CN111617061A - Application of spermidine in preparation of preparation for improving abundance of lachnospirillum bacteria - Google Patents
Application of spermidine in preparation of preparation for improving abundance of lachnospirillum bacteria Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
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Abstract
The invention discloses application of spermidine in preparation of a preparation for improving the abundance of Lachnospiraceae bacteria, wherein exogenous spermidine is taken to remarkably improve the bacterial abundance of Lachnospiraceae NK4A136group in the intestinal tract of an obese mouse and improve the intestinal barrier function damage caused by metabolic related diseases such as obesity.
Description
(I) technical field
The invention relates to an application of a polyamine substance spermidine, mainly an application of spermidine in preparation and development of drugs and functional foods related to increase of lachnospirillum.
(II) background of the invention
The intestinal microorganisms are an important component of a microecosystem of an organism and can regulate the intestinal health of a human body and maintain the immune and metabolic homeostasis of the organism. It was found by sequencing techniques that the enteric microorganisms are mainly composed of bacteria, the predominant species among which belong to the species firmicutes and bacteroidetes. The intestinal microorganisms and the body coexist to form an intestinal microecological system, and when the intestinal microecological system is unbalanced, the intestinal microorganisms can change correspondingly, such as the increase of pathogenic bacteria and the reduction of beneficial bacteria, and the immune function of the body is damaged, so that diseases are caused. Various animal and clinical studies show that the imbalance of the intestinal flora plays an important role in various intestinal diseases and metabolic syndromes, for example, the abundance of klebsiella pneumoniae and proteus vulgaris is remarkably increased in mice suffering from colitis, so that the research on the abundance change of specific intestinal bacteria is beneficial to the research on the specific role of the intestinal flora in the occurrence and development of diseases. In addition, the current effective scheme for treating diseases by targeting intestinal flora mainly comprises probiotic supplementation and coprophilic transplantation (FMT), wherein the FMT is obtained by transplanting functional flora of healthy animals into the gastrointestinal tract of diseased animals after treatment, reconstructing new intestinal flora and treating diseases related to intestinal flora imbalance. Although the current embodiments of FMT lack clear clinical efficacy, the search for effective functional flora also opens the possibility to further develop FMT for disease.
The family Lachnospiraceae (Lachnospiraceae) is an enterosymbiotic bacterium in humans and mammals, belonging to the order Clostridiales, and all species of this family are anaerobic bacteria. The hair spirillum can decompose carbohydrate and dietary fiber ingested by food to generate butyric acid, and the butyric acid is a main energy source of intestinal epithelial cells and has the functions of enhancing the intestinal barrier function and maintaining the intestinal homeostasis. One metagenomic study found two genera of Lachnospiraceae, including Lachnospiraceae bacteria 10_1 and Lachnospiraceae bacteria 28_4, which are key species for obese and obese resistant mice, respectively. Furthermore, a significant increase in the abundance of lachnospira species was also found in the rat model of ulcerative colitis, and it was therefore necessary to explore the role of the abundance changes of different species of the lachnospiraceae family in intestinal micro-ecological disorders of the body.
Spermidine (SPD) is a polyamine compound naturally occurring in nuts, seeds, beans, animal livers, etc., and studies have shown that Spermidine can exert anti-aging and neuroprotective effects as an autophagy inducer. With the intensive research on spermidine, it has been proved that intestinal flora such as lactococcus lactis LKM512 can synthesize spermidine. In view of the benefit of spermidine in improving metabolic health, the invention takes a C57BL6J male mouse as an experimental subject, induces an obese mouse model through 16-week high-fat diet, and gives spermidine intervention in a drinking water mode, explores whether spermidine intervention can change the bacterial abundance of the mouse lachnosporium, and verifies the relationship between exogenous intake of spermidine and the bacterial abundance change of the lachnosporium through FMT.
Disclosure of the invention
The invention aims to provide application of spermidine in preparation of a preparation for improving the abundance of Lavenospira bacteria, so that the bacterial abundance of Lavenospira NK4A136group in the intestinal tract of an obese mouse is increased, and the intestinal barrier function damage caused by metabolic-related diseases such as obesity is improved.
The technical scheme adopted by the invention is as follows:
the invention provides an application of spermidine in preparation of a preparation for improving abundance of bacteria of the genus Lahnospiraceae, wherein the genus Lahnospiraceae is Lachnospiraceae NK4A136 group.
Further, the preparation is a food for improving the impaired barrier function of the intestinal tract.
The method comprises the steps of inducing a mouse obesity model through high-fat diet, constructing an intestinal microecological disorder environment, exogenously ingesting spermidine in a drinking water mode, analyzing abundance change of drospirenone in mouse caecum contents by utilizing bacterial 16S rRNA high-throughput sequencing, discussing whether spermidine intervention can affect drospirenone abundance change, treating a fecal bacteria mixture of a donor mouse (high-fat diet + SPD treatment), and further verifying the relationship between spermidine ingestion and drospirenone abundance change through lavage treatment of a receptor mouse (high-fat diet + antibiotic treatment).
Compared with the prior art, the invention has the following beneficial effects: the intake of exogenous spermidine can obviously improve the bacterial abundance of Lachnospiraceae NK4A136group in the intestinal tract of obese mice and improve the intestinal barrier function damage induced by metabolic related diseases such as obesity and the like.
(IV) description of the drawings
FIG. 1: effect of spermidine on intestinal permeability in obese mice.
FIG. 2: and (3) analyzing the correlation between the intestinal lachnosporium and the intestinal permeability.
FIG. 3: effect of spermidine on abundance of lachnospirillum in the gut of obese mice.
FIG. 4: effect of FMT on abundance of lachnospirillum in the gut of obese mice.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1
1. Animal treatment
Male C57BL/6 mice at 7 weeks of age were housed under standard experimental conditions: 25 + -2 deg.C, 50 + -5% humidity, 12h/12h light/dark cycle. The 40 mice were divided into 5 groups on average (control group, high fat diet group, spermidine low dose group, spermidine medium dose group, and spermidine high dose group). The concentration of the aqueous spermidine solution is calculated based on the weight of the mice, for example, 25g of the mice, 0.125mg of the spermidine powder is dissolved in 5ml of sterile water to obtain 25mg/L of the aqueous spermidine solution, and each mouse is given a daily intake of 5mg/kg of the aqueous spermidine solution based on the weight of the mice. The control group was given normal drinking water and normal standard diet (energy of 3.52kcal/g), and the remaining groups were given high fat diet (energy of 5.24kcal/g), high fat diet group was given normal drinking water, and the spermidine low dose group, the spermidine medium dose group, and the spermidine high dose group were simultaneously ingested with 5mg/kg, 10mg/kg, and 20mg/kg of spermidine aqueous solution per day as drinking water. The treatment was carried out for 16 weeks.
2. Intestinal permeability detection
During the feeding period, the permeability of the mouse intestinal tract was measured by fluorescently labeled dextran. After feeding for 16 weeks in step 1, the mice were fasted and normally drunk, and after 4 hours, the mice were gazed with fluorescently-labeled dextran, and the specific perfusion volume was calculated according to 0.5mg/g body weight, with the perfusion concentration being 50 mg/mL. After 4 hours, the tail vein of the mice was bled and immediately centrifuged (7000rpm, 7min) to collect serum. The content of the fluorescently labeled dextran was measured using a fluorescence microplate reader under excitation 490nm and detection 525nm conditions, and the results are shown in fig. 1 and 2.
3. 16S rRNA sequencing analysis
Step 1 after 16 weeks of treatment, mice were sacrificed and the cecal contents were collected and total DNA of the cecal contents was extracted according to QIAamp DNA Stool MiniKit (qiagen, germany) kit instructions to prepare sequencing samples.
The analysis procedure followed the QIIME2 pipeline flow, first importing FASTQ files using QIIME2 and performing library segmentation on the sequences. After segmentation, all the sequences tested were homologously aligned and clustered into Operational Taxonomic Units (OTUs) based on 97% similarity using DADA2 plug-in. And (3) after obtaining the classification unit, constructing an evolutionary tree by using a PHYLOGENY plug-in, and calculating the alpha and beta diversity of the flora by using an DIVERSITY plug-in. Diversity results the significance of the diversity index changes was analyzed using the chi-square test, and the results are shown in figure 3.
4、FMT
Step 1 after 16 weeks of treatment, mouse feces of the high fat diet group and the spermidine high dose group were collected, washed with physiological saline and centrifuged, and supernatants were collected to obtain a high fat diet group supernatant (FMT-HF) and a spermidine high dose group supernatant (FMT-SPD), respectively, as FMT-treated samples.
Recipient mouse treatment: male C57BL/6 mice at 7 weeks of age were housed under standard experimental conditions: 25 + -2 deg.C, 50 + -5% humidity, 12h/12h light/dark cycle. 16 mice were divided on average into 2 groups (FMT-HF group and FMT-SPD group). The 2 groups of mice are first induced to form conditioned sterile mice by continuous three-day gavage of broad-spectrum antibiotic solution (final concentration composition: ampicillin 1g/L, metronidazole 1g/L, vancomycin 0.5g/L, neomycin 0.5g/L, solvent is physiological saline) and gavage of 200 mul/mouse every day, then both are fed with high-fat diet, after 12 weeks, the high-fat diet group supernatant and the spermidine high-dose group supernatant are respectively passed through gavage to treat FMT-HF group and FMT-SPD group mice, each mouse is continuously gavage for 4 weeks at a dose of 1g/kg every day, the mice are sacrificed, the cecal content of the mice is collected, the total DNA of the cecal content is extracted, a sequencing sample is prepared, and 16SrRNA sequencing analysis is carried out, and the result is shown in figure 4.
5. Results
(1) Spermidine ameliorates intestinal permeability impairment in obese mice
The permeability of the mouse intestinal tract was measured by fluorescently labeled dextran, and the change is shown in FIG. 1. Compared with the control group of mice, the content of the fluorescence labeled dextran is obviously increased, which indicates that the intestinal permeability of the obese mice is increased. After spermidine treatment, intestinal permeability of obese mice decreased in a dose-dependent manner, and high doses of spermidine treatment were most effective in improving intestinal permeability impairment in obese mice.
(2) Correlation analysis of bacterial abundance and intestinal permeability impairment of Lachnospiraceae NK4A136group in obese mice
The correlation analysis of bacterial abundance and mouse intestinal permeability of the intestinal flora Lachnospiraceae NK4a136group is shown in fig. 2. The bacterial abundance of Lachnospiraceae NK4A136group is inversely related to the intestinal permeability of mice, so that increasing the bacterial abundance of Lachnospiraceae NK4A136group may reduce the intestinal permeability of mice and maintain the intestinal barrier integrity of mice.
(3) Spermidine increased bacterial abundance of Lachnospiraceae NK4A136group in obese mice
The changes in bacterial abundance of the gut flora Lachnospiraceae NK4a136group are shown in fig. 3. The bacterial abundance of Lachnospiraceae NK4a136group was significantly reduced in the high fat diet group mice compared to the control group mice. After the treatment of high-dose spermidine, the bacterial abundance of Lachnospiraceae NK4A136group is obviously increased, and is closer to the control group, which shows that spermidine can increase the bacterial abundance of Lachnospiraceae NK4A136group of fat mice induced by high-fat diet.
(4) FMT validation that spermidine increased bacterial abundance of Lachnospiraceae NK4A136group in obese mice
The changes in bacterial abundance of the gut flora Lachnospiraceae NK4a136group after FMT treatment are shown in figure 4. The bacterial abundance of Lachnospiraceae NK4a136group in FMT-spermidine high dose group mice was significantly increased compared to FMT-high fat diet group mice, suggesting that spermidine treatment may indeed increase the bacterial abundance of Lachnospiraceae NK4a136group in high fat diet induced obese mice.
In conclusion, 16S rRNA sequencing data analysis shows that exogenous spermidine intake can increase bacterial abundance of Lachnospiraceae NK4A136group of fat mice induced by high fat diet, and meanwhile, the fecal transplantation treatment further verifies the effectiveness of the implementation case, and provides theoretical basis for application of spermidine in preparation of increasing Lachnospiraceae preparations. In addition, through correlation analysis, the bacterial abundance of Lachnospiraceae NK4A136group and the intestinal permeability of mice are in negative correlation, so that spermidine can improve the intestinal barrier function damage induced by metabolic related diseases such as obesity and the like by increasing the bacterial abundance of Lachnospiraceae NK4A136 group.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention in any way. Any simple modification, equivalent change and modification of the above embodiments according to the technical spirit of the present invention fall within the scope of the present invention.
Claims (3)
1. An application of spermidine in preparation of preparation for improving abundance of Lachnospirillum bacteria is provided.
2. Use according to claim 1, characterized in that said genus lachnospira is lachnospiraceae enk4a136 group.
3. Use according to claim 1, characterized in that the preparation is a food product for ameliorating impaired gut barrier function.
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| CN (1) | CN111617061A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112168822A (en) * | 2020-09-27 | 2021-01-05 | 集美大学 | Application of kynurenic acid in improving hyperlipidemia induced dyslipidemia, obesity and intestinal flora disorder |
| CN113584193A (en) * | 2021-07-06 | 2021-11-02 | 中南大学湘雅医院 | Application of lachnospirillum as marker for evaluating antihistamine drug efficacy of patients with chronic spontaneous urticaria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109771402A (en) * | 2019-03-19 | 2019-05-21 | 浙江工业大学 | Spermidine is damaged the application in drug in preparation treatment intestinal barrier function |
| CN110801446A (en) * | 2019-09-25 | 2020-02-18 | 浙江工业大学 | Application of nitrilamine in preparation of colitis treatment medicine |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109771402A (en) * | 2019-03-19 | 2019-05-21 | 浙江工业大学 | Spermidine is damaged the application in drug in preparation treatment intestinal barrier function |
| CN110801446A (en) * | 2019-09-25 | 2020-02-18 | 浙江工业大学 | Application of nitrilamine in preparation of colitis treatment medicine |
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| 石涛等: "溃疡性结肠炎患者的肠屏障功能观察", 《中华医学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112168822A (en) * | 2020-09-27 | 2021-01-05 | 集美大学 | Application of kynurenic acid in improving hyperlipidemia induced dyslipidemia, obesity and intestinal flora disorder |
| CN112168822B (en) * | 2020-09-27 | 2022-11-08 | 集美大学 | Application of kynurenic acid in improving hyperlipidemia induced dyslipidemia, obesity and intestinal flora disorder |
| CN113584193A (en) * | 2021-07-06 | 2021-11-02 | 中南大学湘雅医院 | Application of lachnospirillum as marker for evaluating antihistamine drug efficacy of patients with chronic spontaneous urticaria |
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