CN117919232A - Application of indopropionic acid in treatment of autoimmune liver injury and liver cirrhosis - Google Patents

Application of indopropionic acid in treatment of autoimmune liver injury and liver cirrhosis Download PDF

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Publication number
CN117919232A
CN117919232A CN202311625949.6A CN202311625949A CN117919232A CN 117919232 A CN117919232 A CN 117919232A CN 202311625949 A CN202311625949 A CN 202311625949A CN 117919232 A CN117919232 A CN 117919232A
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liver
cirrhosis
ipa
pharmaceutically acceptable
mice
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曾欣
林勇
罗媛元
孙春燕
关亚萍
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Shanghai East Hospital Tongji University Affiliated East Hospital
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Shanghai East Hospital Tongji University Affiliated East Hospital
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Abstract

The invention relates to application of indoxyl propionic acid in liver diseases, and in particular discloses application of indoxyl propionic acid or pharmaceutically acceptable salt or derivative thereof in preparation of medicines or medicine compositions or kits for preventing and/or treating liver cirrhosis and acute autoimmune liver diseases caused by chronic liver injury.

Description

Application of indopropionic acid in treatment of autoimmune liver injury and liver cirrhosis
Technical Field
The invention relates to the field of biological medicine. In particular, the invention relates to the use of indolepropionic acid (IPA) in liver diseases.
Background
Cirrhosis is a clinically common chronic progressive liver disease, a diffuse liver lesion formed by long-term or repeated actions of one or more etiologies. Most of the liver cirrhosis is posthepatitic cirrhosis in China, and the small part is alcoholic cirrhosis and schistosome cirrhosis. Histopathology is characterized by extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue hyperplasia and fibrous septa, resulting in destruction of liver lobule structure and formation of prosthetic lobules, which progress to liver fibrosis, and even cirrhosis. Liver cirrhosis is characterized by collagen deposition, the generation of prosthetic leaflets as its core pathological feature, and inflammation and immune abnormality of the liver are important in the course of liver cirrhosis.
Cirrhosis is an important intermediate link for the development of chronic liver diseases into liver cancer, and the number of people suffering from liver cirrhosis is huge in China, which is a high-incidence country of chronic liver diseases, and patients suffering from liver cirrhosis have long disease course, more complications and poor prognosis, and no reversed treatment scheme exists at present.
Autoimmune liver disease (ALD LIVER DISEASE) is a special group of chronic liver diseases caused by immune dysfunction in the body. Different types of autoimmune liver diseases vary in their demographic characteristics, clinical manifestations, and pathological changes of the liver. The mechanism of action and therapeutic action of autoimmune liver disease are not clear.
Accordingly, there has been an urgent development in the art of an effective treatment or prevention of liver cirrhosis and autoimmune liver disease.
Disclosure of Invention
The present invention aims at providing a new method of treating liver cirrhosis and/or acute autoimmune liver disease caused by chronic liver injury.
It is another object of the present invention to provide the use of indomethacin for the treatment or prevention of cirrhosis of the liver and/or acute autoimmune liver disease caused by chronic liver injury.
In a first aspect of the present invention there is provided the use of an active ingredient selected from the group consisting of indolopropionic acid, or a pharmaceutically acceptable salt or derivative thereof, for the manufacture of a medicament or pharmaceutical composition or kit for the prevention and/or treatment of a disease selected from the group consisting of:
(a) Cirrhosis caused by chronic liver injury;
(b) Acute autoimmune liver disease.
In another preferred embodiment, the chronic liver injury is a chemical or poison induced chronic liver injury.
In another preferred embodiment, the chronic liver injury is a chronic liver injury caused by carbon tetrachloride.
In another preferred embodiment, the medicament is also for the following uses:
(y 1) decreasing serum AST and/or ALT levels;
(y 2) decrease the level of pro-inflammatory factors;
(y 3) decrease collagen levels.
In another preferred embodiment, the acute autoimmune liver disease is an acute autoimmune liver disease caused by ConA.
In another preferred embodiment, the amount of drug administered is at least 100mg/kg/d body weight as IPA.
In another preferred embodiment, the pharmaceutical dosage form is an oral dosage form.
In another preferred embodiment, the collagen mRNA comprises: col1a1, col3a1, col6a1, or combinations thereof; the collagen comprises Col1a1, col3a1, col6a1, or a combination thereof.
In another preferred embodiment, the medicament is administered to a subject selected from the group consisting of: rodents or primates.
In another preferred embodiment, the subject is a subject who is not a high fat diet.
In another preferred embodiment, the subject is a non-diabetic patient.
In another preferred embodiment, the subject does not suffer from any of the following diseases:
(x 1) alcoholic fatty liver disease;
(x 2) non-alcoholic fatty liver disease.
In another preferred embodiment, the pro-inflammatory factor is selected from the group consisting of: TNF- α, INF- γ, IL-6, IL-17, or combinations thereof.
In another preferred embodiment, the pharmaceutical composition comprises:
(z 1) a first active ingredient, indopropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof;
(z 2) optionally, a second active ingredient, other agents for preventing and/or treating cirrhosis and/or acute autoimmune liver disease; and
(Z 3) a pharmaceutically acceptable carrier.
In another preferred embodiment, the component (z 1) comprises 1 to 99wt%, preferably 10 to 90wt%, more preferably 30 to 70wt% of the total weight of the pharmaceutical composition.
In another preferred embodiment, the other agent for preventing and/or treating cirrhosis and/or acute autoimmune liver disease is selected from the group consisting of: interferons, nucleotide analogs, nitrates, calcium channel blockers, or combinations thereof.
In another preferred embodiment, the interferon comprises a normal interferon or a long-acting interferon.
In another preferred embodiment, the nucleotide analog comprises lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir dipivoxil, clavulanate, or a combination thereof.
In another preferred embodiment, the nitrate esters include cardiodynia.
In another preferred embodiment, the calcium channel blocker comprises a cardioversion.
In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of compounds.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament or preparation for treating or preventing cirrhosis and/or acute autoimmune liver disease caused by chronic liver injury.
In another preferred embodiment, the pharmaceutical dosage form is an oral or non-oral dosage form.
In another preferred embodiment, the oral administration form is a tablet, powder, granule or capsule, or an emulsion or syrup.
In another preferred embodiment, the non-oral administration form is an injection or an injection.
In another preferred embodiment, the pharmaceutical composition is selected from the group consisting of: injection, inhalant, tincture, powder, granule, capsule, oral liquid, tablet, pill, suspension, emulsion, buccal tablet, or dripping pill.
In another preferred embodiment, the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.
In another preferred embodiment, the pharmaceutical composition is administered to a human or non-human mammal.
In another preferred embodiment, the kit comprises:
(f1) A first pharmaceutical composition comprising (i) indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof, as a first active ingredient; and (ii) a pharmaceutically acceptable carrier; and
(F2) An agent for detecting collagen or mRNA levels thereof.
In another preferred embodiment, the collagen comprises: col1a1, col3a1, col6a1, or combinations thereof.
In another preferred embodiment, the collagen comprises Col1a1.
In another preferred embodiment, the detection of collagen is detection of Col protein content, or activity thereof, or a combination thereof.
In another preferred embodiment, the indolepropionic acid is enterogenic indolepropionic acid.
In another preferred embodiment, the indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof, is used for one or more uses selected from the group consisting of:
(a1) Lowering the expression level of RNA of TNF- α, INF- γ, IL-6, IL-17, col1a1, col3a1, col6a1 or α -SMA in the liver;
(a2) Reducing inflammatory infiltration of liver, and inhibiting increase of autoimmune liver inflammation;
(a3) Reducing collagen deposition or pseudo-lobule formation in the liver;
(a4) Inhibiting liver cirrhosis;
(a5) Inhibit protein expression of Col1a1 and α -SMA;
(a6) Decrease the concentration of ALT and/or AST in serum.
In a second aspect of the present invention, there is provided a method for preventing and/or treating liver cirrhosis caused by chronic liver injury or autoimmune liver disease caused by acute liver injury, comprising the steps of:
Administering to a subject in need thereof a safe and effective amount of indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof.
In another preferred embodiment, the administration comprises oral administration.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent and primate, preferably a mouse, rat, rabbit, monkey.
In another preferred embodiment, the indomethacin, or a pharmaceutically acceptable salt thereof, or derivative thereof, is administered at a frequency of 1 to 7 consecutive days per week, preferably 2 to 5 consecutive days per week, more preferably 2 to 3 consecutive days per week.
In another preferred embodiment, the indomethacin, or a pharmaceutically acceptable salt thereof, or derivative thereof, is administered for a period of time ranging from 1 to 20 weeks, preferably from 2 to 12 weeks, more preferably from 4 to 8 weeks.
In another preferred embodiment, the indomethacin, or a pharmaceutically acceptable salt thereof, or derivative thereof, is administered at a dosage of 50 to 500 mg/kg/day, preferably 80 to 250 mg/kg/day, for example 100 mg/kg/day.
In another preferred embodiment, the indomethacin, or a pharmaceutically acceptable salt thereof, or derivative thereof, is administered at a dosage of 1 to 50 mg/kg/day, preferably 5 to 20 mg/kg/day, for example 8.1 mg/kg/day.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1A shows a general diagram of the liver of mice in which IPA reduced CCl 4 -induced liver cirrhosis.
FIG. 1B shows that Elisa detects IPA in portal venous blood of mice with IPA-reduced CCl 4 -induced cirrhosis.
FIG. 1C shows that RT-PCR detection of IPA reduces RNA level expression of Col1a1, col3a1, col6a1, α -SMA in CCl 4 -induced liver cirrhosis mice.
FIG. 1D shows the results of IPA-reduced CCl 4 -induced HE staining of liver tissue of mice.
FIG. 1E shows the results of IPA-reduced CCl 4 -induced sirius red staining of liver tissue of mice.
FIG. 1F shows the results of immunohistochemical staining of liver tissue in mice induced by IPA-reduced CCl 4.
FIG. 1G shows the Western Blot results of IPA alleviating CCl 4 -induced liver cirrhosis in mice.
Figure 2 shows the survival of IPA treated ConA-induced acute autoimmune liver disease mice.
Fig. 3 shows liver function (ALT) in IPA-treated ConA-induced acute autoimmune liver disease mice, a: 6 hours after ConA injection, B: 18 hours after ConA injection.
Fig. 4A shows a general diagram of the liver of IPA treated ConA-induced liver inflammation in acute autoimmune liver disease mice.
FIG. 4B shows RT-PCR detection of RNA level expression of TNF- α, IFN- γ, IL-6, IL-17 in mice with IPA-treated ConA-induced acute autoimmune liver disease.
Fig. 4C shows the liver tissue HE staining results of IPA-treated ConA-induced acute autoimmune liver disease mice.
Detailed Description
The present inventors have conducted extensive and intensive studies and, for the first time, unexpectedly found that indomethacin is effective in preventing and/or treating cirrhosis and/or acute autoimmune liver disease caused by chronic liver injury. On this basis, the present inventors have completed the present invention.
Terminology
As used herein, the terms "pharmaceutical composition of the invention", "medicament of the invention" are used interchangeably and refer to a formulation or pharmaceutical composition containing IPA, or a pharmaceutically acceptable salt thereof, or a derivative thereof, as an active ingredient.
As used herein, the terms "formulation combination", "formulation combination of the invention", "medicament of the invention" are used interchangeably and refer to a medicament or pharmaceutical composition or kit according to the first aspect of the invention.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …", or "consisting of …".
As used herein, the terms "subject," "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, goats.
Cirrhosis of the liver
Cirrhosis is an important intermediate link in the development of chronic liver disease into liver cancer. Cirrhosis is a pathophysiological process, an important stage of the development of various chronic liver diseases to liver cancer. The number of people suffering from liver cirrhosis is huge in China, the period of the disease is long, the complications are more, the prognosis is poor, and no reversed treatment scheme exists at present. Therefore, the method for treating liver cirrhosis is very important for preventing and treating liver cancer in early stage. Inflammation and immune abnormality of the liver are important in the course of cirrhosis. Research shows that the metabolic products of intestinal flora are absorbed into blood through intestinal tracts, circulate to portal veins and enter the liver, and can directly influence the inflammatory reaction of the liver.
Indolopropionic acid
Indolopropionic acid (indole-3-propionic acid, indole-3-propionic acid, IPA, molecular formula C 11H11 NO) is an important end product of tryptophan metabolism in indole products. Indolopropionic acid is a plant growth regulator with biological activity of auxin, and is mainly used as a medical intermediate and plant growth hormone.
Tryptophan is an essential amino acid, is the only amino acid containing indole structure in human body, and is rich in oat, milk, yoghurt, cheese, red meat, eggs, fish and the like. Tryptophan can be metabolized by intestinal flora to produce indole products, so that the steady-state balance of the organism is regulated. In recent years, important regulatory roles have been played in intestinal and liver diseases. And an increase in the instability of the gut microbiota, pathogen abundance, is associated with an increased susceptibility to various metabolic liver diseases.
The present invention finds that indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof, is capable of inhibiting liver fibrosis, alleviating liver cirrhosis, inhibiting the progression of liver cirrhosis or inhibiting inflammation of the acute autoimmune liver.
Pharmaceutical composition
The pharmaceutical compositions provided by the invention preferably contain 0.1 to 99wt% of the first active ingredient, the remainder being the second active ingredient, a pharmaceutically acceptable carrier, a diluent or solution or a salt solution.
The first active ingredient of the invention is indopropionic acid or pharmaceutically acceptable salt or derivative thereof. In addition, it may also be used in combination with other therapeutic agents, i.e. the second active ingredient.
The second active ingredient may be any pharmaceutical ingredient capable of preventing and/or treating cirrhosis and/or autoimmune liver disease, including, but not limited to, interferons, nucleotide analogs, nitrates, calcium channel blockers, and the like.
If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.
The compounds and pharmaceutical compositions provided herein may be in a variety of forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, and may be presented in a suitable solid or liquid carrier or diluent and in a suitable sterilizing device for injection or infusion.
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 dosage unit of the formulation generally comprises from 0.05 to 1000mg of the active compound of the invention, preferably from 1mg to 500mg of the active compound of the invention.
The pharmaceutical compositions of the present invention may be used clinically in mammals, including humans and animals, by oral, nasal, dermal, pulmonary or gastrointestinal routes of administration. Most preferably orally. Most preferably, the daily dosage is 0.01-400mg/kg body weight, and the medicine is administered once or in divided doses of 0.01-200mg/kg body weight. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Typically starting from a small dose, the dose is gradually increased until the most suitable dose is found.
The agents or inhibitors of the invention may be administered by a variety of different means, for example, by injection, spraying, nasal drops, eye drops, permeation, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped by other materials and introduced into the body.
Typically, the active ingredient of the present invention or pharmaceutical compositions containing it may be administered in unit dosage form by the enteral or parenteral route, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, ocular, pulmonary and respiratory routes, skin, vaginal, rectal and the like.
The dosage form may be a liquid, solid or semi-solid dosage form. The liquid preparation can be solution (including true solution and colloid solution), emulsion (including O/W type, W/O type and multiple emulsion), suspension, injection (including water injection, powder injection and transfusion), eye drop, nasal drop, lotion, liniment, etc.; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like.
The active ingredients of the invention can be prepared into common preparations, slow-release preparations, controlled-release preparations, targeted preparations and various microparticle administration systems.
For the preparation of the active ingredient according to the invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrating agent can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.
The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.
In order to make the administration unit into a capsule, the active ingredient of the present invention may be mixed with a diluent, a glidant, and the mixture may be directly placed in a hard capsule or a soft capsule. Or mixing the effective components with diluent, binder, and disintegrating agent, granulating or micropill, and making into hard capsule or soft capsule. The various diluents, binders, wetting agents, disintegrants and glidants used in the preparation of the tablets of the invention may also be used in the preparation of the capsules of the invention.
For preparing the active ingredient of the present invention into injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof may be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator, osmotic pressure regulator commonly used in the art may be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.
In addition, colorants, preservatives, fragrances, flavoring agents, or other additives may also be added to the pharmaceutical formulation, if desired.
The active ingredients or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.
When the active ingredient of the present invention has a synergistic effect with other therapeutic agents, its dosage should be adjusted according to the actual situation.
The main advantages of the invention include:
the invention provides application of indopropionic acid or pharmaceutically acceptable salt or derivative thereof in treating liver cirrhosis and acute autoimmune liver diseases caused by chronic liver injury, and the application has the following advantages:
(1) The indopropionic acid is the final product of tryptophan metabolism indole product, and is safe and effective.
(2) The administration of the indomethacin can effectively reduce the RNA expression level of Col1a1, col3a1, col6a1 and alpha-SMA, inhibit Col1a1 and alpha-SMA proteins, and reduce inflammatory infiltration and collagen deposition, thereby reducing hepatic fibrosis and liver cirrhosis caused by chronic liver injury and inhibiting the progress of liver cirrhosis.
(3) The administration of indomethacin can inhibit inflammatory reaction of autoimmune liver disease, and has protective effect on acute autoimmune liver disease.
(4) The invention provides a novel method for treating liver cirrhosis caused by chronic liver injury and acute autoimmune liver diseases.
(5) The method has the advantages of simple operation, simple preparation of the medicine and high efficiency.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
Example 1 mouse experiment with CCl 4 -induced liver cirrhosis with IPA
1.1 Mouse CCl 4 -induced liver cirrhosis model and materials
1.1.1 Methods
The C57BL/6 mice were divided into three groups, one group was intraperitoneally injected with olive oil as a control group, and the other two groups were intraperitoneally injected with CCl 4(0.5ml/kg,CCl4:olive oil=1:9, 2 times per week), and the total molding was performed for eight weeks. On the sixth weekend of molding, IPA was dissolved with 5% CMC, and one group of CCl 4 molding groups (hereinafter referred to as CCl 4 +IPA 100) was subjected to intragastric administration at a concentration of 100mg/kg, and the control group CCl 4 and olive Oil group were subjected to intragastric administration with 5% CMC (hereinafter referred to as Oil group and CCl 4 group) for a total of 2 weeks. Eighth week, mice were sacrificed, portal venous blood and inferior vena cava blood were removed, and the liver was exposed and removed intact. The right liver is embedded in paraffin, and the left liver is used for extracting RNA and protein for subsequent RT-PCR and Western blot verification.
1.1.2 Results
Mice were randomly divided into 3 groups by setting up a mouse liver cirrhosis model using cci 4 intraperitoneal injection: olive oil control module (fig. 1A, oil group), CMC control solvent gavage+ccl 4 group (fig. 1A, CCl 4 group), IPA-100mg/kg gavage+ccl 4 group (fig. 1A, CCl 4 +ipa100), five each.
1.2Elisa assay of the concentration of mouse serum IPA
1.2.1 Method
After the collected portal blood was left overnight at 4 ℃, the supernatant was collected by centrifugation at 3000rpm at 4℃for 15 minutes. Using an enzyme-linked biological IPA Elisa kit (YJ 416125), 10. Mu.l of serum was diluted to 50. Mu.l with a sample dilution, incubated with horseradish peroxidase-labeled antibody at 37℃for 1h, and the plate was washed 5 times. 100 μl of the color development liquid was added to each well, incubated at 37℃for 15min in the absence of light, and 50 μl of the stop liquid was added thereto, and the IPA concentration was measured by reading at 450nm with a absorbance photometer.
1.2.2 Results
Mice portal venous blood serum IPA concentration was measured with the Elisa kit.
The results showed that the serum concentration of IPA was reduced in the cci 4 mice compared to the Oil group and there was a statistical difference (p < 0.001), indicating a downward trend in portal serum of the liver cirrhosis mice.
Compared with CCl 4 mice, the total serum IPA concentration of CCl 4 +IPA100 mice is obviously increased, which indicates that IPA-100mg/kg lavage can improve the serum IPA level of the mice (FIG. 1B).
1.3Real time-PCR (RT-PCR) detection of changes in liver cirrhosis index of mouse liver
1.3.1 Method
(1) RNA extraction
1Ml Trizol (RNAiso Plus) was added to the tissue and allowed to stand for 5 minutes, then the tissue was blown with a pipette and collected in an imported Eppendorf centrifuge tube. 0.2ml chloroform (one fifth of the volume of Trizol) was added, capped, and shaken well for 15s until it was well emulsified. The temperature was set at room temperature for 5min (at this time, the centrifuge was set at 4 ℃ C., 12,000g was prepared). Centrifuge at 4℃for 15min at 12,000 g. Taking the upper transparent belt, adding 0.4ml isopropanol (equal volume with the supernatant sucked out from the upper part), slowly mixing up and down for 5 times, and standing at room temperature for 10min. Centrifugation was carried out at 12,000g for 10min at 4℃and the bottom was visually precipitated and the supernatant was discarded.
1Ml of 75% ethanol (DEPC water) was gently added, the RNA pellet was sprung, rinsed, centrifuged at 7,500g for 5min at 4℃to remove the supernatant as thoroughly as possible, and the RNA pellet was prevented from being lost. The EP tube is inverted, the tube wall is dried, 30 mu l of DEPC water is used for dissolving the dried RNA, the OD260/280 value is measured by a spectrophotometer, the working concentration (1 mu g/. Mu.l) is prepared, and the RNA integrity is detected by 1% agarose gel electrophoresis and is reserved at-80 ℃.
(2) Reverse transcription reaction
Reverse transcription Using TAKARA kit, 10. Mu.l system reversed 1000ng RNA, 1ug,20ul system reversed, and reverse transcription kit reagents 1,2,3 were used. Each RNA concentration was measured and the amount required for 1. Mu.g of RNA was calculated. Taking common eight-link PCR tubes, and respectively adding the eight-link PCR tubes into a system: 5*Primescript PT Enzyme MIX (4 ul), total RNARNASE FREE DD H 2 O (up to 20. Mu.l). Reverse transcription was performed on a conventional PCR apparatus at 37℃for 15 minutes, 85℃for 30 seconds, and 4℃for a forever.
(3)real time-PCR
1.5Ml EP tube was used as MIX with 2X TB Green, primer, ddH 2 O. 10ul system: TB Green (5 ul), primer (0.3 ul each in front and back), ddH 2 O (up to 6 ul). MIX was dispensed into real-time plates, 6ul per well, and run on ice.
And adding 4ul of corresponding diluted templates into each hole, and then loading the machine.
(4) Primer sequences
Table 1 shows the primer sequences used for RT-PCR.
TABLE 1
1.3.2 Results
As can be seen from the RT-PCR experiments, the CCl 4 mice exhibited higher levels of RNA levels of Col1a1, col3a1, col6a1 and α -SMA than the Oil group, indicating that CCl 4 was successful in modeling mouse liver cirrhosis (FIG. 1C).
Compared to CCl 4 mice, the RNA levels of Col1a1, col3a1, col6a1 and α -SMA were significantly reduced in CCl 4 +ipa100 mice (fig. 1C), indicating that IPA has an in vivo inhibitory effect on liver cirrhosis in mice.
1.4HE staining
1.4.1 Method
Paraffin embedded tissue was cut into 0.4 μm sections, followed by 3 xylene dewaxes for 15 minutes each. The dewaxed sections were sequentially subjected to gradient alcohol hydration (100%, 95%,85%,75%, 3 minutes each) and washed three times with PBS, then stained with hematoxylin for 1 minute, eosin for 30s. And (3) washing the PBS for three times, drying and sealing the neutral resin.
1.4.2 Results
After CCl 4 molding (compare Oil group), the liver of mice showed obvious infiltration of obvious inflammation, while IPA gavage for 14 days could significantly reduce the infiltration of inflammation caused by CCl 4 molding (FIG. 1D).
1.5 Detection of liver collagen deposition by sirius red staining
1.5.1 Method
Paraffin-embedded tissue was cut into 0.4 μm sections. The sections were subjected to 3 xylene dewaxed times, 15 minutes each. The dewaxed sections were once subjected to gradient alcohol hydration (100%, 95%, 85%, 75% for 3 minutes each) and washed three times with PBS and stained with sirius red drops for 1h. After 1h, PBS is washed once, dried and the neutral resin is sealed.
1.5.2 Results
In comparison to Oil group, CCl 4 mice showed significant collagen deposition, as well as false lobule formation, and semi-quantitative statistics with image j showed significant statistical differences between the two groups (fig. 1E).
IPA treated mice were seen with a clear reduction in collagen deposition, reduced pseudo-leaflet formation, and statistical differences after statistics (fig. 1E).
1.6 Immunohistochemical staining
1.6.1 Method
Paraffin-embedded tissue was cut into 0.4 μm sections. The sections were subjected to 3 xylene dewaxed times, 15 minutes each. Dewaxed sections were once subjected to gradient alcohol hydration (100%, 95%, 85%, 75% for 3 minutes each) and then washed 3 times with PBS. Antigen retrieval (acid retrieval or alkali retrieval) is boiled (300 w power) for 20 minutes; after natural cooling, washing for 2 times by double steaming water, and then using water-blocking strokes to block the water ring. The PBS was washed 3 times for 5 minutes each. 0.3% H 2O2 was added dropwise to the tissue to remove peroxidase from the tissue for 10 minutes. PBST was rinsed and soaked 3 times for 5 minutes each. After blocking with 10% goat serum for 1h, incubation with primary antibody was carried out overnight at 4 ℃. After 3 times of PBST washes on the following day, the incubation with the secondary antibody was performed for 1h. And preparing DAB mixed solution for developing color, and stopping the reaction when brown particles appear under a microscope. After 20S counterstaining with hematoxylin, the alcohol hydrochloride differentiated for several seconds and the warm water reversed for several seconds. Drying and dehydrating in a drying oven at 65 ℃, and sealing the neutral resin.
1.6.2 Results
The results showed that Col1a1 and Acta2 (alias: α -SMA) significantly increased in the interstitium after liver fibrosis in mice, whereas deposition of both in the interstitium was significantly reduced 14 days after IPA administration (FIG. 1F). Taken together, the results indicate that IPA has obvious effect of inhibiting liver cirrhosis in vivo.
1.7Western Blot detection of protein level changes in liver cirrhosis index of mouse liver
1.7.1 Method
(1) WesternBlot procedure
The tissue was lysed with 2Xlysis, vigorously mixed and then water-bath for 15 minutes. 12000rpm, centrifuging at normal temperature for 5 minutes, taking the supernatant for further BCA protein quantification, and calculating the volume of the solution containing 80 mug protein after measuring the protein content to obtain the sample loading amount. After preparing 10% of the separation gel and 4% of the concentration gel, an electrophoresis solution (Tris (MW 121.14) 3.02g, glycine (MW 75.07) 14.4g, SDS1g, distilled water to 1000 ml) was prepared. And after enough electrophoresis liquid is added, the sample is prepared, the voltage is 80V, and after bromophenol blue runs to the lower layer glue, the voltage is adjusted to 120V. And (5) stopping electrophoresis until the bromophenol blue just runs out, and performing transfer. And setting constant current 250mA by a wet rotating method, and finishing film rotating after 90 min. After the transfer, the membranes were transferred to a dish containing a blocking solution (5% w/v skim milk) and blocked by shaking on a decolorizing shaker at room temperature for 1h. Diluting the primary antibody with blocking solution to the appropriate concentration (in a 1.5ml centrifuge tube); the antibody solution was added drop wise to PVF coated glass plates, placed in a wet box and placed with the membrane protein side down on the antibody liquid surface at 4℃overnight. The next day, wash twice with 0.1% pbst (Tween 20+ PBS) at room temperature on a decolorizing shaker for 10min each. The secondary antibody dilutions were prepared and contacted with the membrane, after incubation for 1h at room temperature, washed twice with PBST on a decolorizing shaker at room temperature for 10min each time, and the membrane was swept. Table 2 shows information on the antibodies used.
TABLE 2
1.7.2 Results
The 8 week intraperitoneal injection of cci 4 successfully increased the protein expression of Col1a1 and α -SMA in the mouse liver, while this increase in Col1a1 and α -SMA protein levels caused by cci 4 was inhibited by IPA (fig. 1G).
IPA can thus significantly inhibit the key proteins Col1a1 and α -SMA in the progression of liver cirrhosis in mice, thereby alleviating liver cirrhosis.
The results show that IPA can reduce inflammatory infiltration and collagen deposition on liver cirrhosis mice, and obviously inhibit in-vivo fibrosis, so that liver fibrosis is inhibited from progressing to liver cirrhosis.
EXAMPLE 2IPA experiment on ConA-induced acute autoimmune liver disease in mice
2.1ConA tail intravenous injection to establish a model of autoimmune liver disease in mice
Female mice of 18g c57bl/6 were divided into three groups, a blank group (n=5), a model group (n=5) and an IPA-treated group (n=5). Mice were given 10mg/kg body weight of ConA tail intravenous to establish an AIH model, and a blank group was given an equivalent dose of PBS tail intravenous. Mice in the IPA-treated group were given 200mg/kg body weight IPA (dissolved in 5% CMC) 5 days before and 1 hour after ConA or PBS injection, and the model group and the blank group were given equal doses of 5% CMC for gastric lavage. Mice were sacrificed 6 hours or 18 hours from ConA injection, portal venous blood and inferior vena cava blood were removed, and the liver was exposed and removed intact. The right liver was paraffin-embedded and the left liver was RNA-extracted for subsequent RT-PCR validation.
2.2 Survival detection
2.2.1 Methods
18G of C57BL/6 female mice were divided into two groups, a model group (n=10) and an IPA treatment group (n=10), and 15mg/kg body weight ConA tail intravenous injection was given to the mice to establish an AIH model. Two groups of mice were given IPA and CMC at the same time and dose as described above. Mice survival status was observed.
2.2.2 Results
After tail vein injection of ConA, mice were observed once per hour, up to 24h, and survival and status of the mice were observed.
As shown in fig. 2, mice in the gavage IPA group had a survival rate of 90% (only 1 death within 10, 24 hours) and a good 24-hour feeding. The 24h survival rate of control ConA mice was 30% (10, 7 dying within 24 h) and feeding was poor.
The resulting curve is shown in FIG. 2, where IPA is protective against ConA-induced acute autoimmune liver disease and is statistically different.
2.3ALT detection
2.3.1 Methods
The peripheral blood of the mice was obtained by taking blood from eyeballs of the mice, and after leaving the peripheral blood at 4℃overnight, the supernatant was collected by centrifugation at 3000rpm at 4℃for 15 minutes. An enzyme-linked biological ALT Elisa kit (YJ 063179) was used. Mu.l of serum was diluted to 50. Mu.l with a sample dilution, incubated with horseradish peroxidase-labeled antibody at 37℃for 1h, and the plate was washed 5 times. 100 μl of the color development solution was added to each well, incubated at 37℃for 15min in the absence of light, and then 50 μl of the stop solution was added, and the ALT concentration was measured by reading at 450nm with a absorbance photometer.
2.3.2 Results
As shown in fig. 3, ALT was significantly lower in IPA lavage group than in ConA group, indicating that IPA continued to exert protective effect at both 6h and 18h of acute inflammation.
2.4RT-PCR inflammation index verification
Mouse liver tissue was taken, ground by a grinder, 1ml Trizol (RNAiso Plus) was added for cleavage, RNA was extracted, and reverse transcription was performed to perform RT-PCR verification. Specific steps and primer sequences are as 1.3.1.
2.4.2 Results
The results are shown in FIG. 4B, which demonstrates that the liver of mice in the IPA group is significantly lower in TNF- α, INF- γ, IL-6, and IL-17 than in the control ConA group.
IPA proved to suppress the inflammatory response of the acute autoimmune liver in mice.
2.5HE staining
2.5.1 Methods
Paraffin embedded tissue was cut into 0.4 μm sections, followed by 3 xylene dewaxes for 15 minutes each. The dewaxed sections were sequentially subjected to gradient alcohol hydration (100%, 95%,85%,75% for 3 minutes each) and washed three times with PBS, then stained with hematoxylin for 1 minute, eosin for 30s. And (3) washing the PBS for three times, drying and sealing the neutral resin.
2.5.2 Results
As shown in fig. 4C, in comparison to cmc+pbs group, significant liver edema and necrotic areas and aggregation of a large number of inflammatory cells around the liver HE staining occurred in cmc+cona group mice, and the reduction of inflammatory cells and the significant reduction of necrotic areas after IPA treatment was administered, indicating that IPA has a protective effect on acute necrosis and inflammation of liver caused by ConA.
Discussion of the invention
In recent years, tryptophan metabolic pathway is a relatively studied amino acid pathway in liver diseases, and its metabolic products are closely related to the progress of acute and chronic liver diseases. In 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), found that IPA activated hepatic stellate cells (HEPATIC STELLATE CELLS, HSCs) through the TGF-. Beta.1/Smads signaling pathway, exacerbating CCl 4 -induced liver injury and fibrosis. In this study, liu et al used 20mg/kg/d IPA. In the present invention, 100mg/kg/d of IPA is used, which has preventive and/or therapeutic effects on liver cirrhosis and/or autoimmune liver diseases. Different doses of IPA produced different effects on liver disease.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. Use of an active ingredient selected from the group consisting of indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof, for the preparation of a medicament or a pharmaceutical composition or kit for the prevention and/or treatment of a disease selected from the group consisting of:
(a) Cirrhosis caused by chronic liver injury;
(b) Acute autoimmune liver disease.
2. The use according to claim 1, wherein the chronic liver injury is a chemical or poison induced chronic liver injury.
3. The use of claim 2, wherein the chronic liver injury is a chronic liver injury caused by carbon tetrachloride.
4. The use according to claim 1, wherein the medicament is further for the following use:
(y 1) decreasing AST and/or ALT levels in the liver;
(y 2) decrease the level of pro-inflammatory factors;
(y 3) decrease collagen levels.
5. The use according to claim 1, wherein the acute autoimmune liver disease is an acute autoimmune liver disease caused by ConA.
6. The use according to claim 1, wherein the medicament is administered in an amount of at least 100mg/kg/d body weight as IPA.
7. The use according to claim 1, wherein the pharmaceutical dosage form is an oral dosage form.
8. The use according to claim 1, wherein the pharmaceutical composition comprises:
(z 1) a first active ingredient, indopropionic acid, or a pharmaceutically acceptable salt thereof, or a derivative thereof;
(z 2) optionally, a second active ingredient, other agents for preventing and/or treating cirrhosis and/or acute autoimmune liver disease; and
(Z 3) a pharmaceutically acceptable carrier.
9. The use according to claim 1, wherein the kit comprises:
(f1) A first pharmaceutical composition comprising (i) indolepropionic acid, or a pharmaceutically acceptable salt or derivative thereof, as a first active ingredient; and (ii) a pharmaceutically acceptable carrier; and
(F2) An agent for detecting collagen or mRNA levels thereof.
10. The use of claim 9, wherein the collagen mRNA comprises:
Col1a1, col3a1, col6a1, or combinations thereof; the collagen comprises Col1a1, col3a1, col6a1, or a combination thereof.
CN202311625949.6A 2023-11-30 2023-11-30 Application of indopropionic acid in treatment of autoimmune liver injury and liver cirrhosis Pending CN117919232A (en)

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