Application of osteopontin in regulating intestinal flora homeostasis

Abstract

The present invention relates to the technical field of intestinal flora regulation, in particular to the application of osteopontin in intestinal flora homeostasis regulation; the technical problem to be solved by the present invention is to provide the use of osteopontin in regulating the homeostasis balance of mammalian intestinal flora; the present invention expects to increase the abundance of intestinal probiotics or reduce the abundance of harmful bacteria through the intake or expression of osteopontin; the abundance of probiotics or symbiotic bacteria including Akkermansia muciniphila in the intestine is increased by osteopontin, and the abundance of harmful bacteria including Duncaniella dubosii is reduced, thereby ultimately maintaining the homeostasis of intestinal flora, and overcoming the technical prejudice of the prior art regarding the use of osteopontin in maintaining the homeostasis of intestinal flora.

Description

Technical Field

[0001] The present invention relates to the technical field of intestinal flora regulation, and in particular to the application of osteopontin in intestinal flora homeostasis regulation. Background Art

[0002] The prior art believes that osteopontin (OPN) has an inhibitory effect on the intestinal flora, especially the colonization of probiotics, and the intake or expression of osteopontin may disrupt the steady-state balance of the intestinal flora.

[0003] For example, a Chinese patent discloses the use of osteopontin as a target molecule in regulating intestinal flora colonization, patent number 202010153324.4, which records: The present invention discloses the use of osteopontin as a target molecule in regulating intestinal flora colonization; specifically: inhibiting OPN gene expression or blocking OPN binding to cell receptors, thereby increasing the number of probiotics in the intestine; blocking OPN binding to cell receptors or inhibiting the Notch signaling pathway can increase the adhesion of bacteria to intestinal epithelial cells. The present invention confirms that osteopontin affects the adhesion between cells and bacteria by inhibiting the expression of cell adhesion factors, thereby inhibiting the colonization of flora in the intestine.

[0004] For example, on April 20, 2023, Li Lu published a study on the in vitro digestion and fermentation characteristics of osteopontin in infants and the mechanism of its impact on intestinal barrier function (Master's thesis of South China University of Technology), which recorded: Bovine milk OPN (equivalent to milk osteopontin, osteopontin in milk is also called milk pontin and milk osteopontin) has a slow and stable fermentation rate, but the large accumulation of harmful by-products may be detrimental to maintaining intestinal homeostasis.

[0005] However, the present invention found that the above-mentioned prior art has technical bias. The present invention confirms that osteopontin can be used for the steady-state regulation of intestinal flora and produces a positive effect that is beneficial to the colonization of probiotics. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide the use of osteopontin in regulating the steady-state balance of the intestinal flora of mammals; the present invention hopes to increase the abundance of intestinal probiotics or reduce the abundance of harmful bacteria through the intake or expression of osteopontin.

[0007] The technical solution of the present invention is:

[0008] The application of osteopontin in the regulation of intestinal flora homeostasis is to use osteopontin to prepare medicines, health products or foods for preventing or treating intestinal flora homeostasis imbalance caused by downregulation or blocking of Spp1 gene expression.

[0009] Specifically, the imbalance of microbiota homeostasis includes a decrease in the abundance of probiotics or symbiotic bacteria or an increase in the abundance of harmful bacteria.

[0010] Specifically, the probiotics include Akkermansia muciniphila, which is negatively correlated with obesity, diabetes, cardiovascular disease and low-grade inflammation and is a probiotic.

[0011] Specifically, the symbiotic bacteria include Bacteroldes caccae, Bacteroldes caecimuris, Bacteroldesfaecium, Bacteroldesfaecium and Bacteroldes uniformis, all of which are common intestinal symbiotic bacteria.

[0012] Specifically, the harmful bacteria include Duncaniella dubosii. Duncaniella dubosii promotes inflammation and is a harmful bacterium.

[0013] Specifically, the harmful bacteria include Duncaniella sp C9. Duncaniella sp C9 increases the spleen index and is a harmful bacterium.

[0014] Specifically, the harmful bacteria include Paramuribaculumintestinale. Paramuribaculumintestinale belongs to the Muribaculaceae family of bacteria, and excessive proliferation may induce IDD (immunodeficiency disease), which is a harmful bacterium.

[0015] Specifically, the efficiency of SPP1 gene in expressing osteopontin can be improved, thereby promoting the effective colonization of probiotics in the intestine and reducing the proliferation of some harmful bacteria.

[0016] Specifically, it also includes the repair of the impaired digestion and absorption, lipid metabolism and immune response functions caused by the loss of the Spp1 gene in colon tissues induced by the intake of OPN. The specific knockout of the OPN gene (Spp1) changes the expression pattern of intestinal genes, especially affecting lipid metabolism and intercellular adhesion. These changes may lead to functional disruption of colon tissues, affect immune response and barrier function, and thus increase the risk of colitis and other intestinal diseases. Obviously, the above problems can be repaired or alleviated by taking OPN.

[0017] Specifically, it also includes maintaining the homeostasis of specific bacterial flora (such as Akkermansia muciniphila) by ingesting OPN or upregulating the expression of OPN, thereby regulating intestinal digestion and absorption, lipid metabolism, and immune feedback.

[0018] The beneficial effects of the present invention are: osteopontin is used to increase the abundance of probiotics or symbiotic bacteria in the intestine, including Akkermansia muciniphila, and to reduce the abundance of harmful bacteria, including Duncanialla dubosii, thereby ultimately maintaining the homeostasis of the intestinal flora, and overcoming the technical prejudice of the prior art regarding osteopontin in maintaining the homeostasis of the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Representative photomicrographs of AB-PAS (Alcian blue-periodic acid-Schiff) stained colon tissue sections.

[0020] Figure 2 Figure 2 is a graph of differential gene expression and functional enrichment in the intestine induced by CKO, where: (a) Volcano plot of differentially expressed genes in the intestine of CKO mice: the x-axis represents log2FoldChange, and the y-axis represents the negative log10 of the significance level. The two vertical dotted lines represent the threshold for the fold change in gene expression, and the horizontal dotted line represents the significance threshold. Red indicates that the gene is upregulated in the CKO group, while blue indicates genes that are downregulated or not significantly differentially expressed. b) Changes in differentially expressed genes in the intestine of CKO mice. In (b), the x-axis shows the comparison group used for differential analysis, and the y-axis represents the number of differentially expressed genes. Red represents the total number of upregulated genes in the CKO group, while blue represents the total number of downregulated genes. c) GO functional enrichment of differentially expressed genes in the intestine of CKO mice: the x-axis represents GO terms, and the y-axis represents the -log10 (p-value) of GO term enrichment. Different colors represent KEGG categories: cellular component (CC), biological process (BP), and molecular function (MF). (d) KEGG functional enrichment of differentially expressed genes in the intestine of CKO mice; the x-axis represents pathways enriched in pathways, and the y-axis represents -log10 (p-value) of pathway enrichment. Blue, organismal system; red, metabolism; green, human disease; purple, environmental information processing.

[0021] Figure 3Figure 1 shows the microbial diversity and species level of the intestinal microbiota of CKO mice, where: (a) Changes in the Shannon index and Simpson index of the intestinal microbiota of the two groups: The box plot in (a) shows the interquartile range of the values ​​of each group. The line in the box represents the median, and the dotted line represents the minimum and maximum values ​​within 1.5 times the interquartile range. Points above or below the upper and lower lines are outliers. (b) Changes in intestinal β diversity, PC1, and PCoA between the two groups. In (b), the symbols representing the samples of the two groups are displayed in different colors, and the results of the Adonis test are shown in the lower right corner of the PCoA graph. (c) Changes in the structure of the intestinal microbiota between the two groups of mice. In (c), species with significantly different abundances between the two groups are shown, and different colors represent different species. (d) Changes in the abundance of Akkermansia muciniphila in the intestine between the two groups of mice; the y-axis represents species abundance, and the x-axis represents each group. e) Bar graph of species composition and abundance of the intestinal microbiota between the two groups; the Y-axis represents species abundance, and the X-axis represents the group. Statistical significance was assessed using a two-sided Wilcoxon rank-sum test, with *P < 0.05 considered statistically significant.

[0022] Figure 4 Figure 2 shows the differential metabolic pathways between CKO mice and WT control groups, where: Differential metabolic pathways between CKO mice and WT control groups: Predicted fecal metagenomic metabolic pathways, which differed significantly between the two groups. PLS-DA generated projected variable importance (VIP) scores to identify metabolites that contribute to population differentiation, and P values ​​were calculated using a two-sided Wilcoxon rank sum test (threshold: VIP>2; P<0.05).

[0023] Figure 5 Figure 2 is a correlation network diagram, where: (a) Correlation network of gut microbiota and differentially expressed genes in CKO mice. (b) Correlation between Akkermansia muciniphila and differentially expressed genes in CKO mice. The solid line indicates the positive correlation between species abundance and differentially expressed genes, while the dotted line indicates the negative correlation. The thickness of the line indicates the strength of the correlation, and the size and depth of the point color indicate the number of related entities. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings:

[0025] Experimental animals

[0026] In this study, we used the intestinal-specific knockout of Spp1 gene (Spp1 [flox / flox，Vil1-cre] ) specific pathogen-free (SPF) C57BL / 6 mice. Male Spp1 [flox / flox] Mouse and female Spp1 [flox / flox；Vil1-cre] Mouse. In Spp1 [flox / flox]In the genotype, LoxP sites are located on both sides of the Spp1 gene, and Spp1 [Vil1-Cre] The genotype specifically expresses Cre recombinase in the intestinal epithelium. The offspring were genotyped, and those that showed intestinal-specific OPN knockout were designated as the CKO group, while their wild-type littermates served as the WT control group. All animals were housed in an SPF-grade facility under controlled environmental conditions: 12 h / 12 ​​h light-dark cycle, temperature of 22 ± 2 °C, and humidity of 50 ± 10%. Food and water were provided daily throughout the study. Mice were monitored daily and euthanized at the end of the study period. Colonic contents and tissues were collected for metagenomic and transcriptomic sequencing, and additional colon samples were collected for histopathological analysis. This study was conducted in strict accordance with the GB / T35823-2018 Guide for Animal Research and strictly adhered to ethical standards. This study was approved by the Animal Welfare and Ethics Committee of Beijing Huafukang Biotechnology Co., Ltd. (approval number: HFK-AP-20230523).

[0027] Mouse genotyping

[0028] Use RNaseA Genomic DNA was extracted from mouse tail tissue using a genomic DNA kit (TransGenBiotech, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using two sets of primers: set 1 (F1: 5'-GTAGAGCGTCATTGCAGTTTCAT-3' and R1: 5'-TTATTGTAGTTGAGCACCCATGTTC-3') and set 2 (Vil1-cre-F: 5'-CCATAGGAAGCCAGTTTCCCTTC-3' and Vil1-cre-R: 5'-TTCCAGGTATGCTCAGAAAACGC-3'). The 207 bp target bands of primer set 1 and 304 bp of primer set 2 in the PCR product indicated that Spp1 [flox / flox，Vil1-cre] homozygous knockout state.

[0029] Histological analysis

[0030] Mouse colon tissues were fixed in paraformaldehyde, embedded in paraffin, and sliced. Subsequently, the tissue sections were stained with Alcian Blue-Periodic Acid-Schiff (AB-PAS). The distribution of mucin and polysaccharides was observed using a 3DHISTECH P250 FLASH panoramic scanner (3DHISTECH, Hungary).

[0031] Transcriptomic analysis

[0032] Colon tissues extracted from WT and CKO mice were immediately snap-frozen in liquid nitrogen and then stored at -80 °C for subsequent RNA-seq analysis. Total RNA was extracted using the Agilent 2100 Bioanalyzer RNA 6000Nano Kit (Cat. No. 5067-1511, Agilent Technologies, USA). After quality control, paired-end sequencing (PE150) was performed on the Illumina Novaseq X Plus platform using the Mus musculus genome (GRCm39) for gene annotation. Differential gene expression analysis was performed using DESeq (v1.38.3), and the thresholds for differentially expressed genes were set to |log2FoldChange|>1 and P<0.05.

[0033] Gene ontology (GO) enrichment analysis was performed using topGO (v2.50.0). The hypergeometric distribution method was applied to calculate the p-value, and significant enrichment was defined as p < 0.05 to identify significantly enriched GO terms and determine the main biological functions of differentially expressed genes. To investigate changes at the pathway level, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using clusterProfiler (v4.6.0), in which pathways were considered significantly enriched at p < 0.05. Gene set enrichment analysis (GSEA, v4.1.0) was used to further explore related gene sets.

[0034] Metagenomic sequencing

[0035] Metagenomic sequencing analysis was performed on samples of mouse colon contents. Total DNA was extracted from colonic fecal samples using QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany). Sequencing libraries were prepared using the NEB Next Ultra DNA Library Preparation Kit (New England Biolabs Inc., Ipswich, MA, USA). The Illumina HiSeq 2500 sequencing platform generated raw data, and low-quality and host-contaminated reads were subsequently deleted using Kneaddata software (http: / / huttenhower.sph.harvard.edu / kneaddata). The HUMAn N2 + Kraken 2 platform was used to perform species annotation and functional analysis on the resulting data.

[0036] Statistical analysis

[0037] All experimental data are reported as mean ± standard deviation (SD). Statistical analysis was performed using the R programming language (v4.1.1), and the differences between the two groups were assessed using the Wilcoxon test. Statistical significance was set at p < 0.05. Graphical representations were generated using GraphPad Prism 9.5 with the R package and STAMP software (v2.1.3).

[0038] result:

[0039] Knockout of the Spp1 gene leads to a decrease in the colonic mucus layer in mice (e.g. Figure 1 )

[0040] AB-PAS staining was used to visualize mucus distribution and integrity to assess changes in the colonic mucus layer and its composition in mice. The results showed that colon sections of wild-type mice (WT group) exhibited a reddish-purple hue after AB-PAS staining, indicating PAS-positive and abundant acidic mucus, which was mainly secreted by goblet cells and evenly distributed along the epithelial cell surface with a healthy thickness. In contrast, OPN intestinal-specific knockout mice (CKO group) exhibited a significant reduction in the colonic mucus layer and reduced staining intensity, indicating that the number and function of goblet cells were impaired. These findings indicate that OPN is essential for maintaining the structure and function of the intestinal mucus layer. The reduction in the mucus layer observed in CKO mice may have impaired the intestinal barrier and may increase susceptibility to infection and inflammation.

[0041] Changes in gene expression patterns in colon tissue of Spp1 knockout mice (e.g. Figure 2 )

[0042] To investigate the effects of OPN deficiency on gene expression in colonic tissue, transcriptome sequencing was performed on colonic samples. Analysis of the sequencing data against the reference genome resulted in annotations for 21,959 expressed genes. Comparison between WT and CKO mice revealed significant differences in the expression of 169 genes in colonic tissue of CKO mice (p<0.05, |log2FoldChange|>1). Of these, 115 genes were upregulated and 54 genes were downregulated in the CKO group ( Figure 2 a and 2b). These findings suggest that loss of the gene encoding OPN induces differential gene expression in colonic tissue.

[0043] To determine the functional significance of these differentially expressed genes, GO functional enrichment and KEGG analysis were performed. GO analysis showed that the differentially expressed genes were mainly related to processes such as cell adhesion (GO: 0007155), biological adhesion (GO: 0022610), acute inflammatory response (GO: 0002526), ​​extracellular matrix organization (GO: 0030198), extracellular space (GO: 0005615), cell surface (GO: 0009986), extracellular structure organization (GO: 0043062), intercellular signaling (GO: 0007267) and multicellular development (GO: 0007275) (p < 0.05, Figure 2 c). In addition, KEGG enrichment analysis showed that the differentially expressed genes in CKO mice relative to the WT group were significantly associated with immune and metabolic functions. The pathways involved included fat digestion and absorption (pathway ID: mmu04975), antigen processing and presentation (pathway ID: mmu04612), regulation of adipocyte lipolysis (pathway ID: mmu04923), ether lipid metabolism (pathway ID: mmu00565), α-linolenic acid metabolism (pathway ID: mmu00592), mucin-type O-sugar biosynthesis (pathway ID: mmu00512), niacin and nicotinamide metabolism (pathway ID: mmu00760), linoleic acid metabolism (pathway ID: mmu00591), pyrimidine metabolism (pathway ID: mmu00240), cell adhesion molecules (pathway ID: mmu04514), and cGMP-PKG signaling pathway (pathway ID: mmu04022). Notably, pathways closely related to immune response included antigen processing and presentation, mucin O-glycan biosynthesis, cell adhesion, and cGMP-PKG signaling pathway (p < 0.05, Figure 2 d).

[0044] Studies have shown that changes in colonic tissue induced by Spp1 gene deletion lead to impaired digestion and absorption, lipid metabolism, and immune response functions. Specifically, genes related to inflammation and signaling, including C3, Adoral, and Cdh3, were significantly upregulated in the CKO group, while genes related to lipid metabolism and cell adhesion, such as Pla2g12b, Gcnt3, and Adra2a, were significantly downregulated (Table 1). Intestinal-specific OPN gene knockout changed the expression pattern of intestinal genes, especially lipid metabolism and cell-cell adhesion. These changes may lead to the destruction of colon tissue function, affect immune response and barrier function, and thus increase the risk of colitis and other intestinal diseases.

[0045] Based on these observations, we can infer that the expression of OPN in colonic tissue has a great impact on the development of intestinal diseases in mice, as well as signal transduction, cell adhesion, and lipid metabolism processes. This effect may work through two mechanisms: first, endogenous OPN regulates the expression of intestinal-related genes; second, OPN may affect the intestinal commensal microbiota, thereby indirectly affecting intestinal health. To further investigate this mechanism, metagenomic sequencing was used to comprehensively analyze the intestinal flora of the two groups.

[0046] Table 1 Candidate gene screening

[0047]

[0048] Table Notes: “Gene” refers to the genes screened from the differentially expressed genes, “Description” provides the description of the gene, and “Expression pattern in CKO” indicates the expression pattern of the gene in the CKO group.

[0049] Effects of Spp1 gene knockout on the structure and composition of intestinal flora in mice (e.g. Figure 3 )

[0050] 1. Microbial diversity analysis showed no significant differences in Shannon and Simpson indices between the two groups of mice; however, the CKO group showed a trend of increased α diversity compared with the control group ( Figure 3 a).

[0051] 2. Bray-Curtis distance analysis showed that there were significant differences in the intestinal microbiota structure between the CKO group and the control group, indicating that there were potential structural differences in the microbial composition ( Figure 3 b).

[0052] 3. The abundance of Akkermansia muciniphila, Bacteroldes caccae, Bacteroldes caecimuris, Bacteroldesfaecium and Bacteroldes uniformis in the CKO group was significantly lower than that in the control group (p < 0.05, Figure 3 e).

[0053] Comparative analysis of the abundance of Duncaniella dubosii, Duncaniella sp C9 and Paramuriba culumintestinale showed that the abundance of the CKO group was significantly higher than that of the control group (p < 0.05, Figure 3 e).

[0054] In short, when the intestine lacks OPN, many probiotics are difficult to survive and many harmful bacteria proliferate. Therefore, when the intestinal flora is unbalanced due to downregulation or blocking of Spp1 gene expression, the intestinal flora homeostasis can be regulated by taking OPN (such as consuming milk osteopontin, which is also called milk pontin or milk osteopontin) or upregulating the expression of OPN. Therefore, the present invention can be applied to medicines, health products or foods that regulate the homeostasis of intestinal flora.

[0055] Alterations in the intestinal microbiota may induce changes in intestinal metabolic pathways. Intestinal function was annotated using HUMAnN2 software, and 274 metabolic pathways within the microbiota of all mice were identified, of which 25 pathways showed significant differences between groups (p < 0.05) (e.g. Figure 4 ). Notably, eight of the differential pathways were related to fatty acid biosynthesis and metabolism, including stearic acid biosynthesis, saturated fatty acid elongation, oleic acid biosynthesis IV (anaerobic), palmitic acid biosynthesis I (from (5Z)-dodecenoic acid), (5Z)-dodecenoic acid biosynthesis, hexadecanoic acid synthesis, cis-octadecenoic acid biosynthesis, and Escherichia coli-induced super fatty acid biosynthesis pathways. In addition, the functional differences of the gut microbiota involved pathways related to glycolysis, amino acid biosynthesis, nucleotide and phospholipid biosynthesis, and vitamin and cofactor biosynthesis. These findings suggest that OPN may act as a signaling molecule to indirectly regulate host metabolism and physiological status through its effects on the structure and function of the gut microbiota.

[0056] Notably, the observed functional differences in the gut microbiota between CKO and WT mice were closely associated with digestion and absorption, lipid metabolism, and synthesis, consistent with the functional alterations detected in the transcriptomic profiles of colonic tissue. These findings highlight the multifaceted role of OPN in the regulation of intestinal function, including its direct effects on host cell activity and indirect effects on host metabolism through the gut microbiota. Furthermore, the observed changes in host-microbe composition showed synergistic interactions.

[0057] In other words, the homeostasis of specific flora (such as Akkermansia muciniphila) can be maintained by ingesting OPN (such as consuming milk osteopontin, the osteopontin in milk is also called lactopontin or milk osteopontin) or upregulating the expression of OPN, thereby regulating intestinal digestion and absorption, lipid metabolism, and immune feedback.

[0058] Spp1 knockout induces coordinated changes in gene expression and composition of the microbiota in the intestinal tissue of mice (e.g. Figure 5 )

[0059] The study showed that modifications in the expression of the OPN encoding gene (Spp1) have both direct effects on host cell function and potential changes in the intestinal environment, thereby affecting the composition and function of the intestinal microbiota. These findings highlight the intricate interactions between host genes and intestinal microbes. To elucidate the relationship between intestinal microbes and host gene expression, the researchers performed Spearman correlation analysis to quantify the association between the relative abundance of intestinal microbes and differentially expressed genes in mouse colon tissue. Figure 5 As shown in a, C3 and Abi3bp were positively correlated with Pseudomonas faecalis, while Gcnt3 was negatively correlated with Duncaniella dubosii. Alistipes indistinctus was negatively correlated with Prelp, while Ruminococcus torque was positively correlated with mt-Nd5 and Grem1. Interestingly, A. muciniphila showed a strong negative correlation with Pcsk9 (rho = -0.94) and negatively correlated with Ly6c2 and Fcamr, although these correlations were not statistically significant ( Figure 5 b) Overall, these results highlight the complex interplay between gut microbiota and host gene expression.

[0060] The above embodiments and descriptions are only for illustrating the principles and best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, all of which fall within the scope of the present invention to be protected.

Claims

1. Application of osteopontin in regulating intestinal flora homeostasis.

2. The use of osteopontin in regulating intestinal flora homeostasis according to claim 1, characterized in that: Using osteopontin to prepare products for preventing or treating intestinal flora homeostasis imbalance caused by downregulation or blocking of Spp1 gene expression; The imbalance of microbiota homeostasis includes a decrease in the abundance of probiotics or symbiotic bacteria or an increase in the abundance of harmful bacteria.

3. The use of osteopontin in regulating intestinal flora homeostasis according to claim 2, characterized in that: The probiotics include Akkermansia muciniphila.

4. The use of osteopontin in regulating intestinal flora homeostasis according to claim 2, characterized in that: The symbiotic bacteria include Bacteroldes caccae, Bacteroldes caecimuris, Bacteroldes faecium or Bacteroldes uniformis.

5. The use of osteopontin in regulating intestinal flora homeostasis according to claim 2, characterized in that: The harmful bacteria include Duncaniella dubosii or Duncaniella sp C9.

6. The use of osteopontin in regulating intestinal flora homeostasis according to claim 2, characterized in that: The harmful bacteria include Paramuribaculumintestinale.

7. The use of osteopontin in regulating intestinal flora homeostasis according to claim 1, characterized in that: It also includes a method for regulating intestinal flora colonization using osteopontin as a target molecule, comprising the following steps: upregulating the Spp1 gene to express osteopontin, thereby increasing the abundance of Akkermansia muciniphila, Bacteroldes caccae, Bacteroldescaecimuris, Bacteroldesfaecium and Bacteroldes uniformis in the intestine.

8. The use of osteopontin in regulating intestinal flora homeostasis according to claim 7, characterized in that: Upregulation of the Spp1 gene expressed osteopontin, thereby reducing the abundance of Duncaniella dubosii, Duncaniella sp C9, and Paramuriba culumintestinale in the intestine.

9. The use of osteopontin in regulating intestinal flora homeostasis according to claim 1, characterized in that: It also includes the repair of the impaired digestion and absorption, lipid metabolism and immune response functions caused by the ingestion of OPN on the colon tissue changes induced by Spp1 gene deficiency.

10. The use of osteopontin in regulating intestinal flora homeostasis according to claim 1, characterized in that: It also includes maintaining the homeostasis of Akkermansia flora by ingesting OPN or upregulating the expression of OPN, thereby regulating intestinal digestion and absorption, lipid metabolism, and immune feedback.

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