the colon of the mouse is increased. In order to further verify the colonization effect of the strain, a lactobacillus reuteri LN0214 colonization experiment is also carried out in a sterile mouse, and after lactobacillus reuteri LN0214 is colonized in the sterile mouse, an Elisa detection result shows that the melatonin level in serum is remarkably changed.
The application of a Lactobacillus reuteri LN0214 strain according to the present invention will be described in further detail with reference to the following embodiments, and the technical solution of the present invention includes, but is not limited to, the following embodiments.
In the following examples, Lactobacillus reuteri LN0214 strain was deposited at the institute of microbiology, national academy of sciences (accession number: CGMCC No. 24586). Ampicillin, colistin, metronidazole, neomycin and vancomycin were purchased from alatin bioreagent limited (shanghai, china) with the following cargo numbers in order: n329566, a187417, D333704, B300250, V301569. The mouse melatonin Elisa detection kit was purchased from shanghai enzyme-linked biotechnology limited (shanghai, china). The lactobacillus liquid culture medium is MRS broth with a product number of 11307, purchased from Nippon Biotechnology Limited (Shandong, China); the solid medium was MRS medium, having a product number of M8330, purchased from Solebao technologies, Inc. (Beijing, China). Other reagents are ordinary commercial products unless otherwise specified.
The mixed antibiotic-treated mice were ICR female mice (12 mice per group, 6 weeks old) purchased from liaoning biotechnology, ltd (liaoning, china). The lactobacillus reuteri LN0214 field planting sterile mouse is a KM mouse (6 males and females in each group and 6 weeks old) in the experimental animal center of Huazhong university of agriculture (Wuhan, China). The environment temperature for breeding the mice is 20-26 ℃, the relative humidity is 50-60%, the illumination period is 12 hours per day, and food and water can be freely obtained.
Example 1
Effect of lactobacillus reuteri LN0214 on melatonin metabolism after mixed antibiotics eliminate intestinal microorganisms of mice
After the mice are treated for 1 week by adding mixed antibiotics containing ampicillin (1g/L), metronidazole (1g/L), neomycin (1g/L) and vancomycin (0.5g/L) into drinking water, a control group and a lactobacillus reuteri LN0214 group are respectively arranged, and the lactobacillus reuteri LN0214 group is respectively perfused with PBS suspension (1 x 10) containing the lactobacillus reuteri LN0214 into each stomach every day 8 CFU/mL, 200 μ L), the same amount of PBS as the control group, and after 1 week of continuous gavage, mouse serum and colon samples (a in fig. 1, flowchart of colonization experiment of lactobacillus reuteri LN0214 in mice treated with mixed antibiotics) were collected, and melatonin content was detected by using targeted metabonomics technology.
Metabonomics sample pretreatment: after a colon tissue sample (40.0mg) was accurately weighed in a centrifuge tube, 400. mu.L of ultrapure water was added, vortex-shaking was carried out for 4.5 minutes, and then 200. mu.L of homogenate was added to 800. mu.L of a methanol-acetonitrile solution (50:50, V/V). For serum samples, 200. mu.L of the sample was added directly to 800. mu.L of methanol-acetonitrile. The mixture was vortexed in ice water at 4 ℃ for 10 minutes, then centrifuged at 19,000g at 4 ℃ for 15 minutes, the supernatant was taken, vacuum dried at 60 ℃ for 90 minutes, and then blown dry with nitrogen to give a dry substance. The dry material was dissolved in 200. mu.L of methanol-water (50:50, V/V), sonicated with ice water at 4 ℃ for 10 minutes, centrifuged at 4 ℃ at 19,000g for 15 minutes and the supernatant was passed through a 0.22 μm membrane filter, filtered and transferred to a sample vial for storage at-20 ℃ until detection on a UPLC-Orbitrap-MS/MS machine.
As a result, it was found that the serum melatonin level of the mice colonized with Lactobacillus reuteri LN0214 was decreased (B in FIG. 1, a graph showing the change in the serum melatonin level of the mice colonized with Lactobacillus reuteri LN 0214), and the melatonin level in the colon was increased (C in FIG. 1, a graph showing the change in the melatonin level of the colon of the mice colonized with Lactobacillus reuteri LN 0214). The expression of the colon AANAT protein of the lactobacillus reuteri LN0214 group mouse is detected by a Westernblot technology, and the result shows that the expression of the AANAT protein of the lactobacillus reuteri LN0214 group is increased (D in figure 1, a Westernblot result chart of the mouse colon AANAT protein colonized by the lactobacillus reuteri LN 0214), which is consistent with the increase and change of the content of the colon melatonin.
Example 2
Influence of lactobacillus reuteri LN0214 colonized in sterile mice on melatonin metabolism
In order to further verify the colonization effect of lactobacillus reuteri, the invention uses a sterile mouse to carry out a colonization experiment of lactobacillus reuteri LN 0214. Experimental setup control and Lactobacillus reuteri LN0214 groups, the Lactobacillus reuteri LN0214 group was treated with PBS suspension containing Lactobacillus reuteri LN0214 on days 1 and 3 (1X 10) 8 CFU/mL, 200 μ L), gavage equal amounts of PBS on day 1 and day 3 in the control group, and collecting mouse large intestine content and serum samples on day 7 (a in fig. 2, experimental flowsheet for colonization of sterile mouse lactobacillus reuteri LN 0214). 16S rRNA gene amplicon sequencing analysis is carried out on the content of the large intestine, and the result shows that the lactobacillus reuteri LN0214 group genus level is dominantTo be of the genus lactobacillus (fig. 2, B, plot of relative abundance of gene amplicon sequencing of 16S rRNA from colon contents colonized by sterile mouse lactobacillus reuteri LN 0214), it was shown that lactobacillus reuteri LN0214 was successfully colonized in mice. The results of the detection of the serum melatonin content of the mice by using the Elisa kit show that the serum melatonin level of the mice in the Lactobacillus reuteri LN0214 group is reduced (C in figure 2, a graph of the change result of the melatonin level in the serum of the mice fixedly planted by the Lactobacillus reuteri LN 0214), which is consistent with the change result of the serum melatonin of the mice fixedly planted by the Lactobacillus reuteri LN0214 after the mixed antibiotic is used for removing the intestinal microorganisms.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. The application of Lactobacillus reuteri LN0214 in preparing a medicament for regulating and controlling melatonin, wherein the preservation number of the Lactobacillus reuteri LN0214 is CGMCC No. 24586.
2. The application of lactobacillus reuteri LN0214 in preparing a medicament for reducing the level of serum melatonin, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
3. The application of lactobacillus reuteri LN0214 in preparing a medicine for improving the level of colonic melatonin is disclosed, wherein the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
4. The lactobacillus reuteri LN0214 is applied to the preparation of the medicine for improving the expression level of the colon AANAT protein, and the preservation number of the lactobacillus reuteri LN0214 is CGMCC No. 24586.
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