Application of 6-ketolithocholic acid in the preparation of drugs to reduce fat deposition

Abstract

This invention relates to the field of biomedical technology, specifically to the application of 6-ketolithocholic acid in the preparation of drugs that reduce fat deposition. The 6-ketolithocholic acid in this invention inhibits the accumulation of lipid droplets in adipocytes and suppresses gene expression in the PPARγ pathway involved in lipid synthesis, thereby achieving the function of reducing fat deposition. The structural formula of the 6-ketolithocholic acid is: [Insert structural formula here].

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of 6-ketolithocholic acid in the preparation of drugs that reduce fat deposition. Background Technology

[0002] Lipid deposition refers to the accumulation of lipids such as triglycerides in adipose tissue or non-adipocytes within an organism. Under physiological conditions, appropriate lipid deposition plays an important role in maintaining energy balance, cell membrane construction, and endocrine regulation. However, with changes in modern lifestyles, excessive or ectopic lipid deposition has become a global health concern. In the field of human medicine, excessive accumulation of fat in tissues such as the abdomen, liver, and muscles is a significant contributing factor to obesity, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes, and cardiovascular disease. Particularly in hepatocytes, excessive lipid accumulation not only leads to hepatocyte steatosis but may also further induce oxidative stress and inflammatory responses, progressing to NASH, cirrhosis, and even liver cancer.

[0003] In livestock and aquaculture, fat deposition is also a key factor affecting product quality. Excessive abdominal and intramuscular fat not only reduces feed conversion ratio and increases farming costs, but also seriously affects the taste of meat and consumer acceptance. Therefore, developing strategies to safely and effectively regulate fat metabolism and inhibit excessive fat deposition is of significant practical importance and application value, both for maintaining human health and improving animal quality. Summary of the Invention

[0004] To develop a strategy for inhibiting fat deposition, this invention provides the application of 6-ketolithocholic acid in the preparation of drugs that reduce fat deposition. The 6-ketolithocholic acid in this invention inhibits lipid synthesis in cells, reduces triglyceride levels in cells, and inhibits genes involved in the PPARγ pathway, which is involved in fat synthesis, thereby achieving the function of reducing fat deposition.

[0005] This invention provides the application of 6-ketolithocholic acid in the preparation of drugs for reducing fat deposition, wherein the structural formula of 6-ketolithocholic acid is: .

[0006] The 6-ketolithocholic acid in this invention is used to inhibit lipid synthesis in cells, reduce triglyceride content in cells, and inhibit genes involved in the PPARγ pathway of lipid synthesis, thereby achieving the function of reducing fat deposition.

[0007] Furthermore, the 6-ketolithocholic acid is used to inhibit the accumulation of lipid droplets in adipocytes.

[0008] Furthermore, the 6-ketolithocholic acid is used to reduce triglyceride levels in cells.

[0009] Furthermore, the 6-ketolithocholic acid is used to specifically activate genes in the integrated stress response pathway. ATF4 , CHOP and CHAC1 It inhibits gene expression in the PPARγ pathway, which is involved in lipid synthesis.

[0010] Furthermore, the drug dosage form is a solution.

[0011] Furthermore, the effective concentration of 6-ketolithocholic acid in the drug is 25 μM to 100 μM.

[0012] Furthermore, the preparation of the 6-ketolithocholic acid includes the following steps: strain Anaerobutyricumsoehngenii LT-7 was inoculated into fermentation medium, and HDCA was added to the fermentation medium at a final concentration of 100 μg / ml to 125 μg / ml. Anaerobic fermentation was carried out at 37°C for 24 h to 30 h. The fermentation supernatant was collected, and samples containing 6-ketolithocholic acid were obtained by centrifugation and filtration. Each liter of the fermentation medium contains: 5.60–5.66 g casein peptone, 0.8–1.0 g soybean peptone, 3.0–5.0 g yeast extract, 5.0 g mixed peptone, 5.73–5.83 g glucose, 0.73–0.83 g K₂HPO₄, 1.60–1.66 g NaCl, 2.0–3.0 g hydroxymethylaminomethane, 0.001–0.002 g resazurin, 0.01–0.02 g heme, and 0.3–0.4 g cysteine ​​hydrochloride, with the remainder being water; The strain Anaerobutyricumsoehngenii The accession number for LT-7 is DSM 17630.

[0013] Furthermore, each liter of the fermentation medium contains: 5.66 g casein peptone, 1.0 g soybean peptone, 5.0 g yeast extract, 5.0 g mixed peptone, 5.83 g glucose, 0.83 g K2HPO4, 1.66 g NaCl, 3.0 g hydroxymethylaminomethane, 0.001 g resazurin, 0.01 g heme and 0.4 g cysteine ​​hydrochloride.

[0014] Furthermore, the volume ratio of gases H2, CO2, and N2 in the anaerobic fermentation environment is 5:10:85.

[0015] This invention also provides a method for preparing 6-ketolithocholic acid, comprising the following steps: strain AnaerobutyricumsoehngeniiLT-7 was inoculated into fermentation medium, and HDCA was added to the fermentation medium at a final concentration of 100 μg / ml to 125 μg / ml. Anaerobic fermentation was carried out at 37°C for 24 h to 30 h. The fermentation supernatant was collected, and samples containing 6-ketolithocholic acid were obtained by centrifugation and filtration. Each liter of the fermentation medium contains: 5.60–5.66 g casein peptone, 0.8–1.0 g soybean peptone, 3.0–5.0 g yeast extract, 5.0 g mixed peptone, 5.73–5.83 g glucose, 0.73–0.83 g K₂HPO₄, 1.60–1.66 g NaCl, 2.0–3.0 g hydroxymethylaminomethane, 0.001–0.002 g resazurin, 0.01–0.02 g heme, and 0.3–0.4 g cysteine ​​hydrochloride, with the remainder being water; The strain Anaerobutyricumsoehngenii The accession number for LT-7 is DSM 17630.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The 6-ketolithocholic acid in this invention inhibits genes involved in the PPARγ pathway, which is involved in lipid synthesis, thereby suppressing lipid synthesis in cells and inhibiting the accumulation of lipid droplets in adipocytes, thus reducing fat deposition. Therefore, 6-ketolithocholic acid not only holds promise for preventing diseases caused by fat deposition but also for improving the quality of meat in animal husbandry.

[0017] This invention employs natural probiotic fermentation technology to prepare key active substances. This method is gentle, maximizing the retention and enrichment of active ingredients derived from natural products. Simultaneously, the fermentation process itself may generate new beneficial metabolites, ensuring the product's natural properties and biocompatibility. Furthermore, the active ingredient screening in this invention is not solely based on in vitro experiments but is achieved through a systematic in vivo discovery strategy. Therefore, the subsequent commercialization of this invention will be safer and faster. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, 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 the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 HDCA-AS bacteria in the in vivo test of Example 1 of this invention ( Anaerobutyricumsoehngenii A schematic diagram illustrating the lipid-lowering effects of the LT-7 and 6-ketolithocholic acid (6-ketoLCA) pathways; In the figure, A represents a comparison of tail fat weight (TF) between the HC group and the HDCA group; B is a comparison of the tail fat percentage (TF%) between the HC group and the HDCA group; C represents a comparison of 6-ketoLCA content in the ileum of the HC group and the HDCA group; D represents a comparison of 6-ketoLCA content in the colon of the HC group and the HDCA group; E represents a comparison of 6-ketoLCA content in the livers of the HC group and the HDCA group; F represents a comparison of 6-ketoLCA levels in the blood of the HC group and the HDCA group; G represents the results of LEfse analysis of colonic microbiota; H represents the mediating analysis of the microbiome-metabolite-phenotype relationship; I represents the qPCR quantitative analysis of AS bacteria in the HC and HDCA groups; K represents the result of in vitro fermentation of HDCA by AS bacteria.

[0020] Figure 2 The effects of different concentrations of HDCA and 6-ketoLCA on the survival rate of 3t3-L1 cells; In the figure, A represents the effect of HDCA and 6-ketoLCA on triglyceride (TG) content in 3t3-L1 cells; B represents the effect of different concentrations of HDCA on the survival rate of 3t3-L1 cells; C represents the effect of different concentrations of 6-ketoLCA on the survival rate of 3t3-L1 cells.

[0021] Figure 3 Image showing the lipid-lowering effect of 6-ketoLCA; In the figure, A shows the Oil Red O staining results of 3t3-L1 cells after treatment with HDCA and its microbial metabolites; B represents the effect of HDCA and its microbial metabolites on the lipid surface area of ​​3t3-L1 cells. C represents the molecular mechanism by which HDCA exerts its lipid-lowering function; D represents the molecular mechanism by which 6-ketoLCA exerts its lipid-lowering function. Detailed Implementation

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0023] Example 1: Correlation study between 6-ketoLCA content and tail fat ratio.

[0024] I. Experimental Methods 1. Laboratory animals and grouping Thirty male Tan sheep, approximately 6 months old, with an average weight of 25.0 kg (±1.30 SD).

[0025] Thirty male Tan sheep were randomly assigned to six blocks, with five sheep in each block. Then, the six blocks were randomly divided into two groups on average. Group I served as the control group: fed a high-concentrate diet with a concentrate-to-roughage ratio of 8:2 and a dry matter (DM) basis.

[0026] Group II was the treatment group: In addition to the high-energy diet of the control group, 0.1% of rumen-protected bile acids were added. These rumen-protected bile acids were prepared by mixing a rumen-protecting substance and bile acids (HDCA). The mass ratio of the rumen-protecting substance (composed of palmitic acid, stearic acid, and oleic acid in a mass ratio of 60:30:5) to the bile acids (HDCA) was 6:4. (Palmitic acid CAS: 57-10-3, stearic acid CAS: 57-11-4, oleic acid CAS: 112-80-1, bile acid CAS: 83-49-8). The rumen-protected bile acids (rumen-protected HDCA) were directly mixed into the feed for feeding.

[0027] The experiment lasted 84 days, including a 14-day pre-feeding period. The lambs were fed twice daily, at 9:00 AM and 5:00 PM. Throughout the experiment, all lambs had free access to feed and water. The weight of each lamb was recorded every 35 days. At the end of the feeding experiment (70 days), six lambs were randomly selected from each group (two from each block) for blood collection, slaughter, and sampling. Tail fat weight was determined using the slaughter and fractionation method; the specific procedure was as follows: The lambs were slaughtered, bled, decapitated, and hooves removed. The tail fat and carcass were separated and weighed separately. The tail fat weight was divided by the carcass weight to obtain the tail fat ratio. Serum: Venous blood was collected and incubated in coagulation tubes at room temperature for 1 hour, centrifuged at 3000 rpm for 15 minutes at 4 °C, aliquoted, and stored at −80 °C. Liver molecular samples: The middle portion of the left lobe was taken, divided into three portions, and placed in 1.5 ml cryovials, stored at −80 °C. Intestinal contents: The ileum and proximal colon were divided into three portions and placed in 1.5 ml cryovials and stored at −80 °C.

[0028] Table 1. Composition of High-Concentrate Diets *The premix contains 200,000 IU of Vitamin A, 16,000 IU of Vitamin D3, 2,500 IU of Vitamin E, 150 g NaCl, 20 g Ca, 20 g P, 1,750 mg Zn, 15 mg Se, 50 mg I, 2,000 mg Fe, 20 mg Co, 1,500 mg Mn, and 600 mg Cu per kilogram of dry matter. See: ZHANG B, SUN Z, YU Z, LI H, LUOH, WANG B. Transcriptome and targeted metabolome analysis provide insights into bile acids' new roles and mechanisms on fat deposition and meat quality in lamb [J]. Food Research International, 2022, 162: 111941.

[0029] The amount of bile acids (HDCA) fed is calculated based on the previous day's feed intake for each pen. It is spread on the surface of the feed every morning and fed to ensure that the feed is consumed.

[0030] 2. Targeted metabolism (bile acids) was detected in tail fat, tail fat weight, ileal contents, colonic contents, liver, and blood of different experimental groups. The content of porcine deoxycholic acid in feces and serum was detected using a multiple reaction monitoring (MRM) targeted assay. The specific implementation method is as follows: First, 25 mg of sample was weighed and 1000 μL of extraction buffer (methanol:acetonitrile:water = 2:2:1, containing isotope internal standard) was added. The mixture was vortexed for 30 s, followed by the addition of steel beads. The mixture was then ground at 35 Hz for 4 min, sonicated for 5 min (ice-water bath), and repeated 3 times. The mixture was then incubated at -40℃ for 1 h, and centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was then transferred to an LC vial for UHPLC-MS / MS analysis.

[0031] Preparation of standards: Accurately weigh the appropriate amount of standard into volumetric flasks and prepare 1 mg / mL standard stock solutions. Take the appropriate amount of standard stock solution into volumetric flasks and prepare mixed standard solutions. Dilute this standard solution sequentially to obtain a series of calibration solutions (containing an isotope-labeled internal standard mixture with the same concentration as in the samples).

[0032] Mobile phase conditions: The target compounds were separated chromatographically using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph with a Waters ACQUITY UPLC BEH C18 (150 * 2.1 mm, 1.7 μm, Waters) column. Phase A of the liquid chromatography was 5 mmol / L ammonium acetate aqueous solution, and phase B was acetonitrile. The column oven temperature was 45 ℃, the sample tray was set to 4 ℃, and the injection volume was 2 μL.

[0033] Mass spectrometry conditions: An Orbitrap Exploris 120 high-resolution mass spectrometer was used for mass spectrometry analysis in Parallel Reaction Monitoring (PRM) mode. Ion source parameters were as follows: Spray voltage = +3500 / -3200 V, Sheath gas (N2) flow rate = 40, Aux gas (N2) flow rate = 15, Sweep gas (N2) flow rate = 0, Aux gas (N2) temperature = 350 ℃, Capillary temperature = 320 ℃.

[0034] Before performing UHPLC-MS / MS analysis, standard solutions of the target compounds were introduced into the mass spectrometer. The PRM parameters for each target compound were optimized; since most bile acid compounds lacked sufficiently strong daughter ions for quantification in their daughter ion spectra, the precursor ion under high-resolution conditions was selected for quantification. The calibration solutions were then analyzed by UPLC-PRM-MS / MS. y represents the peak area ratio of the target compound to the internal standard, and x represents the concentration of the target compound (nmol / L). Least squares regression analysis was performed, with the best accuracy and correlation coefficient (R²) when the weight was set to 1 / x. If the recovery rate of a certain calibration concentration exceeded the range of 80%–120%, that calibration concentration was excluded.

[0035] Quantitative parameters of the method: The calibration solution was repeatedly diluted 2-fold before analysis by UHPLC-PRM-MS. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using the signal-to-noise ratio (SNR). The limit of detection (LLOD) was defined as the compound concentration at an SNR of 3, and the limit of quantitation (LLOQ) was defined as the compound concentration at an SNR of 10 (US FDA guideline for bioanalytical method validation). The precision of the method was assessed by the standard relative deviation (RSD) of repeated injections of QC samples. The accuracy was assessed by the recovery of the spiked samples; the percentage of the measured concentration to the spiked concentration was the recovery. The LODs ranged from 0.12 to 62.50 nmol / L, and the LLOQs ranged from 0.24 to 125.00 nmol / L, with regression coefficients all above 0.9840. The average recovery of QC samples ranged from 80.8% to 101.1%, with standard relative deviations (RSDs) all less than 14.5%.

[0036] The final concentration (cF, nmol / L) of the sample is the concentration directly measured by the instrument. CThe calculated concentration (nmol / L) is multiplied by the dilution factor (Dil), in nmol / L. The target metabolite concentration (cM) in the sample is equal to the final measured concentration (cF) multiplied by the final volume (VF) (μL), divided by the sample weight (m) (mass, mg), or for liquid samples, divided by the sample volume (VS) (μL). 0 indicates that the target metabolite was not detected in the sample. The calculation formula for solid samples is:

[0037] ; Where cM is the target metabolite concentration, nmol / kg; cF is the final measured concentration of the sample, nmol / L; VF is the final sample volume, μL; and m is the sample weight, mg.

[0038] 16S rDNA V3-V4 region high-throughput sequencing: Primer sequences were: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT. The purified amplicon was ligated into sequencing adapters to construct a sequencing library, which was then sequenced using Illumina. DADA2 was used to filter and correct the reads, outputting non-redundant reads and their corresponding abundance information. The reads were then assembled into tags, and chimeric tags were removed to obtain the tag sequences and abundance information for subsequent analysis, namely, ASV sequences and ASV abundance information.

[0039] The results are as follows Figure 1 As shown, significantly increased levels of 6-ketoLCA were found in all four sites. 16S rRNA sequencing of gut microbiota revealed that... Eubacteriumhalliigroup The significantly increased bacterial content revealed that this bacterium is a mediator of HDCA's lipid-lowering function through mediation analysis. Subsequent qPCR quantification further confirmed this. Eubacteriumhalliigroup AS strains in the genus ( Anaerobutyricumsoehngenii The content of LT-7 (accession number DSM17630) increased significantly.

[0040] Example 2: Preparation of 6-ketolithocholic acid (6-ketoLCA).

[0041] Commercial strains AnaerobutyricumsoehngeniiLT-7 (accession number DSM 17630, Gray Algae Biotechnology, catalog number HZB53843) underwent in vitro fermentation experiments in medium 1669 (containing 5.66 g / L casein peptone, 1.0 g / L soybean peptone, 5.0 g / L yeast extract, 5.0 g / L mixed peptone, 5.83 g / L glucose, 0.83 g / L K2HPO4, 1.66 g / L NaCl, 3.0 g / L hydroxymethylaminomethane, 0.001 g / L resazurin, 0.01 g / L heme, and 0.4 g / L cysteine ​​hydrochloride). First, activation and enrichment were performed under strictly anaerobic conditions (H2 / CO2 / N2 = 5:10:85, v / v / v), pH 7.5 ± 0.2, and 37°C. McFarland turbidimetric assay was used to determine the bacterial concentration, and an inoculum of 10^8 CFU / mL was prepared. HDCA was added to a fresh 1669 medium at a final concentration of 0.0125% (w / v) (125 μg / mL). The inoculum was inoculated at 3% (v / v). A medium without HDCA was used as a control. After 24 hours of incubation, the fermentation supernatant was collected, centrifuged at 5000 rpm for 10 min at 4°C, and then filtered through a 0.22 μm aqueous filter to obtain the sample. The sample was stored at -80°C for subsequent targeted metabolomics analysis to identify and quantify the HDCA transformation products. The final results showed that the concentration of 6-ketoLCA increased to 945.3 nmol / L (0.3692 μg / mL) compared to the control group.

[0042] Example 3: Application of 6-ketolithocholic acid (6-ketoLCA) in reducing fat deposition.

[0043] 3T3-L1 preadipocytes at 2×10 5 Cells were seeded at a density of cells / cm² and cultured at 37°C with 5% CO₂ until approximately 90% confluence (medium purchased from Keycell Biotechnology, QS-M018A). Adipogenic differentiation was then initiated using a three-step induction method (reagents purchased from the QS-S506 series): cells were first cultured at 37°C for 3 days in induction medium A (QS-S506-A), then incubated at 37°C for 1 day in induction medium B (QS-S506-B), and finally maintained at 37°C for 10 days in induction medium C (QS-S506-C) until mature lipid droplets were formed visible to the naked eye.

[0044] After successful induction, cells were treated in the following groups: Group A: blank control; Group B: blank control + DMSO solvent; Group C: 25 μM HDCA (Sigma, catalog number H3878, 98% purity); Group D: 50 μM HDCA; Group E: 100 μM HDCA; Group F: 200 μM HDCA; Group G: 25 μM 6-ketoLCA (MCE, catalog number 35373, 98% purity); Group H: 50 μM 6-ketoLCA; Group I: 100 μM 6-ketoLCA; Group J: 200 μM 6-ketoLCA (MCE, catalog number 35373, 98% purity). Cell supernatants were transferred to 96-well plates for analysis at 0, 12, 24, and 36 hours. Add 600 μL of fresh culture medium containing 10% CCK-8 reagent to each well and incubate at 37°C for 2 hours. Then, measure the absorbance at 450 nm using a microplate reader (BIOBASE, model BK-EL10C) and calculate the cell proliferation rate according to the formula "proliferation rate (%) = (OD treatment group - OD blank) / (OD control group - OD blank) × 100%".

[0045] Triglycerides (TG) in group A (blank control), group B (blank control + DMSO solvent), group F (200 μM HDCA), and group J (200 μM 6-ketoLCA) were detected. First, protein was extracted from the samples and the protein concentration was calculated using the BCA protein assay kit (G3522-2). Cell collection: The prepared cell suspension (1 × 10⁻⁶ cells / mL) was collected. 6 Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and retain the cell pellet. Wash twice with isotonic buffer (0.1 mol / L, pH 7-7.4 phosphate buffer), centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and retain the cell pellet. Cell disruption: Add 0.3 mL of homogenization medium (0.1 mol / L, pH 7-7.4 phosphate buffer or physiological saline) for homogenization, and sonicate under ice-water bath conditions (power: 300 W, 5 seconds / time, 30-second interval, repeated 5 times). The prepared homogenate is directly measured without centrifugation.

[0046] Completely dissolve the protein standard in PBS solution, and dilute 20 μL to 100 μL to a final concentration of 1 mg / mL. Dilute the samples with PBS solution as well. Based on the number of samples, prepare an appropriate amount of BCA working solution by mixing 50 volumes of BCA reagent A with 1 volume of BCA reagent B (50:1), and mix thoroughly. The working solution is valid for the day of preparation. For example, add 5 mL of BCA reagent A to 100 μL of BCA reagent B, mix well, and prepare 5.1 mL of BCA working solution. Add 0, 4, 8, 12, 16, and 20 μL of the protein standard solution to the wells of a 96-well plate, and add PBS (or standard diluent) to bring the total to 20 μL to establish a standard curve. Add 20 μL of the PBS-diluted protein sample to each well of the 96-well plate. It is recommended to set up parallel wells for each sample. Prepare the BCA working solution according to the BCA reagent A:B ratio of 50:1 and mix thoroughly. Add 200 μL of BCA working solution to each well and gently vortex to mix, avoiding the formation of air bubbles. Incubate the 96-well plate at 37 ℃ in the dark for 13 min. Measure the absorbance (OD value) of each well at 568 nm using a microplate reader, and calculate the sample protein concentration based on the standard curve. The final standard curve for the day is: y = 1.9585x + 0.0564, R0 2 = 0.9902.

[0047] The TG content of the cell extract was measured using a triglyceride (TG) assay kit (A110-1-1, Nanjing Jiancheng Biotechnology Institute), and the sample was added to a 96-well plate as shown in Table 2.

[0048] Table 2 Sampling Settings Finally, the TG content of each sample is calculated based on the measured OD value. The calculation formula is: TG content (mmol / gport) = (sample OD value - blank OD value) / (standard OD value - blank OD value) * standard concentration (mmol / L) / protein concentration of the sample to be tested (gport / L).

[0049] The results showed that, compared with other concentrations, the triglyceride (TG) content of HDCA and 6-ketolithocholic acid (6-ketoLCA) was significantly decreased at a concentration of 200 µM, but the cell proliferation rate was significantly reduced in both cases. This indicates that 100 µM of 6-ketoLCA had no effect on cell proliferation but could inhibit lipid synthesis in cells; therefore, a safe and effective concentration of 100 µM was used in subsequent studies. Figure 2 ).

[0050] Subsequently, the cells were divided into five treatment groups for further experiments: (1) untreated control group, (2) solvent control group (DMSO), (3) 100 μM HDCA (purchased from Sigma, catalog number H3878, purity 98%), (4) 100 μM 6-ketoLCA (purchased from MCE, catalog number 35373, purity 98%), (5) 100 μM MDCA (purchased from Sigma, catalog number 700270P, purity 98%), and (6) 100 μM HCA (purchased from Sigma, catalog number 700159P, purity 98%).

[0051] Lipid staining was performed on cells 24 hours after treatment. The specific steps for Oil Red O staining were as follows: cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes; fixed cells were then rapidly rinsed twice with 60% isopropanol (5 seconds each time), followed by staining with Oil Red O working solution (Flagcell, catalog number BT-P104) for 15 minutes; 60% isopropanol was used for separation to remove excess dye until the background was clear; the nuclei were then counterstained with Mayer hematoxylin (Flagcell, catalog number BT-P107) for 10 minutes and then blued in PBS for 5 minutes; after washing with distilled water, the samples were mounted with aqueous mounting medium (SouthernBiotech, catalog number 0100-01), and finally images were acquired using a Leica FLEXACAM C1 microscope and LAS X software. Total RNA was extracted from cell and tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA quality and integrity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, PaloAlto, CA, USA) and further validated by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was enriched using Oligo(dT) magnetic beads and fragmented. The mRNA was then reverse transcribed into cDNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The resulting double-stranded cDNA underwent end repair, A-tailing, and adapter ligation. After purification using AMPure XP magnetic beads (1.0×), a library was constructed via PCR amplification. High-throughput sequencing was then performed on the Illumina NovaSeq 6000 platform (Gene Denovo Biotechnology Co., Guangzhou, China). The raw sequencing data were first processed using Fastp (v0.18.0) to remove adapter sequences and low-quality sequences. Bowtie2 (v2.2.8) was then used to align with an rRNA database to remove rRNA contamination. The filtered high-quality sequences (clean reads) were then aligned to sheep reference genome Ovis aries (Oar_rambouillet_v1.0) using HISAT2 (v2.2.4). Transcript assembly was performed using StringTie (v1.3.1).Gene expression levels are initially expressed as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). To further normalize transcript abundance at the whole-genome level, TPM (Transcripts Per Million) values ​​are calculated. For gene i in sample j, the number of reads per kilobase (RPK) is first calculated:

[0052] Among them, RPK ij This represents the number of reads per 1,000 bases for gene i in sample j; count ij This represents the sequencing read of gene i in sample j (i.e., the number of fragments aligned to that gene); length i Indicates the transcript length of gene i (unit: base pairs).

[0053] The scaling factor for each sample is then calculated, which is the sum of all RPK values ​​divided by 10. 6 And calculate TPM accordingly: ; Among them, TPM ij This represents the TPM value (number of transcripts per million) of gene i in sample j; scaling j This represents the scaling factor for sample j.

[0054] This method effectively normalizes transcript abundance across different samples, resulting in a gene × sample expression matrix (including raw counts and TPM values). Differential expression analysis was performed using DESeq2 software, with a selection criterion of P < 0.05.

[0055] The final results showed that 6-ketoLCA had the strongest lipid-suppressing effect. Compared with HDCA, 6-ketoLCA specifically activated genes in the integrated stress response pathway. ATF4 , CHOP (DDIT3) and CHAC1 It also inhibited genes involved in the PPARγ pathway, which is involved in lipid synthesis. This transcriptional signature suggests that 6-ketoLCA induces metabolic stress signaling and may be involved in persistent metabolic reprogramming in adipocytes. Figure 3 ).

[0056] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

The application of 1,6-ketolithocholic acid in the preparation of drugs for reducing fat deposition, characterized in that, The structural formula of the 6-ketolithocholic acid is: 。 2. The application of 6-ketolithocholic acid according to claim 1 in the preparation of drugs for reducing fat deposition, characterized in that, The 6-ketolithocholic acid is used to inhibit lipid droplet accumulation in adipocytes.

3. The application of 6-ketolithocholic acid according to claim 2 in the preparation of drugs for reducing fat deposition, characterized in that, The 6-ketolithocholic acid is used to reduce triglyceride levels in cells.

4. The application of 6-ketolithocholic acid according to claim 3 in the preparation of drugs for reducing fat deposition, characterized in that, The 6-ketolithocholic acid described herein is used to specifically activate genes in the integrated stress response pathway. ATF4 , CHOP and CHAC1 It inhibits gene expression in the PPARγ pathway, which is involved in lipid synthesis.

5. The application of 6-ketolithocholic acid according to claim 1 in the preparation of drugs for reducing fat deposition, characterized in that, The drug dosage form is a solution.

6. The application of 6-ketolithocholic acid according to claim 5 in the preparation of drugs for reducing fat deposition, characterized in that, The effective concentration of 6-ketolithocholic acid in the drug is 25 μM to 100 μM.

7. The application of 6-ketolithocholic acid according to claim 1 in the preparation of drugs for reducing fat deposition, characterized in that, The preparation of the 6-ketolithocholic acid includes the following steps: strain Anaerobutyricumsoehngenii LT-7 was inoculated into fermentation medium, and HDCA was added to the fermentation medium at a final concentration of 100 μg / ml to 125 μg / ml. Anaerobic fermentation was carried out at 37°C for 24 h to 30 h. The fermentation supernatant was collected, and samples containing 6-ketolithocholic acid were obtained by centrifugation and filtration. Each liter of the fermentation medium contains: 5.60–5.66 g casein peptone, 0.8–1.0 g soybean peptone, 3.0–5.0 g yeast extract, 5.0 g mixed peptone, 5.73–5.83 g glucose, 0.73–0.83 g K₂HPO₄, 1.60–1.66 g NaCl, 2.0–3.0 g hydroxymethylaminomethane, 0.001–0.002 g resazurin, 0.01–0.02 g heme, and 0.3–0.4 g cysteine ​​hydrochloride, with the remainder being water; The strain Anaerobutyricumsoehngenii The accession number for LT-7 is DSM 17630.

8. The use of 6-ketolithocholic acid according to claim 7 in the preparation of drugs for reducing fat deposition, characterized in that, Each liter of the fermentation medium contains: 5.66 g casein peptone, 1.0 g soybean peptone, 5.0 g yeast extract, 5.0 g mixed peptone, 5.83 g glucose, 0.83 g K2HPO4, 1.66 g NaCl, 3.0 g hydroxymethylaminomethane, 0.001 g resazurin, 0.01 g heme and 0.4 g cysteine ​​hydrochloride.

9. The use of 6-ketolithocholic acid according to claim 7 in the preparation of drugs for reducing fat deposition, characterized in that, The volume ratio of gases H2, CO2, and N2 in the anaerobic fermentation environment is 5:10:85.

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