CN110368405A - A kind of method for reconstructing of intestinal flora - Google Patents

Abstract

The invention relates to a reconstruction method of intestinal flora, which is pretreated with antibiotics and intervened by probiotics. The invention promotes the reconstruction of intestinal flora, and plays a certain role in improving glucose metabolism by reducing intestinal permeability, enhancing intestinal mucosal barrier, and changing bile acid components.

Description

A Reconstruction Method of Gut Microbiota

technical field

The invention belongs to the field of type 2 diabetes, and particularly relates to a method for reconstructing intestinal flora.

Background technique

The prevalence of type 2 diabetes mellitus (T2DM) is gradually increasing globally with changes in lifestyle and an aging population. According to data from the International Diabetes Federation survey, an estimated 425 million people (20-79 years old) were living with diabetes in 2017, with a quarter of those with diabetes over the age of 65, and in high- and middle-income countries, The incidence of diabetes peaks in the 75-79 age group and the 60-74 age group, respectively. It is estimated that by 2045, the total number of diabetic patients will exceed 629 million, of which T2DM accounts for 90% of diabetes. Therefore, T2DM is a major burden of diseases in the elderly, and finding and reducing the risk of diabetes is essential to control T2DM.

Among the many risk factors for obesity and T2DM, evidence from recent studies on the gut microbiome supports Hippocrates' 2,500-year-old statement that "all diseases begin in the gut." As the main place of digestion and absorption, the intestine contains tens of thousands of intestinal symbiotic bacteria, which play an important role in the absorption of host nutrients and the regulation of energy metabolism. During the development of T2DM, the structure and function of gut microbiota changed significantly. In recent years, it has been found that the co-development and co-evolution of intestinal commensal bacteria and the host, and the core species of the intestinal flora will change with age. Lactobacillus and Prevotella are initially found in neonates. Intestinal colonization, both Lactobacillus and Bifidobacterium were enriched in the gut of lactating infants and young children, and were gradually replaced by Bacteroides and Prevotella with age. However, whether these age-related gut microbiota signatures are associated with the development of T2DM and other age-related metabolic diseases has not been elucidated. There have been a lot of studies on the relationship between intestinal flora disturbance and T2DM. Metagenomics analysis has found that the occurrence of T2DM is related to the disturbance of intestinal flora. During the development of T2DM, the structure and function of the intestinal flora have obvious changes. Variety. Therefore, the disturbance of gut microbiota plays an important role in the occurrence of T2DM.

However, at present, there is still a lack of research on the improvement of gut microbiota related to T2DM.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for the reconstruction of intestinal flora, which promotes the reconstruction of intestinal flora, plays a certain role by reducing the intestinal permeability, enhancing the intestinal mucosal barrier, and changing the bile acid composition. Improves glucose metabolism.

The invention provides a reconstruction method of intestinal flora through antibiotic pretreatment and probiotic intervention.

Preferably, the antibiotic is gentamicin sulfate.

Preferably, the probiotics are Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Bifidobacterium breve, and Bifidobacterium longum. one or more.

Preferably, the probiotics are lyophilized powder.

Preferably, the probiotics also include maltodextrin.

beneficial effect

The invention uses antibiotics in combination with probiotics for the purpose of intestinal flora reconstruction, promotes the reconstruction of intestinal flora, and plays a certain role in improving glucose metabolism by reducing intestinal permeability, enhancing intestinal mucosal barrier, and changing bile acid components. It has certain market application prospects.

Description of drawings

Figure 1 shows the change of fasting blood glucose; among them, A is the change curve of fasting blood glucose, and B is the absolute value of fasting blood glucose after the experiment.

Figure 2 shows the glucose tolerance test and fasting insulin levels; among them, A is the intraperitoneal glucose tolerance test, B is the area under the glucose tolerance curve, and C is the fasting blood insulin level after the experiment.

Figure 3 shows the changes of epididymal fat and liver mass; A is the absolute value of epididymal fat mass, B is the absolute value of liver mass, C is the ratio of epididymal fat mass to body weight, and D is the ratio of liver mass to body weight.

Figure 4 shows the changes of liver and plasma lipids; where A is the level of triglyceride in liver tissue, B is the level of total cholesterol in liver tissue, C is the level of plasma triglyceride, D is the level of total plasma cholesterol, and E is the total free plasma level fatty acid levels.

Figure 5 shows the HE staining of intestinal tissue; wherein, A is the HE staining of the jejunum tissue, B is the HE staining of the ileum tissue, and C is the HE staining of the colon tissue.

Figure 6 is the intestinal permeability test; wherein, A is the change of FITC-D concentration in plasma at 1h and 4h; B is the area under the curve of the change of plasma FITC-D concentration between 1h and 4h.

Figure 7 shows the AB/PAS staining of colon tissue and the mRNA expression level of MUC2 gene; among them, A is the AB/PAS staining of colon tissue (the black arrow points to the inner mucus layer, the white arrow points to the goblet cells), and B is the goblet of the colonic crypt The number of cells, C is the mRNA expression level of MUC2 gene in colon tissue.

Figure 8A is the relative abundance change of gut flora phyla level;

Figure 8B shows the relative abundance changes at the level of gut microbiota.

Figure 9A shows the relative abundance of intestinal flora in the CT group compared with the DM group;

Figure 9B shows the relative abundance of intestinal flora in the DM+AB group compared with the DM group;

Figure 9C shows the relative abundance of intestinal flora in the DM+AB+PRO group compared with the DM group;

Figure 9D shows the relative abundance of intestinal flora in the DM+AB+PRO group compared with the DM+AB group.

Figure 10 shows the changes of bile acid components in feces; where A is the percentage of each component of fecal bile acids after probiotic intervention; B is the total bile acid content in feces; C is the ratio of bile acids TCA to TβMCA in feces; D is The ratio of bile acid components in feces.

Figure 11 shows the changes of bile acid components in plasma; among them, A is the percentage of each component of plasma bile acid after probiotic intervention; B is the content of total bile acid in plasma; C is the ratio of bile acid TCA to TβMCA in plasma; D is The ratio of the components of bile acids in plasma.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

100 16-month-old male SPF C57BL/6 mice, weighing 30±5 g, were provided and raised by Nanjing Baishijin Biological Model Animal Center, 10 of which were used for normal control (CT) and 90 were used for model construction.

The antibiotic gentamicin sulfate was purchased from Guangzhou Baiyunshan Tianxin Pharmaceutical.

The probiotic powder or its placebo is provided by Shanghai Jiaotong University Only Co., Ltd. The probiotic powder is 9 strains (Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus fermentum , Lactobacillus gasseri, Bifidobacterium breve, Bifidobacterium longum) mixed freeze-dried powder, which also contains maltodextrin, and the probiotic placebo is mainly composed of maltodextrin, all of which are stored at 4°C.

Example 1

1. Processing method

After the successful establishment of the T2DM model, in order to maintain the hyperglycemic state, HFD feeding was continued, and 10 diabetic mice were selected as the control group (DM) without any treatment, and the rest were given 1 week of antibiotic pretreatment (gentamicin sulfate) Injection 80mg: 80,000 units/piece), dissolved in drinking water at a concentration of 1g/L, and then randomly divided into 2 groups according to fasting blood glucose and body weight, with at least 10 mice in each group: antibiotic pretreatment + probiotic placebo ( DM + AB), antibiotic pretreatment + probiotics experimental group (DM + AB + PRO) were administered by intragastric administration once a day for a total of 4 weeks. The probiotic solvent was 1×PBS (pH7.4), the dose was 10 ⁹ CFU/mice, and each mouse was given 0.2 ml by gavage. Equal volume of PBS was administered by gavage.

2. Experimental results

①Antibiotic pretreatment + probiotics improved fasting and postprandial blood glucose in aged diabetic mice, but had no effect on lipid metabolism

HFD combined with STZ-induced aged diabetic mice were pretreated with gentamicin for 1 week, and then entered a 4-week specific probiotic gavage experimental period. During the whole experimental process, the weight and weight of mice were measured weekly. Fasting blood sugar. The fasting blood glucose of the mice was measured before antibiotic treatment. Compared with the normal control group, the fasting overnight blood glucose of the diabetic mice was higher than 11.1 mmol/l, and there was a significant difference (P<0.001, DM vs CT). The model was successfully established; after antibiotic treatment, there was no difference in fasting blood glucose between the diabetic mouse groups; after 2 weeks of probiotic intervention, the fasting blood glucose of the antibiotic pretreatment + probiotic experimental group was significantly lower than that of the diabetic control group (P< 0.05, DM+AB+PRO vs DM) (Fig. 1A), the difference in fasting blood glucose remained significant from the 3rd week of the intervention until the end of the intervention, while there was no significant improvement in DM+AB fasting blood glucose (Fig. 1B).

The results of intraperitoneal injection of glucose tolerance (IPGTT) in mice showed that in addition to significantly reducing fasting blood glucose, the mice in the antibiotic pretreatment + probiotic group also significantly reduced blood glucose 120 minutes after glucose injection (P<0.05, DM+AB+PRO vs DM). ), there was no difference in blood glucose between the 30-60min time point and the diabetic control group (Fig. 2A). The AUC of the area under the glucose tolerance curve showed no difference between the diabetic mice in the experimental group and the mice in the non-experimental group (Fig. 2B). The fasting plasma insulin of the mice was measured, and the fasting insulin level of the aged diabetic mice was significantly lower than that of the normal glucose tolerance mice (P<0.01, DM vs CT). It did not affect insulin secretion in diabetic mice (P<0.01, P<0.05, DM+AB vs DM, DM+AB+PRO vs DM) (Fig. 2C).

Further evaluation of the changes in epididymal fat and liver mass in mice found that epididymal fat (P<0.001, DM vs CT) and liver mass (P<0.01, DM vs CT) were significantly increased in all diabetic mice compared with normal controls , after pretreatment with antibiotics, probiotics placebo or probiotics intervention were given, and there was no statistical difference in epididymal fat and liver mass between the three groups of mice (Figure 3A, B). The ratio of epididymal fat/body weight was not statistically different (Fig. 3C), while the ratio of liver/body weight in the antibiotic pretreatment + probiotic experimental group was decreased compared with the diabetic control group (P=0.071), and compared with the antibiotic group There was a significant decrease in the pretreatment+probiotic placebo group (P<0.05, DM+AB+PRO vs DM+AB) (Figure 3D).

Liver tissue and plasma total cholesterol and triglycerides and plasma free fatty acid levels were also measured. After long-term HFD diet feeding, the liver tissue and plasma total cholesterol and triglyceride levels of mice in the diabetic control group were significantly higher than those in the normal control group (P<0.001, DM vs CT). After drug treatment, only antibiotic pretreatment + probiotics Compared with the diabetic control group, the level of triglyceride in the liver of the placebo group was significantly decreased (P<0.05, DM+AB vs DM), and there was no significant change in the total cholesterol and triglyceride in the liver of the other mice (Fig. 4A, B); from the change of plasma lipid level, the plasma total cholesterol level of the diabetic control group was significantly higher than that of the normal control group, and the plasma triglyceride level was not statistically different from that of the normal control group (Fig. 4C, D); the probiotics placebo group or probiotics intervention were given after antibiotic pretreatment, and there was no statistical difference in plasma total cholesterol levels among the three groups (Fig. 4C), while plasma triglyceride and free fatty acid levels were associated with diabetes Compared with the control group mice, there was a downward trend, and the probiotic experimental group had a more obvious downward trend in free fatty acids (P=0.054) (Figure 4D, E).

②Antibiotic pretreatment + probiotics improve intestinal villus structure and intestinal permeability

To observe the effect of probiotics on intestinal tissue morphology, HE staining was performed on mouse intestinal tissue after probiotic intervention. HE staining showed typical submucosal hemorrhage, edema, severe vacuolar lesions, goblet cell hyperplasia, and villous shedding lesions in the intestinal wall of each intestinal segment including the jejunum, ileum and colon in the diabetic control group (Fig. The fossa disappeared and the structure was seriously disordered (Fig. 5B); antibiotic pretreatment + probiotic placebo significantly improved the changes in the above intestinal tissue morphology, but there were still obvious submucosal edema at the top of some villi, and the structure of intestinal villi was incompletely restored, while antibiotics Pretreatment + probiotics improved the intestinal villus structural disorder more significantly, which was closer to the normal control group.

In order to explore the effect of probiotics on intestinal permeability, an intestinal permeability test was performed on mice after the probiotics experiment, and Fluorescein isothiocyanate-dextran (FITC- D). After gavage, blood was collected from the tail vein at 1h and 4h after gavage, the concentration of FITC-D in plasma was detected, and the change curve of plasma FITC-D at 1h and 4h was made. 1h and 4h after gavage, the concentration of FITC-D in the plasma of the diabetic control group was significantly increased, which was different from that of the normal control group (P<0.001, DM vs CT) (Fig. 6A); according to the 1h and 4h plasma FITC-D The concentration change curve was calculated, and the corresponding area under the curve AUC was calculated. The results showed that the intestinal permeability of the antibiotic pretreatment + probiotics experimental group was significantly lower than that of the other two groups of diabetic mice (P<0.001, P<0.01, DM+AB+PRO vs DM+AB+PRO vs. DM, DM+AB+PRO vs DM+AB) (FIG. 6B).

③Antibiotic pretreatment + probiotics reduce the number of goblet cells and restore the shape of the mucus layer

Goblet cells are mucus-secreting cells present in the mucosal epithelium, and the mucus they secrete plays an important role in maintaining the integrity of the intestinal mucus layer. The results of AB/PAS staining of colon tissue showed that compared with the normal control group, the number of goblet cells in the colon tissue of the diabetic control group increased significantly, the volume increased, the mucus in the goblet cell was filled, and the morphology of the mucus layer changed; antibiotic pretreatment The number of goblet cells in colon tissue decreased slightly after + probiotics placebo intervention, while the number of goblet cells decreased significantly after antibiotic pretreatment + probiotics intervention, compared with the other two groups of diabetic mice, the difference was significant (P<0.001, DM +AB+PRO vs DM, DM+AB+PRO vs DM+AB) (Fig. 7A,B). In addition, the morphology of the intestinal mucus layer also changed. The morphology of the mucus layer in the antibiotic pretreatment + probiotic experimental group was regular and clearly visible, and it was more inclined to the normal control mice (Fig. 7A). Intestinal permeability results were consistent. In addition, the results of colon tissue RT-PCR showed that the transcription level of mucin MUC2 gene, an important component of the intestinal mucus layer, changed significantly. Compared with normal control mice, the mRNA expression level of colon MUC2 gene in diabetic control mice was significantly Compared with the diabetic control group, the mRNA expression level of colon MUC2 gene decreased significantly, and there was a statistical difference (P<0.01, DM+ AB+PRO vs DM, DM+AB vs DM) (Fig. 7C).

④ Effects of antibiotic pretreatment + probiotics on intestinal flora

Judging from the changes in the abundance of different species, compared with the normal control group, the intestinal flora of the mice in the diabetic control group showed a significant increase in abundance except for Bacteroidetes (P <0.01, DM vs CT); and after drug intervention in diabetic mice, i.e. antibiotic pretreatment + probiotic placebo and antibiotic pretreatment + probiotic, the intestinal flora of the two groups of mice was similar to that of normal control. The changes in mice are mainly manifested in the phylum Firmicutes, Proteobacteria, Verrucomicrobia, Deferribacteres, Candidatus Saccharibacteria, Actinobacteria at the phylum level. ) decreased and the abundance of Bacteroidetes increased. However, comparing the two groups, it was found that the effect of antibiotic pretreatment + probiotic intervention on the significantly altered intestinal flora after antibiotics was mainly manifested in the increase in the abundance of Firmicutes and Actinobacteria, and the relative decrease in the abundance of Bacteroidetes and Proteobacteria (Figure 8A). From the analysis of changes at the family level, compared with normal control mice, the abundance of these 18 differentially dominant species in the gut of diabetic mice all increased, while the abundance of Bifidobacteriaceae decreased significantly (P=0.001, the heat map was not shown). shown); antibiotic pretreatment + probiotic placebo or probiotic intervention both reduced the increased abundance of enterobacteria in diabetic mice (Fig. 8B). Comparing the two groups, it was found that probiotic intervention could significantly increase the abundance of Lactobacillaceae, Peptostreptococcaceae and Prevotellaceae (P<0.01, P<0.05, P<0.05, P<0.01). <0.05, DM+AB+PRO vs DM+AB).

Further subordinate level analysis showed that compared with normal control mice, the abundance of most measurable gut flora in diabetic mice was increased, but Bifidobacterium and Pseudo-Prevotella were found. The abundance of Alloprevotella was significantly decreased (P<0.01, DM vs CT) (Fig. 9A); compared with the diabetic control group, the intestinal flora of the antibiotic-pretreated mice in both groups showed the presence of Bacteroides. (P<0.01, DM+AB vs DM, DM+AB+PRO vs DM) and Clostridium XlVa (P<0.05, DM+AB+PRO vs DM) abundance (Fig. 9B, C). Compared with the antibiotic pretreatment + probiotic placebo group and the antibiotic pretreatment + probiotic experimental group, it was found that the abundance of Lactobacillus and Bifidobacterium increased after probiotic intervention (P= 0.054, DM+AB+PRO vs DM+AB) (Figure 9D), verifying that probiotics can be effectively enriched in the gut after intervention. It was also found that the abundance of Clostridium XI was also significantly increased compared to placebo (P<0.05, DM+AB+PRO vs DM+AB).

⑤ Effects of antibiotic pretreatment + probiotics on bile acid metabolism in intestinal flora

The changes in the levels of BAs metabolites in the intestinal flora of mice after probiotic intervention were further analyzed. The results from the determination of total bile acids in feces and plasma showed that compared with the diabetic control mice, the levels of total bile acids in the feces of the mice in the antibiotic pretreatment + probiotic placebo group and the antibiotic pretreatment + probiotic experimental group were The decrease trend was more obvious in the probiotic group (P=0.054, DM+AB+PRO vs DM), but there was no statistical difference (Figure 10B). A variety of primary bile acid components were significantly altered in both groups of mice, mainly in primary bile acids CA, βMCA, TCA, TαMCA, TβMCA and primary bile acids/secondary bile acids (PBA/SBA), conjugated bile acids The acid/unconjugated bile acid (CBA/UCBA) and 12-hydroxy/non-12-hydroxy bile acid (12αBA/non12αBA) ratios all increased significantly, while the secondary bile acid ωMCA ratio decreased (Fig. 10A, D). Comparing these two groups, we found that TβMCA and TωMCA were no longer significantly increased in the antibiotic pretreatment + probiotic experimental group. In addition, since the TCA levels in the two groups were basically the same, and the TβMCA level was no longer significantly increased after probiotic intervention, the ratio of TCA/TβMCA in the antibiotic pretreatment + probiotic experimental group also increased significantly (P<0.01, DM+AB+PRO vs. DM) (FIG. 10C).

Different from the changes of bile acid components in feces, the changes in plasma bile acid components were small after the intervention. Compared with the diabetic control group, the total bile acid levels and each bile acid components in plasma of the two treatment groups only saw antibiotic-preventive changes. The level of TCA in the plasma of the treatment + probiotic experimental group was significantly increased (P<0.05, DM + AB + PRO vs DM) (Figure 11A, B), and the ratio of TCA/TβMCA decreased in the antibiotic pretreatment + probiotic placebo group (P<0.05, DM+AB vs DM), the difference disappeared after the probiotic experiment (Fig. 11C), while other bile acid composition indexes, such as PBA/SBA, CBA/UBA and 12αBA/non12αBA, were not changed in both groups (Fig. 11C). Figure 11D).

3. Discussion of results

In this example, antibiotics combined with probiotics were applied to aged diabetic mice for the first time, and the effects of the gut microbiota reconstruction on the metabolic phenotype, intestinal permeability, intestinal permeability of aged diabetic mice by antibiotic pretreatment + probiotics intervention were observed. The effects of mucosal barrier, intestinal flora and bile acids were compared with the antibiotic pretreatment + probiotic placebo group to analyze the effect of short-term antibiotic pretreatment on the above research contents. It was found that antibiotic pretreatment + probiotics can significantly reduce fasting and postprandial blood glucose in aged diabetic mice, improve intestinal permeability, enhance intestinal mucosal barrier, promote colonization of beneficial bacteria, change bile acid composition, and its metabolic phenotype. The improvement may be related to changes in gut permeability and bile acid composition after colonization with beneficial bacteria.

Claims (5)

1. A method for reconstructing intestinal flora, characterized in that: pretreatment with antibiotics and probiotics intervention. 2. The reconstruction method of a kind of intestinal flora according to claim 1, is characterized in that: described antibiotic is gentamicin sulfate. 3. the reconstruction method of a kind of intestinal flora according to claim 1, is characterized in that: described probiotic is Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus crispatus, Lactobacillus casei, One or more of Lactobacillus fermentum, Lactobacillus gasseri, Bifidobacterium breve, and Bifidobacterium longum. 4 . The method for reconstructing intestinal flora according to claim 1 , wherein the probiotics are freeze-dried powder. 5 . 5 . The method for reconstructing intestinal flora according to claim 1 , wherein the probiotics further comprise maltodextrin. 6 .