Use of indole propionic acid in treatment of autoimmune liver injury and cirrhosis

Abstract

The present invention relates to the use of indole propionic acid in liver disease. Specifically, the present application discloses the use of indole propionic acid or a pharmaceutically acceptable salt thereof or a derivative thereof in preparing a medicine or a pharmaceutical composition or a kit for preventing and / or treating cirrhosis and acute autoimmune liver disease caused by chronic liver injury.

Description

Application of indolepropionic acid in the treatment of autoimmune liver damage and cirrhosis Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of indolepropionic acid (IPA) in treating liver diseases. Background Art

[0002] Liver cirrhosis is a common, chronic, progressive liver disease in clinical practice, characterized by diffuse liver damage caused by the long-term or repeated effects of one or more etiologies. In my country, most cases are post-hepatitis cirrhosis, with a smaller proportion being alcoholic cirrhosis and schistosomal cirrhosis. Histopathologically, there is extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue proliferation, and fibrous septa formation, leading to structural destruction of the hepatic lobule and the formation of pseudolobules, which progress to liver fibrosis and even cirrhosis. Cirrhosis is characterized by collagen deposition and pseudolobule formation as its core pathological features. Liver inflammation and immune abnormalities are crucial in the development of cirrhosis.

[0003] Cirrhosis is an important intermediate link in the development of chronic liver disease into liver cancer. As a country with a high incidence of chronic liver disease, my country has a large population of people with cirrhosis. In addition, patients with cirrhosis have a long course of disease, many complications, and poor prognosis. There is currently no reversal treatment option.

[0004] Autoimmune liver disease (ALD) is a specific group of chronic liver diseases caused by immune dysfunction. Different types of ALD vary in demographics, clinical manifestations, and liver pathological changes. The mechanisms of action and treatment of ALD remain unclear.

[0005] Therefore, there is an urgent need to develop an effective method for treating or preventing liver cirrhosis and autoimmune liver diseases. Summary of the Invention

[0006] The purpose of the present invention is to provide a new method for treating cirrhosis and / or acute autoimmune liver disease caused by chronic liver damage.

[0007] Another object of the present invention is to provide use of indolepropionic acid for treating or preventing cirrhosis and / or acute autoimmune liver disease caused by chronic liver damage.

[0008] In a first aspect, the present invention provides a use of an active ingredient selected from indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, for preparing a medicament, pharmaceutical composition, or kit for preventing and / or treating a disease selected from the group consisting of:

[0009] (a) Cirrhosis caused by chronic liver damage;

[0010] (b) Acute autoimmune liver disease.

[0011] In another preferred embodiment, the chronic liver damage is chronic liver damage caused by chemical drugs or poisons.

[0012] In another preferred embodiment, the chronic liver injury is chronic liver injury caused by carbon tetrachloride.

[0013] In another preferred embodiment, the drug is also used for the following purposes:

[0014] (y1) reduce serum AST and / or ALT levels;

[0015] (y2) reduce the levels of pro-inflammatory factors;

[0016] (y3) Reduce collagen levels.

[0017] In another preferred embodiment, the acute autoimmune liver disease is acute autoimmune liver disease caused by ConA.

[0018] In another preferred embodiment, the dosage of the drug is at least 100 mg / kg / d body weight calculated as the dosage of IPA.

[0019] In another preferred embodiment, the dosage form of the drug is an oral dosage form.

[0020] In another preferred embodiment, the collagen mRNA includes: Col1a1, Col3a1, Col6a1, or a combination thereof; the collagen includes Col1a1, Col3a1, Col6a1, or a combination thereof.

[0021] In another preferred embodiment, the drug is administered to a subject selected from the group consisting of a rodent or a primate.

[0022] In another preferred embodiment, the subject is a subject not on a high-fat diet.

[0023] In another preferred embodiment, the subject is a non-diabetic patient.

[0024] In another preferred embodiment, the subject does not suffer from any of the following diseases:

[0025] (x1) Alcoholic fatty liver disease;

[0026] (x2) Non-alcoholic fatty liver disease.

[0027] In another preferred embodiment, the pro-inflammatory factor is selected from the group consisting of TNF-α, INF-γ, IL-6, IL-17 or a combination thereof.

[0028] In another preferred embodiment, the pharmaceutical composition comprises:

[0029] (z1) a first active ingredient, indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof;

[0030] (z2) optionally, a second active ingredient, other drugs for preventing and / or treating cirrhosis and / or acute autoimmune liver disease; and

[0031] (z3) a pharmaceutically acceptable carrier.

[0032] In another preferred embodiment, in the pharmaceutical composition, the component (z1) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0033] In another preferred embodiment, the other drugs for preventing and / or treating cirrhosis and / or acute autoimmune liver disease are selected from the following group: interferon, nucleotide analogs, nitrates, calcium channel blockers, or a combination thereof.

[0034] In another preferred embodiment, the interferon includes conventional interferon or long-acting interferon.

[0035] In another preferred embodiment, the nucleotide analogs include lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoproxil, clavudine, or a combination thereof.

[0036] In another preferred embodiment, the nitrates include isoflurane.

[0037] In another preferred embodiment, the calcium channel blocker includes nifedipine.

[0038] In another preferred embodiment, the pharmaceutical composition may contain a single compound or a mixture of multiple compounds.

[0039] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for treating or preventing cirrhosis and / or acute autoimmune liver disease caused by chronic liver damage.

[0040] In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

[0041] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0042] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0043] In another preferred embodiment, the pharmaceutical composition is selected from the group consisting of injection, inhalant, tincture, powder, granule, capsule, oral solution, tablet, pill, suspension, emulsion, lozenge, or pill.

[0044] In another preferred embodiment, the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.

[0045] In another preferred embodiment, the subject to which the pharmaceutical composition is administered is a human or non-human mammal.

[0046] In another preferred embodiment, the kit comprises:

[0047] (f1) a first pharmaceutical composition comprising (i) indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof as a first active ingredient; and (ii) a pharmaceutically acceptable carrier; and

[0048] (f2) A reagent for detecting the level of collagen or its mRNA.

[0049] In another preferred embodiment, the collagen includes: Col1a1, Col3a1, Col6a1, or a combination thereof.

[0050] In another preferred embodiment, the collagen includes Col1a1.

[0051] In another preferred embodiment, the detection of collagen is the detection of Col protein content, or its activity, or a combination thereof.

[0052] In another preferred embodiment, the indolepropionic acid is enteric indolepropionic acid.

[0053] In another preferred embodiment, the indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is further used for one or more purposes selected from the following groups:

[0054] (a1) reducing the expression levels of TNF-α, INF-γ, IL-6, IL-17, Col1a1, Col3a1, Col6a1, or α-SMA RNA in the liver;

[0055] (a2) Reduce inflammatory infiltration of the liver and inhibit the increase of autoimmune liver inflammation;

[0056] (a3) reduce collagen deposition or pseudolobule formation in the liver;

[0057] (a4) inhibiting liver cirrhosis;

[0058] (a5) inhibiting the protein expression of Col1a1 and α-SMA;

[0059] (a6) Reduction of serum ALT and / or AST concentrations.

[0060] In a second aspect, the present invention provides a method for preventing and / or treating cirrhosis caused by chronic liver damage or autoimmune liver disease caused by acute liver damage, comprising the steps of:

[0061] A safe and effective amount of indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is administered to a subject in need thereof.

[0062] In another preferred embodiment, the administration comprises oral administration.

[0063] In another preferred embodiment, the subject includes humans or non-human mammals.

[0064] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0065] In another preferred embodiment, the indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is administered 1-7 times a week, preferably 2-5 times a week, and more preferably 2-3 times a week.

[0066] In another preferred embodiment, the administration time of the indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is 1-20 weeks, preferably 2-12 weeks, and more preferably 4-8 weeks.

[0067] In another preferred embodiment, the dosage of indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is 50 to 500 mg / kg / day, preferably 80 to 250 mg / kg / day, for example 100 mg / kg / day.

[0068] In another preferred embodiment, the dosage of indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is 1 to 50 mg / kg / day, preferably 5 to 20 mg / kg / day, for example 8.1 mg / kg / day.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG1A shows a gross image of mouse liver showing that IPA alleviates CCl 4 -induced mouse liver cirrhosis.

[0071] Figure 1B shows that IPA alleviated the IPA level in portal vein blood of mice with CCl4-induced cirrhosis detected by ELISA.

[0072] Figure 1C shows that IPA alleviated the expression of RNA levels of Col1a1, Col3a1, Col6a1, and α-SMA in mice with CCl4-induced liver cirrhosis detected by RT-PCR.

[0073] FIG1D shows the HE staining results of mouse liver tissue in which IPA alleviated CCl4-induced leukemia.

[0074] Figure 1E shows the results of Sirius red staining of mouse liver tissues in which IPA alleviated CCl4-induced inflammatory reactions.

[0075] FIG1F shows the immunohistochemical staining results of IPA-induced CCl4-induced mouse liver tissue.

[0076] Figure 1G shows the Western Blot results showing that IPA alleviates CCl4-induced liver cirrhosis in mice.

[0077] FIG2 shows the survival rate of mice with ConA-induced acute autoimmune liver disease treated with IPA.

[0078] FIG3 shows the liver function (ALT) of mice with ConA-induced acute autoimmune liver disease treated with IPA, A: 6 hours after ConA injection, B: 18 hours after ConA injection.

[0079] FIG4A shows a gross image of mouse liver showing liver inflammation in mice with ConA-induced acute autoimmune liver disease treated with IPA.

[0080] Figure 4B shows the expression of RNA levels of TNF-α, IFN-γ, IL-6, and IL-17 in mice with ConA-induced acute autoimmune liver disease treated with IPA as detected by RT-PCR.

[0081] FIG4C shows the results of HE staining of liver tissues from mice with ConA-induced acute autoimmune liver disease treated with IPA. DETAILED DESCRIPTION

[0082] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that indolepropionic acid can effectively prevent and / or treat cirrhosis and / or acute autoimmune liver disease caused by chronic liver damage. Based on this, the inventors completed the present invention.

[0083] the term

[0084] As used herein, the terms "pharmaceutical composition of the present invention" and "drug of the present invention" are used interchangeably and refer to a preparation or pharmaceutical composition containing IPA, or a pharmaceutically acceptable salt thereof, or a derivative thereof as an active ingredient.

[0085] As used herein, the terms "preparation combination", "preparation combination of the present invention" and "drug of the present invention" are used interchangeably and all refer to the drug or pharmaceutical composition or kit described in the first aspect of the present invention.

[0086] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0087] As used herein, the terms "subject" and "subject in need thereof" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, and goats.

[0088] Cirrhosis

[0089] Liver cirrhosis is an important intermediate link in the development of chronic liver disease into liver cancer. Liver cirrhosis is a pathophysiological process and an important stage in the development of various chronic liver diseases into liver cancer. my country is a country with a high incidence of chronic liver disease. The population of people with cirrhosis is large, the disease course is long, there are many complications, the prognosis is poor, and there is currently no reversal treatment. Therefore, methods for the treatment of cirrhosis are very important for the early prevention and treatment of liver cancer. Inflammation and immune abnormalities of the liver are very important in the process of cirrhosis. Studies have shown that the metabolites of intestinal flora are absorbed into the blood through the intestine, circulate to the portal vein, enter the liver, and can directly affect the liver's inflammatory response.

[0090] Indolepropionic acid

[0091] Indole-3-propionic acid (IPA, molecular formula C 11 H 11 NO) is an important end product of tryptophan metabolism, the indole product. Indolepropionic acid is a plant growth regulator with auxin activity, primarily used as a pharmaceutical intermediate and plant growth stimulant.

[0092] Tryptophan is an essential amino acid and the only amino acid in the human body that contains an indole structure. It is abundantly found in oats, milk, yogurt, cheese, red meat, eggs, and fish. Tryptophan is metabolized by the intestinal microbiome to produce indole products, which regulate the body's homeostasis. In recent years, tryptophan has played a crucial regulatory role in intestinal and liver diseases. Furthermore, instability in the intestinal microbiome and increased pathogen abundance are associated with increased susceptibility to various metabolic liver diseases.

[0093] The present invention finds that indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof can inhibit liver fibrosis, alleviate liver cirrhosis, inhibit the progression of liver cirrhosis, or inhibit acute autoimmune liver inflammation.

[0094] Pharmaceutical composition

[0095] The pharmaceutical composition provided by the present invention preferably contains 0.1-99 wt% of the first active ingredient, with the remainder being the second active ingredient, a pharmaceutically acceptable carrier, a diluent, a solution or a saline solution.

[0096] The first active ingredient of the present invention is indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof. In addition, it can also be used in combination with other therapeutic agents, i.e., the second active ingredient.

[0097] The second active ingredient may be any pharmaceutical ingredient capable of preventing and / or treating liver cirrhosis and / or autoimmune liver disease, including but not limited to interferon, nucleotide analogs, nitrates, calcium channel blockers, etc.

[0098] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of the present invention, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0099] The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or infusion.

[0100] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the formulation generally contains 0.05-1000 mg of the active compound of the present invention, preferably 1 mg-500 mg of the active compound of the present invention.

[0101] The pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered via the oral, nasal, dermal, pulmonary, or gastrointestinal routes. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight taken as a single dose, or 0.01-200 mg / kg body weight taken in divided doses. Regardless of the route of administration, the optimal individual dose will depend on the specific treatment being used. Typically, a low dose is started and gradually increased until the most suitable dose is found.

[0102] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, or mucosal tissues; or can be mixed or encapsulated in other substances and introduced into the body.

[0103] Typically, the active ingredient of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0104] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0105] The active ingredient of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems.

[0106] In order to prepare the active ingredients of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; adhesives can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0107] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0108] To prepare the dosing unit as a capsule, the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard or soft capsule. Alternatively, the active ingredient can be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard or soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

[0109] To prepare the active ingredient of the present invention as an injection, water, ethanol, isopropanol, propylene glycol, or a mixture thereof can be used as the solvent, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Examples of solubilizers or cosolvents include poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin; pH adjusters include phosphates, acetates, hydrochloric acid, and sodium hydroxide; and osmotic pressure regulators include sodium chloride, mannitol, glucose, phosphates, and acetates. For lyophilized powder injections, mannitol, glucose, and the like can also be added as support agents.

[0110] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings or other additives may be added to the pharmaceutical preparations.

[0111] The active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0112] When the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0113] The main advantages of the present invention include:

[0114] The present invention provides the use of indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof in the treatment of cirrhosis caused by chronic liver damage and acute autoimmune liver disease, which has the following advantages:

[0115] (1) Indolepropionic acid is the final product of indole products in tryptophan metabolism and is safe and effective.

[0116] (2) Administration of indolepropionic acid can effectively reduce the RNA expression levels of Col1a1, Col3a1, Col6a1 and α-SMA, inhibit Col1a1 and α-SMA proteins, reduce inflammatory infiltration and collagen deposition, thereby alleviating liver fibrosis and cirrhosis caused by chronic liver damage and inhibiting the progression of cirrhosis.

[0117] (3) The administration of indolepropionic acid can inhibit the inflammatory response of autoimmune liver disease and has a protective effect on acute autoimmune liver disease.

[0118] (4) The present invention provides a new method for treating cirrhosis and acute autoimmune liver disease caused by chronic liver damage.

[0119] (5) The method of the present invention is simple to operate, simple to prepare the drug, and highly efficient.

[0120] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0121] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0122] Example 1 IPA Experiment on CCl4-Induced Liver Cirrhosis in Mice

[0123] 1.1 CCl4-induced liver cirrhosis model in mice and sample collection

[0124] 1.1.1 Method

[0125] C57BL / 6 mice were divided into three groups: one control group received an intraperitoneal injection of olive oil, while the other two groups received intraperitoneal injections of CCl4 (0.5 ml / kg, CCl4:olive oil = 1:9, twice weekly) for eight weeks. At the end of the sixth week of modeling, IPA (100 mg / kg) dissolved in 5% CMC was administered orally to one of the CCl4-treated groups (hereinafter referred to as CCl4+IPA100). The control CCl4 and olive oil groups received 5% CMC orally (hereinafter referred to as the Oil and CCl4 groups) daily for two weeks. At the eighth week, mice were sacrificed, and portal and inferior vena cava blood samples were collected. The livers were exposed and removed intact. The right livers were paraffin-embedded, and the left livers were used for RNA and protein extraction for subsequent RT-PCR and Western blot analysis.

[0126] 1.1.2 Results

[0127] The mouse cirrhosis model was established by intraperitoneal injection of CCl4, and the mice were randomly divided into three groups: olive oil control modeling group (Figure 1A, Oil group), CMC control solvent gavage + CCl4 group (Figure 1A, Figure CCl4 group), IPA-100 mg / kg gavage + CCl4 group (Figure 1A, Figure CCl4+IPA100), with five mice in each group.

[0128] 1.2 Elisa assay for IPA concentration in mouse serum

[0129] 1.2.1 Method

[0130] After collecting portal venous blood overnight at 4°C, centrifuge it the next day at 3000 rpm for 15 minutes at 4°C, and collect the supernatant. Using the ELISA kit (YJ416125), dilute 10 μl of serum with sample diluent to 50 μl. Incubate with horseradish peroxidase-labeled antibody at 37°C for 1 hour, then wash the plate five times. Add 100 μl of colorimetric solution to each well, incubate at 37°C in the dark for 15 minutes, then add 50 μl of stop solution. Determine the IPA concentration using an absorbance microplate reader at 450 nm.

[0131] 1.2.2 Results

[0132] The IPA concentration in mouse portal vein serum was detected using an Elisa kit.

[0133] The results showed that the IPA concentration in the serum of mice in the CCl4 group was reduced compared with that in the Oil group, and the difference was statistically significant (p<0.001), indicating that IPA had a downward trend in the portal vein serum of cirrhotic mice.

[0134] Compared with the mice in the CCl4 group, the whole blood serum IPA concentration of the mice in the CCl4 + IPA100 group was significantly increased, indicating that oral administration of IPA-100 mg / kg can increase the serum IPA level of mice (Figure 1B).

[0135] 1.3 Real-time PCR (RT-PCR) detection of changes in liver cirrhosis markers in mice

[0136] 1.3.1 Method

[0137] (1) RNA extraction

[0138] Add 1 ml of Trizol (RNAiso Plus) to the tissue, let it sit for 5 minutes, then pipette up and down, then collect in an imported Eppendorf centrifuge tube. Add 0.2 ml of chloroform (one-fifth the volume of Trizol), cover tightly, and shake thoroughly for 15 seconds until fully emulsified. Incubate at room temperature for 5 minutes (at this time, adjust the centrifuge to 4°C and 12,000g). Centrifuge at 4°C and 12,000g for 15 minutes. Remove the upper transparent band, add 0.4 ml of isopropanol (equal to the volume of the supernatant aspirated from the top), mix gently up and down 5 times, and incubate at room temperature for 10 minutes. Centrifuge at 4°C and 12,000g for 10 minutes. When a precipitate is visible at the bottom, discard the supernatant.

[0139] Gently add 1 ml of 75% ethanol (prepared with DEPC water) to flick the RNA pellet and rinse. Centrifuge at 7,500 g for 5 min at 4°C. Aspirate the supernatant as thoroughly as possible to prevent loss of the RNA pellet. Invert the EP tube to dry any water droplets on the tube wall. Dissolve the dried RNA in 30 μl of DEPC water. Measure the OD260 / 280 value using a spectrophotometer to prepare a working concentration (1 μg / μl). Check RNA integrity by electrophoresis on a 1% agarose gel. Store at -80°C until ready to use.

[0140] (2) Reverse transcription reaction

[0141] Reverse transcription was performed using the TAKARA kit. 10 μl of the system was sufficient to reverse 1000 ng of RNA. For a 1 μg, 20 μl system, use reagents 1, 2, and 3 from the reverse transcription kit. Measure the RNA concentration and calculate the required amount for 1 μg of RNA. To a standard eight-tube PCR strip, add the following: 5x Primescript PT Enzyme MIX (4 μl) and Total RNA (RNase-Free) dd H2O (up to 20 μl). Reverse transcription was performed using a standard PCR instrument at 37°C for 15 minutes, 85°C for 30 seconds, and 4°C forever.

[0142] (3) real time-PCR

[0143] Prepare a mix of 2*TB Green, primers, and ddH2O in a 1.5ml EP tube. A 10µl system includes: 2*TB Green (5µl), primers (0.3µl each for forward and reverse), and ddH2O (up to 6µl). Aliquot the mix into a real-time plate, 6µl per well, and work on ice.

[0144] Then add 4ul of the corresponding diluted template to each well and put it on the machine.

[0145] (4) Primer sequence

[0146] Table 1 shows the primer sequences used for RT-PCR.

[0147] Table 1

[0148] 1.3.2 Results

[0149] RT-PCR experiments showed that compared with the Oil group, the mice in the CCl4 group showed higher RNA levels of Col1a1, Col3a1, Col6a1, and α-SMA, indicating that CCl4 successfully established a mouse cirrhosis model ( Figure 1C ).

[0150] Compared with the mice in the CCl4 group, the RNA levels of Col1a1, Col3a1, Col6a1, and α-SMA were significantly decreased in the mice in the CCl4 + IPA100 group ( Figure 1C ), indicating that IPA has an inhibitory effect on liver cirrhosis in mice in vivo.

[0151] 1.4 HE staining

[0152] 1.4.1 Method

[0153] Paraffin-embedded tissue was cut into 0.4 μm sections and dewaxed in xylene three times for 15 minutes each. Dewaxed sections were then rehydrated with a gradient of ethanol (100%, 95%, 85%, and 75%, 3 minutes each), washed three times with PBS, and stained with hematoxylin for 1 minute and eosin for 30 seconds. The sections were then washed three times with PBS, dried, and mounted with neutral resin.

[0154] 1.4.2 Results

[0155] After CCl4 modeling (compared with the Oil group), the mouse liver showed obvious inflammatory infiltration, and IPA gavage for 14 days could significantly alleviate the inflammatory infiltration caused by CCl4 modeling (Figure 1D).

[0156] 1.5 Sirius red staining to detect liver collagen deposition

[0157] 1.5.1 Method

[0158] Paraffin-embedded tissue was cut into 0.4 μm sections. The sections were dewaxed in xylene three times for 15 minutes each. The dewaxed sections were then rehydrated with a gradient of ethanol (100%, 95%, 85%, and 75%, 3 minutes each), washed three times with PBS, and stained with Sirius Red for 1 hour. After 1 hour, the sections were washed once more with PBS, dried, and mounted with neutral resin.

[0159] 1.5.2 Results

[0160] Compared with the Oil group, mice in the CCl4 group showed obvious collagen deposition and pseudolobule formation, and semi-quantitative statistics using Image J showed significant statistical differences between the two groups (Figure 1E).

[0161] IPA-treated mice showed a significant decrease in collagen deposition and pseudolobule formation, which were statistically significant ( Figure 1E ).

[0162] 1.6 Immunohistochemical staining

[0163] 1.6.1 Method

[0164] Paraffin-embedded tissue was cut into 0.4 μm sections. The sections were dewaxed in xylene three times, each for 15 minutes. Dewaxed sections were then rehydrated with a gradient of alcohol (100%, 95%, 85%, and 75%, each for 3 minutes), followed by three washes with PBS. Antigen retrieval (acid or alkaline) was performed by boiling (300W power) for 20 minutes; after cooling naturally, the sections were washed twice with double-distilled water, and then a water-blocking circle was drawn with a water-blocking pen. The sections were washed with PBS three times, each for 5 minutes. 0.3% H₂O₂ was applied dropwise to the tissue to remove peroxidase for 10 minutes. The sections were rinsed and soaked in PBST three times, each for 5 minutes. After blocking with 10% goat serum for 1 hour, the sections were incubated with the primary antibody at 4°C overnight. The following day, the sections were washed three times with PBST and incubated with the secondary antibody for 1 hour. A DAB mixture was prepared for color development, and the reaction was stopped when brown particles appeared under a microscope. After counterstaining with hematoxylin for 20 seconds, the sections were differentiated with hydrochloric acid and alcohol for a few seconds, and then blued with warm water for a few seconds. The samples were dried and dehydrated in a 65°C oven and sealed with neutral resin.

[0165] 1.6.2 Results

[0166] The results showed that Col1a1 and Acta2 (also known as α-SMA) levels increased significantly in the interstitial tissue of mice with liver fibrosis, and that 14 days of IPA administration significantly reduced their interstitial deposition (Figure 1F). These results suggest that IPA has a significant inhibitory effect on liver cirrhosis in vivo.

[0167] 1.7 Western Blot analysis of protein levels of liver cirrhosis markers in mice

[0168] 1.7.1 Method

[0169] (1) Western Blot steps

[0170] Use 2Xlysis to lyse the tissue, mix vigorously, and place in a water bath for 15 minutes. Centrifuge at 12000rpm, room temperature for 5 minutes, take the supernatant for further BCA protein quantification, and after measuring the protein content, calculate the volume of the solution containing 80μg protein as the sample loading amount. After preparing 10% separation gel and 4% concentration gel, prepare the electrophoresis fluid (Tris (MW121.14) 3.02g, glycine (MW75.07) 14.4g, SDS1g, add distilled water to 1000ml). After adding enough electrophoresis fluid, start preparing for sample loading, the voltage is 80V, and after bromophenol blue runs to the lower layer of gel, adjust the voltage to 120V. Stop electrophoresis when bromophenol blue just runs out and transfer to the membrane. Use the wet transfer method, set a constant current of 250mA, and the transfer is completed in 90min. After transfer, transfer the membrane to a plate containing blocking solution (5% w / v skim milk) and block the membrane by shaking at room temperature for 1 hour. Dilute the primary antibody in blocking solution to the appropriate concentration in a 1.5ml centrifuge tube. Add the antibody solution dropwise to a PVF-coated glass plate, place it in a humidified chamber, and place the membrane protein-side down on the antibody solution overnight at 4°C. The next day, wash the plate twice with 0.1% PBST (Tween 20 + PBS) for 10 minutes each on a shaker at room temperature. Prepare the secondary antibody dilution and contact the membrane. After incubation at room temperature for 1 hour, wash the plate twice with PBST for 10 minutes each on a shaker at room temperature, and scan the membrane. Table 2 provides information on the antibodies used.

[0171] Table 2

[0172] 1.7.2 Results

[0173] Intraperitoneal injection of CCl4 for 8 weeks successfully increased the protein expression of Col1a1 and α-SMA in the mouse liver, and this CCl4-induced increase in Col1a1 and α-SMA protein levels could be inhibited by IPA (Figure 1G).

[0174] Therefore, IPA can significantly inhibit the key proteins Col1a1 and α-SMA in the progression of cirrhosis in mice, thereby alleviating cirrhosis.

[0175] The above results indicate that IPA can reduce inflammatory infiltration and collagen deposition in cirrhotic mice, significantly inhibit fibrosis in the body, and thus inhibit the progression of liver fibrosis to cirrhosis.

[0176] Example 2 Experimental study on IPA against ConA-induced acute autoimmune liver disease in mice

[0177] 2.1 Establishment of a mouse autoimmune liver disease model by tail vein injection of ConA

[0178] 18g C57BL / 6 female mice were divided into three groups: blank control group (n=5), model group (n=5) and IPA treatment group (n=5). The mice were injected with 10mg / kg body weight ConA through the tail vein to establish the AIH model, and the blank control group was injected with an equal dose of PBS through the tail vein. Every day for 5 days before the injection of ConA or PBS and 1 hour after the injection, the mice in the IPA treatment group were given 200mg / kg body weight IPA (dissolved in 5% CMC), and the model group and the blank control group were gavaged with an equal dose of 5% CMC. 6 hours or 18 hours after the ConA injection, the mice were killed, and the portal vein blood and inferior vena cava blood were collected. The liver was exposed and completely removed. The right liver was embedded in paraffin, and the left liver was used for RNA extraction for subsequent RT-PCR verification.

[0179] 2.2 Survival rate detection

[0180] 2.2.1 Methods

[0181] 18g female C57BL / 6 mice were divided into two groups: a model group (n=10) and an IPA-treated group (n=10). ConA (15 mg / kg body weight) was injected into the tail vein to establish an AIH model. IPA and CMC were administered to both groups at the same time and dosage as described above. Survival of the mice was observed.

[0182] 2.2.2 Results

[0183] After the tail vein injection of ConA, the mice were observed every hour for 24 h to observe their survival and status.

[0184] As shown in Figure 2, the survival rate of mice in the IPA group was 90% (10 mice, only 1 died within 24 hours), and they ate well over 24 hours. The survival rate of mice in the control ConA group was 30% (10 mice, 7 died within 24 hours), and they ate poorly over 24 hours.

[0185] The generated curve is shown in Figure 2, which shows that IPA has a protective effect on ConA-induced acute autoimmune liver disease, and the difference is statistically significant.

[0186] 2.3 ALT detection

[0187] 2.3.1 Methods

[0188] Peripheral blood was obtained by drawing blood from the mouse eyeballs. After standing overnight at 4°C, the blood was centrifuged the next day at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected. An ELISA kit (YJ063179) was used. 10 μl of serum was diluted to 50 μl with sample diluent and incubated with horseradish peroxidase-labeled antibody at 37°C for 1 hour. The plate was then washed five times. 100 μl of colorimetric solution was added to each well, incubated at 37°C in the dark for 15 minutes, and then 50 μl of stop solution was added. ALT concentration was determined using an absorbance microplate reader at 450 nm.

[0189] 2.3.2 Results

[0190] As shown in Figure 3 , the ALT level in the IPA gavage group was significantly lower than that in the ConA group, indicating that IPA can continue to exert a protective effect at both 6 and 18 h after acute inflammation.

[0191] 2.4 Verification of inflammatory markers by RT-PCR

[0192] Mouse liver tissue was obtained and ground using a grinder. 1 ml of Trizol (RNAiso Plus) was added for lysis. RNA was extracted and reverse transcribed for verification by RT-PCR. The specific steps and primer sequences are as described in 1.3.1.

[0193] 2.4.2 Results

[0194] The results are shown in Figure 4B , demonstrating that the levels of TNF-α, INF-γ, IL-6, and IL-17 in the liver of mice in the IPA group were significantly lower than those in the control ConA group.

[0195] It was demonstrated that IPA could inhibit the inflammatory response of acute autoimmune liver in mice.

[0196] 2.5 HE staining

[0197] 2.5.1 Methods

[0198] Paraffin-embedded tissue was cut into 0.4 μm sections and dewaxed in xylene three times for 15 minutes each. Dewaxed sections were then rehydrated with a gradient of ethanol (100%, 95%, 85%, and 75%, 3 minutes each), washed three times with PBS, and stained with hematoxylin for 1 minute and eosin for 30 seconds. The sections were then washed three times with PBS, dried, and mounted with neutral resin.

[0199] 2.5.2 Results

[0200] As shown in Figure 4C, compared with the CMC+PBS group, the HE staining of the livers of mice in the CMC+ConA group showed obvious liver edema and necrotic areas, as well as a large number of inflammatory cells gathered around them. After IPA treatment, the number of inflammatory cells and the area of ​​necrotic area were significantly reduced, indicating that IPA has a protective effect on ConA-induced acute liver necrosis and inflammation.

[0201] discuss

[0202] In recent years, the tryptophan metabolic pathway is a related amino acid pathway that has been widely studied in liver disease, and its metabolites are closely related to the progression of acute and chronic liver disease. In a study by Liu et al. (Indole-3-propionic Acid-aggravated CCl4-induced Liver Fibrosisvia the TGF-β1 / Smads Signaling Pathway, Journal of Clinical and Translational Hepatology 2021vol.9(6).917–930), it was found that IPA activated hepatic stellate cells (HSCs) through the TGF-β1 / Smads signaling pathway, aggravating CCl4-induced liver damage and fibrosis. In this study, Liu et al. used 20 mg / kg / d of IPA. The present invention uses 100 mg / kg / d of IPA, which has a preventive and / or therapeutic effect on cirrhosis and / or autoimmune liver disease. Different doses of IPA have different effects on liver disease.

[0203] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of an active ingredient, wherein the active ingredient is selected from indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof, characterized in that: Used for preparing a medicine or a pharmaceutical composition or a kit for preventing and / or treating liver cirrhosis caused by chronic liver damage.

2. The use according to claim 1, characterized in that The chronic liver damage is caused by chemical drugs or poisons.

3. The use according to claim 2, characterized in that The chronic liver damage is chronic liver damage caused by carbon tetrachloride.

4. The use according to claim 1, characterized in that The drug is also used for the following purposes: (y1) reducing the levels of AST and / or ALT in the liver; (y2) reduce the level of pro-inflammatory factors; (y3) Reduce collagen levels.

5. The use according to claim 1, characterized in that The medicine or pharmaceutical composition or kit is also used for treating acute autoimmune liver disease.

6. The use according to claim 5, characterized in that The acute autoimmune liver disease is an acute autoimmune liver disease caused by ConA.

7. The use according to claim 1, characterized in that The dosage of the drug is at least 100 mg / kg / d body weight according to the dosage of IPA.

8. The use according to claim 1, characterized in that The dosage form of the drug is an oral dosage form.

9. The use according to claim 1, characterized in that The pharmaceutical composition comprises: (z1) a first active ingredient, indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof; (z2) optionally, a second active ingredient, other drugs for preventing and / or treating liver cirrhosis and / or acute autoimmune liver disease; and (z3) a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that The kit comprises: (f1) a first pharmaceutical composition comprising (i) indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof as a first active ingredient; and (ii) a pharmaceutically acceptable carrier; and (f2) A reagent for detecting the level of collagen or its mRNA.

11. The use according to claim 10, characterized in that The mRNA of the collagen includes: Col1a1, Col3a1, Col6a1, or a combination thereof; the collagen includes Col1a1, Col3a1, Col6a1, or a combination thereof.

12. A method for preventing and / or treating liver cirrhosis caused by chronic liver damage, comprising the steps of: A safe and effective amount of indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is administered to a subject in need thereof.

13. The method according to claim 12, characterized in that The dosage of the indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is 50 to 500 mg / kg / day.

14. The method according to claim 12, characterized in that The dosage of the indolepropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof is 1 to 50 mg / kg / day.

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