Application of indolepropionic acid in preparation of product for preventing and treating alcoholic liver injury

Abstract

The invention provides application of indolepropionic acid (IPA) in preparation of a product for preventing and treating alcoholic liver injury, and relates to the technical field of medicines. The invention discloses indolepropionic acid or starch ester indolepropionylated high amylose corn starch (HAMSIPA) for the first time, which can be used for remarkably relieving alcoholic liver injury, improving liver functions, liver fatty degeneration and liver oxidative stress of individuals with alcoholic liver injury, protecting intestinal barrier injury of the individuals with alcoholic liver injury, and improving the intestinal barrier injury of the individuals with alcoholic liver injury. And intestinal lipopolysaccharide (LPS) is prevented from leaking out and entering the hepatic portal vein, so that the activation of a liver inflammatory pathway is inhibited, and finally, the intestinal liver axis homeostasis is maintained. The indolepropionic acid has a wide application prospect in the aspect of alcoholic liver injury, and the application scene of indolepropionic acid is widened.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to the use of indolepropionic acid in the preparation of products for the prevention and treatment of alcoholic liver injury. Background Technology

[0002] Alcoholic liver injury (ALD) is a leading cause of liver-related death worldwide. Its course typically begins with alcoholic fatty liver disease and can progress to alcoholic hepatitis, cirrhosis, and liver cancer. In recent years, with rapid economic growth and frequent social activities, alcohol consumption in China has continued to increase, leading to a rising prevalence of ALD and a very serious situation for prevention and control. However, existing clinical drugs for ALD often require combination with alcohol abstinence therapy, and long-term treatment is often ineffective. Besides alcohol abstinence, safe and effective intervention strategies for ALD are still lacking.

[0003] Increasing clinical and experimental evidence suggests that alcohol-induced gut-hepatic axis disruption is a significant factor contributing to the progression of ALD. After alcohol ingestion, it is initially absorbed in the intestines. Prolonged alcohol exposure damages the intestinal mucosal barrier, increasing intestinal permeability and allowing gut-derived stimuli such as pathogenic microorganisms and pathogen-associated molecular patterns (PAMPs) to translocate into the portal vein, causing hepatic inflammation and abnormal lipid metabolism. This further damages the liver beyond the direct toxicity of alcohol. Once liver damage occurs, the liver's initial tolerance to mild gut-derived stimuli is completely lost, and its sensitivity to stimuli increases, accelerating the progression of liver damage. Therefore, targeting and maintaining gut-hepatic axis homeostasis has become a potential approach for the prevention and treatment of ALD.

[0004] In recent years, with the deepening research on the gut microbiome, gut microbiota metabolites have provided a new option for disease treatment due to their good biocompatibility and diverse biological functions. Among them, indolepropionic acid, as one of the products of gut microbiota metabolism of tryptophan, has multiple activities, but its potential in the treatment of ALD and its regulatory role in the gut-hepatic axis in ALD remain unclear. Summary of the Invention

[0005] The purpose of this invention is to provide the application of indolepropionic acid (IPA) or its salts, esters, and complexes, especially indolepropionylated high amylose corn starch (HAMSIPA), in the prevention and treatment of alcoholic liver injury. This invention demonstrates that indolepropionic acid and HAMSIPA have significant protective effects against alcoholic liver injury, showing remarkable effects in improving liver function, reducing hepatic steatosis, mitigating hepatic oxidative stress, and maintaining gut-hepatic axis homeostasis, thus contributing to the development of drugs for the prevention and treatment of alcoholic liver disease.

[0006] On the one hand, this application provides the use of indolepropionic acid or its (physiologically acceptable) salts, esters, or complexes in the preparation of products for the prevention and / or treatment of alcoholic liver injury.

[0007] As used herein, the term "physiologically acceptable" means a molecular entity and composition that is physiologically tolerable and, when administered to humans, generally does not produce toxicity or sensitization or similar adverse reactions (such as stomach upset, dizziness, etc.). Optionally, as used herein, the term "physiologically acceptable" means approved by a government regulatory agency or approved in a pharmacopoeia or other generally recognized manner for use in animals or humans.

[0008] Furthermore, the products for the prevention and / or treatment of alcoholic liver injury include pharmaceuticals and / or functional foods.

[0009] This application demonstrates for the first time that indolepropionic acid or its salts, esters, or complexes can be used to prevent and / or treat alcoholic liver injury.

[0010] Furthermore, the indolepropionate exists in the form of its physiologically acceptable salt, including potassium indolepropionate, sodium indolepropionate, calcium indolepropionate, magnesium indolepropionate, ammonium indolepropionate, and / or amino acid indolepropionate; preferably, the indolepropionate is sodium indolepropionate (IPA-Na).

[0011] Further, the indole propionate is formed by covalently linking indole propionic acid with inulin, polysaccharide and / or starch; preferably, the indole propionate is formed by covalently linking indole propionic acid with starch; more preferably, the indole propionate is indole propionic acid starch ester.

[0012] Furthermore, the starch is selected from one or more of corn starch, wheat starch, mixed bean starch, sweet potato starch, cassava starch, potato starch, and yam starch; preferably, the starch is high amylose corn starch.

[0013] Furthermore, the indole propionate is indolepropionylated high amyl corn starch (HAMSIPA).

[0014] This application demonstrates for the first time that the use of indolepropionylated high-amyl corn starch for the prevention and / or treatment of alcoholic liver injury is more effective than the direct use of an equivalent dose of indolepropionate.

[0015] Furthermore, the degree of substitution of indolepropionic acid in the indolepropionylated high amyl corn starch is 0.08-0.54.

[0016] The degree of substitution of indolepropionic acid can be any value or range from 0.08, 0.09, 0.1, 0.12, 0.2, 0.24, 0.3, 0.36, 0.4, 0.48, 0.5, to 0.54.

[0017] Preferably, the degree of substitution of the indolepropionic acid is 0.24-0.36.

[0018] A substitution degree of 0.24-0.36 can achieve the effect of highly efficient targeted release of indolepropionic acid from the intestine.

[0019] Furthermore, the method for preparing the indolepropionylated high amylose corn starch includes: esterification reaction of the carboxyl group of indolepropionic acid (IPA) with the hydroxyl group of high amylose corn starch (HAMS).

[0020] Preferably, the reaction further includes a condensing agent and / or a catalyst.

[0021] More preferably, the condensing agent includes carbodiimide (EDCI).

[0022] More preferably, the catalyst comprises 1-methylimidazole (N-MIM).

[0023] The carbodiimide coupling reaction is catalyzed by 1-methylimidazole, which plays a dual role: on the one hand, it acts as a base to deprotonate the hydroxyl group, and on the other hand, it accelerates the formation of reaction intermediates through a proton shuttle mechanism.

[0024] Preferably, the reaction is carried out in a solvent, and more preferably, the solvent may be dimethyl sulfoxide (DMSO).

[0025] DMSO was chosen as the reaction solvent because of its dual function: (1) it can effectively destroy the hydrogen bond crystallization region in starch, thereby dissolving the HAMS polymer chain; (2) by increasing the fluidity of the macromolecular chain, it enhances the accessibility of the reagent to the starch hydroxyl groups.

[0026] Preferably, the molar mass ratio of indolepropionic acid (IPA) to high amylose corn starch (HAMS) is (0.10-0.80):1.

[0027] Preferably, the reaction conditions are 20℃-80℃, and the reaction time is 20-30 h.

[0028] Taking HAMSIPA with a degree of substitution of 0.36 as an example, the preparation method includes: 12.3 mol of high amylose corn starch (HAMS) was added to 10 L of dimethyl sulfoxide (DMSO) and stirred until the solution was clear. Then, 6.17 mol of indolepropionic acid (IPA), 7.41 mol of carbodiimide (EDCI), and 15.4 mol of 1-methylimidazole (N-MIM) were added sequentially. After the addition was completed, the mixture was stirred at room temperature for 24 h.

[0029] In this application, indolepropionylated high amyl corn starch can be prepared using the method described in Chinese Patent CN116854830B.

[0030] Further, the indolepropionic acid complex comprises A1) indolepropionic acid and A2) inulin, polysaccharide and / or starch, wherein the indolepropionic acid complex is formed by a non-covalent compound of A1) and A2); preferably, the indolepropionic acid complex comprises indolepropionic acid and starch, wherein the indolepropionic acid complex is formed by a non-covalent compound of indolepropionic acid and starch; more preferably, the starch is selected from one or more of corn starch, wheat starch, mixed bean starch, sweet potato starch, cassava starch, potato starch and yam starch.

[0031] Furthermore, the intervention dose of indolepropionate is 0.28 mg / kg-350 mg / kg, preferably 2.8 mg / kg-35 mg / kg.

[0032] Further, the intervention dose of the indolepropionate is 1.6 mg / kg to 2000 mg / kg; preferably, 16 mg / kg to 200 mg / kg.

[0033] Those skilled in the art may adjust the intervention dosage of indolepropionic acid or its salts, esters, or complexes according to the actual situation, without making specific limitations here.

[0034] Furthermore, the prevention and / or treatment of alcoholic liver injury includes improving liver function, reducing hepatic steatosis, and / or reducing hepatic oxidative stress.

[0035] Preferably, the prevention and / or treatment of alcoholic liver injury includes one or more of the following: (B1) Improve liver function; preferably, the improvement of liver function includes inhibiting the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels; more preferably, the alanine aminotransferase or aspartate aminotransferase is alanine aminotransferase or aspartate aminotransferase in peripheral blood. (B2) Reduce hepatic steatosis; preferably, the reduction of hepatic steatosis includes inhibiting alcohol-induced lipid droplet formation and / or lipid accumulation in liver tissue, and reducing total cholesterol (TC) and triglyceride (TG) levels; more preferably, the total cholesterol or triglycerides are total cholesterol or triglycerides in the liver and / or peripheral blood. (B3) Reducing hepatic oxidative stress; preferably, reducing hepatic oxidative stress includes increasing the activity of antioxidant enzymes and / or increasing the content of antioxidants; more preferably, the antioxidant enzymes include catalase (CAT) and / or superoxide dismutase (SOD); more preferably, the antioxidants include reduced glutathione (GSH); preferably, reducing hepatic oxidative stress also includes reducing the content of lipid peroxides; more preferably, the lipid peroxides are malondialdehyde (MDA).

[0036] Furthermore, the indolepropionic acid or its starch ester can prevent and / or treat alcoholic liver injury by maintaining gut-hepatic axis homeostasis.

[0037] Furthermore, maintaining gut-liver axis homeostasis includes repairing the intestinal mucosal epithelial barrier, reversing gut microbiota dysbiosis, and / or inhibiting liver inflammation.

[0038] Preferably, the repair of the intestinal mucosal epithelial barrier includes increasing the concentration of IPA in intestinal contents, inhibiting the reduction of tight junction proteins in ileal tissue, and / or reducing the translocation of enterogenic pathogen-associated molecular patterns to the liver; More preferably, the tight junction proteins include claudin-1, occludin, E-cadherin, and / or ZO-1; More preferably, the pathogen-associated molecular pattern is LPS.

[0039] More preferably, the reduction of translocation of enterogenic pathogen-associated molecular patterns to the liver includes: HAMSIPA releases IPA in the colon to protect the integrity of the intestinal mucosal barrier by activating the Ahr signaling pathway, thereby inhibiting the extravasation of enterogenic LPS to the liver.

[0040] More preferably, the activation of the Ahr signaling pathway includes promoting an increase in the levels of Ahr and its downstream CYP1A1.

[0041] Preferably, reversing gut microbiota imbalance includes reversing alcohol-induced changes in gut microbiota, enriching beneficial bacteria, and / or inhibiting the growth of harmful bacteria.

[0042] Preferably, the indolepropionic acid or its starch ester inhibits liver inflammation and reduces liver damage by suppressing the liver TLR4 / NF-κB inflammatory signaling pathway.

[0043] More preferably, inhibiting the hepatic TLR4 / NF-κB inflammatory signaling pathway includes inhibiting the increase in TLR4, MyD88, p-NF-κB, and p-IκBα protein levels.

[0044] On the other hand, this application also provides a product for the prevention and / or treatment of alcoholic liver injury, the product comprising indolepropionic acid or its salts, esters, or complexes.

[0045] Preferably, the products for preventing and / or treating alcoholic liver injury include pharmaceuticals and / or functional foods.

[0046] Preferably, the indolepropionate is indolepropionylated high-amyl corn starch.

[0047] Preferably, the indolepropionate is sodium indolepropionate.

[0048] The product of this application may also contain excipients, which may include appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0049] The product of this application can be prepared by a general method, wherein one or more diluents or carriers may be added, such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0050] The present invention has the following beneficial effects: This invention discloses for the first time that indolepropionic acid (IPA) and its intestinal targeted delivery system, indolepropionylated high amyl corn starch (HAMSIPA), can significantly alleviate alcoholic liver injury, improve liver function, hepatic steatosis, and hepatic oxidative stress in individuals with alcoholic liver injury, and protect against intestinal barrier damage in individuals with alcoholic liver injury, reduce extravasation of enterogenic lipopolysaccharide (LPS) into the portal vein, thereby inhibiting the activation of hepatic inflammatory pathways and ultimately maintaining intestinal-hepatic axis homeostasis. This invention has broad application prospects in alcoholic liver injury and also expands the application scenarios of indolepropionic acid. Attached Figure Description

[0051] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 NMR spectra of HAMS and HAMSIPA with different degrees of substitution; Figure 2 A statistical chart showing the content of IPA in intestinal contents after treatment with HAMSIPA and IPA-Na; Figure 3 Statistical graph showing the effects of HAMSIPA and IPA-Na on body weight in ALD mice; Figure 4 The effects of HAMSIPA and IPA-Na on the liver function indicators ALT and AST activities in ALD mice are shown in the figure. The left figure is a statistical chart of AST activity, and the right figure is a statistical chart of ALT activity. Figure 5 Figure 1 shows the results of HE staining and Oil Red O staining of ALD mouse liver with HAMSIPA and IPA-Na. Figure 6The effects of HAMSIPA and IPA-Na on liver TC and peripheral blood TC and TG levels in ALD mice are shown in the graphs from left to right. Figure 7 The effects of HAMSIPA and IPA-Na on liver oxidative stress levels in ALD mice are shown in the following graphs, from left to right: CAT, SOD, GSH, and MDA levels. Figure 8 The content of IPA in the intestinal contents of ALD mice after HAMSIPA treatment; Figure 9 The figure shows the effect of HAMSIPA on the expression of tight junction protein in the intestine of ALD mice. The top figure is the Western blot result, and the bottom figure is the qRT-PCR analysis result. Figure 10 A statistical graph showing serum LPS levels in ALD mice after HAMSIPA treatment; Figure 11 Western blot results of Ahr and its downstream CYP1A1 protein in the intestine of ALD mice after HAMSIPA treatment; Figure 12 A diagram showing the β diversity analysis of gut microbiota in ALD mice treated with HAMSIPA, where MK refers to the blank control group, ME refers to the alcohol model group, and IPA refers to the HAMSIPA intervention group. Figure 13 The graph shows the abundance of gut microbiota at the phylum level in ALD mice after HAMSIPA treatment. MK represents the blank control group, ME represents the alcohol model group, and IPA represents the HAMSIPA intervention group. The left graph shows the abundance changes at the phylum level and the ratio of Firmicutes to Bacteroidetes. Figure 14 This is a statistical chart showing the abundance of gut microbiota genera in ALD mice after HAMSIPA treatment. MK represents the blank control group, ME represents the alcohol-induced model group, and IPA represents the HAMSIPA intervention group.

[0052] Figure 15 Western blot results of TLR4-NF-κB signaling pathway-related proteins in liver tissue of ALD mice after HAMSIPA treatment. Detailed Implementation

[0053] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0054] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0055] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0056] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0057] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0058] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0059] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0060] The following specific embodiments further illustrate the above-mentioned contents of the present invention, involving the synthesis of HAMSIPA, the identification of IPA efficiency in targeted intestinal delivery, the construction of an animal model of alcoholic liver injury, drug administration intervention, sample collection, efficacy evaluation and mechanism exploration.

[0061] Example 1: Synthesis and IPA delivery efficiency verification of indolepropionylated high-amyl corn starch HAMSIPA for intestinal-targeted IPA release. 1.1 Synthesis of HAMSIPA HAMSIPA is synthesized via an esterification reaction of the carboxyl group of IPA with the hydroxyl group of HAMS mediated by EDCI. Specific steps include: 12.3 mol of high-amylose corn starch (HAMS) was added to 10 L of dimethyl sulfoxide (DMSO) and stirred until the solution became clear. Indolepropionic acid (IPA), 7.41 mol of carbodiimide (EDCI), and 15.4 mol of 1-methylimidazole (N-MIM) were then added sequentially. After the addition was complete, stirring was continued at room temperature for 24 h. After the reaction was complete, the reaction solution was added dropwise to ethanol (EtOH) or H2O, precipitating a solid. The solid was filtered, and the filter cake was washed with EtOH or H2O and dried.

[0062] A series of structurally modified HAMSIPA derivatives with controllable degree of substitution (DS 0.08-0.54) were synthesized by systematically adjusting the stoichiometric ratio of indolepropionic acid (IPA) to high amylose corn starch (HAMS) (0.10-0.80). Specific parameters are shown in Table 1.

[0063] The carbodiimide coupling reaction is catalyzed by 1-methylimidazole, which plays a dual role: on the one hand, it acts as a base to deprotonate the hydroxyl group, and on the other hand, it accelerates the formation of reaction intermediates through a proton shuttle mechanism.

[0064] DMSO was chosen as the reaction solvent because of its dual function: (1) it can effectively destroy the hydrogen bond crystallization region in starch, thereby dissolving the HAMS polymer chain; (2) by increasing the fluidity of the macromolecular chain, it enhances the accessibility of the reagent to the starch hydroxyl groups.

[0065] The preparation method can be found in Chinese patent CN116854830B.

[0066] 1.2 NMR and Substitution Determination of HAMSIPA Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1The sample was characterized by 1H NMR (Bruker AV III 400 MHz). The starch sample HAMSIPA (20 mg) was dissolved in DMSO-d6 (0.6 mL), and chemical shifts are reported in ppm. Characteristic proton signals between 4.00 and 6.00 ppm were attributed to the dehydrated glucose unit, while the indole NH proton signal appeared at 10.71 ppm. Results are as follows: Figure 1 As shown.

[0067] Degree of substitution (DS) is calculated using the formula: DS = N × 4 / A; In the formula, N is the integral area of ​​NH protons (10.85 ppm); A is the sum of the integral areas of four dehydrated glucose unit protons (4.00-6.00 ppm). The results are shown in Table 1.

[0068] Figure 1 The NMR spectra of HAMS and HAMSIPA with different degrees of substitution are shown. Compared with HAMS, the HAMSIPA spectrum shows a characteristic peak of indole NH at 10.83 ppm, five aromatic proton peaks of the indole ring at 7.50, 7.35, 7.27, 7.08, and 6.99 ppm, and a proton peak of the indole propionyl side chain methylene group at 2.96 and 2.68 ppm. These characteristic resonance peaks demonstrate that IPA was successfully incorporated into the HAMS backbone via acylation, and the signal intensity increases with increasing DS value.

[0069] Quantitative analysis by proton nuclear magnetic resonance spectroscopy (Table 1) showed that the degree of substitution of HAMSIPA was positively correlated with the molar ratio of IPA, indicating that the substitution efficiency had a dose-dependent characteristic.

[0070] Table 1. Degree of Substitution (DS) of HAMSIPA

[0071] 1.3 HAMSIPA Intestinal Targeted Delivery Efficiency of IPA C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a controlled environment with 55%-60% humidity, 23±2℃, and a 12-hour light-dark cycle. After one week of environmental acclimatization, the mice were randomly divided into two groups: a blank control group fed with normal AIN-93G rodent diet, and a HAMSIPA group fed with AIN-93G rodent diet containing 1.0% HAMSIPA of different substitution degrees, as shown in Table 2. The IPA-Na group was fed normal AIN-93G rodent diet with 0.01 mmol / mL IPA-Na added to their drinking water. After 7 days, colonic contents were collected and the IPA content was measured.

[0072] Table 2. Composition of experimental rat diet based on AIN-93G formulation

[0073] Quantitative analysis of IPA was performed using HPLC-MS / M according to established methods. The sample pretreatment process was as follows: 50 mg of intestinal contents was accurately weighed and added to 12.5 μL of a 20 ppm deuterated internal standard mixture (IAA-d5 and IPA-d2). This was followed by two extractions with 500 μL of methanol / water / formic acid (15:4:1, v / v / v) extraction solution. The extraction process included vortex mixing, 10 min of ultrasonic extraction, and centrifugation (10,000 rpm, 10 min, 4℃). The supernatant was collected and purified using an HLB solid-phase extraction column. This included activation of the column with 4 mL of methanol and 4 mL of MQ water, followed by the addition of 600 μL of sample supernatant, rinsing with 4 mL of MQ water, drying, and then eluting with 3 mL of 0.1% formic acid-methanol eluent. Finally, the eluent was collected, filtered through a 0.22 μm microporous membrane, and injected for analysis. The IPA content determination results are shown in Table 3. Figure 2 As shown.

[0074] Table 3 and Figure 2 To compare the efficiency of HAMSIPA and IPA-Na solutions in targeting intestinal IPA delivery, the IPA content in the colonic contents of mice treated with HAMSIPA was significantly higher than that in the blank control group and the sodium indolepropionate (IPA-Na) group. Moreover, when the DS value was between 0.24 and 0.36, HAMSIPA exhibited the best colon-targeted delivery efficiency, with the IPA content in the colonic contents increasing by about 80 times compared to the control group, confirming the high efficiency of HAMSIPA in targeting intestinal IPA delivery.

[0075] Table 3. Efficiency of HAMSIPA and IPA-Na in targeting intestinal release of IPA

[0076] Example 2: Alleviating effects of HAMSIPA and IPA-Na on alcoholic liver injury HAMSIPA prepared by the method in Example 1 (with a degree of substitution of 0.36 as an example) was used as a sample to conduct an experiment on the relief of alcoholic liver injury.

[0077] 2.1 Establishment of a mouse model of alcoholic liver injury (ALD) (NIAAA method) Forty-eight 8-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of environmental acclimatization, the mice were randomly divided into a control group, an alcohol modeling group (EtOH), a HAMSIPA group (EtOH+HAMSIPA), and an IPA-Na group (EtOH+IPA-Na). During the experiment, mice were acclimatized for 5 days with Lieber Decarli control liquid diet (purchased from Shenzhen Ruide Biotechnology Co., Ltd.), while the control group mice were fed Lieber Decarli control liquid diet until the end of the experiment. Mice in the modeling group were fed Lieber Decarli alcohol liquid diet, with the alcohol concentration increasing by 1% daily. After a 4-day transition period, the modeling period began, and the mice were finally fed a 4% alcohol liquid diet until the end of the experiment. Mice in the drug treatment group and the model group were fed the same way, but during the modeling period, HAMSIPA and sodium indolepropionate (IPA-Na) were added to the Lieber Decarli alcohol liquid diet so that the amount of HAMSIPA ingested by the mice was equivalent to the amount of HAMSIPA ingested when fed 0.1% HAMSIPA solid diet, i.e., 200 mg / kg, which is equivalent to a human dose of approximately 16.2 mg / kg. At the same time, the molar mass of IPA ingested by the IPA-Na group (0.1 g / L, mouse dose of 35 mg / kg, equivalent to a human dose of approximately 2.8 mg / kg) and the 0.1% HAMSIPA group was the same to facilitate comparison of the effects between the two groups. Throughout the experiment, mice in each group were fed according to the paired feeding principle. Eighteen days after modeling, from 7 to 9 am, the model group and the drug treatment group were administered alcohol (5 g / kg) by gavage, while the control group was administered dextrin with the same calories by gavage. After 9 hours, various samples were collected for weight measurement, biochemical index detection, and tissue staining.

[0078] Weight test results as follows Figure 3 As shown, during ALD modeling, mice in the alcohol intake group experienced a significant decrease in body weight, while treatment with HAMSIPA and IPA-Na was able to inhibit the weight loss of mice to some extent.

[0079] 2.2 Liver function index testing The levels of liver function-related indicators, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in peripheral blood serum were measured using a detection kit from Nanjing Jiancheng Bioengineering Institute, according to the instructions. The results are shown in Table 4. Figure 4 As shown, HAMSIPA significantly reduced the levels of ALT and AST in the peripheral blood of ALD mice, indicating that HAMSIPA can significantly restore liver function in ALD mice.

[0080] Table 4 Effects of HAMSIPA and IPA-Na on ALT and AST activities, liver function indicators, in ALD mice

[0081] 2.3 Detection of markers for hepatic steatosis Histological examination of liver tissue was performed using tissue staining. HE staining: Liver tissue was paraffin-embedded and sectioned to a thickness of approximately 2-3 μm. After soaking in xylene and anhydrous ethanol, the tissue samples were thoroughly hydrated with graded ethanol solutions. Hematoxylin staining was performed, and after 10 min, excess staining was removed. Cell nuclei were counterstained with a blue staining solution, followed by eosin staining for 3 min. The tissue was then dehydrated with ethanol, soaked in xylene, air-dried, and mounted. Oil Red O staining: Frozen sections of liver tissue, 10-15 μm thick, were dried and briefly washed with 50% ethanol. They were then stained with diluted Oil Red O staining solution for 8-10 min, separated with 50% ethanol, washed with water, and the nuclei were stained with Harris hematoxylin for 30 s. The sections were rinsed with water until they turned blue and mounted with glycerol gelatin. After scanning the HE and Oil Red O stained sections, pathological changes were observed blindly using CaseViewer, mainly observing the distribution, extent, and area of ​​lipid droplets in the liver. The results are as follows: Figure 5 As shown.

[0082] like Figure 5 HE staining and Oil Red staining results of the liver showed that both HAMSIPA and IPA-Na could inhibit alcohol-induced lipid droplet formation and lipid accumulation in mouse liver tissue, and HAMSIPA had a significantly better inhibitory effect than IPA-Na.

[0083] The levels of total cholesterol (TC) and triglycerides (TG) in liver tissue and peripheral blood were further measured using a test kit from Nanjing Jiancheng Bioengineering Institute, following the instructions. The results of the liver and peripheral blood lipid level tests are shown in Table 5. Figure 6 As shown, HAMSIPA significantly reduced the levels of TC in the liver, TG and TC in the peripheral blood of ALD mice, while IPA-Na only significantly reduced the level of TG in the peripheral blood of ALD mice.

[0084] Table 5 Effects of HAMSIPA and IPA-Na on hepatic TC and peripheral blood TC and TG levels in ALD mice

[0085] 2.4 Detection of liver oxidative stress indicators The levels of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) in liver tissue were determined using a detection kit from Nanjing Jiancheng Bioengineering Institute, following the instructions. The results are shown in Table 6. Figure 7As shown, HAMSIPA significantly increased the activity of antioxidant enzymes CAT and SOD in the liver tissue of ALD mice, increased the content of antioxidant GSH, and decreased the content of lipid peroxide MDA, confirming that HAMSIPA can alleviate alcohol-induced liver oxidative stress. In contrast, IPA-Na increased the levels of SOD and GSH in the liver of ALD mice, but had no effect on CAT and MDA.

[0086] Table 6 Effects of HAMSIPA and IPA-Na on liver oxidative stress levels in ALD mice

[0087] In summary, IPA supplementation can improve alcohol-induced liver damage by addressing liver function, hepatic steatosis, and hepatic oxidative stress. Furthermore, targeted intestinal delivery of propionic acid (HAMSIPA) is more effective in improving ALD than oral sodium propionate (IPA-Na). These results also suggest that IPA improves ALD by regulating gut-hepatic axis homeostasis.

[0088] Example 3: Regulatory effect of HAMSIPA on the gut-hepatic axis in ALD mice 3.1 Detection of IPA content in intestinal contents The IPA content in the intestinal contents of mice in each group after HAMSIPA treatment in Example 2 was detected using the same method as in Example 1. The results are shown in Table 7. Figure 8 As shown, alcohol exposure leads to a decrease in IPA concentration in intestinal contents, while HAMSIPA intervention significantly increases it, confirming that HAMSIPA exerts a hepatoprotective effect in ALD by acting on the intestine.

[0089] Table 7. Indolepropionic acid (IPA) content in the intestinal contents of mice in each experimental group.

[0090] 3.2 Expression of intestinal tight junction proteins To detect the integrity of the intestinal mucosal barrier, the expression of tight junction proteins in intestinal tissue was detected by Western blot and qRT-PCR, respectively.

[0091] Western blot analysis: Ileum from mice used in Example 2 was lysed by grinding with RIPA strong lysis buffer containing 1 mM PMSF, centrifuged at 12000 rpm at 4℃ for 15 min, and the supernatant was collected. The protein concentration was determined, and the mixture was mixed with 5× protein loading buffer and boiled in a metal bath at 95℃ for 10 min. Proteins were separated using 6%-12% separating gels according to their molecular weight, transferred to PVDF membranes, blocked, and incubated overnight at 4℃ with primary antibody (claudin-1, occludin, E-cadherin, ZO-1) dilution buffer. After washing, the membranes were incubated at room temperature for 1 h with secondary antibody dilution buffer. Finally, the target protein was detected using ECL chemiluminescence buffer and a G: BOX Chemi XX9 gel imaging system.

[0092] qRT-PCR detection: Total RNA was extracted from the ileum tissue of the mice used in Example 2 using TRIzol reagent. Using 1 μg of RNA as a template, cDNA was synthesized using the SPARKscript II RT Plus Kit. Subsequently, real-time quantitative PCR was performed using the SYBR Green qPCRMix Kit on a Roche Light Cycler 480 system. (The last sentence appears to be incomplete and requires further context.) -ΔΔCT The relative expression levels of target gene (claudin-1, occludin, ZO-1) mRNA were calculated, with GAPDH used as an internal reference gene.

[0093] The results are shown in Table 8 and Figure 9 As shown, Western blot results indicated that supplementation with HAMSIPA could reverse the reduction of tight junction proteins claudin-1, occludin, E-cadherin, and ZO-1 in ileal tissue caused by alcohol. qRT-PCR results further confirmed that HAMSIPA could increase the mRNA levels of claudin-1, occludin, and ZO-1 in ileal tissue of ALD mice, thus demonstrating that HAMSIPA can repair alcohol-induced intestinal epithelial damage.

[0094] Table 8. Effects of HAMSIPA on intestinal tight junction protein mRNA levels in ALD mice

[0095] 3.3 Endotoxin Level Detection The concentration of endotoxin (LPS) in the serum of mice in Example 2 was determined using the endotoxin detection kit from Shanghai Beyotime Biotechnology Co., Ltd., according to the instructions. The results are as follows: Figure 10As shown in Table 9, endotoxin translocation, such as LPS translocation, is a direct consequence of intestinal barrier damage. Compared with the control group, the serum LPS level in the alcohol-fed group was significantly increased, indicating that the intestinal mucosal barrier of ALD mice was significantly damaged. Conversely, HAMSIPA intervention significantly reduced serum LPS levels, further confirming the repairing effect of HAMSIPA on the intestinal mucosal barrier.

[0096] Table 9. Effects of HAMSIPA on intestinal LPS translocation in ALD mice.

[0097] 3.4 Detection of Ahr and its downstream targets As described in section 3.2, the expression levels of Ahr and its downstream CYP1A1 proteins in ileal tissue were detected using Western blot. The results are as follows: Figure 11 As shown. Previous studies have shown that the IPA receptor Ahr is closely related to the maintenance of intestinal epithelial barrier function. Figure 11 The results showed that alcohol caused a decrease in the expression of Ahr and CYP1A1 in the ileum, but this change was significantly restored after HAMSIPA intervention. This indicates that IPA released by HAMSIPA in the colon can protect the integrity of the intestinal mucosal barrier by activating the Ahr signaling pathway, thereby inhibiting LPS translocation to the liver.

[0098] 3.5 Gut microbiota analysis Example 2: One day before the end of the experimental intervention period, 3-4 fresh mouse feces, 100 μL of hepatic serum, and 0.1 g of liver tissue were collected under sterile conditions. Total DNA was extracted using the DNeasy PowerSoil kit. After testing the DNA concentration and integrity, the V3-V4 region of the 16S rRNA gene was amplified using universal primer pairs (343F, TACGGRAGGCAGCAG; 798R, AGGGTATCTAATCCT). A DNA sequencing library was constructed using the purified amplification products, and paired-end sequencing was performed on the Illumina MiSeq platform. The original sequences were quality controlled and clustered using DADA2 to obtain amplicon variants (ASVs). Species annotation of ASVs at different taxonomic levels was performed based on the Greengenes database. Subsequently, bacterial diversity and structure analysis were conducted, and the abundance of each group of taxa was statistically compared at the phylum, genus, and species levels to analyze differential genera.

[0099] The results are as follows Figure 12As shown, gut microbiota dysbiosis is a significant factor exacerbating intestinal barrier damage in ALD. PCoA and NMDS analyses revealed that the data points in the HAMSIPA-treated group showed more similar clustering compared to the alcohol-induced model group, suggesting that HAMSIPA regulates the β-diversity of the gut microbiota in ALD mice. Further microbiota composition analysis showed that, at the phylum level, HAMSIPA significantly reversed the alcohol-induced increase in Firmicutes and Dethiobacteria, the decrease in Bacteroidetes, and the increase in the Firmicutes / Bacteroidetes ratio. Figure 13 At the genus level, such as Figure 14 As shown, HAMSIPA is significantly enriched with Parabacteroides , Bacterroides , Butyricimonas Along with beneficial bacteria such as Oscillibacte, it simultaneously inhibits Monoglobus , Blautia , Harryflinti a and Lachnoclostridium The growth of harmful bacteria was inhibited. These results collectively indicate that HAMSIPA can regulate the structure and composition of the gut microbiota in ALD mice, a role that is crucial for maintaining gut-liver axis homeostasis.

[0100] 3.6 Detection of proteins related to the liver TLR4 / NF-κB inflammatory signaling pathway As described in section 3.2, total protein was extracted from liver tissue using Western blot, and the expression levels of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα proteins were detected. The results are as follows: Figure 15 As shown, the hepatic TLR4 / NF-κB signaling pathway plays a crucial role in the development of liver inflammatory damage as a key downstream signal of enterogenic LPS. Figure 15 Results of analysis on key proteins in this signaling pathway showed that, compared with the control group, alcohol exposure induced a significant increase in the levels of TLR4, MyD88, p-NF-κB, and p-IκBα proteins in mouse liver, while HAMSIPA intervention significantly reduced the expression of these proteins compared with the ethanol group. These results indicate that HAMSIPA can inhibit the activation of the downstream TLR4-NF-κB signaling pathway of LPS, thereby exerting a protective effect against liver injury.

Claims

1. Use of indolepropionic acid or its salts, esters, complexes in the preparation of a product for preventing and / or treating alcoholic liver injury.

2. Use according to claim 1, characterized in that, The product for preventing and / or treating alcoholic liver injury includes a pharmaceutical product and / or a functional food product.

3. Use according to claim 1, characterized in that, The indolepropionic acid salt includes an indolepropionic acid potassium salt, an indolepropionic acid sodium salt, an indolepropionic acid calcium salt, an indolepropionic acid magnesium salt, an indolepropionic acid ammonium salt, and / or an indolepropionic acid amino acid salt; preferably, the indolepropionic acid salt is an indolepropionic acid sodium salt.

4. Use according to claim 1, characterized in that, The indolepropionic acid ester is formed by covalently linking indolepropionic acid to inulin, polysaccharide, and / or starch; preferably, the indolepropionic acid ester is formed by covalently linking indolepropionic acid to starch; more preferably, the starch is selected from one or more of corn starch, wheat starch, bean starch, sweet potato starch, cassava starch, potato starch, and yam starch; more preferably, the starch is high-amylose corn starch; more preferably, the indolepropionic acid ester is indolepropionylated high-amylose corn starch.

5. Use according to claim 4, characterized in that, In the indolepropionylated high-amylose corn starch, the degree of substitution of indolepropionic acid is 0.08-0.54; preferably, the degree of substitution of indolepropionic acid is 0.24-0.

36.

6. Use according to claim 1, characterized in that, The indolepropionic acid complex includes A1) indolepropionic acid and A2) inulin, polysaccharide, and / or starch, and the indolepropionic acid complex is formed by non-covalent complexing of A1) and A2); preferably, the indolepropionic acid complex includes indolepropionic acid and starch, and the indolepropionic acid complex is formed by non-covalent complexing of indolepropionic acid and starch; more preferably, the starch is selected from one or more of corn starch, wheat starch, bean starch, sweet potato starch, cassava starch, potato starch, and yam starch.

7. Use according to claim 1, characterized in that, The intervention dose of the indolepropionic acid salt is 0.28 mg / kg-350 mg / kg, preferably 2.8 mg / kg-35 mg / kg.

8. The use according to claim 1, characterized in that, The intervention dose of the indolepropionic acid ester is 1.6 mg / kg-2000 mg / kg; preferably 16 mg / kg-200 mg / kg.

9. The use according to claim 1, characterized in that, The prevention and / or treatment of alcoholic liver injury includes at least one or more of improving liver function, reducing liver steatosis, and reducing liver oxidative stress.

10. The use according to claim 1, characterized in that, The indolepropionic acid or its starch ester prevents and / or treats alcoholic liver injury by maintaining gut-liver axis homeostasis; preferably, the maintenance of gut-liver axis homeostasis includes at least one or more of repairing intestinal mucosal epithelial barrier, reversing intestinal dysbiosis, and inhibiting liver inflammatory pathway activation.

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