Application of osteopontin as AMPK activator

Abstract

The invention relates to the field of biological medicine, in particular to application of osteopontin as an AMPK activator, experiments prove that intake of osteopontin cannot inhibit AMPK activity, and AMPK activity can be improved by up-regulating expression of AMPK gamma subunit coding gene Prkag3; in addition, by activating the AMPK gamma subunit coding gene Prkag3, the stability of the tight junction protein can be enhanced by phosphorylating a downstream target Sgk1, so that the effect of repairing the intestinal mucosal barrier function is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of osteopontin as an AMPK activator. Background Art

[0002] AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase), an AMP-dependent protein kinase, is a key regulator of bioenergy metabolism and a central component of research into diabetes and other metabolic diseases. It is expressed in various metabolically relevant organs and is activated by various stimuli, including cellular stress, exercise, and numerous hormones and substances that influence cellular metabolism. Genetic and pharmacological studies have demonstrated that AMPK is essential for maintaining glucose homeostasis.

[0003] AMPK proteins from different species exist in the form of a heterotrimeric complex consisting of an α-catalytic subunit, a β-regulatory subunit and a γ-regulatory subunit.

[0004] Prior art generally believes that the expression of osteopontin (OPN) will lead to the inhibition of AMPK activity. For example, the article "Osteopontin is Critical for Hyperactive mTOR-Induced Tumorigenesis in Oral Squamous Cell Carcinoma (J Cancer. 2017 May 12; 8(8):1362-1370. doi:10.7150 / jca.18031.eCollection 2017.)" believes that the activity of mTOR complex 1 (mTORC1) is positively correlated with the expression of OPN. It is well known that after mTORC1 is activated, it will phosphorylate TSC2, thereby inhibiting TSC2's inhibitory effect on RHEB, thereby reducing RHEB activation and ultimately inhibiting AMPK activity.

[0005] However, the present invention confirms that exogenous OPN intervention can upregulate the expression of AMPKγ subunit encoding gene Prkag3, thereby overcoming the technical bias of the prior art. Summary of the Invention

[0006] In view of the deficiencies of the prior art, one of the objectives of the present invention is to provide the use of osteopontin as an AMPK activator.

[0007] One of the objectives of the present invention is achieved through the following technical solutions:

[0008] Application of osteopontin as an AMPK activator. The present invention demonstrates through OPN intervention the regulation of key molecules in the metabolism-immunity axis that a certain amount of exogenous osteopontin intake significantly increases the expression of the AMPKγ subunit encoding gene Prkag3 in animal cells compared to the wild type.

[0009] Specifically, the osteopontin is bovine milk-derived OPN. Bovine milk is the most widely used milk source, and its safety has been widely proven. Using bovine milk-derived OPN ensures the safety of the present invention as a food and drug. Osteopontin in milk is also known as milk osteopontin or lactopontin.

[0010] Specifically, the dosage of osteopontin for mice is 33-130 mg / kg / day. Converted to this, the dosage for adults is 2.68-10.57 mg / kg / day, and the dosage for infants is 4.875-19.5 mg / kg / day. The OPN content in milk powder is 33-130 mg / L. Specific conversion method: Based on the body surface area conversion, the Km value of a 60kg adult is compared with the Km value of a mouse, that is, 37 divided by 3 is approximately equal to 12.3. The intervention dose of mice is about 130mg / kg / day, which is converted to about 10.57mg / kg / day for adults (divided by 12.3), that is, a 60kg body weight (adult) eats 634.2mg per day; for infants and young children, it is calculated based on the weight and surface area of ​​infants and young children aged 0-1 in my country. Similarly, the Km value of infants and young children is 20, that is, 20 divided by 3 is approximately 6.67, that is, the mouse dose is divided by 6.67. Therefore, the intake of infants and young children is 19.5mg / kg / d. Based on the milk drinking volume of 150mL / kg, the OPN content of milk powder is calculated to be 130mg / L

[0011] Specifically, the dosage of osteopontin for mice is 65 mg / kg / day. By conversion, the dosage for infants and young children is 9.75 mg / kg / day, and the dosage for adults is 5.28 mg / kg / day.

[0012] A second object of the present invention is to provide a use of an AMPK activator in the preparation of intestinal homeostasis foods or medicines.

[0013] The second object of the present invention is achieved through the following technical solutions:

[0014] Specifically, the above-mentioned AMPK activator is used to phosphorylate AMPK to inhibit mTORC1 activity, downregulate glycolysis-related transcription factors, thereby inhibiting Th17 differentiation, and ultimately achieving the regulation of Th17 / Treg balance.

[0015] Specifically, the above-mentioned AMPK activator is used to activate forkhead box protein P3 (FoxP3) to maintain its immunosuppressive function; and phosphorylation activation of Akt Ser473 site promotes intestinal epithelial cell migration and crypt regeneration.

[0016] Specifically, the above-mentioned AMPK activators are used to upregulate β-defensins and antimicrobial peptides to inhibit the colonization of pathogenic bacteria, enhance the thickness of the intestinal mucus layer and MUC2 synthesis, and inhibit host glycolysis.

[0017] A third object of the present invention is to provide a use of an AMPK activator in the preparation of food or medicine that promotes the development or repair of intestinal mucosa.

[0018] The second object of the present invention is achieved through the following technical solutions:

[0019] The above-mentioned AMPK activators were used to simultaneously upregulate Prkag3 and Sgk1 expression, promoting connexin stability.

[0020] Specifically, the dosage of osteopontin in the AMPK activator for infants and young children is 9.75 mg / kg / day, and the dosage for adults is 5.28 mg / kg / day.

[0021] The beneficial effects of the present invention are as follows: the present invention has experimentally confirmed that the intake of osteopontin not only does not inhibit AMPK activity, but can also enhance AMPK activity by upregulating the expression of the AMPKγ subunit encoding gene Prkag3; in addition, activation of the AMPKγ subunit encoding gene Prkag3 may enhance the stability of tight junction proteins by phosphorylating the downstream target Sgk1, thereby playing a role in repairing the intestinal mucosal barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Design diagram for the exogenous OPN intervention experiment;

[0023] Figure 2 This is a diagram of the regulation of key molecules in the metabolism-immune axis by OPN intervention. DETAILED DESCRIPTION

[0024] The following is further explained in conjunction with specific implementation methods:

[0025] I. Preparation of CKO mice:

[0026] This study used specific pathogen-free (SPF) C57BL / 6 mice with an intestinal epithelium-specific knockout of the Spp1 gene (Spp1[flox / flox, Vil1-cre]) as an animal model. First, using CRISPR-Cas9 technology, Spp1[flox / +] mice were generated, with lox P sites inserted on both sides of the Spp1 gene. Homozygous Spp1[flox / flox] male mice were obtained by self-crossing. Furthermore, Spp1[flox / +] mice were crossed with intestinal villus tool mice, Vil1-cre+ / + mice, to generate Spp1[flox / +, Vil1-cre] female mice. Homozygous Spp1[flox / flox] male mice were crossed with Spp1[flox / +, Vil1-cre] female mice to generate Spp1[flox / flox, Vil1-cre] mice, representing the intestinal epithelium-specific OPN knockout (CKO) mouse model.

[0027] Second, grouping

[0028] like Figure 1 As shown, exogenous OPN intervention began in mice 7 days after birth and lasted until 21 days (3 weeks of age), covering the critical window period for the development of the intestinal immune system (equivalent to 6 months after birth in human infants). Intervention at this stage can simulate the early supplementation strategy of active ingredients in infant formula feeding, and provide time-dependent evidence for the analysis of the mechanism of action of OPN on intestinal barrier maturation. Three dose gradients of low, medium and high were set: 33, 65, and 130 mg / kg / day, respectively named OPN-L group, OPN-M group and OPN-H group, and the control group was gavaged with normal saline and named CKO group. In addition, wild-type (WT) mice of the same strain, age and sex were used as the control group.

[0029] 3. Colon Transcriptome Analysis

[0030] Mice in each group were sacrificed and colon tissues were extracted and immediately snap-frozen in liquid nitrogen for transcriptome analysis.

[0031] Total RNA was extracted from the samples and sequenced using the Illumina Novasek Xplus sequencing platform in PE150 mode after quality control. Gene annotation was performed using the Mus musculus (GRCm39) reference genome. HTSeq (v0.9.1) was used to calculate the read count value of each gene as the raw gene expression level, and FPKM (Fragments Per Kilobases per Million fragments) was used for normalization. Differential gene expression analysis was performed using DESeq (v1.38.3) software, with a threshold of |log2FoldChange|>1 and P < 0.05 for differential gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Cluster Profiler (v4.6.0) software, and significant enriched pathways with P < 0.05 were screened.

[0032] like Figure 2 As shown, the expression level of Prkag3, the gene encoding the AMPKγ subunit, was significantly upregulated, with the WT group at -1.5, while the OPN-L, OPN-M, and OPN-H groups were 0, 1.5, and 1, respectively. In addition, the expression level of Sgk1 was also significantly upregulated, with the WT group at 0.5, while the OPN-L, OPN-M, and OPN-H groups were -1, 1, and 1.5, respectively.

[0033] Upregulated Prkag3 expression inevitably leads to an increase in AMPKγ subunits, which in turn promotes AMPK activity. This discovery of the present invention breaks the prior art's preconception that OPN necessarily inhibits AMPK activity and provides a new approach for preventing or treating AMPK insufficiency.

[0034] Notably, AMPK's metabolic regulatory function is deeply embedded in immune homeostasis. It is well known that the polarization state of macrophages (pro-inflammatory M1 versus anti-inflammatory M2) directly determines the direction of inflammatory progression. M1 relies on glycolysis for rapid energy supply and releases pro-inflammatory cytokines such as TNF-α and IL-6, while M2 maintains anti-inflammatory function through fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). AMPK drives the transition of macrophages to the M2 phenotype through dual mechanisms: phosphorylation of HIF-1α inhibits the expression of key glycolytic enzymes, blocking M1 energy supply; activation of PGC-1α promotes mitochondrial biogenesis, and upregulates CPT1A to enhance fatty acid β-oxidation. Within T cell subsets, AMPK regulates the Th17 / Treg balance through metabolic pathways. Th17 cells rely on glycolysis and glutaminolysis to maintain function, while Treg cells obtain energy through FAO and OXPHOS. AMPK phosphorylation inhibits mTORC1 activity and downregulates glycolysis-related transcription factors, thereby suppressing Th17 differentiation.

[0035] At the same time, AMPK provides metabolic substrates for Tregs by promoting fatty acid uptake mediated by carnitine palmitoyltransferase 1A (CPT1A) and FAO, and activates forkhead box protein P3 (FoxP3) to maintain its immunosuppressive function. Furthermore, AMPK phosphorylation at AktSer473 promotes intestinal epithelial cell migration and crypt regeneration, suggesting that AMPK has metabolic microenvironment-dependent regulatory characteristics. When energy stress is relieved, it provides bioenergetic support for intestinal mucosal repair by enhancing fatty acid oxidation.

[0036] Furthermore, AMPK, as a core hub of energy metabolism, plays a key regulatory role by sensing metabolic signals from the microbiome and reshaping the immune microenvironment. Microbial metabolites, including SCFAs, tryptophan derivatives, and secondary bile acids, constitute important upstream signals for AMPK activation. SCFAs, such as butyrate, activate AMPK, inhibiting histone deacetylases to promote Treg differentiation and suppress Th17 proliferation. This mechanism involves FoxP3 deacetylation to enhance transcriptional activity and histone modification in the IL-17A promoter region. Tryptophan metabolites inhibit mTORC1 signaling through the aryl hydrocarbon receptor-AMPK axis, blocking Th17 overactivation. Secondary bile acids, in turn, promote intestinal mucus secretion by activating AMPK. AMPK, in turn, upregulates bile acid metabolizing enzymes, forming a positive feedback loop between microbiome and host metabolism. Notably, the interaction between AMPK and the microbiome is bidirectional. AMPK inhibits pathogenic bacterial colonization by upregulating β-defensins and antimicrobial peptides, and provides a niche for probiotics by enhancing intestinal mucus thickness and MUC2 synthesis. Metabolomics studies have shown that AMPK activation reduces the availability of glucose in the intestinal lumen by inhibiting host glycolysis, forcing the microbiota to switch to fiber fermentation metabolism to increase SCFAs production, forming the metabolic basis for "host-microbiota" collaborative repair.

[0037] On the other hand, exogenous OPN significantly upregulated the expression of Sgk1. It is well known that Prkag3 can enhance the stability of tight junction proteins by phosphorylating its downstream target Sgk1. This means that exogenous OPN intervention not only upregulated the expression of Prkag3 and Sgk1, but also promoted the synergistic effect of these two factors on the stability of tight junction proteins, thereby repairing the intestinal mucosal barrier function.

[0038] The above embodiments and descriptions are only for explaining 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, which shall fall within the scope of the invention to be protected.

Claims

1. Use of osteopontin as an AMPK activator, characterized in that: Osteopontin upregulates the expression of Prkag3, the gene encoding the AMPKγ subunit.

2. The use according to claim 1, characterized in that The osteopontin is bovine milk-derived OPN.

3. The use according to claim 2, characterized in that The dosage of osteopontin for adults is 2.68-10.57 mg / kg / day, and the dosage for infants is 4.875-19.5 mg / kg / day.

4. The use according to claim 3, characterized in that The dosage of osteopontin for adults is 5.28 mg / kg / day, while the dosage for infants is 9.75 mg / kg / day.

5. Use of the AMPK activator according to claim 1 in the preparation of immune homeostatic food or medicine, characterized in that: AMPK activators are used to inhibit mTORC1 activity, downregulate glycolysis-related transcription factors, and thus inhibit Th17 differentiation.

6. Use of the AMPK activator according to claim 5 in the preparation of immune homeostatic food or medicine, characterized in that: AMPK activators are used to activate forkhead box protein P3 to maintain its immunosuppressive function; phosphorylation activation of Akt Ser473 site promotes intestinal epithelial cell migration and crypt regeneration.

7. Use of the AMPK activator according to claim 5 in the preparation of immune homeostatic food or medicine, characterized in that: The above-mentioned AMPK activators are used to upregulate β-defensins and antimicrobial peptides to inhibit pathogenic bacterial colonization, enhance intestinal mucus layer thickness and MUC2 synthesis, and inhibit host glycolysis.

8. Use of the AMPK activator according to claim 1 in the preparation of a food or medicine for promoting intestinal mucosal development or repair, characterized in that: AMPK activators were used to simultaneously upregulate Prkag3 and Sgk1 expression, promoting connexin stability.

9. The use according to claim 8, characterized in that: The dosage of osteopontin in the AMPK activator for adults is 5.28 mg / kg / day, while the dosage for infants is 9.75 mg / kg / day.

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