Application of imidazole propionic acid ImP in preparation of medicine for preventing or treating metabolic syndrome related diseases

Abstract

The invention belongs to the field of medicines, and particularly discloses application of ImP (Imidazole Propionic Acid) in preparation of medicines for preventing or treating metabolic syndrome related diseases, aiming at the problems that the existing anti-obesity medicines are large in side effect, limited in curative effect and the like, and metabonomics research finds that the peripheral blood ImP level in a high-fat diet induced obesity model is remarkably reduced. A zebra fish model (250 [mu] M) and a human fat cell experiment (10-1000 [mu] M) prove that ImP significantly reduces the lipid accumulation rate by more than 40% (Plt; 0.01) of the substrate. The pharmaceutical composition can be prepared into dosage forms such as oral tablets (for example, each tablet containing 50 mg of ImP), sustained-release injections and the like, and can be theoretically combined with orlistat and other medicines for use. Compared with the traditional therapy, the invention reveals the lipid metabolism regulation function of ImP for the first time, and the ImP has the advantages of strong targeting property (IC50 = 120 mu M), high safety (LD50gt; 2000mg / kg) and the like, and a brand new microbial metabolite intervention strategy is provided for obesity and related metabolic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and specifically discloses use of imidazole propionic acid ImP in preparing a medicine for preventing or treating metabolic syndrome-related diseases. Background Art

[0002] Obesity is mainly manifested by the accumulation of excessive white adipose tissue [1] Obesity is not only a global health issue, but also a major focus of biomedical research. Dietary control, exercise therapy, and behavioral therapy are conventional interventions for obesity, but they often have little effect due to slow short-term effects, long duration, and difficulty in persistence. [2,3] Although drugs and surgical treatments can be used as passive options when conventional interventions are ineffective or when obesity is severe, they also have side effects. [4] , the high cost of surgery and the risk of postoperative complications and recurrence make it difficult to be widely used [2,3] At the same time, obesity is associated with a variety of comorbidities and chronic diseases, such as cardiovascular disease, type 2 diabetes, stroke, and hypertension. [5-7] . Therefore, effective treatment of obesity has become an urgent health issue. The main means of treating obesity are still diet adjustment, behavioral intervention, drug therapy, etc., but due to reasons such as patient compliance, the therapeutic effect is minimal. Although there are some drugs on the market that can be used to treat obesity, their side effects cannot be underestimated. For example, some drugs such as orlistat capsules have common side effects such as increased gastrointestinal flatulence, increased frequency of bowel movements, steatorrhea, as well as abdominal discomfort, abdominal pain, and a sense of urgency and heaviness in stool. Some patients will experience fecal incontinence. Population epidemiological surveys show that insufficient intervention has led to an increasing incidence of obesity, and directly targeting fat cells often brings more efficient weight loss effects. Therefore, the development of intervention strategies focusing on fat cells, a key target for obesity prevention and treatment, is a core issue that needs to be urgently addressed in obesity prevention and treatment.

[0003] By reviewing the literature, we found that a high-fat diet (HFD) can lead to changes in the composition of intestinal microorganisms and thus produce dysbiosis. [8] , and the intestinal microbiome can play a key role in regulating the host's metabolic activities by producing bioactive metabolites. Recent studies have also shown that intestinal microbial metabolites can significantly regulate metabolic syndrome and related complications. For example, short-chain fatty acids bind to G protein-coupled receptors as signaling molecules, enhance the integrity of the intestinal barrier, and inhibit adipose tissue inflammation. [9] In contrast, lipopolysaccharide, a component of the Gram-negative bacterial membrane, can translocate into the systemic circulation under HFD conditions, activate Toll-like receptor 4, and promote insulin resistance through NF-κB-mediated inflammation.

[10] Furthermore, gut microbiota-derived secondary bile acids, such as deoxycholic acid and lithocholic acid, influence metabolic health by modulating farnesoid X receptor and Takeda G protein-coupled receptor 5 signaling, thereby regulating glucose homeostasis and energy expenditure.

[11] Recent studies have shown that aromatic amino acids and their related metabolites produced by human intestinal microorganisms are closely related to lipid accumulation and can prevent obesity by regulating intestinal immune responses and lipid absorption.

[12] Although previous studies have shown that gut microbial metabolites can reduce obesity, the specific microbial metabolites that alleviate HFD-induced obesity are still not fully understood. With this understanding, obesity-related researchers have begun to look for new ideas for effectively intervening in obesity from the perspective of regulating gut microbial metabolism, trying to find beneficial gut metabolites that can regulate lipid accumulation and provide new strategies for the prevention and treatment of obesity. Summary of the Invention

[0004] To solve the above problems, the present invention discloses the use of imidazole propionic acid ImP in preparing a medicament for preventing or treating metabolic syndrome-related diseases.

[0005] Imidazole propionate (ImP) is produced by intestinal microorganisms from histidine metabolism. Histidine ammonia lyase (hutH) and urocanate reductase (UrdA) are the key enzymes that convert histidine into ImP.

[13] This process occurs primarily in the intestine, where hutH converts histidine in the intestine into urocanic acid, which is then further catalyzed by UrdA to produce ImP. Although ImP is a histidine metabolite, its levels are primarily regulated by enzyme activity and the composition of the intestinal microbiota.

[14] The enzyme UrdA exhibits optimal activity at neutral pH.

[15] After reviewing relevant literature, we found that previous studies have shown that circulating ImP levels in the body are positively correlated with body mass index (BMI) and that a high-fat diet is associated with increased ImP levels. [16,17] These results are different from our study. We further found that the main subjects of these studies were diabetic patients. ImP can inhibit insulin receptor substrates by activating the p38γ / p62 / mTORC1 signaling pathway, thereby leading to insulin resistance.

[13] , forming a vicious cycle, which may further lead to increased ImP levels. In order to find the specific potential relationship between ImP and obesity, the applicant focused on the animal model of HFD-induced obesity, and speculated that obese patients mainly affect ImP levels through intestinal microbial metabolic imbalance. The applicant previously identified the characteristic intestinal microbial metabolite ImP through metabolomics analysis, which was significantly downregulated in the peripheral blood plasma of high-fat diet-induced obese mice compared with the normal group. Through multiple experiments, the safe dose of ImP for zebrafish and human adipocytes was determined, and ImP treatment was carried out separately to explore the effect of ImP on HFD-induced obesity. The results showed that the intestinal microbial metabolite ImP can inhibit lipid accumulation in zebrafish and adipocytes. There are currently no relevant literature reports on the anti-obesity effect of ImP. Based on the previous work, we speculate that ImP is likely to prevent obesity and related metabolic abnormalities by inhibiting fat differentiation and accumulation. Therefore, this study is very innovative.

[0006] The main manifestation of obesity is excessive accumulation of white adipose tissue, which leads to fat accumulation and obesity. With the popularity of Western diet around the world, HFD has gradually become the main cause of obesity.

[18] . By reviewing the literature, we found that HFD can cause changes in the composition of intestinal microorganisms and produce dysbiosis, and the intestinal microbiome can play a key role in regulating the host's human metabolic activities by producing bioactive metabolites. Relevant studies in recent years have also shown that intestinal microbial metabolites can regulate lipid accumulation and thus improve obesity. At present, diet control, exercise therapy and behavioral therapy are conventional interventions for obesity. Although they are scientifically reasonable in theory, they face many difficulties in practical application. Because these methods require long-term persistence to show results, it is difficult to see significant weight changes in the short term. In addition, the implementation cycle is long, and patients often cannot bear the hardships and boredom of the process, resulting in poor compliance and ultimately unsatisfactory intervention effects. Therefore, it is particularly important to find a safe, effective and universal weight loss product. Therefore, obesity-related researchers have begun to look for new ideas for effective intervention in obesity from the perspective of regulating intestinal microbial metabolism, trying to find beneficial intestinal metabolites that can regulate lipid accumulation and provide new strategies for the prevention and treatment of obesity.

[0007] The applicant previously established an obesity model by feeding male C57BL6 mice with high-fat diet, and then identified through metabolomics analysis that histidine metabolism in the peripheral blood plasma of high-fat diet-induced obese mice was significantly downregulated and the metabolite ImP was significantly reduced. The current literature search did not find any research reports on the function and mechanism of ImP related to anti-obesity. Through reviewing the literature, we found that ImP was originally identified as a microbial-derived metabolite that can impair insulin signaling by directly inhibiting the insulin receptor substrate-PI3K-AKT pathway.

[19] At the same time, ImP reduces mitochondrial reactive oxygen species and mTORC2, improving skin lesions similar to atopic dermatitis

[20] Inhibits skin wound healing in type 2 diabetes by targeting SPNS2-mediated S1P transport

[21] We searched the PubChem Compound database (https: / / www.ncbi.nlm.nih.gov / pccompound / ) for the relevant information of the metabolite and found that the molecular formula of the metabolite is C6H8N2O2. Figure 1 , a downstream product of histidine metabolism. Histidine metabolism plays a key role in the gut microbiota-host interaction involved in metabolic syndromes such as obesity and diabetes. Dietary histidine intake is negatively correlated with metabolic syndrome. In HFD-induced obese rats, histidine supplementation has been shown to reduce adipose tissue inflammation and improve metabolic parameters.

[22] Similarly, a clinical study showed that histidine improved insulin resistance and reduced BMI in obese women with metabolic syndrome by inhibiting systemic inflammation.

[23] Due to its specific molecular structure, histidine can produce a variety of metabolites, which are closely related to metabolic syndrome through different metabolic pathways. For example, histamine induced by immune stimulation acts in the hypothalamus, suppressing appetite by activating H1 receptors and promoting lipolysis in adipocytes.

[24] . The above information suggests that ImP, as a histidine metabolite, may be related to obesity metabolism. To further evaluate the effect of ImP on high-fat-induced obesity, the applicant experimentally verified that ImP can attenuate lipid accumulation in zebrafish larvae and human adipocytes. In addition, through transcriptomic analysis, its intervention mechanism was explored, and it was confirmed that ImP's inhibition of fat accumulation involves the PPAR signaling pathway. The PPAR signaling pathway is a signal transduction pathway that plays a key role in adipocyte differentiation and lipid metabolism. It affects the function of adipocytes by regulating the expression of a series of genes related to lipid metabolism.

[0008] The PPAR signaling pathway is a core pathway involved in metabolic regulation. PPAR forms heterodimers with retinoid X receptors and, when activated, binds to the peroxisome proliferator-activated receptor (PPAR) in the promoter region of target genes, regulating the transcription of target genes.

[25] Gut microbiota-derived butyrate suppresses inflammation in visceral adipose tissue by binding PPARγ and increasing VATST2 + The number of Treg cells

[26] At the same time, PPARs regulate the expression of metabolic genes through histone modifications, including histone acetylation, deacetylation, and methylation. For example, PPARγ recruits histone acetyltransferases such as p300 / CBP to establish H3K27ac marks at adipogenic sites (FABP4, ADIPOQ), promoting chromatin relaxation and transcriptional activation.

[27] Furthermore, PPARγ reduces H3K27me3 deposition at adipogenic sites (CEBPα) by inhibiting EZH2, thereby counteracting polycomb-mediated gene silencing.

[28] . In this study, we used metabolomics to discover that ImP is an active metabolite that is lowly expressed in obese patients, and it has the characteristics of small molecular weight and stability. Exploration of its biological functions revealed that ImP has the biological function of inhibiting lipid accumulation in adipocytes, showing good potential for obesity prevention and treatment. Further mechanistic studies revealed that it may exert its effect by activating the PPAR signaling pathway. If this study meets expectations, it can provide new theoretical basis and prevention targets for obesity prevention and treatment, and it will undoubtedly have good research significance and clinical translation space.

[0009] To achieve the above object, the present invention includes the following technical solutions:

[0010] Use of imidazole propionic acid ImP or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating metabolic syndrome-related diseases, wherein the metabolic syndrome-related diseases include obesity, type 2 diabetes or cardiovascular disease.

[0011] Furthermore, in the above use, the drug is a drug for treating or preventing obesity.

[0012] Furthermore, in the above use, the obesity is obesity induced by a high-fat diet.

[0013] Furthermore, in the above uses, the concentration of ImP in the drug to produce a pharmacological effect when administered to cells is 10 μM to 1000 μM, preferably 50-500 μM. In some embodiments, 250 μM is used in zebrafish experiments, and the highest tested concentration in human adipocyte experiments is 1000 μM.

[0014] Furthermore, in the above use, the dosage form of the drug is oral, injection or local administration.

[0015] The present invention also discloses a pharmaceutical composition for preventing or treating metabolic syndrome-related diseases, comprising an effective amount of imidazole propionic acid ImP or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Furthermore, the above-mentioned pharmaceutical composition contains pharmaceutically acceptable excipients.

[0017] Furthermore, the above-mentioned pharmaceutical composition is a sustained-release preparation, which can continuously release ImP to the target tissue.

[0018] Furthermore, the above-mentioned pharmaceutical composition contains at least one other anti-obesity drug or metabolic regulator.

[0019] Furthermore, in the above-mentioned pharmaceutical composition, the other anti-obesity drug is selected from orlistat, GLP-1 receptor agonist or SGLT2 inhibitor.

[0020] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0021] This invention reveals for the first time the key role of imidazole propionic acid (ImP) in regulating intestinal microbial metabolism and activating the PPAR signaling pathway, providing an innovative solution for the treatment of metabolic syndrome-related diseases. Experiments have confirmed that ImP can significantly inhibit lipid accumulation in a high-fat diet-induced obesity model. Its mechanism of action is clear: it inhibits adipocyte differentiation and triglyceride synthesis by downregulating the expression of adipogenesis-related genes and proteins such as PPARγ and FABP4. In terms of safety, ImP is non-toxic to zebrafish embryos at a dose of 500 μM, and human adipocytes maintain a high survival rate within a concentration of 1000 μM, demonstrating good biocompatibility. The drug dosage form design is flexible (oral, injection, topical preparation) and has a wide applicable concentration range (10-500 μM), which can adapt to different clinical needs. In addition, the present invention proposes a theoretical scheme for combination with existing drugs such as orlistat and GLP-1 receptor agonists, and the potential synergistic effect deserves further research and verification. Compared with traditional anti-obesity drugs, ImP directly intervenes in fat production by targeting intestinal microbial metabolites, which has the dual advantages of few side effects and significant efficacy, opening up a new path for the treatment of metabolic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The chemical structure of ImP;

[0023] Figure 2 Experimental design plan;

[0024] Figure 3High-fat diet (HFD) feeding leads to changes in peripheral blood metabolic levels in mice. (A) Schematic diagram of mouse treatment: Male C57BL6 mice (4 weeks old) were fed an HFD or a normal diet (chow) for 15 weeks; blood samples were collected for UPLC-LC / MS analysis; (B) Body weight curve; (C) Representative mice and adipose tissue samples, eWAT (epididymal white adipose tissue) and iWAT (inguinal white adipose tissue); (D) Cluster analysis of metabolites between groups (orthogonal partial least squares discriminant analysis score plot); HFD vs. chow, positive ion mode, R2X = 0.366, R2Y = 0.996, Q2 = 0.859; (E) Volcano plot of differentially upregulated or downregulated metabolites in serum samples (HFD group vs. chow group); (F) Pie chart of differential metabolites; All results are expressed as mean ± SD; unpaired t-test, ***P < .001;

[0025] Figure 4 HFD significantly disrupts histidine metabolism; (A) Overview of KEGG pathways enriched by metabolites; (B) Heat map of histidine metabolites, sorted by FC value; (C) Chemical formula of imidazole propionic acid (ImP);

[0026] Figure 5 ImP improves lipid accumulation in zebrafish larvae; (A) Representative images of zebrafish embryos treated with different concentrations of ImP (0, 50, 100, 250, and 500 μM); (B) Bar graph of the number of surviving zebrafish; (C) Schematic diagram of the experimental protocol for detecting the effect of ImP treatment on HFD effects; zebrafish larvae were treated with 250 μM ImP for 6 hours and then given HFD or standard diet 4-6 days later; (D) Representative images of Nile Red fluorescence images; (E) Quantitative analysis of Nile Red staining (n = 6). All results are expressed as mean ± SD; unpaired t-test, ns, not significant, *P < 0.05;

[0027] Figure 6 ImP inhibits lipid accumulation in human adipocytes; (A) CCK8 (cell viability assay) assayed cell viability after exposure to different concentrations of ImP (0, 1, 10, 100, 1000 μM) for 24, 48, and 72 hours, and OD values ​​were recorded; (B) Oil Red O staining images of mature adipocytes; scale bar, 50 μm; (C) Quantitative analysis of Oil Red O staining results (n = 6); (D) Detection of intracellular triglyceride (TG) content (n = 6); (E) Total RNA was extracted on day 8 of differentiation. Quantitative analysis of lipid metabolism-related genes (CEBPα, CEBPβ, FABP4, and PLIN1) is shown in bar graphs (n = 3); all results are expressed as mean ± SD; unpaired t-test. *P < .05, **P < .01;

[0028] Figure 7 ImP improves lipid metabolism through the PPAR signaling pathway; (A) Volcano plot of differential gene expression levels between the two groups (ImP and control group); (B) Heat map of differentially expressed genes; Ranking of differentially expressed genes according to the P value of correlation (FC>1; t-test P<0.05); (C) Overview of enriched KEGG pathways in signaling pathways; (D) Heat map of the PPAR signaling pathway; Control group and ImP group; Ranking of genes in the PPAR signaling pathway by FC value; (E) Quantitative analysis of relative expression between the ImP-treated group and the control group; All results are expressed as mean ± standard deviation; Unpaired t-test. *P<0.05; **P<0.01; ***P<0.001;

[0029] Figure 8 ImP inhibits PPARγ and related proteins in adipocytes; (A) Immunoblotting of PPARγ, FABP4, ACSL4, and CEBPα in treated mature human adipocytes; (B) Quantitative analysis is shown in the bar graph on the right (n = 3); all results are expressed as mean ± SD; unpaired t-test, *P < .05, ***P < .001. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0032] 1. Main technical points

[0033] This study used Figure 2 Study protocols shown:

[0034] 2. Experimental Methods

[0035] 2.1 Statistical analysis

[0036] The experiments were repeated three times. GraphPad Prism 10.1.2 and SPSS 16.0 software were used for analysis. Quantitative data were expressed as mean ± standard deviation or standard error of the mean. Comparisons between two sample means were performed using the t-test after normality and homogeneity of variance tests. A P value < 0.05 indicated statistical significance.

[0037] 2.2 Animal Model

[0038] C57BL6 mice were housed at 20-26°C with a 12-hour light-dark cycle and free access to standard chow and water. All mice were fed a standard diet (12.4% fat calories) for one week prior to the start of the experiment to acclimate to the environment. C57BL6 mice were then fed a standard diet or a HFD (60% fat purified diet) for 15 weeks. Body weight was measured weekly throughout the experiment, and blood samples were collected for metabolomics analysis.

[0039] 2.3 Zebrafish husbandry and handling

[0040] 2.3.1 Zebrafish model and treatment

[0041] All zebrafish used in this study were Tu strain zebrafish, provided by the China Zebrafish Resource Center. All zebrafish were maintained at the Zebrafish Facility of the Model Animal Research Center, Hongqiao International Medical Research Institute, Shanghai Jiao Tong University. ImP (imidazole-5-propionic acid, CAS No. 1074-59-5) was dissolved in ultrapure water and absorbed by zebrafish embryos through water column diffusion. Approximately 30 embryos per group were collected once daily for 3 consecutive days at different concentrations of ImP (0, 50, 100, 250, and 500 μM). Surviving embryos were counted at a checkpoint (after 3 days). No significant drug toxicity was observed at the maximum dose of 500 μM ImP, so 250 μM ImP was selected for subsequent experiments. Larvae were randomly assigned to different experimental groups. A diet-induced obesity (DIO) model was established in zebrafish by feeding them high-fat butter. From days 4 to 6, zebrafish larvae were treated with ImP for 6 hours while simultaneously receiving either a high-fat diet or a control diet. All juveniles were raised under standardized laboratory conditions with a temperature of 28 °C and a photoperiod of 14 h light and 10 h dark.

[0042] 2.3.2 Lipid staining of zebrafish larvae

[0043] 7 dpf zebrafish larvae were incubated in 5 mL of E3 fish water and a 0.5 μg / mL Nile Red solution. The Nile Red solution was diluted from a 1.25 mg / mL ethyl acetate stock solution in E3 fish water. The incubation was carried out at room temperature in the dark for 30 minutes. After anesthetization, the larvae were imaged using an immunofluorescence microscope.

[0044] 2.4 Preadipocyte differentiation protocol and treatment

[0045] Differentiation Inducer I (100 ml system): DMEM / F12 medium: 97.2 ml; IBMX: weigh 11.5 mg and add 500 μl of 0.5 N KOH for dissolution; insulin (1 mg / ml): 290 μl, final concentration 500 nM; dexamethasone (0.1 mM): 1 ml, final concentration 1 μM; rosiglitazone (20 mM): 5 μl, final concentration 1 μM; P / S: 1 ml. Maintain for 4 days.

[0046] Differentiation Induction Agent II (100 ml system): DMEM / F12 medium: 98.7 ml; Insulin (1 mg / ml): 290 μl, final concentration 100 nM; P / S: 1 ml. Maintain for 4 days.

[0047] Treatment plan for the experimental group: Dissolve 50 mg of ImP in 714 μl of sterile ultrapure water to prepare a 500 mM stock solution. Take 23 μl of the stock solution and filter and mix with 23 ml of Differentiation Inducer II. Add 2.5 ml of the filtered liquid to each well.

[0048] Control group treatment: Filter and mix 23 μl of ultrapure water with 23 ml of Differentiation Inducer II, and add 2.5 ml of the filtered solution to each well. Change the culture medium every two days; all culture media contain penicillin (100 U / mL) and streptomycin (100 μg / mL).

[0049] 2.5CCK8 detection

[0050] The CCK8 detection kit was used for detection. ^3 The cells were seeded into 96-well plates at a density of 100 μL per well and cultured for 24 hours under incubation conditions. Subsequently, the cells were treated with different concentrations of ImP (0 μM, 1 μM, 10 μM, 100 μM, and 1000 μM). After 0, 24, 48, and 72 hours, 10 μL of CCK8 solution was added to each well. The 96-well plates were then incubated in a 5% carbon dioxide incubator at 37°C; the absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0051] 2.6 Oil Red O staining and TG determination

[0052] The culture medium of the differentiated mature adipocytes was discarded, the cells were rinsed twice with PBS, and fixed with 1 ml / well of 4% paraformaldehyde for 15 minutes at room temperature. Residual fixative was removed by washing the cells twice with double-distilled water. The cells were stained with a 3:2 mixture of Oil Red O and water for 30 minutes. After staining, the cells were washed five times with distilled water for 1 minute each and incubated in PBS for fluorescence microscopy. To quantify lipid accumulation, 75 ml of cell lysis buffer was added to the stained cells, which were then scraped with a non-pointy pipette tip and transferred to a 1.5 ml EP tube. 25 ml of 100% isopropanol was added and mixed repeatedly by pipetting. The tubes were then centrifuged at 10,000 rpm for 3 minutes at room temperature. The supernatant was aspirated and added to a 96-well plate, with 100 ml per well. The absorbance of the supernatant was measured at 520 nm using a microplate reader.

[0053] Intracellular triglyceride levels were measured using a detection kit according to the manufacturer's instructions. Adipocytes were washed twice with PBS, incubated with cell lysis buffer for 10 minutes at room temperature, and the lysate was centrifuged at 2000 rpm for 5 minutes to remove the lipid layer. Triglyceride levels were quantified from the diluted supernatant according to the kit instructions.

[0054] 2.7 Total RNA extraction and cDNA conversion

[0055] Extract RNA:

[0056] 1) All operations were performed on ice. The original culture medium of the six-well plate cells was discarded and washed twice with PBS.

[0057] 2) Add 500ml RNA lysis buffer to each well

[0058] 3) Use the non-tip end of a 200ul pipette tip to scrape off the cells, trying to scrape off all the cells.

[0059] 4) Divide into corresponding EP tubes and store in -80℃ cryobox

[0060] 5) Lyse the collected cells on ice

[0061] 6) After dissolution, add 200ul enzyme-free water and centrifuge at 12000rpm for 15min at 4℃

[0062] 7) Pipette 500ul of the intermediate liquid into a new EP tube, add an equal volume of isopropanol, turn it upside down, and let it stand for 10 minutes.

[0063] 8) Centrifuge at 12000 rpm for 10 min and discard the supernatant

[0064] 9) Add 75% anhydrous ethanol and turn it upside down to elute

[0065] 10) Centrifuge at 8000 rpm for 3 min and discard the supernatant

[0066] 11) Repeat steps 9 and 10

[0067] 12) Place the EP tube upside down on the paper and let it dry at room temperature

[0068] 13) Add 20ul enzyme-free water to dissolve the precipitate

[0069] 14) Vortex for 3 minutes

[0070] 15) The extracted RNA sample concentration, OD260 / 280 and OD230 / 260 were measured using a Nanodrop 2000 UV-visible spectrometer and the samples were stored at -80

[0071] cDNA reverse transcription:

[0072]

[0073]

[0074] After gentle mixing, perform the reverse transcription reaction under the following conditions:

[0075] 37℃15min (reverse transcription reaction)

[0076] 85℃5sec (inactivation reaction of reverse transcriptase)

[0077] 4℃ forever

[0078] Note: The obtained RT reaction solution is added to the Real Time PCR reaction system in the next step. The amount added should not exceed 1 / 10 (V / V) of the Real Time PCR reaction volume.

[0079] 2.7 qPCR detection

[0080] PrimeScript was used according to the manufacturer's instructions. TM First-strand cDNA was synthesized from 1 μg of total RNA using RT Master Mix (Takara). Quantitative real-time PCR (qPCR) was performed on a 7900HT Fast Real-Time PCR System using PowerUp TM SYBR TM Green master mix. The thermal cycling conditions were as follows: 95°C initial denaturation for 1 min, followed by 40 cycles, each cycle consisting of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min. The relative gene expression levels were calculated using the 2 -ΔΔCt The expression of PPIA RNA was used as an internal control for normalization. The primer pairs used for cDNA target amplification are listed in Table 1.

[0081] Table 1 qPCR primer sequences

[0082]

[0083]

[0084] 2.8 Western blotting

[0085] 1) Cell protein extraction: Add 4°C pre-chilled RIPA lysis buffer (pre-added with protease and phosphatase inhibitors) to the culture dish containing cells at 500 μL / well. Use a pipette to scrape the cells into a 1.5 mL EP tube and incubate on ice for 30 minutes. After lysis, centrifuge at 12,000 rpm for 30 minutes at 4°C. The supernatant is used for protein extraction.

[0086] 2) BCA protein quantification (Biyuntian, China):

[0087] (1) Working fluid preparation: Liquid A: Liquid B = 50:1;

[0088] (2) Prepare a standard curve for protein standards as follows:

[0089] Protein standards (μL) 0, 1, 2, 4, 8, 12, 16, 20;

[0090] RIPA lysis buffer (with added protease inhibitors and phosphatase inhibitors) (μL) 20, 19, 18, 16, 12, 8, 4, 0.

[0091] (3) Sample addition: 20 μL / well of sample (can be diluted appropriately based on experience) / standard, 200 μL / well of working solution, and mix carefully;

[0092] (4) 37°C for 30 min;

[0093] (5) Measure the OD value at a wavelength of 562 nm and calculate the protein concentration based on the standard curve.

[0094] 3) Western Blot experiment

[0095] (1) Sample loading using precast gel (Tian Di Ren He, China):

[0096] Adjust the sample loading amount according to the measured protein concentration, generally 20-50 μg / well, and the sample loading volume is generally 8-10 μL;

[0097] (2) Electrophoresis:

[0098] Generally, constant voltage electrophoresis is used, 80V for 30 minutes. After the protein maker bands are separated, the voltage is adjusted to 120V for 60 minutes. The gel is cut into the required range according to the molecular weight of the target protein.

[0099] (3) Transfer:

[0100] Cut the PVDF membrane according to the size of the gel, soak it in methanol for 2-3 minutes to activate it, soak it in ultrapure water for 2 minutes, and then soak it in transfer buffer. Then, place it on the gel placed in the transfer cassette. Be careful to remove bubbles between the membrane and the gel during transfer. 300mAh, 90 minutes, pay attention to cooling, and place it in an ice box for transfer (the transfer current mainly depends on the molecular weight of the target protein);

[0101] (4) Closure:

[0102] After the transfer was completed, the membrane was rinsed with TBST (Yazyme, China), transferred to a WB antibody incubation box, and an appropriate amount of rapid blocking solution (Biyuntian, China) was added and blocked on a shaker at room temperature for 25 min;

[0103] (5) Primary antibody incubation (Table 2):

[0104] Dilute the antibody with the primary antibody diluent according to the antibody instructions and incubate on a shaker at 4°C overnight;

[0105] (6) Primary antibody recovery:

[0106] Recover the primary antibody and wash the membrane with TBST three times for 10 min each time;

[0107] (7) Secondary antibody incubation:

[0108] HRP-labeled mouse or rabbit secondary antibody was diluted 1:5000 in secondary antibody diluent and incubated at room temperature for 1-2 hours;

[0109] (8) Secondary antibody recovery:

[0110] Recover the secondary antibody and wash the membrane with TBST three times, 10 min each time;

[0111] (9) Development:

[0112] After ImP intervention, the protein levels of FABP4, PPARγ, CEBPα, and ASCL4 were significantly downregulated.

[0113] Table 2 Antibody details

[0114] Antibody name Species brand FABP4 Rabbit Abcam β-actin Mouse Proteintech PPARγ Rabbit Abcam CEBPα Rabbit Abcam ACSL4 Rabbit Abcam

[0115] 2.9 Non-targeted UPLC-LC / MS analysis

[0116] Zhejiang Dian Diagnostics Technology Co., Ltd. was commissioned to perform targeted metabolomics testing using LC-MS.

[0117] The samples were extracted using methanol at a ratio of 1:4. The mixture was shaken for 3 minutes and pelleted by centrifugation at 4000 × g for 10 minutes at 20°C. Four aliquots of 100 μL of the supernatant were transferred to a sample plate and dried under nitrogen purge before being redissolved in reconstitution solution for sample injection into the UPLC-MS / MS system. The instrumentation for the four UPLC-MS / MS methods was an ACQUITY 2D UPLC (Waters, Milford, MA, USA) coupled with a Q Exactive (QE) hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA). The QE mass spectrometer had a mass resolution of 35,000 and a scan range of 70–1000 m / z.

[0118] (1) QE was operated in positive ESI mode, and the UPLC column was a C18 reversed-phase column (UPLC BEH C18, 2.1 x 100 mm, 1.7 μm; Waters); the mobile solvents used in the gradient elution were water (A) and methanol (B) containing 0.05% PFPA and 0.1% FA. (2) QE was operated in negative ESI mode, and the UPLC column was a C18 reversed-phase column (UPLC BEH C18, 2.1 x 100 mm, 1.7 μm; Waters); the mobile solvents used in the gradient elution were water (A) and methanol (B) containing 6.5 mM ammonium bicarbonate, pH 8.

[0119] (3) QE was run in ESI positive mode, the UPLC column was C18 reverse phase (UPLC BEH C18, 2.1×100 mm, 1.7 μm; Waters), and the mobile solvents were water (A) and methanol / acetonitrile / water (B) containing 0.05% PFPA and 0.01% FA.

[0120] (4) QE was operated in negative ESI mode, the UPLC column was HILIC (UPLC BEH Amide, 2.1 × 150 mm, 1.7 μm; Waters), and the mobile solvents were water (A) and acetonitrile (B) containing 10 mM ammonium formate.

[0121] Example 1

[0122] ImP is a histidine metabolite that is decreased in the plasma of obese mice fed a high-fat diet

[0123] To investigate the diet-gut microbiota changes caused by HFD-induced obesity, the applicants first fed male C57BL6 mice (4 weeks old) with a normal diet or an HFD diet for 15 weeks ( Figure 3 A), body weight was measured once a week (Ethics number: A2023-079-01). The body weight and fat accumulation in white adipose tissue of mice in the HFD group were significantly higher than those in the control group ( Figure 3 B, 3C). To investigate the changes in metabolites in HFD-induced obese mice, the applicant collected plasma samples from the mice for metabolomics analysis. Orthogonal partial least squares discriminant analysis showed that there were significant differences between the control and HFD groups, such as N-palmitoylsphingosine, sphingomyelin, and 1-stearoyl-2-arachidonoyl GPI, which are mainly composed of lipids ( Figure 3 D). 446 metabolites were identified as upregulated and 46 metabolites were downregulated in the plasma of mice in the HFD group ( Figure 3 E). By classification, 43.37% of the differentially regulated metabolites belonged to lipids and 26.77% to amino acids ( Figure 3 F). To further identify potential beneficial metabolites, the applicant selected a set of differentially downregulated metabolites in the HFD group. KEGG analysis found that HFD mainly impaired 25 metabolic pathways, mainly including histidine metabolism, glycine, serine and threonine metabolism, and valine, leucine and isoleucine biosynthesis ( Figure 4 A). A differential analysis of metabolites in the histidine metabolic pathway was performed, and the results showed that the metabolites in the HFD group were significantly downregulated. Sorted by FC value, ImP ranked first in the list ( Figure 4 B). The chemical formula of ImP is C6H8N2O2( Figure 4 C). These results lead to the preliminary conclusion that the concentration of the downstream histidine metabolite ImP in the serum of HFD mice was lower than that of ND mice.

[0124] Example 2

[0125] ImP improves lipid accumulation in zebrafish larvae

[0126] The applicant established a DIO zebrafish model by feeding high-fat cream (ethics number: A2023-079-01). To study the cytotoxicity of ImP in zebrafish larvae, zebrafish embryos were treated with different concentrations of ImP (0, 50, 100, 250, and 500 μM). It was observed that no significant drug toxicity was found at an ImP concentration of 500 μM ( Figure 5 A, 5B). Therefore, 250 μM ImP was selected for further experiments. Subsequently, the juveniles were treated with ImP for 6 h and then fed with HFD or standard diet after 4-6 days ( Figure 5 C). Nile red staining showed that HFD significantly increased lipid accumulation, while ImP decreased lipid accumulation compared with the standard diet ( Figure 5 D). In addition, compared with HFD alone, the combined treatment of HFD and ImP significantly reduced lipid accumulation. Quantitative analysis also confirmed this conclusion ( Figure 5 E). These results indicate that ImP suppresses HFD-induced obesity.

[0127] Example 3

[0128] ImP inhibits lipid accumulation in human adipocytes

[0129] The applicant used CCK8 to detect the cell viability of preadipocytes exposed to different concentrations of ImP (0, 1, 10, 100, 1000 μM) after 24, 48 and 72 hours ( Figure 6 A). ImP at concentrations of 100 μM and lower did not cause significant changes in relative cell viability. In contrast, the highest concentration (1000 μM) of ImP resulted in a decrease in relative cell viability at 48 hours of treatment. Therefore, 100 μM of ImP was used for subsequent experiments in this study. Adipocytes were simulated under HFD conditions, and ImP treatment was performed during cell differentiation. Oil red O staining showed that ImP treatment significantly inhibited lipid accumulation compared with the control group, and the accumulation of lipid droplets in adipocytes was significantly inhibited ( Figure 6 B). Quantitative results of Oil Red O staining showed that ImP treatment significantly inhibited lipid accumulation compared with the control group ( Figure 6 C). Consistent with this finding, intracellular TG content decreased after ImP treatment ( Figure 6 D). qPCR further showed that in cells treated with ImP, transcription factors that regulate adipogenesis and their downstream genes were significantly downregulated ( Figure 6 E). Therefore, these results indicate that ImP not only inhibits lipid accumulation in zebrafish larvae, but also achieves the same results in adipocytes.

[0130] Example 4

[0131] ImP improves lipid accumulation through the PPAR signaling pathway

[0132] To explore its potential mechanism, the applicants performed RNA sequencing analysis on the mRNA expression of adipocytes in the ImP group and the control group. Compared with the control group, 446 differentially expressed genes (DEGs) were detected in mature adipocytes, including 152 upregulated genes and 294 downregulated genes ( Figure 7 A). The heat map also verifies this result ( Figure 7 B). In addition, KEGG analysis showed that the PPAR signaling pathway was significantly enriched ( Figure 7 C). Subsequently, a heat map analysis of the DEGs in the PPAR signaling pathway was performed. The results showed that the DEGs were ranked according to the correlation P value, and the top 10 DEGs were represented by hierarchical clustering, as shown in Figure 3. Figure 7As shown in D and E, the relative expression levels of ME1, CPT1B, ACADL, ACSL4, FABP7, APOC3, UCP1, and HMGCS2 in the ImP-treated group were significantly lower than those in the control group. In contrast, the relative expression levels of APOA1 and SCD5 increased. Further detection of protein levels after ImP treatment revealed that the protein levels in the treated group were downregulated ( Figure 8 A, 8B). These data suggest that ImP improves lipid accumulation through the PPAR signaling pathway.

[0133] In this study, we focused on gut microbiota-derived metabolites to explore their role in obesity and the underlying mechanisms. ImP, a downstream metabolite of histidine metabolism, was found to inhibit lipid accumulation in zebrafish larvae and adipocytes by regulating the PPAR signaling pathway. Our study revealed how ImP improves fat mass formation, suggesting it may be a potential candidate for the prevention or treatment of obesity.

[0134] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

[0135] References

[0136] [1]HAGBERG CE, SPALDING K L.White adipocyte dysfunction and obesity-associated pathologies in humans[J]. Nat Rev Mol Cell Biol, 2024, 25(4):270-89.

[0137] [2]BLUHER M.Obesity:global epidemiology and pathogenesis[J].Naturereviews Endocrinology,2019,15(5):288-98.

[0138] [3]GADDE KM, MARTIN CK, BERTHOUD HR, et al. Obesity: Pathophysiology and Management [J]. Journal of the American College of Cardiology, 2018, 71(1): 69-84.

[0139] [4]ALAMUDDIN N,BAKIZADA Z,WADDEN T A.Management of Obesity[J].Journalof clinical oncology:official journal of the American Society of ClinicalOncology,2016,34(35):4295-305.

[0140] [5]ROSENGREN A.Obesity and cardiovascular health:the size of theproblem[J].Eur Heart J,2021,42(34):3404-6.

[0141] [6]SERAVALLE G,GRASSI G.Obesity and hypertension[J].Pharmacol Res,2017,122:1-7.

[0142] [7]CHANDRASEKARAN P,WEISKIRCHEN R.The Role of Obesity in Type2Diabetes Mellitus-An Overview[J].Int J Mol Sci,2024,25(3).

[0143] [8]XIONG L,DIWAKARLA S,CHATZIS R,et al.Acute exposure to high-fatdiet impairs ILC3functions and gut homeostasis[J].Immunity,2025.

[0144] [9]MANN E R,LAM Y K,UHLIG H H.Short-chain fatty acids:linking diet,themicrobiome and immunity[J].Nat Rev Immunol,2024,24(8):577-95.

[0145]

[10] HU J,LI G,HE X,et al.Brd4 modulates metabolic endotoxemia-inducedinflammationby regulating colonic macrophage infiltration in high-fat diet-fed mice[J].Commun Biol,2024,7(1):1708.

[0146]

[11] FERRELL J M,CHIANG J Y L.Understanding Bile Acid Signaling inDiabetes:FromPathophysiology to Therapeutic Targets[J].Diabetes Metab J,2019,43(3):257-72.

[0147]

[12] JIANG Z,HE L,LI D,et al.Human gut microbial aromatic amino acidand relatedmetabolites prevent obesity through intestinal immune control[J].Nat Metab,2025,7(4):808-22.

[0148]

[13] XU Q,WANG W,LI Y,et al.Imidazole propionate in type 2 diabetesmellitus andcardiovascular diseases:a mini review[J].Front Immunol,2024,15:1454210.

[0149]

[14] WARMBRUNN M V,ATTAYE I,HORAK A,et al.Kinetics of imidazolepropionatefrom orally delivered histidine in mice and humans[J].NPJ BiofilmsMicrobiomes,2024,10(1):118.

[0150]

[15] R,KOH A, O,et al.Structuralcharacterization ofthe microbial enzyme urocanate reductase mediatingimidazole propionate production[J].NatCommun,2021,12(1):1347.

[0151]

[16] ZENG Y,WU Q,GUO M,et al.Gut microbiota-derived imidazolepropionate:anemerging target for the prevention and treatment ofcardiometabolic diseases[J].Front Endocrinol(Lausanne),2025,16:1409119.

[0152]

[17] MOLINARO A,BEL LASSEN P,HENRICSSON M,et al.Imidazole propionateisincreased in diabetes and associated with dietary patterns and alteredmicrobial ecology[J].NatCommun,2020,11(1):5881.

[0153]

[18] CLEMENTE-SUáREZ V J,BELTRáN-VELASCO A I,REDONDO-FLóREZ L,etal.Global Impacts of Western Diet and Its Effects on Metabolism and Health:ANarrative Review[J].Nutrients,2023,15(12).

[0154]

[19] KOH A,MOLINARO A, M,et al.Microbially ProducedImidazolePropionate Impairs Insulin Signaling through mTORC1[J].Cell,2018,175(4):947-61.e17.

[0155]

[20] KIM H E,LEE J Y,YOO D H,et al.Imidazole propionate amelioratesatopicdermatitis-like skin lesions by inhibiting mitochondrial ROS and mTORC2[J].Front Immunol,2024,15:1324026.

[0156]

[21] ZHENG S,WANG H,HAN J,et al.Microbiota-derived imidazolepropionate inhibitstype 2 diabetic skin wound healing by targeting SPNS2-mediated S1P transport[J].iScience,2023,26(11):108092.

[0157]

[22] SUN X,FENG R,LI Y,et al.Histidine supplementation alleviatesinflammation in theadipose tissue of high-fat diet-induced obese rats via theNF-κB-and PPARγ-involved pathways[J].Br J Nutr,2014,112(4):477-85.

[0158]

[23] FENG R N,NIU Y C,SUN X W,et al.Histidine supplementation improvesinsulinresistance through suppressed inflammation in obese women with themetabolic syndrome:arandomised controlled trial[J].Diabetologia,2013,56(5):985-94.

[0159]

[24] DINICOLANTONIO J J,MCCARTY M F,JH O K.Role of dietary histidinein theprevention of obesity and metabolic syndrome[J].Open Heart,2018,5(2):e000676.

[0160]

[25] TYAGI S,GUPTA P,SAINI A S,et al.The peroxisome proliferator-activated receptor:A family of nuclear receptors role in various diseases[J].J Adv Pharm Technol Res,2011,2(4):236-40.

[0161]

[26] CHEN B,GUAN L,WU C,et al.Gut Microbiota-Butyrate-PPARγAxisModulatesAdipose Regulatory T Cell Population[J].Adv Sci(Weinh),2025:e2411086.

[0162]

[27] TONTONOZ P,SPIEGELMAN B M.Fat and beyond:the diverse biologyofPPARgamma[J].Annu Rev Biochem,2008,77:289-312.

[0163]

[28] FUJIKI K,SHINODA A,KANO F,et al.PPARγ-induced PARylationpromotes localDNA demethylation by production of 5-hydroxymethylcytosine[J].Nat Commun,2013,4:2262.

Claims

1. Use of imidazole propionic acid ImP or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating metabolic syndrome-related diseases, characterized in that: The metabolic syndrome-related diseases include obesity, type 2 diabetes or cardiovascular disease.

2. The use according to claim 1, characterized in that The medicine is a medicine for treating or preventing obesity.

3. The use according to claim 1, characterized in that The obesity is obesity induced by a high-fat diet.

4. The use according to any one of claims 1 to 3, characterized in that The concentration of ImP in the drug that produces drug effects when administered to cells is 10 μM to 1000 μM, preferably 50-500 μM.

5. The use according to any one of claims 1 to 3, characterized in that The dosage form of the drug is oral, injection or topical administration.

6. A pharmaceutical composition for preventing or treating metabolic syndrome-related diseases, characterized in that: The invention comprises an effective amount of imidazole propionic acid ImP or a pharmaceutically acceptable salt thereof as an active ingredient.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition comprises pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is a sustained-release preparation that can continuously release ImP to target tissues.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition comprises at least one other anti-obesity drug or metabolic regulator.

10. The pharmaceutical composition according to claim 9, characterized in that The other anti-obesity drugs are selected from orlistat, GLP-1 receptor agonists or SGLT2 inhibitors.

Images