Novel Uses and Screening Methods for Small Molecules that Enhance Intestinal Fatty Acid Absorption

Abstract

This invention discloses novel uses and screening methods for small molecule substances that enhance intestinal fatty acid absorption. The method is applied to mice fed a high-fat diet via gavage. Romboutsia ilealis Probiotics ， Serum OAA levels were found to be closely related to intestinal fatty acid absorption in mice. In vitro experiments using OAA-treated intestinal epithelial cells verified that this small molecule had no cytotoxicity within the 100 μM concentration range, significantly increased the expression levels of fatty acid transporters CD36 and FATP4 in intestinal epithelial cells, and promoted the absorption of medium- and long-chain fatty acids by porcine intestinal epithelial cells. In vivo experiments in mice via gavage with OAA further validated this invention. This invention proposes for the first time the functional role of OAA in lipid metabolism; on the one hand, it may be directly applied as a feed additive in livestock production; on the other hand, this small molecule may become a novel drug target for treating obesity or metabolic-related diseases.

Description

Technical Field

[0001] This invention relates to the field of feed additives and health products research and development, specifically to new uses and screening methods for small molecule substances that enhance intestinal fatty acid absorption. Background Technology

[0002] Different animals or different growth stages have different energy requirements. Insufficient energy intake can seriously affect animal production performance and directly harm economic benefits. Fatty acids are one of the important sources of energy. Not only does the energy produced during fatty acid oxidation support animal physiological activities, growth, and body temperature regulation, but it also plays an important role in cell growth and development. Fatty acids regulate immune function, improve animal resistance to pathogens, maintain cell membrane permeability, alleviate inflammatory responses, and also help improve the taste, tenderness, and nutritional value of meat. Studies have found that weaning stress severely affects fatty acid absorption, and impaired lipid digestion and absorption are also one of the important causes of diarrhea in piglets. However, no feed additives have yet been developed that can effectively improve intestinal fatty acid absorption. Therefore, ensuring healthy lipid absorption in the animal gut is crucial for ensuring healthy livestock farming and promoting the development of animal husbandry.

[0003] 2-Oxoindole-3-acetate (OAA) is an isomer of indole-3-acetic acid. Related studies have indicated the existence of a metabolic pathway known as the "indole-3-acetic acid pathway." In this pathway, microorganisms utilize L-tryptophan as a starting material, converting it to indole-3-acetic acid through a series of enzymatic catalysis. Other studies have reported that indole-3-acetic acid is significantly reduced in mice fed a high-fat diet, and that 3-indoleacetic acid produced from tryptophan metabolism by gut microbiota activates aryl hydrocarbon receptors, lowers fasting blood glucose levels, improves abnormal glucose and insulin metabolism, helps mice resist diet-induced obesity, and alleviates inflammation. Furthermore, 3-indoleacetic acid can enhance intestinal barrier function by increasing the expression of root apical junction proteins that mediate intestinal epithelial permeability, thereby improving colitis-related pathologies. However, research on its isomer OAA in animal health is currently very rare. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides new uses and screening methods for small molecules that enhance the absorption of fatty acids in the intestine.

[0005] A screening method for small molecules that enhance intestinal fatty acid absorption.

[0006] 1) Feeding mice probiotics Romboutsia ilealis Mice were not fed probiotics as a control group;

[0007] 2) Metabolomics detection of differentially metabolites to obtain... Romboutsia ilealis The relative abundance of bacteria was negatively correlated with small molecules, while it was positively correlated with fatty acid absorption in the mouse intestine.

[0008] The small molecule is 2-Oxoindole-3-acetate (OAA).

[0009] A novel use of 2-oxo-indole-3-acetic acid: preparation of feed additives for pigs and rats; preparation of animal feed.

[0010] Another novel use of 2-oxo-indole-3-acetic acid is in the preparation of health products for pigs and rats that increase the absorption of fatty acids in the intestines.

[0011] Another novel use of 2-oxo-indole-3-acetic acid is for the preparation of drugs that increase the absorption of fatty acids in the intestines of pigs and mice. These drugs are used to increase the expression levels of fatty acid transport proteins CD36 and FATP4 in intestinal epithelial cells, thereby promoting the absorption of fatty acids by intestinal epithelial cells in mice and pigs.

[0012] Another novel use of 2-oxo-indole-3-acetic acid is as a drug target for treating obesity-related metabolic diseases, including reducing serum levels by targeting and inhibiting its synthases and activating its degradative enzymes.

[0013] A feed additive that enhances intestinal fatty acid absorption, the active ingredient of which includes OAA; or includes components that can be metabolized to produce OAA. Romboutsia ilealis And the carrier.

[0014] The carrier is at least one of maltodextrin, wheat bran, soybean meal, limestone powder, and corn flour.

[0015] A feed that enhances intestinal fatty acid absorption and promotes growth, comprising the aforementioned feed additive.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention discloses a novel function of the small molecule compound 2-Oxoindole-3-acetate (OAA), which can significantly increase the expression levels of fatty acid transport proteins CD36 and FATP4 in intestinal epithelial cells, effectively promoting the absorption of fatty acids by intestinal epithelial cells in mice and pigs. This provides a new target for effectively regulating fat deposition in the body. On the one hand, it can be directly applied to livestock production as a feed additive. On the other hand, since this small molecule drug was found in mouse serum, it may become a novel drug target for treating obesity or metabolic-related diseases. For example, it could reduce serum levels by targeting and inhibiting its synthases or activating its degradative enzymes, or alleviate obesity and related diseases by inhibiting its binding to receptor proteins. Further research is needed in this area. Attached Figure Description

[0018] Figure 1 for Romboutsia ilealis Differential metabolites in mouse serum during single-strain colonization assays and Romboutsia ilealis Correlation heatmap of relative abundance (left), serum triglyceride level and total cholesterol level (right). Red indicates positive correlation, blue indicates negative correlation. Significance analysis: * indicates p<0.05, ** indicates p<0.01.

[0019] Figure 2 The effects of different concentrations of 2-oxoindole-3-acetate (OAA) on the activity of porcine intestinal epithelial cells were investigated. * indicates p < 0.05, ** indicates p < 0.01.

[0020] Figure 3A This represents the amount of Bodipy-labeled fatty acids absorbed by intestinal epithelial cells treated with different concentrations of OAA.

[0021] Figure 3B This is a fluorescence image of intestinal epithelial cells treated with 25 μM OAA absorbing Bodipy-labeled fatty acids; significance analysis: * indicates p<0.05, ** indicates p<0.01.

[0022] Figure 4 The effect of 2-Oxoindole-3-acetate (OAA) on the expression of fatty acid transporters in porcine intestinal epithelial cells was investigated. * indicates p < 0.05, *** indicates p < 0.001.

[0023] Figure 5A This refers to the changes in the levels of triglycerides or fatty acids in the serum, intestinal tissue, and feces of mice administered the same concentration of OAA via gavage.

[0024] Figure 5BThis is an analysis of Oil Red O staining of the intestines of mice administered the same concentration of OAA by gavage;

[0025] Figure 5C This is a fluorescence image of Bodipy-labeled fatty acids absorbed by intestinal epithelial cells of mice administered the same concentration of OAA by gavage; significance analysis: * indicates p<0.05, ** indicates p<0.01. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Example 1 Correlation Analysis

[0028] Spearman correlation analysis was used to analyze differentially metabolites and... Romboutsia ilealis The correlation between serum TG and TChol was analyzed, and correlation heatmaps and correlation scatter plots were plotted using R version 4.0.5. P <0.05 indicates a significant difference. For example... Figure 1 As shown, serum OAA levels and Romboutsia ilealis Bacterial abundance was significantly negatively correlated with serum triglyceride levels and significantly positively correlated with serum triglyceride levels.

[0029] Example 2: Initial screening of the concentration of 2-oxo-indole-3-acetic acid (OAA) (Shanghai Haoyuan Pharmaceutical Co., Ltd.) in IPEC-J2 cells.

[0030] 104 IPEC-J2 cells were seeded into 96-well plates, with a cell-free control group included. When the cells reached approximately 50-60% confluence, they were treated with 0, 2, 5, 25, 50, 100, 200, 500, and 1000 μM OAA for 24 h. During treatment, the cells were cultured in DMEM / F12 medium containing 2% FBS. After treatment, cell viability was measured according to the CCK-8 assay kit instructions.

[0031] like Figure 2 As shown, OAA concentrations below 100 μM (including 100 μM) have no toxic side effects on cells and do not affect cell survival. However, concentrations above 200 μM will cause a decrease in cell survival.

[0032] Example 3: Screening of OAA concentrations affecting fatty acid uptake in IPEC-J2 cells

[0033] 1) 104 IPEC-J2 cells were seeded into 96-well black / clear culture plates, with blank wells used as cell-free controls;

[0034] 2) When the cells have grown to about 50-60%, treat them with DMEM / F12 medium containing 2% FBS and 0, 2, 5, 25, 50, or 100 μM OAA for 16 h.

[0035] 3) Then, replace the medium with FBS-free DMEM / F12 containing 0, 2, 5, 25, 50, and 100 μM OAA and starve the culture for 8 h.

[0036] 4) Discard the original culture medium, wash twice with PBS, add 50 μL of Bodipy C16 solution (add Bodipy C16 and fatty acid-free bovine serum albumin (BSA) to PBS in a molar ratio of 2:1, incubate for 10 min, and then incubate for 5 min in a constant temperature cell culture incubator;

[0037] 5) After 5 min, discard the Bodipy C16 solution and wash twice with 0.5% BSA in PBS, 2 min each time;

[0038] 6) To suppress extracellular fluorescence, add 50 μL of 0.4% trypan blue to each well. Under light-protected conditions, immediately measure intracellular fluorescence using a SpectraMax M5 microplate reader. Set the microplate reader to "fluorescence mode" with an excitation wavelength of 488 nm, an absorption wavelength of 515 nm, and a cutoff wavelength of 495 nm, and read the data.

[0039] 7) Analyze the data to determine the effect of OAA on fatty acid uptake in IPEC-J2 cells and the optimal concentration.

[0040] like Figure 3A OAA concentrations of 5-100 μM effectively promoted fatty acid uptake in the IPEC-J2 cell line, with the strongest effect at 25 μM.

[0041] Example 4: Laser Confocal Experiment

[0042] 1) Seed IPEC-J2 cells into laser confocal microplates. When the cells reached 50-60% confluence, treat the cells with DMEM / F12 medium containing 25 μmol / L OAA and 2% FBS for 16 h.

[0043] 2) Subsequently, the medium was replaced with DMEM / F12 without FBS containing 25 μmol / L OAA and starved for 8 h;

[0044] 3) Discard the original culture medium, wash twice with PBS, add 50 μL of Bodipy C16 solution (add Bodipy C16 and fatty acid-free BSA to PBS in a molar ratio of 2:1, incubate for 10 min), and incubate in a constant temperature cell culture incubator for 5 min;

[0045] 4) After 5 min, discard the Bodipy C16 solution and wash twice with 0.5% BSA in PBS, 2 min each time;

[0046] 5) After washing, take pictures under a confocal microscope and use ImageJ software to count the cell fluorescence intensity.

[0047] like Figure 3B By inducing with BodipyC16 (a fatty acid analog), IPEC-J2 cells treated with 25 μM OAA showed a significant increase in BodipyC16 uptake compared to the control group, further demonstrating the promoting effect of OAA on cellular fatty acid uptake.

[0048] Example 5: Determination of gene expression

[0049] 1) qPCR: Total RNA was extracted from cells after treatment with Trizol, and the relative expression levels of mRNA were detected by reverse transcription and quantitative real-time PCR. Primer sequences are shown in the table below:

[0050] The Primers Sequences of Genes for qPCR

[0051]

[0052] 2) The expression levels of relevant proteins were detected using Western blot. The antibody sources are shown in the table below:

[0053]

[0054] like Figure 4 As shown, the differences in transcription and translation levels of fatty acid transport-related genes in IPEC-J2 cells treated with 25 μM compared with the control group were analyzed. The results showed that OAA effectively promoted the transcription and translation of fatty acid transport proteins CD36 and FABP2.

[0055] Example 6 Triglyceride Detection

[0056] Sample collection

[0057] Mice were administered equal volumes of PBS, 5 μg / ml, and 10 μg / ml OAA via gavage for two weeks, and the following samples were collected afterward:

[0058] 1) Collect approximately 500 μL of blood from the eye veins of mice and let it stand at room temperature for 1 hour to allow the serum to separate. Transfer the serum to a new EP tube for later use.

[0059] 2) Collect fresh feces from mice, weigh them, add 500 μL of 0.57 mg / ml BSA solution, break them up thoroughly, centrifuge at 12000 rpm for 10 min, and collect the supernatant for later use.

[0060] 3) Collect 2-3 mm of the jejunum from mice, rinse with PBS, add 500 μL of RIPA lysis buffer (containing PMSF), lyse thoroughly, centrifuge at 12000 rpm for 10 min, collect the supernatant for later use, and measure the protein concentration in the supernatant using the BCA method.

[0061] The above samples were tested using a triglyceride content detection kit (catalog number BC0625, Solarbio, Beijing). For detailed operation instructions, please refer to the supplier's operation guide.

[0062] like Figure 5A As shown, mice induced by OAA (especially at a concentration of 5 μg / ml) for 2 weeks had significantly higher levels of triglycerides (TG) in their serum and intestines compared to the control group. Differences in fatty acid absorption in mice were detected by gavage with Bodipy C16. The results showed that after OAA induction, the fluorescence intensity of Bodipy C16 in mouse feces was significantly reduced, consistent with the conclusion that OAA promotes fatty acid absorption; however, further detection of Bodipy C16 levels in the intestines is needed.

[0063] Example 7: Tissue Sectioning and Staining

[0064] After fixing the proximal jejunum and epididymal fat in 4% paraformaldehyde for more than 24 hours, the tissue blocks were removed and trimmed. Subsequently, the tissues underwent dehydration and clearing, paraffin embedding, sectioning and mounting, dewaxing, and HE staining to obtain sections. The specific operational steps are as follows:

[0065] 1) Dehydration and clearing: The tissue sample was dehydrated in a gradient of 70%, 80%, and 90% ethanol for 30 min each time, and then dehydrated twice with 95% and 100% ethanol for 20 min each time. After dehydration, the tissue sample was cleared with an equal volume of ethanol and xylene mixture for 15 min each time, and then cleared twice with pure xylene for 15 min each time until it became clear.

[0066] 2) Paraffin embedding: The transparent tissue block is placed in an equal volume of xylene and paraffin mixture for 15 min, and then immersed in pure paraffin I and paraffin II for 60 min each; the paraffin-embedded tissue sample is placed in the center of a container containing paraffin solution, and after cooling, a paraffin block is obtained.

[0067] 3) Sectioning and mounting: Fix the wax block on the microtome, cut it into 5-7 µm thin sections, flatten them and mount them on glass slides, and dry them in an oven at 45℃;

[0068] 4) Dewaxing: Dewaxing was performed using xylene, pure ethanol, and a series of ethanols until the product was washed with distilled water.

[0069] 5) Oil Red O staining: Use Oil Red O to stain the sections according to the supplier's (Beyotime) instructions.

[0070] 6) Observation: After the slides are prepared, use a microscope to obtain images of the slides for observation.

[0071] like Figure 5B As shown, the intestinal lipid levels in mice induced by OAA were significantly higher than those in the control group. Figure 5C The results showed that after gavage administration of Bodipy C16, its accumulation in the intestinal tissue of OAA-induced mice was higher than that in the control group. All of these findings indicate that OAA-induced mice exhibit significantly enhanced fatty acid absorption.

[0072] The technical features of the embodiments described above can be further combined. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The embodiments described above only illustrate several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, all of which fall within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalent technical solutions.

Claims

1. Use of 2-oxo-indole-3-acetic acid, characterized in that, Prepare feed additives for pigs and rats to increase the absorption of fatty acids in the intestines of pigs and rats.

2. The use according to claim 1, characterized in that, The active ingredient also includes a carrier.

3. The use according to claim 2, characterized in that, The carrier is at least one of maltodextrin, wheat bran, soybean meal, limestone powder, and corn flour.

4. Uses of 2-oxo-indole-3-acetic acid, characterized in that, Used to prepare health products for pigs and rats to increase the absorption of fatty acids in their intestines.

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