A method for modulating gut microbiota to improve physiological effects caused by re-feeding a high-fat diet after mild caloric restriction and applications thereof

Abstract

The application discloses a method for adjusting intestinal flora to improve physiological influence caused by light time-limited and then high-fat diet, and application thereof. By means of fecal transplantation technology, the application can improve negative influence suffered by mice of different ages after time-limited and then high-fat diet, including change of intestinal phenotype, expression of inflammatory factors and change of intestinal flora structure. The specific method comprises the following steps: grouping mice, implementing time-limited and high-fat diet, collecting fecal samples of a control group, delivering the fecal samples into the mice of an experimental group by means of gavage, and detecting intestinal flora change and related physiological indexes by means of molecular biology technology. The application comprises the following aspects: improving intestinal phenotype, reducing pathological damage, strengthening intestinal barrier function, reducing expression of pro-inflammatory factors, relieving inflammation state of the liver, regulating LPS level, and significantly changing levels of intestinal microbial populations Lachnospiraceae, Bacteroides and Alistipes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method and its application for regulating the gut microbiota to improve the physiological effects caused by a mild time-restricted high-fat diet followed by a heavy feeding. Background Technology

[0002] Overweight and obesity affect nearly 2 billion adults worldwide, with more than half being obese. Through dietary strategies that restrict arbitrary food intake, people can lose weight and improve their health. The gut microbiota and its health are well-known to be critically influenced by food quantity (managing food intake) and are considered a key mediator connecting diet and host physiology. Due to the effects of food restriction and refeeding, the host's food intake depth is disrupted, and the composition of the gut microbiota changes. Whether these microbiota oscillations and their associated negative effects can be mitigated is an interesting question. Furthermore, a significant issue in treating obesity is weight rebound after weight loss and how the gut microbiota alters.

[0003] Compensatory weight gain after the end of dietary restriction can lead to increased awareness of diet and dietary restriction. Weight rebound after weight loss may play a key role in disease development. The degree of food restriction-refeeding in a dietary regimen, as well as the gut-diet interaction during food delivery, may have multifaceted effects on individuals. Consistently, similar behaviors have been observed in mice in numerous studies. A recent study showed a link between weight gain after food restriction in mice and a more efficient metabolic phenotype involving a unique gut microbiota structure for energy harvesting. However, the effects of the depth of food restriction and refeeding on host metabolic profile, gut microbiota, and inflammation remain unclear.

[0004] To determine the effects of varying depths of food restriction and refeeding on host physiology and gut microbiota effectiveness, this invention administered the same two food restriction regimens to 12-week-old adult and 6-week-old young mice, examining their metabolic phenotypes, gut microbiota, and correlation analyses to explore the long-term effects of these two levels of food restriction-refeeding on hosts of different ages. Background research revealed that mice subjected to different levels of food restriction—restricting free-feed intake by 15% and 40% of their body weight—followed by a high-fat diet. The study found that mildly food-restricted-refeeding mice experienced significantly different physiological changes, including fat accumulation, liver inflammation, enteritis, and changes in energy intake. Furthermore, the gut microbiota of mildly food-restricted-refeeding mice remained significantly different from that of severely food-restricted-refeeding mice and two control groups over a prolonged period. This invention describes the changes in gut microbiota and metabolic phenotype in mice refeeding after food restriction, inferring that refeeding a high-fat diet after mild food restriction leads to significant abnormal fat accumulation, liver damage, increased recruitment of intestinal inflammatory factors and immune cells in mice of different ages, involving multiple types of alterations in the gut microbiota. This invention describes a method for modulating the gut microbiota to improve the physiological effects of a mild time-restricted high-fat diet followed by a heavy feeding, characterized by significantly altering the levels of the gut microbiota species Lachnospiraceae, Bacteroides, and Alistipes.

[0005] Currently, there is no research or clinical focus on the long-term negative effects of refeeding a high-fat diet after mild dietary restriction, such as significant abnormal fat accumulation, liver damage, increased recruitment of intestinal inflammatory factors and immune cells, and changes in the gut microbiota in hosts of different ages. Furthermore, there is a lack of research on whether these negative effects are plastic and how to improve them. There is also a lack of a method for regulating the gut microbiota to improve the physiological effects caused by refeeding a high-fat diet after mild dietary restriction and its application. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the purpose of this invention is to use 16S rRNA sequencing to compare changes and composition of the gut microbiota, determine the negative impacts on the host and specific changes in the microbiota caused by a high-fat diet after mild food restriction, and provide a method and its application for regulating the gut microbiota to improve the physiological effects caused by a high-fat diet after mild food restriction.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solutions: A first aspect of the present invention is to provide a method for regulating the gut microbiota to improve the physiological effects caused by a mildly time-restricted high-fat diet followed by a heavy-feed diet. A second aspect of the present invention is to provide the application of said method in assessing the effects of the gut microbiota on host lipid metabolism and inflammatory responses. A third aspect of the present invention is to provide an apparatus for carrying out said method, characterized by comprising devices for gavage and devices for molecular biological detection. A fourth aspect of the present invention is to provide a kit for preparing fecal samples for said method.

[0008] The present invention discloses a method for regulating the gut microbiota to improve the physiological effects caused by a mild time-restricted high-fat diet followed by a refeeding diet, comprising the following steps:

[0009] S1 mice were divided into groups according to different degrees of food restriction and heavy feeding: normal control group, high-fat control group, mild food restriction group, severe food restriction group, mild food restriction-heavy feeding group, and severe food restriction-heavy feeding group. Fecal samples were collected.

[0010] S2 sent fecal samples from different groups to test 16S rRNA to determine the composition of the gut microbiota and the changes in specific bacterial groups in different groups;

[0011] S3 divided the donor and recipient mice into groups: adult control group, juvenile control group, adult restricted-feed group, and juvenile restricted-feed group.

[0012] S4 involved a treatment of mild food restriction followed by a high-fat diet in both the adult and juvenile food-restricted heavy feeding groups.

[0013] S5 collected fresh fecal samples from adult and juvenile control animals;

[0014] S6 processed fecal samples by homogenization, dilution, centrifugation and filtration, and then delivered them to the experimental group animals by gavage.

[0015] S7 implemented a treatment of mild food restriction followed by heavy feeding of a high-fat diet in both adult and juvenile food-restricted heavy feeding groups.

[0016] Furthermore, in step S2, to eliminate confounding time factors, fecal samples were collected from each group during week 2 of food restriction and week 6 after transitioning to free feeding, and the gut microbiota at the same time points was compared. The study was conducted using sequencing of the V3-V4 region of the 16S rRNA gene.

[0017] Furthermore, in steps S1 and S4, mild food restriction involves limiting the mice's free food intake to 15% of their total food weight for 14 days, followed by a 40-day period of free access to a high-fat diet.

[0018] Further, in steps S1 and S5, the method for collecting fecal samples is to collect fresh feces from each group of donor mice, homogenize them, dilute them with sterile physiological saline to a final concentration of 1 mg feces / 10 μL, centrifuge at 3000 rpm for 5 minutes, and collect the supernatant through a 70 μm filter.

[0019] Furthermore, in step S6, fecal samples are delivered to the experimental group animals via gavage at a volume of 10 mL / kg.

[0020] Furthermore, in step S7, the molecular biology techniques include quantitative polymerase chain reaction (QPCR), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA).

[0021] The method of this invention is applied in evaluating the effects of the gut microbiota on host lipid metabolism and inflammatory responses.

[0022] An apparatus for carrying out the method according to the present invention is characterized by comprising a device for gavage and a device for molecular biological detection.

[0023] A kit for preparing fecal samples for the method of the present invention comprises sterile saline and a 70 μm filter.

[0024] Furthermore, the present invention describes a method for modulating the gut microbiota to improve the physiological effects of a mild time-restricted high-fat diet followed by a heavy feeding, characterized by significantly altering the levels of the gut microbiota species Lachnospiraceae, Bacteroides, and Alistipes.

[0025] Beneficial effects: The present invention includes improving intestinal phenotype, reducing pathological damage, strengthening intestinal barrier function, reducing the expression of pro-inflammatory factors, alleviating liver inflammation, and regulating LPS levels, providing precise obesity prevention and treatment strategies for clinical practice.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) Detection of changes in gut microbiota: Using 16S rRNA sequencing technology, this invention determined the changes in the gut microbiota composition of mice subjected to different degrees of food restriction-restriction-reinforcement. Among them, the gut microbiota structure of mildly restricted-reinforcement mice showed significant changes, and the total amount of isolated gut microbiota was also significantly different from other groups. Further analysis revealed that the levels of gut microbes Lachnospiraceae, Bacteroides, and Alistipes in this group of mice were significantly altered, with higher levels of Lachnospiraceae and Bacteroides, and increased expression of Alistipes.

[0028] (2) Improvement of intestinal phenotype: Through fecal transplantation, this invention successfully improved the intestinal phenotype of recipient mice of different ages that were subjected to mild food restriction and heavy feeding. Specifically, the small intestine length of recipient mice in both age groups increased, and compared with donor mice, it showed that the reintroduction of feces from the control group could structurally restore some intestinal function.

[0029] (3) Reduction of pathological damage: Fecal transplantation significantly reduced the intestinal pathological damage score in recipient mice subjected to mild food restriction-replenishment. Compared with donor mice, recipient mice showed reduced intestinal damage, indicating that fecal transplantation helps repair intestinal tissue.

[0030] (4) Enhanced intestinal barrier function: Immunofluorescence homology double labeling experiment showed that the intestinal permeability of the recipient mice was improved and the expression levels of two key tight junction proteins, ZO-1 and occludin, were significantly increased, indicating that the intestinal barrier function was enhanced.

[0031] (5) Decreased expression of pro-inflammatory factors: QPCR experiments revealed that the mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 were significantly reduced in recipient mice, indicating that fecal transplantation helps to alleviate intestinal inflammatory response.

[0032] (6) Relief of liver inflammation: Although the effect of improving liver pro-inflammatory factors was not significant, fecal transplantation alleviated the liver inflammation in mildly restricted-refeed mice, suggesting that liver damage may be reversible to some extent.

[0033] (7) Regulation of LPS levels: ELISA experiments revealed that fecal transplantation can reduce LPS levels in the serum and liver of recipient mice, which is negatively correlated with the expression of PPAR-α in the liver. This suggests that the regulation of the gut microbiota may affect host lipid metabolism and inflammatory response through the LPS-PPAR-α pathway.

[0034] (8) Potential for clinical applications: This invention provides new perspectives and methods for clinical practice, particularly in developing obesity prevention and treatment strategies for different age groups. By modulating the gut microbiota, new therapeutic strategies can be provided for managing the physiological effects of dietary restriction and refeeding on the host. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0037] Figure 1 The following is a diagram showing the changes in mouse fecal microbiota according to the present invention: (1) The gut microbiota of fecal samples from two mildly restricted-refeeding groups was investigated by sequencing the V3-V4 region of the 16S rRNA gene. (2) Principal coordinate analysis (PCoA) was performed on the two restricted groups. Principal coordinate analysis (PCoA) was performed on the two restricted-refeeding groups. (3) Alpha diversity changed during food restriction and refeeding. (4) The levels of Chao1 and Simpson indices changed during food restriction. (5) Alpha diversity changed during feeding. (6) The levels of Chao1 and Simpson indices changed during food restriction. (7) Phylogenetic composition of the microbiota in the two restricted groups. Genus composition of the microbiota in the two restricted groups. Species composition of the microbiota in the two restricted groups. (8) Phylogenetic composition of the microbiota in the two restricted-refeeding groups. Genus composition of the microbiota in the two restricted-refeeding groups. Species composition of the microbiota in the two restricted-refeeding groups. CON: Animals were allowed free access to food throughout the experiment; HFD: Animals were fed 60% high-fat diet throughout the experiment; 15%-restricted-feed and 40%-restricted-feed groups had their free food intake restricted by 15% and 40% respectively for 2 weeks, followed by a 6-week high-fat diet. n = 6 mice per group.

[0038] Figure 2 The following is a diagram showing the effects of food restriction and refeeding on the composition of fecal microbiota as discovered in this invention; (1) The top ten species-level differences in microbiota between the two food restriction groups. The top ten species-level differences in microbiota between the two food restriction-refeeding groups. (2) Correlation analysis of microbiota between the two food restriction-refeeding groups. (3) Random forest analysis. (4) Association between microbiota and inflammatory factors. CON, animals were allowed free access to food throughout the experiment; HFD, animals were fed 60% high-fat diet throughout the experiment; In the 15% food restriction-refeeding group and the 40% food restriction-refeeding group, animals were restricted to 15% and 40% of their free access to food, respectively, for 2 weeks, and then fed high-fat diet for 6 weeks. n = 6 mice per group.

[0039] Figure 3The intestinal length and phenotype of the mice in this invention are shown below; (1) Adult control group (n=6): Mice were allowed free access to water and food for 2 months; (2) Juvenile control group (n=6): Mice were allowed free access to water and food for 2 months; (3) Adult mildly restricted-heavy feeding group (n=6): Adult mice were allowed free access to water for 7 days, restricted to 15% of their diet for 14 days, and then fed a high-fat diet for 40 days; (4) Juvenile mildly restricted-heavy feeding group (n=6): Juvenile mice were allowed free access to water for 7 days, restricted to 15% of their diet for 14 days, and then fed a high-fat diet for 40 days. Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-heavy feeding and received fecal samples from the control group donor mice, and two age-differential control group recipient mice received fecal samples from the mildly restricted-heavy feeding group donor mice.

[0040] Figure 4 HE staining and pathological scoring of mouse jejunum and colon for this invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mild food restriction-replenishment group (n=6) (4) Juvenile mild food restriction-replenishment group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mild food restriction-replenishment group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mild food restriction-replenishment group donor mice.

[0041] Figure 5 This is a diagram showing the expression of intestinal permeability-related proteins detected by immunofluorescence double staining according to the present invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0042] Figure 6The graph shows the expression of intestinal inflammatory factors in mice according to the present invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0043] Figure 7 The expression diagram of mouse liver inflammatory factors and PPAR-α in this invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) After two months of modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0044] Figure 8 The ELISA experiment for determining LPS content in mouse serum and liver is shown in the figure. (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) After two months of modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups. Two groups of recipient mice underwent mildly restricted-refeeding and received fecal samples from donor mice in the control group. Two groups of recipient mice underwent age difference and received fecal samples from donor mice in the mildly restricted-refeeding group.

[0045] Figure 9 The regulatory method and clinical application of the present invention: metabolism, inflammatory response and gut microbiota mapping. Detailed Implementation

[0046] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0047] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0048] A first aspect of the present invention is to provide a method for regulating the gut microbiota to improve the physiological effects caused by a mild time-restricted high-fat diet followed by a heavy-feed diet, comprising the following steps:

[0049] S1: Mice with different degrees of food restriction and heavy feeding were divided into groups: normal control group, high-fat control group, mild food restriction group, severe food restriction group, mild food restriction-heavy feeding group, and severe food restriction-heavy feeding group. Fecal samples were collected.

[0050] S2: Fecal samples from different groups were sent for 16S rRNA testing. The gut microbiota at the same time points were compared to determine the composition of the gut microbiota and the changes in specific bacterial groups in different groups.

[0051] S3: The donor and recipient mice were divided into groups: adult control group, juvenile control group, adult restricted-feed group, and juvenile restricted-feed group.

[0052] S4: In the adult and juvenile restricted-feed groups, a mild food restriction followed by a high-fat diet was implemented. Mild food restriction was achieved by limiting the mice's free access to food by 15% of their weight for 14 days, followed by a 40-day period of free access to a high-fat diet.

[0053] S5: Collect fresh fecal samples from adult control group and juvenile control group animals; the method of collecting fecal samples is to collect fresh feces from each group of donor mice, homogenize them, dilute them with sterile physiological saline to a final concentration of 1 mg feces / 10 μL, centrifuge at 3000 rpm for 5 minutes, and collect the supernatant through a 70 μm filter.

[0054] S6: Fecal samples were homogenized, diluted, centrifuged and filtered, and then delivered to the experimental group animals by gavage; the fecal sample was delivered to the experimental group animals by gavage at a volume of 10 mL / kg.

[0055] S7: Changes in the gut microbiota and related physiological indicators in the experimental group animals were detected using molecular biological techniques such as quantitative polymerase chain reaction (QPCR), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Molecular biological techniques were used to detect changes in the composition of the gut microbiota, the expression levels of pro-inflammatory factors, the expression levels of intestinal permeability-related proteins, and the LPS content in serum and liver.

[0056] A second aspect of the present invention is to provide the application of the method in assessing the effects of the gut microbiota on host lipid metabolism and inflammatory responses.

[0057] A third aspect of the invention is to provide an apparatus for carrying out the method, characterized in that it includes devices for gavage and devices for molecular biological detection.

[0058] A fourth aspect of the present invention is to provide a kit for preparing a fecal sample for the method, comprising sterile saline and a 70 μm filter.

[0059] Example 1

[0060] Changes in mouse fecal microbiota

[0061] like Figure 1 As shown, fecal microbiota was analyzed using 16S rRNA sequencing. The key feature was the identification of changes in the gut microbiota composition in mice subjected to different degrees of food restriction followed by heavy feeding. Specifically, the gut microbiota structure of mice subjected to mild food restriction followed by heavy feeding showed significant changes, and the total amount of isolated gut microbiota was also significantly different from other groups.

[0062] (i) DNA extraction and amplification

[0063] Fecal samples were immediately frozen and stored at -80°C after collection. In this study, six mice were used in each group. Fecal samples from two mice in each group were pooled before sequencing. Bacterial DNA was isolated from the fecal contents using the MagPure Soil DNA LQ Kit (Magen Pharmaceuticals, Guangdong, China) according to the manufacturer's instructions. DNA concentration and integrity were determined by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed using universal primer pairs (343F: 5'-TACGGRAGGCAGCAG-3'; 798R: 5'-AGGTATCTAATCCT-3') in a 25 μl reaction. Reverse primers contained a sample barcode, and both primers were linked to an Illumina sequencing adapter.

[0064] (ii) Library construction and sequencing

[0065] The quality of the amplicon was assessed by gel electrophoresis. PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, USA) and quantified using the Qubit dsDNA assay kit. The concentration was then adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq 6000 with two paired-end read cycles, each cycle containing 250 bases. (Illumina Inc., San Diego, CA; OE Biotech Company, Shanghai, China)

[0066] (iii) Bioinformatics Analysis

[0067] The raw sequencing data were in FASTQ format. Paired-end reads were then preprocessed using the cutadapt software to detect and cleave adapters. After pruning, low-quality sequences in the paired-end reads were filtered, denoised, merged, and chimeric reads were detected and cleaved using default parameters of DADA2 and QIIME2. Finally, the software output representative reads and an ASV abundance table. Representative reads for each ASV were selected using the QIIME 2 package. All representative reads were annotated using the q2-feature-classifier and aligned with the Silva version 138 database (or Unite) (16S / 18S / ITS rDNA) using default parameters. Alpha diversity, including the Chao1 index and Shannon index, was used to estimate microbial diversity in cecal contents samples. The Unifrac distance matrix from the QIIME2 software was used for unweighted Unifrac principal coordinate analysis (PCoA) and phylogenetic tree construction.

[0068] Example 2

[0069] The method in Example 2 is the same as in Example 1.

[0070] The effects of food restriction and refeeding on fecal microbiota composition. The key feature was that further analysis revealed significant changes in the levels of the gut microbiota Lachnospiraceae, Bacteroides, and Alistipes in this group of mice, with higher levels of Lachnospiraceae and Bacteroides, and increased expression of Alistipes.

[0071] like Figure 2 As shown, Figure 2The following is a diagram showing the effects of food restriction and refeeding on the composition of fecal microbiota as discovered in this invention; (1) The top ten species-level differences in microbiota between the two food restriction groups. The top ten species-level differences in microbiota between the two food restriction-refeeding groups. (2) Correlation analysis of microbiota between the two food restriction-refeeding groups. (3) Random forest analysis. (4) Association between microbiota and inflammatory factors. CON, animals were allowed free access to food throughout the experiment; HFD, animals were fed 60% high-fat diet throughout the experiment; In the 15% food restriction-refeeding group and the 40% food restriction-refeeding group, animals were restricted to 15% and 40% of their free access to food, respectively, for 2 weeks, and then fed high-fat diet for 6 weeks. n = 6 mice per group.

[0072] Experimental Example 1

[0073] Intestinal length and phenotype in mice. The key feature is the improvement in intestinal phenotype. The improvement of intestinal phenotype in recipient mice of different ages subjected to mild food restriction followed by heavy feeding was successfully achieved. Specifically, the small intestine length increased in both age groups of recipient mice, and compared to donor mice, it showed that the reintroduction of feces from the control group structurally restored some intestinal function.

[0074] The animal was euthanized using a RWD anesthesia ventilator (product catalog number: R540-48), and blood was then collected. After blood collection, the stomach, small intestine, and large intestine tissues were carefully and quickly separated, weighed (to 1 mg), measured, and photographed.

[0075] like Figure 3 As shown, Figure 3 The intestinal length and phenotype of the mice in this invention are shown below; (1) Adult control group (n=6): Mice were allowed free access to water and food for 2 months; (2) Juvenile control group (n=6): Mice were allowed free access to water and food for 2 months; (3) Adult mildly restricted-heavy feeding group (n=6): Adult mice were allowed free access to water for 7 days, restricted to 15% of their diet for 14 days, and then fed a high-fat diet for 40 days; (4) Juvenile mildly restricted-heavy feeding group (n=6): Juvenile mice were allowed free access to water for 7 days, restricted to 15% of their diet for 14 days, and then fed a high-fat diet for 40 days. Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-heavy feeding and received fecal samples from the control group donor mice, and two age-differential control group recipient mice received fecal samples from the mildly restricted-heavy feeding group donor mice.

[0076] Experimental Example 2

[0077] HE staining and pathological scoring of mouse jejunum and colon. The key feature is the reduction of pathological damage. Fecal transplantation significantly reduced intestinal pathological damage scores in mildly restricted-refeed recipient mice. Compared to donor mice, recipient mice showed reduced intestinal damage, indicating that fecal transplantation helps repair intestinal tissue.

[0078] The central portions of each intestine and liver were preserved in paraformaldehyde for 24 hours for pathological evaluation. Then, 3 μm paraffin-embedded cross sections (H and E) stained with hematoxylin and eosin were prepared. These H&E sections were scored using a scoring system proposed by Vieira et al., scored by a researcher who was not aware of the experimental protocol. Inflammation degree (0: none, 1: mild, 2: moderate, 3: severe), lesion depth (0: none, 1: mucosa, 2: mucosa and submucosa, 3: transmural), and crypt damage were used to determine the histological score (0: none, 1: basal third damaged, 2: basal two-thirds damaged, 3: only surface epithelium intact, 4: loss of thrush cells throughout the crypt and epithelium). The score for each parameter was multiplied by the percentage of tissue-related factors (1: 1–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%). In short, the histological score = (inflammation + lesion depth + crypt destruction) × lesion width.

[0079] like Figure 4 As shown, Figure 4 HE staining and pathological scoring of mouse jejunum and colon for this invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mild food restriction-replenishment group (n=6) (4) Juvenile mild food restriction-replenishment group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mild food restriction-replenishment group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mild food restriction-replenishment group donor mice.

[0080] Experimental Example 3

[0081] Immunofluorescence double staining of paraffin sections was used to detect the expression of intestinal permeability-related proteins. The key feature was the enhancement of intestinal barrier function. Immunofluorescence double staining experiments showed improved intestinal permeability in recipient mice, with significantly increased expression levels of two key tight junction proteins, ZO-1 and occludin, indicating enhanced intestinal barrier function.

[0082] The central portion of each intestine was preserved in paraformaldehyde for 24 hours. Then, after deparaffining to water in paraffin sections, antigen retrieval, serological blocking, addition of primary antibody and HRP secondary antibody, fluorescent dye reaction, antibody elution, and repeating the second round of labeling steps, DAPI restaining of cell nuclei, quenching of tissue autofluorescence, mounting, and finally microscopic examination and photography.

[0083] like Figure 5 As shown, Figure 5 This is a diagram showing the expression of intestinal permeability-related proteins detected by immunofluorescence double staining according to the present invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0084] Test Example 4

[0085] qPCR was used to detect the expression of intestinal inflammatory factors in mice. The key feature was the reduction in the expression of pro-inflammatory factors. Through qPCR experiments, it was found that the mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly reduced in recipient mice, indicating that fecal transplantation helps alleviate intestinal inflammation.

[0086] Real-time quantitative polymerase chain reaction (qPCR): Total RNA was extracted from jejunal and ileal samples using TRIzol reagent (Invit-Rogen, Carlsbad, CA, USA). cDNA was synthesized using random primers oligo(dT)18 and AMV reverse transcriptase (TAKARA) in a final reaction volume of 20 μL. 2 μL of cDNA sample was taken and PCR was performed using gene-specific primers (Table 1). The final reaction volume was 20 μL, containing 10 μL of 2×SYBR Premix EX Tag™, 0.4 μL of forward and reverse primers (final concentration of 0.2 M per primer), 2 μL of cDNA template, and 7.2 μL of DEPC. Quantitative real-time PCR was performed on a CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA, USA) using SsoFastEvaGreenSupermix. Actin was used as an internal standard. The relative gene expression levels in the samples were quantified.

[0087] like Figure 6 As shown, Figure 6The graph shows the expression of intestinal inflammatory factors in mice according to the present invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) Two months after modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0088] Experimental Example 5

[0089] Expression of inflammatory factors and PPAR-α in mouse liver. A key feature was the alleviation of liver inflammation. Although the improvement in liver pro-inflammatory factors was not significant, fecal transplantation alleviated liver inflammation in mildly restricted-refeed mice, suggesting that liver damage may be reversible to some extent.

[0090] Real-time quantitative polymerase chain reaction (PCR) was performed. Statistical analysis was conducted using GraphPad Prism 9.0 and SPSS statistical software (version 21.0). One-way ANOVA was used to analyze differences between groups, and two-tailed Student's t-tests were used to analyze the significance of differences between the two groups. All data are expressed as mean ± SEM. Statistical significance was defined as P < 0.05.

[0091] like Figure 7 As shown, Figure 7 The expression diagram of mouse liver inflammatory factors and PPAR-α in this invention; (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) After two months of modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups, of which two groups underwent mildly restricted-refeeding group recipient mice transplanted with fecal samples from control group donor mice, and two age-difference control group recipient mice transplanted with fecal samples from mildly restricted-refeeding group donor mice.

[0092] Experimental Example 6

[0093] ELISA assays were used to determine LPS levels in mouse serum and liver. The key feature was the regulation of LPS levels. ELISA experiments revealed that fecal transplantation reduced LPS levels in the serum and liver of recipient mice, which was negatively correlated with PPAR-α expression in the liver, suggesting that gut microbiota regulation may affect host lipid metabolism and inflammatory responses through the LPS-PPAR-α pathway.

[0094] Alterations in the gut microbiota and the endotoxin LPS produced by gut bacteria play a crucial role in the development and progression of liver diseases. Mouse lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA): This kit is a one-step sandwich ELISA using double antibodies. The sample, standard, and HRP-labeled detection antibody are added sequentially to wells pre-coated with LPS / LPS antibodies, followed by incubation and thorough washing. The substrate TMB is used for color development; TMB is converted to blue under the catalysis of peroxidase, and finally to yellow under acidic conditions. The color intensity is positively correlated with the LPS / LPS concentration in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0095] like Figure 8 As shown, Figure 8 The ELISA experiment for determining LPS content in mouse serum and liver is shown in the figure. (1) Adult control group (n=6) (2) Juvenile control group (n=6) (3) Adult mildly restricted-refeeding group (n=6) (4) Juvenile mildly restricted-refeeding group (n=6) After two months of modeling, fresh fecal samples were collected daily and transplanted into fecal microbiota transplant recipient mice. Recipient mice were also divided into four groups. Two groups of recipient mice underwent mildly restricted-refeeding and received fecal samples from donor mice in the control group. Two groups of recipient mice underwent age difference and received fecal samples from donor mice in the mildly restricted-refeeding group.

[0096] Experimental Example 7

[0097] Regulation Methods and Clinical Applications: This invention utilizes AI to visualize and connect the relationships between host lipid metabolism, inflammatory responses, and the gut microbiota. Its key feature is its potential for clinical application. This invention provides a new perspective and methodology for clinical practice, particularly in developing obesity prevention and treatment strategies for different age groups. By modulating the gut microbiota, new therapeutic strategies can be provided for managing the physiological effects of dietary restriction and refeeding on the host.

[0098] This embodiment presents a method for regulating the gut microbiota to improve the physiological effects of a mildly time-restricted followed by a high-fat diet. Using fecal transplantation, it improved intestinal phenotype length, intestinal injury score, intestinal permeability, and pro-inflammatory cytokine expression in recipient mice of different ages undergoing mild food restriction followed by a high-fat diet. Although the improvement in liver pro-inflammatory cytokines was not significant, it indicates that liver damage may be irreversible. Furthermore, the discovery of the LPS-PPAR-α regulation method provides an application in clinical practice for developing more precise obesity prevention and treatment strategies for different age groups. Figure 9 As shown, Figure 9 The regulatory method and clinical application of the present invention: metabolism, inflammatory response and gut microbiota mapping.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A research method for regulating the gut microbiota to improve the physiological effects of a high-fat diet following mild food restriction, characterized in that... Includes the following steps: S1 mice were divided into groups based on different degrees of food restriction and heavy feeding: a normal control group, a high-fat control group, a mild food restriction group, a severe food restriction group, a mild food restriction-heavy feeding group, and a severe food restriction-heavy feeding group. Fecal samples were collected. In the normal control group, animals had free access to food throughout the experiment. In the high-fat control group, animals were fed 60% high-fat diet throughout the experiment. In the mild food restriction-heavy feeding group and the severe food restriction-heavy feeding group, animals had their free food intake restricted by 15% and 40% respectively for 2 weeks, and then were fed a high-fat diet for 6 weeks. S2 sent fecal samples from different groups for 16S rRNA testing to determine the composition and specific changes in the gut microbiota of different groups. To eliminate confounding time factors, fecal samples from each group were collected in the second week of food restriction and the sixth week after switching to free feeding. The gut microbiota at the same time points were compared, and the study was conducted by sequencing the V3-V4 region of the 16S rRNA gene. In the mild food restriction-heavy feeding group, the levels of the gut microbiota Lachnospiraceae, Bacteroides, and Alistipes were significantly altered. S3 divided donor and recipient mice into four groups: adult control group, juvenile control group, adult mildly restricted-refeed group, and juvenile mildly restricted-refeed group. Adult control group: mice had free access to water and food for 2 months; juvenile control group: mice had free access to water and food for 2 months; adult mildly restricted-refeed group: adult mice had free access to water and food for 7 days, then restricted their diet by 15% for 14 days, and then were fed a high-fat diet for 40 days; juvenile mildly restricted-refeed group: juvenile mice had free access to water and food for 7 days, then restricted their diet by 15% for 14 days, and then were fed a high-fat diet for 40 days. Two months after S4 modeling, fresh fecal samples were collected from adult and juvenile control animals. S5 processed fecal samples by homogenization, dilution, centrifugation and filtration, and then delivered them to the recipient group animals by gavage at a volume of 10 mL / kg. S6 uses molecular biology techniques to detect changes in the gut microbiota and related physiological indicators.

2. The research method for regulating gut microbiota to improve the physiological effects caused by a high-fat diet following mild food restriction, as described in claim 1, is characterized in that: In steps S1 and S4, the fecal samples were collected by gathering fresh feces from each group of donor mice, homogenizing them, diluting them with sterile saline to a final concentration of 1 mg feces / 10 μL, centrifuging at 3000 rpm for 5 minutes, and collecting the supernatant through a 70 μm filter.

3. The research method for regulating gut microbiota to improve the physiological effects caused by a high-fat diet following mild food restriction, as described in claim 1, is characterized in that: In step S6, molecular biology techniques include quantitative polymerase chain reaction (qPCR), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA).

4. The application of the method according to any one of claims 1 to 3 in assessing the effects of the gut microbiota on host lipid metabolism and inflammatory response.

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