Use of ethyl lactate for liver damage and alcohol-associated liver disease

Abstract

A use of ethyl lactate in the prevention and treatment of liver damage and / or alcohol-associated liver disease. Ethyl lactate inhibits alcohol-induced lipid synthesis, and regulates liver metabolism levels by means of increasing SIRTI and FGF21 activity, thereby alleviating alcohol-associated liver disease.

Description

Use of ethyl lactate in liver injury and alcohol-associated liver disease TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to use of ethyl lactate in liver injury and alcohol-associated liver disease. BACKGROUND

[0002] With the increasing global alcohol consumption, the global burden of alcohol-related diseases is also increasing year by year, especially alcohol-related liver diseases. More than 90% of people with long-term chronic alcohol consumption will develop alcohol-related fatty liver, and with long-term chronic alcohol consumption or occasional heavy drinking, it will gradually progress to alcohol-related fatty hepatitis, fibrosis, cirrhosis, etc. At the same time, alcohol-associated liver disease (ALD) is a major factor in the incidence and death of chronic liver disease. So far, there is no drug approved by the U.S. Food and Drug Administration for the treatment of ALD.

[0003] Therefore, there is an urgent need in the art to provide new drugs or methods for improving alcoholic liver injury or treating ALD. SUMMARY

[0004] The purpose of the present application is to provide a drug or a pharmaceutical composition for improving alcoholic liver injury or treating ALD.

[0005] In a first aspect of the present application, the use of ethyl lactate or a pharmaceutically acceptable salt thereof for the preparation of a medicament or a pharmaceutical composition for one or more uses selected from the group consisting of:

[0006] (a1) preventing or treating liver injury;

[0007] (a2) preventing or treating alcohol-associated liver disease;

[0008] (a3) SIRT1 agonist;

[0009] (a4) FGF21 agonist;

[0010] (a5) improving hangover.

[0011] In another preferred embodiment, the alcohol-associated liver disease comprises chronic alcohol-associated liver disease, acute alcohol-associated liver disease, or acute-on-chronic mixed alcohol-associated liver disease.

[0012] In another preferred embodiment, the alcohol-associated liver disease is selected from the group consisting of liver injury, liver steatosis, alcoholic steatohepatitis, liver oxidative stress, alcoholic fatty liver, or a combination thereof.

[0013] In another preferred embodiment, the FGF21 agonist increases the activity of FGF21 in hepatocytes.

[0014] In another preferred embodiment, the agonist of SIRT1 increases the activity of SIRT1 in hepatocytes.

[0015] In another preferred embodiment, the medicament or pharmaceutical composition is further used for a purpose selected from the group consisting of:

[0016] (a1) ameliorating liver injury

[0017] (a2) ameliorating liver fat deposition;

[0018] (a3) an inhibitor of alcohol-related liver disease inflammatory factor;

[0019] (a4) inhibiting alcohol-induced lipid synthesis;

[0020] (a5) an inhibitor of lipid synthesis-related factor.

[0021] In another preferred embodiment, the alcohol-related liver disease inflammatory factor is selected from the group consisting of IL-1 β, IL-6, MCP1, ICAM1, CD11b, or a combination thereof.

[0022] In another preferred embodiment, the lipid synthesis-related factor is selected from the group consisting of SREBP-1c, ACC1, FAS, SCD1, DGAT1, ASCL4, or a combination thereof.

[0023] In another preferred embodiment, the medicament or pharmaceutical composition is administered to a subject selected from the group consisting of a mammal or a rodent.

[0024] In another preferred embodiment, the subject has one or more characteristics selected from the group consisting of:

[0025] (c1) liver injury

[0026] (c2) long-term drinking of alcohol;

[0027] (c3) suffering from alcohol-related liver disease;

[0028] (c4) being drunk or suffering from hangover;

[0029] (c5) high expression of alcohol-related liver disease inflammatory factor;

[0030] (c6) high expression of lipid synthesis-related factor;

[0031] (c7) high content of fatty acid synthase.

[0032] In another preferred embodiment, the high expression means that the relative expression level of mRNA (Z1) of the alcohol-related liver disease inflammatory factor or lipid synthesis-related factor is ≥1.2, preferably ≥1.5, more preferably ≥2.0, compared with a reference value (Z0), and the ratio of the two (Z1 / Z0) is ≥1.2, preferably ≥1.5, more preferably ≥2.0.

[0033] In another preferred embodiment, the high content of fatty acid synthase refers to the content of fatty acid synthase (C1) compared to a reference value (C0), and the ratio of the two (C1 / C0) is ≥ 1.2, preferably ≥ 1.5, more preferably ≥ 2.0.

[0034] In another preferred embodiment, the reference value refers to the relative mRNA expression level of an inflammation factor or a lipid synthesis related factor or the content of fatty acid synthase in a healthy person.

[0035] In another preferred embodiment, the pharmaceutical or pharmaceutical composition can be used alone or in combination in the prevention and treatment of liver injury and alcoholic liver disease.

[0036] In another preferred embodiment, the combination use includes the use in combination with other drugs for the prevention and treatment of liver injury and alcoholic liver disease.

[0037] In another preferred embodiment, the pharmaceutical composition further comprises an additional drug for the treatment of liver injury and alcoholic liver disease.

[0038] In another preferred embodiment, the additional drug for the treatment of liver injury and alcoholic liver disease is selected from the group consisting of encephalophytol, glycyrrhizin, glutathione, N-acetylcysteine, tiopronin, silymarin, bicyclol, ursodeoxycholic acid, S-adenosyl methionine, cholestyramine, coenzyme A, coenzyme Q10, water-soluble vitamins (such as vitamin C, vitamin B complex), inosine, ornithine aspartate, alprostadil, or a combination thereof.

[0039] In another preferred embodiment, the pharmaceutical composition is a solid or liquid preparation.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of oral preparations, injection preparations, enteric sustained-release preparations, and lyophilized preparations.

[0041] In another preferred embodiment, the carrier of the injection preparation is selected from the group consisting of physiological saline, glucose, stabilizers, preservatives, suspending agents, emulsifying agents, or a combination thereof.

[0042] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, powders, pastes, powders, injections, and water preparations.

[0043] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, preferably a tablet, a capsule, or a granule.

[0044] In another preferred embodiment, the administration mode of the pharmaceutical composition is selected from the group consisting of subcutaneous, intravenous, and anorectal.

[0045] In another preferred embodiment, the pharmaceutical composition comprises: (i) ethyl lactate or a pharmaceutically acceptable salt thereof as an active ingredient; and (ii) a pharmaceutically acceptable carrier.

[0046] In another preferred embodiment, the pharmaceutical or pharmaceutical composition improves liver injury and alcoholic liver disease by inhibiting hepatic lipid synthesis.

[0047] In another preferred embodiment, the pharmaceutical or pharmaceutical composition improves liver injury and alcoholic liver disease by inhibiting fatty acid synthase.

[0048] In a second aspect of the present application, there is provided a wine product for preventing and treating liver injury and alcoholic liver disease and / or improving hangover, the wine product comprising ethyl lactate.

[0049] In another preferred embodiment, the content of ethyl lactate in the wine product is 0.01-15 g / L, preferably 0.1-10 g / L, and more preferably 0.2-5 g / L.

[0050] In a third aspect of the present application, there is provided a method for promoting SIRT1 expression in hepatocytes, comprising the steps of:

[0051] contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby promoting SIRT1 expression in hepatocytes.

[0052] In another preferred embodiment, the method is in vitro.

[0053] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0054] In another preferred embodiment, the concentration of ethyl lactate is 0.01-15 g / L, preferably 0.1-10 g / L, and more preferably 0.2-5 g / L.

[0055] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0056] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0057] In another preferred embodiment, the non-human mammal includes rodents (e.g., rats, mice), primates (e.g., monkeys).

[0058] In a fourth aspect of the present application, there is provided a method for promoting FGF21 expression in hepatocytes, comprising the steps of:

[0059] contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby promoting FGF21 expression in hepatocytes.

[0060] In another preferred embodiment, the method is in vitro.

[0061] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0062] In another preferred embodiment, the concentration of ethyl lactate is 0.01-15 g / L, preferably 0.1-10 g / L, more preferably 0.2-5 g / L.

[0063] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0064] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0065] In another preferred embodiment, the non-human mammal includes rodents (e.g., rats, mice), primates (e.g., monkeys).

[0066] In a fifth aspect, the present application provides a method for inhibiting lipid synthesis in hepatocytes, comprising the steps of:

[0067] contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby inhibiting lipid synthesis in hepatocytes.

[0068] In another preferred embodiment, ethyl lactate or a pharmaceutically acceptable salt thereof inhibits lipid synthesis by inhibiting fatty acid synthase.

[0069] In another preferred embodiment, the method is in vitro.

[0070] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0071] In another preferred embodiment, the concentration of ethyl lactate is 0.01-15 g / L, preferably 0.1-10 g / L, more preferably 0.2-5 g / L.

[0072] In another preferred embodiment, the hepatocytes are primary hepatocytes.

[0073] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0074] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0075] In another preferred embodiment, the non-human mammal includes rodents (e.g., rats, mice), primates (e.g., monkeys).

[0076] In a sixth aspect, the present application provides a method for preventing or treating liver injury and / or alcohol-related liver disease, comprising the steps of:

[0077] administering to a subject in need thereof a prophylactically or therapeutically effective amount of ethyl lactate or a pharmaceutically acceptable salt thereof, thereby preventing or treating liver injury and / or alcohol-related liver disease.

[0078] In another preferred embodiment, the alcohol-related liver disease comprises chronic alcohol-related liver disease, acute alcohol-related liver disease, or acute-on-chronic alcohol-related liver disease.

[0079] In another preferred embodiment, the alcohol-related liver disease is selected from the group consisting of liver injury, liver steatosis, alcoholic steatohepatitis, liver oxidative stress, alcoholic fatty liver, or a combination thereof.

[0080] It should be understood that, in the scope of the present application, all the technical features described above and the technical features described in detail hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 shows that ethyl lactate dose-dependently ameliorates liver injury and alcohol-related liver disease. (A) Representative pictures of mouse liver H&E and oil red O staining. (B) Mouse plasma ALT and AST activity levels. (C) Mouse plasma triglyceride and total cholesterol levels. (D) Representative pictures of mouse liver 4-HNE and MDA immunohistochemical staining. (E) Representative pictures of mouse liver MPO and F4 / 80 immunohistochemical staining. (F) Mouse liver inflammation-related gene expression levels. Data are presented as mean ± SEM. *p < 0.05 compared with the EtOH+PBS group.

[0082] Figure 2 shows that ethyl lactate directly acts on hepatocytes to inhibit alcohol-induced lipid synthesis. (A) Representative pictures of mouse liver sections of FAS and GLUL immunofluorescence staining. The central vein and portal vein positions have been marked in the figure. (B) Mouse liver lipid synthesis gene expression levels. *p < 0.05 compared with the EtOH+PBS group. (C) Representative pictures of mouse liver primary cells BODIPY staining. (D) BODIPY 493 / 503 staining area quantification results (n = 8). *p < 0.05 compared with the Control group. #p < 0.05 compared with the Vehicle group.

[0083] Figure 3 shows that ethyl lactate increases the expression of SIRT1 and FGF21 in hepatocytes. (A) FGF21 expression level in mouse liver and plasma FGF21 content. (B) SIRT1 expression level in mouse liver. * p < 0.05 compared with EtOH+PBS group. C) SIRT1 protein level in mouse liver, n = 4. (D) Correlation analysis of SIRT1 expression level with FGF21 expression level, plasma FGF21 content and plasma triglyceride content. (E) FGF21 gene expression. (F) SIRT1 gene expression. * p < 0.05 compared with control group, # p < 0.05 compared with 100 mM EtOH. (G) SIRT1 protein level.

[0084] Figure 4 shows that ethyl lactate induces liver FGF21 to inhibit lipid degeneration under starvation.

[0085] Figure 5 shows that ethyl lactate improves hangover induced by acute alcohol gavage in mice.

[0086] Figure 6 shows that lactic acid does not affect alcohol-induced liver steatosis. DETAILED DESCRIPTION

[0087] The present inventors have made extensive and in-depth research, and through a large number of experiments and screening, for the first time, it is accidentally found that ethyl lactate can effectively improve liver damage and / or alcohol-related liver disease. Animal experiments show that ethyl lactate dose-dependently improves alcohol-induced liver steatosis, damage, oxidative stress and inflammatory response. And ethyl lactate inhibits alcohol-induced lipid synthesis by directly acting on hepatocytes, regulates liver metabolism level by increasing the activity of SIRT1, promotes the expression and secretion of liver FGF21 to improve alcoholic steatohepatitis. On this basis, the present application is completed.

[0088] TERMS

[0089] For the purpose of facilitating the understanding of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0090] As used herein, the term "comprises" or variations such as "comprising" or "comprises" is understood to include the elements or components recited, without excluding other elements or components.

[0091] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0092] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range or within a subrange, as well as fractions of the value of any integer to the extent that such fractions are technically feasible.

[0093] As used herein, the term "and / or," refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0094] As used herein, the terms "prevention" and "treatment and / or prevention" are used interchangeably and generally refer to a comprehensive response to a certain disease or health problem, including both preventive and therapeutic measures.

[0095] Ethyl lactate

[0096] As used herein, the terms "active ingredient of the invention," "ethyl lactate of the invention" are used interchangeably and refer to an active ingredient capable of preventing and / or treating liver injury and / or alcohol-related liver disease.

[0097] Ethyl lactate (C5H 10 O3) is a natural ester flavoring substance, mainly synthesized by lactic acid and ethanol under the catalysis of microbial esterification enzyme, and usually has a higher content (0.5-3 g / L) in liquor produced by solid-state fermentation. In liquor, it can increase the full-bodied feeling of liquor, enrich the liquor body, adjust the taste of liquor, and prolong the aftertaste of liquor. Due to the flavoring properties of ethyl lactate, it is also added in wine beverages, chewing gum, baked foods, or cold drinks, because of its non-toxicity, good solubility, non-volatility, and biodegradability. However, there is no report on whether ethyl lactate has in vivo biological activity and regulates biological processes.

[0098] Fibroblast growth factor 21

[0099] Fibroblast growth factor 21 (FGF21) is a stress-induced hormone that plays an important role in regulating energy balance and glucose and lipid homeostasis through a heterodimeric receptor complex composed of FGF receptor 1 (FGFR1) and β-klotho. A number of long-acting FGF21 analogs and agonistic monoclonal antibodies to the FGFR1-β-klotho receptor complex have been developed and entered clinical trials. In these trials, substantial improvements in blood lipids, liver fat content, and serum markers of liver fibrosis were observed in patients with nonalcoholic steatohepatitis (NASH). Thus, drugs targeting hepatic FGF21 have great potential for application in metabolic-related diseases.

[0100] SIRT1

[0101] Sirtuin 1 (SIRT1) is a ubiquitously expressed protein that plays a complex role in the pathology, progression, and treatment of a variety of diseases. SIRT1 is an NAD + dependent deacetylase that regulates gene expression through histone deacetylation modification. SIRT1 has been reported to play a regulatory role in a variety of diseases, such as aging, obesity, fatty liver, etc. In the fasting state, SIRT1 also mediates the expression and secretion of hepatic FGF21.

[0102] Fatty acid synthase

[0103] The biosynthesis of fatty acids affects a variety of cellular functions, and its dysfunction is associated with diseases such as cancer, obesity, and nonalcoholic fatty liver disease. The biosynthesis of cellular fatty acids is carried out by fatty acid synthase (FAS). It catalyzes the production of long-chain fatty acids from acetyl-CoA and malonyl-CoA. The activity of FAS is regulated by a variety of factors, including nutritional status, hormone levels, and gene expression. Abnormal levels of its activity are closely related to the occurrence and development of a variety of diseases.

[0104] Intoxication and hangover

[0105] Intoxication refers to the symptoms of ataxia, coma, etc. that occur in the body within a short period of time after drinking, which is positively correlated with blood ethanol concentration and alleviated as ethanol is metabolized and cleared.

[0106] Hangover refers to the symptoms of physical and psychological discomfort, such as fatigue, lack of concentration, nausea, etc. that occur for a long duration when ethanol is completely metabolized and cleared.

[0107] Pharmaceutical compositions and methods of administration

[0108] The present application also provides a pharmaceutical composition comprising (a) a safe and effective amount of the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application; and (b) a pharmaceutically acceptable carrier or excipient. The severity of the condition being treated, the weight and immune status of the patient, the mode of administration, and the like can affect the appropriate amount of the active ingredient to be used. Generally, satisfactory results are indicated to be obtained when the active ingredient of the present application is administered at a dosage of from about 0.00001 mg to 1 g per kg of animal body weight (preferably from 0.0001 mg to 0.5 g per kg of animal body weight) per day (measured for laboratory mice at 25 mg / kg) in single or divided doses. For example, the dosage can be increased or decreased depending on the response of the treated subject. In addition, the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application can be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0109] The pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means a carrier for administration of a therapeutic agent. This term refers to carriers that are nontoxic to the subject to be treated and do not interfere with the effectiveness of the biological activity of the underlying therapeutic composition. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0110] The pharmaceutically acceptable carrier in the therapeutic composition can contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances can be present in the carrier such as wetting or emulsifying agents, pH buffering substances, and the like.

[0111] Generally, the therapeutic composition can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles; or as lyophilized powders. The therapeutic composition can be prepared for storage as lyophilized powders or air dried powders.

[0112] Once the composition of the present application has been formulated, it can be administered by any conventional route, including, but not limited to, intratumorally, intramuscularly, intravenously, subcutaneously, intradermally, or topically. The subject to be prevented or treated can be an animal; preferably a human.

[0113] When the pharmaceutical composition of the present application is used for actual treatment, various dosage forms of the pharmaceutical composition can be used depending on the use. Preferably, an intravenous preparation is used.

[0114] These pharmaceutical compositions can be formulated according to a conventional method by mixing, diluting or dissolving, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be performed in a conventional manner according to the dosage form.

[0115] For example, the formulation of an eye drop can be performed by dissolving the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application together with a base material in sterile water (in which a surfactant is dissolved), adjusting the osmotic pressure and the pH to a physiological state, and optionally adding suitable pharmaceutical additives such as a preservative, a stabilizer, a buffer, an isotonicity, an antioxidant and a viscosity-increasing agent, and then allowing it to be completely dissolved.

[0116] The pharmaceutical composition of the present application can also be administered in the form of a sustained-release agent. For example, the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application can be incorporated into a pellet or a microcapsule using a sustained-release polymer as a carrier, and then the pellet or the microcapsule can be implanted into a tissue to be treated by surgery. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, etc. can be exemplified, and preferably, biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be exemplified.

[0117] When the pharmaceutical composition of the present application is used for actual treatment, the dose of the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application as an active ingredient can be reasonably determined according to the body weight, age, sex and degree of symptoms of each patient to be treated.

[0118] The main advantages of the present application include:

[0119] (1) Ethyl lactate dose-dependently improves liver injury and / or alcohol-related liver disease, and has a great potential for drug development.

[0120] (2) Ethyl lactate, as a food-derived small molecule having a metabolic regulation function, can be applied to the development of food fields to regulate the metabolic homeostasis of the body.

[0121] (3) Ethyl lactate dose-dependently up-regulates the expression of SIRT1.

[0122] (4) Ethyl lactate can be used as an effective FGF21 inducer.

[0123] The application will be further described in connection with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0124] Materials

[0125] All wild-type C57BL / 6J mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd. by relying on the institute-level animal platform of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences.

[0126] Methods

[0127] (1) Mouse ALD modeling

[0128] The mouse ALD was induced by slow-acute alcohol feeding. Specifically, 12-14 week-old male or female mice were fed with 5-day liquid alcohol feed first for adaptation, and the alcohol volume content was gradually increased from 0% to 4%, and then fed with 10-day 5% volume alcohol content liquid alcohol feed. On the 16th day at 9:00, the mice were given 5g / kg alcohol by gavage, and the mice were sacrificed 9 hours after gavage, and the blood samples and liver samples were collected quickly. The control mice were fed with isocaloric malt dextrin instead of ethanol. During the alcohol feeding and gavage, ethyl lactate (52 vol% alcohol containing ethyl lactate 1g, 3g, 10g / L) was added to the alcohol, and the control mice were fed with PBS.

[0129] (2) Extraction of total RNA from mouse liver tissues and cells

[0130] Put 20-50 mg mouse liver into a 1.5 ml centrifuge tube containing 2 steel beads and 1 ml Trizol, then put it into a pre-cooled grinder for 120 s, 60 Hz grinding to fully lyse the tissue. After centrifuging the lysed tissue at 12,000 rpm for 15 min at 4°C, take 800 μl of supernatant into a new 1.5 ml centrifuge tube. Add 200 ml of chloroform and shake vigorously for 15 s, then stand at room temperature for 3 min, centrifuge at 9,000 rpm for 15 min at 4°C, and take 400 μl of supernatant into a new 1.5 ml centrifuge tube. Add 400 μl of isopropanol, shake gently up and down for 15 s, stand at room temperature for 10 min, centrifuge at 9,000 rpm for 10 min at 4°C, and the white precipitate at the bottom is the RNA. Discard the isopropanol in the centrifuge tube, add 1 ml of 70% ethanol (DEPC water preparation), and centrifuge at 8,000 rpm for 10 min at 4°C to wash the RNA. Discard the ethanol in the centrifuge tube, stand at room temperature for a while to evaporate the ethanol in the centrifuge tube as much as possible, and then add 100-200 μl of DEPC water to dissolve the RNA. After the RNA is fully dissolved, perform concentration detection.

[0131] (3) RNA reverse transcription

[0132] Dilute 12 μl of 3000 ng total RNA with DEPC water in all samples. Use the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit to reverse transcribe the previously extracted RNA:

[0133] • Genomic DNA removal RNA 12 μl 4×gDNA wiper Mix 0.2 μl

[0134] After mixing according to the above proportions, shake well, centrifuge to remove bubbles in the PCR tube, incubate at 42°C for 2 min in the PCR instrument, and remove the genomic DNA.

[0135] • RNA reverse transcription

[0136] Directly add 5×HiScript III qRTSuperMix to the reaction tube of the previous step to configure a 20 μl reverse mixed system: 5×HiScript III qRTSuperMix 4 μl The reaction system of the previous step 16 μl

[0137] Shake well, centrifuge to remove bubbles in the PCR tube, incubate at 37°C for 15 min in the PCR instrument, and at 85°C for 5 s to complete the RNA transcription to obtain cDNA.

[0138] • cDNA dilution

[0139] In the above step reaction tube, directly add 180 μl of dd water to dilute 10 times, and reserve for use.

[0140] (4) Realtime-qPCR

[0141] • qPCR reaction system SYBR MIX 5 μl Forward primer (10 μM) 0.5 μl Reverse primer (10 μM) 0.5 μl

[0142] After the prepared system is shaken and centrifuged, it is added to a qPCR special 384-well plate. 4 μl of diluted cDNA is added to each well of the plate, and after addition, the plate is sealed with a special film. Centrifuge at 2000 rpm for 2 min at room temperature. Place the plate in a qPCR instrument and perform amplification detection according to the following reaction conditions.

[0143] • Reaction conditions

[0144] (5) Isolation of mouse primary hepatocytes

[0145] After normal feeding C57BL / 6 mice are anesthetized with isoflurane, a 24G intravenous needle is inserted into the portal vein of the mice; the mouse liver is perfused with 50 ml of PBS (calcium and magnesium free) containing 0.5 mM EDTA; the mouse liver is perfused with collagenase Buffer (66.7 mM NaCI, 6.7 mM KCI, 6.3 mM CaCI2, 0.05% collagenase 2, 0.226 mM BSA, 100 mM HEPES, pH = 7.4) for 3-5 min; after the liver is fully digested, the liver is transferred to a 10 cm culture dish, the hepatocytes in the liver are released, and then filtered through a 40 micron filter; after the hepatocytes are washed with PBS, centrifuge at 500 rpm for 5 min in a centrifuge at 4°C. After centrifugation, resuspend with 10% FBS low glucose medium, and according to experimental requirements, plate in a 6-well plate, and culture in a 5% CO2 incubator at 37°C.

[0146] (6) BODIPY staining of mouse liver primary cells

[0147] Freshly isolated mouse liver primary cells were evenly spread in 6-well plates with cover glass at the bottom, and were cultured in low-sugar medium overnight. Serum-free low-sugar medium containing 100 mM ethanol was prepared, and the cells were treated with ethanol and ethyl lactate. Fresh serum-free medium containing ethanol and ethyl lactate were replaced every 24 hours, and the treatment lasted for 48 hours. 5 μM BODIPY dye working solution was prepared by diluting the stock solution 2500 times. The stock solution had a concentration of 5 mM (1.3 mg BODIPY was added to 1 mL DMSO), and was stored at -20°C. After 48 hours of treatment, the cells were washed with PBS 3 times, 3 mL PBS per well. 3 ml of staining solution was added to each well, and the cells were incubated at 37°C for 15 min. From this time on, light was avoided as much as possible. After the staining was completed, the cells were washed with 3 ml of PBS 2 times. The cells were fixed with 3 ml of 4% paraformaldehyde solution at room temperature for 30 min. The fixing solution was removed, and the cells were washed with 3 ml of PBS 3 times, 5 min each time. 2 ml of 2 ug / ml DIPY solution was incubated for 10 min, and then the cells were washed with PBS 3 times. After the cells were mounted with glycerol gelatin, they were stored at 4°C, and were photographed.

[0148] (7) Measurement of ALT and AST activities in mouse plasma

[0149] The mouse plasma ALT and AST activity assay kit was used according to the instructions. 7.5 μl of plasma and 150 μl of reagent one were first added to a 96-well plate, and were incubated at 37°C for 10 min. Then, 50 μl of reagent two was added, and the absorbance was detected at 340 nm for 15 cycles.

[0150] The absorbance values of the 4th and 14th cycles were selected for calculation. The ALT and AST activities were calculated using the following formula: ALT / AST (U / L) = ΔOD 340 / ΔT s × 60 × 207.5 / 6.22 / 7.5 / 0.6 × 1000.

[0151] (8) Measurement of TG and TC contents in mouse plasma

[0152] The mouse plasma TG and TC content assay kit was used according to the instructions. 2.5 μl of plasma or standard solution was added to a 96-well plate, followed by the addition of 250 μl of working solution. After incubation at 37°C for 10 min, the wavelength was detected using an enzyme label instrument at a wavelength of 500 nm, and distilled water was used as a blank control. The content in the sample was calculated using the standard concentration.

[0153] (9) Data statistics and analysis

[0154] The experimental data results were mean ± standard error. Excel was used for two-tailed unpaired t test to compare the differences between two groups. When P was less than or equal to 0.05, it was considered to have statistical difference.

[0155] Example 1 Ethyl lactate ameliorates alcohol-related liver disease in a dose-dependent manner

[0156] 1.1 Method

[0157] The model of alcohol-related liver disease in mice induced by slow-acute alcohol feeding (NIAAA or Gao-Binge model) was used to study the effect of ethyl lactate on ALD, which simulates the drinking pattern of long-term heavy drinking plus recent binge drinking in humans and reproduces the pathological features of human ALD. To study the effect of ethyl lactate on ALD in mice, the Gao-Binge model was used, and ethyl lactate was added to the alcohol at a dose of 1 g / L, 3 g / L, and 10 g / L during slow-acute alcohol feeding (group), and whether ethyl lactate has a dose effect on ALD was verified.

[0158] Modeling and related experiments are described in the “Method” section of this application.

[0159] 1.2 Results

[0160] As shown in Figure 1A, mice developed liver steatosis (see white circular vacuoles in H&E staining and red positive areas in oil red O staining) after slow-acute alcohol feeding, while ethyl lactate treatment ameliorated liver steatosis in ALD mice in a dose-dependent manner. At a dose of 1 g / L, ethyl lactate had a significant effect on reducing liver lipid deposition in mice, and at a dose of 3 g / L and 10 g / L, ethyl lactate had a significant effect on ameliorating liver steatosis in ALD mice.

[0161] As shown in Figure 1B, ethyl lactate had a tendency to improve liver injury in ALD mice and had a certain dose effect.

[0162] As shown in Figure 1C, ethyl lactate significantly reduced hyperlipidemia in mice caused by long-term alcohol feeding, and the effect was more significant with increasing doses of ethyl lactate.

[0163] Oxidative stress and inflammation are considered two key features of ALD. Long-term alcohol feeding leads to a compensatory increase in the microsomal alcohol metabolism pathway, which metabolizes ethanol through CYP2E1 and produces a large amount of reactive oxygen species (ROS), thereby causing oxidative stress in hepatocytes. These reactive oxygen species directly cause lipid peroxidation, resulting in an increase in lipid peroxidation products 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) in hepatocytes, thereby forming adducts with intracellular proteins, producing cytotoxicity, and exacerbating oxidative stress. Therefore, liver sections can be immunostained for 4-HNE and MDA by immunohistochemistry to determine liver ROS production and oxidative stress.

[0164] As shown in Figure 1D, ethyl lactate dose-dependently reduced the formation of 4-HNE and MDA protein adducts in the livers of ALD mice, i.e., improved liver ROS production and oxidative stress.

[0165] Neutrophils and macrophages are the most important infiltrating immune cells in the liver of ALD, responding to the inflammatory immune response of the liver. Myeloperoxidase (MPO) is highly expressed in neutrophils and is a common marker protein for detecting neutrophils. F4 / 80 is a specific marker protein for macrophages, and there are certain resident macrophages (Kupffer cells) in hepatocytes, and activated macrophages are clustered.

[0166] As shown in Figure 1E, ethyl lactate dose-dependently reduced the infiltration of neutrophils and macrophages in the livers of ALD mice, improving liver inflammation.

[0167] Inflammatory cells and pro-inflammatory factor-related genes include pro-inflammatory cytokines such as IL-1β (Interleukin-1β) and IL-6 (Interleukin-6), chemokines such as MCP1 (Monocyte Chemoattractant Protein-1, MCP1) (also known as CCL2, (Chemokine (CC-motif) ligand 2), adhesion molecules required for immune cell function such as ICAM1 (Intercellular Adhesion Molecule 1) and CD11b.

[0168] As shown in Figure 1F, ethyl lactate dose-dependently down-regulated the expression of inflammation-related genes in the livers of ALD mice. At a dose of 1 g / L, ethyl lactate had a very significant effect on improving liver inflammation.

[0169] The inhibition of lipid synthesis by ethyl lactate in hepatocytes reduces the lipid toxicity caused by excessive lipid accumulation, thereby reducing the damage and death of hepatocytes. In turn, the release of Damage-Associated Molecular Patterns (DAMPs) by dead hepatocytes reduces the immune response of the liver to some extent, thereby improving the inflammation of the liver of ALD mice.

[0170] The above results show that ethyl lactate has a good effect on improving alcohol-related liver disease, and has a significant dose effect. At a dose of 1 g / L of ethyl lactate, ALD can be significantly improved, and at doses of 3 g / L and 10 g / L, ALD can be significantly improved, which has the potential to treat liver damage and / or alcohol-related liver disease.

[0171] Example 2 Ethyl lactate directly acts on hepatocytes to inhibit alcohol-induced lipid synthesis

[0172] 2.1 Method

[0173] Simple lipid deposition is an early stage of the development of ALD, and excessive lipid accumulation can cause liver steatosis, thereby developing into alcoholic fatty liver (AFL). With continuous drinking, hepatocyte death and inflammation occur in the liver, and AFL gradually develops into alcoholic steatohepatitis (ASH). Therefore, excessive alcohol metabolism-induced lipid synthesis is a key link in the early development of ALD. Ethanol is ultimately metabolized to acetyl-CoA in the liver, which directly participates in the de novo synthesis of fatty acids. This process is regulated by the transcription factor Sterol Regulatory Element-Binding Protein 1c (SREBP-1c). Key proteins involved in lipid synthesis downstream include Acetyl-CoA Carboxylase 1 (ACC1), fatty acid synthase (FAS), Stearoyl-CoA Desaturase 1 (SCD1), Diglyceride Acyltransferase 1 (DGAT1), and long chain-fatty-acid-CoA ligase 4 (ASCL4). The effects of ethyl lactate on lipid synthesis can be explored at the protein and mRNA levels by immunofluorescence and RT-qPCR.

[0174] Modeling and related experiments are described in the "Methods" section of this application.

[0175] 2.1 Results

[0176] As shown in Figure 2A, the FAS level in the liver of non-alcohol fed mice is very low and almost undetectable. However, the FAS level in the liver of mice fed with slow-acute alcohol is significantly increased and shows spatial distribution.

[0177] The expression of FAS gradually decreases from the portal vein (PV) to the central vein (CV) and is the most abundant in the PV area where the nutrient is more abundant. Because alcohol is mostly absorbed in the small intestine and then enters the liver through the PV for catabolism.

[0178] After treatment with ethyl lactate, the FAS level in the liver of mice is significantly reduced and the spatial distribution is also weakened. This indicates that ethyl lactate can inhibit the key genes and proteins of lipid synthesis at the transcriptional and protein levels, thereby reducing the de novo synthesis of lipids and improving liver steatosis.

[0179] As shown in Figure 2B, ethyl lactate significantly down-regulates the expression of the above-mentioned lipid synthesis-related genes and has a dose effect, indicating that ethyl lactate reduces liver lipid deposition by inhibiting alcohol metabolism-induced de novo synthesis of fatty acids, thereby improving slow-acute induced mouse ALD.

[0180] In order to prove that ethyl lactate exerts its inhibitory effect on lipid synthesis by directly interacting with hepatocytes, the applicant isolated liver primary cells of normally fed mice and verified the improvement of ethyl lactate by in vitro cell experiments.

[0181] The liver primary cells were cultured in serum-free low-glucose DMEM medium with the addition of 100 mM ethanol for 48 hours, while being treated with 1 mM and 10 mM ethyl lactate. Then BODIPY 493 / 503 staining was performed to determine the lipid synthesis.

[0182] As shown in Figure 2C, after 48 hours of incubation with 100 mM ethanol, a large number of lipid droplets of different sizes distributed around the nucleus appeared in the liver cells, indicating that the liver cells synthesized a large amount of lipids after ethanol exposure.

[0183] When treated with a gradient of ethyl lactate, the lipid droplets around the liver cells were significantly reduced (Figure 2D) and still had a dose effect.

[0184] The above results show that ethyl lactate inhibits alcohol-induced de novo synthesis of lipids by directly acting on liver cells.

[0185] In summary, the in vivo and in vitro experimental data demonstrate that ethyl lactate inhibits alcohol-induced lipid synthesis by reducing the levels of lipid synthesis-related proteins in hepatocytes, thereby improving liver steatosis in ALD mice.

[0186] Example 3 Ethyl lactate increases the expression of SIRT1 and FGF21 in hepatocytes

[0187] Fibroblast growth factor 21 (FGF21) is a liver-secreted factor with metabolic regulatory effects, which is mainly secreted by the liver and acts on various metabolic organs such as adipose tissue and liver. After drinking, FGF21 is significantly induced in a short time, and regulates the metabolic stress caused by excessive alcohol intake. Therefore, the applicant hypothesized whether FGF21 is a potential downstream target for ethyl lactate to improve ALD.

[0188] The experimental methods in this example are described in the "Methods" section of this application.

[0189] 3.1 In vivo experiment

[0190] As shown in Figure 3A, slow-acute alcohol feeding induced an increase in the expression and secretion of FGF21 in the liver of mice (plasma FGF21 levels). After ethyl lactate treatment, the expression and secretion of FGF21 in the liver of mice were further significantly increased, and showed a dose-dependent effect.

[0191] Under fasting conditions, the transcription of FGF21 is regulated by SIRT1, and liver-specific knockout of SIRT1 reduces FGF21 expression and exacerbates physiological steatosis in the liver induced by fasting.

[0192] The applicant detected the expression of SIRT1 in the liver of mice by RT-qPCR, and the results are shown in Figure 3B. After ethyl lactate treatment, the expression of SIRT1 in the liver of mice was increased in a dose-dependent manner.

[0193] At the same time, the protein level of SIRT1 in the liver of mice was also significantly increased (Figure 3C), which suggests that the increased SIRT1 may promote the transcription of FGF21.

[0194] Correlation analysis showed that after ethyl lactate treatment, the expression of SIRT1 in the liver of mice was significantly positively correlated with the expression of FGF21 and the content of plasma FGF21, and was significantly negatively correlated with the TG level in the plasma of mice (Figure 3D).

[0195] Further, ethyl lactate improves ALD in mice by dose-dependently increasing the expression of SIRT1 and FGF21 in the liver, and the increased FGF21 acts on the liver itself to regulate metabolism and energy homeostasis.

[0196] 3.2 In vitro experiment

[0197] Using mouse normal hepatocyte line AML12, the ethanol exposure in vitro was simulated by incubating the cells with 100 mM ethanol-containing medium for 48 hours, and the mice were treated with ethyl lactate (1 mM or 10 mM) for 48 hours.

[0198] As shown in Figure 3E, ethanol exposure increased the expression of FGF21 in hepatocytes, and ethyl lactate treatment further increased the expression of FGF21, and had a dose effect.

[0199] Ethyl lactate treatment increased the gene expression and protein level of SIRT1 down-regulated after alcohol exposure (Figures 3F and 3G).

[0200] The in vitro experimental data again confirmed that ethyl lactate has the effect of inducing the expression of FGF21 and SIRT1 in hepatocytes.

[0201] In summary, the above in vivo and in vitro experimental data reveal the molecular mechanism of ethyl lactate in improving ALD. That is, ethyl lactate increases the expression of SIRT1 in the liver, promotes the expression and secretion of FGF21. FGF21 as a metabolic regulator improves liver steatosis and reduces liver fat. After ethyl lactate treatment, increased FGF21 down-regulates the transcription of genes related to liver lipid synthesis, thereby reducing liver lipid synthesis and reducing liver lipid deposition, thereby improving liver damage and inflammation.

[0202] Example 4 Ethyl lactate induces liver FGF21 to inhibit lipid degeneration under starvation

[0203] 4.1 Method

[0204] FGF21 is an energy homeostasis regulating hormone in response to metabolic stress, and its induction factors include not only alcohol but also long-term starvation. Long-term fasting will cause adipose tissue lipolysis, and a large amount of free fatty acids will appear in the plasma in a short period of time, and the liver will absorb and utilize these fatty acids, so that the liver also appears short-term physiological fat accumulation. Using a 24-hour fasting mouse model to explore whether ethyl lactate can also induce FGF21 in the liver of mice under physiological conditions.

[0205] Wild-type mice were fed with normal standard feed, and were allowed to drink water containing 3 g / L ethyl lactate freely for one month. In the last week, the mice were additionally injected intraperitoneally with 30 mg / kg ethyl lactate per day. Before sacrifice, the mice were subjected to 24-hour fasting with free water. The liver tissue of the mice was collected, and H&E and oil red O staining were performed to determine the liver steatosis.

[0206] 4.2 Results

[0207] As shown in Figure 4, compared with the liver of normal fed mice, a large number of small lipid droplets were observed in the liver cells of mice after 24 hours of fasting, indicating that physiological steatosis occurred in the liver after long-term starvation.

[0208] However, after ethyl lactate treatment, the lipid droplets in the liver after 24 hours of fasting were significantly reduced, and the reduction of lipid droplets in H&E and the reduction of ORO red positive staining area were observed. The quantitative results of ORO staining area are shown in Figure 4A.

[0209] At the same time, the liver weight index of fasting mice treated with ethyl lactate was significantly lower than that of the control (Figure 4B). This indicates that most of the absorbed free fatty acids in the liver of mice treated with ethyl lactate have been decomposed and utilized, so there are fewer lipid droplets in the liver, and the liver is also lighter.

[0210] After fasting, the TG in the plasma significantly increased due to the lipolysis of adipose tissue, while the TG in the plasma significantly decreased after ethyl lactate treatment (Figure 4D). This indicates that after ethyl lactate treatment, the liver absorbs more fatty acids, and due to the increased fatty acid oxidation level in the liver, more fatty acids are decomposed, thereby reducing the TG content in the plasma.

[0211] As shown in Figures 4E and F, the expression and secretion of FGF21 in the liver of mice after fasting were significantly increased, while the expression and secretion of FGF21 in the liver were further increased after ethyl lactate treatment. And ethyl lactate also significantly increased the expression of FGF21 in the liver of mice in the fed state, which indicates that ethyl lactate can effectively induce FGF21 in the liver, thereby exerting a metabolic improvement effect.

[0212] The expression of SIRT1 in the liver after fasting was significantly increased, which increased the transcription of FGF21. The expression of SIRT1 in the liver after ethyl lactate treatment was further significantly increased, corresponding to the further increased expression of FGF21. However, there was no significant difference in the expression of SIRT1 in the liver of mice in the fed state.

[0213] To determine the functional results of increased FGF21 in the liver after ethyl lactate treatment, key genes involved in lipid metabolism were determined by RT-qPCR, including fatty acid decomposition genes such as carnitine palmitoyl transferase 1α (CPT1α) and medium-chain acyl-coa dehydrogenase (MCAD), and lipid synthesis genes FAS and SCD1.

[0214] As shown in Fig. 4G, the fasting-induced increase in fatty acid oxidation-related genes in mouse liver was significantly increased by 2.5-fold or more, which increased the oxidation of fatty acids absorbed by the liver from adipose tissue lipolysis, thereby providing important energy support for maintaining normal liver metabolism.

[0215] After ethyl lactate treatment, the fatty acid oxidation genes were further significantly increased, which corresponded to the increased FGF21 expression, further indicating that ethyl lactate inhibited the steatosis of mouse liver induced by starvation by increasing the expression and secretion of liver FGF21, thereby increasing downstream fatty acid oxidation, so that the free fatty acids absorbed by the liver were fully utilized, and then the accumulation of lipid droplets in hepatocytes was reduced.

[0216] As shown in Fig. 4H, the fasting-induced significant down-regulation of liver lipid synthesis genes reduced energy loss in the energy-deficient state. After ethyl lactate treatment, the expression of FAS and SCD1 genes in the liver of mice in the fasting state was further significantly down-regulated, thereby further reducing the synthesis of liver lipids.

[0217] Even in the fed state, the expression of FAS and SCD1 in the liver of mice after ethyl lactate treatment was significantly down-regulated. This indicates that even in the fed state, ethyl lactate treatment can inhibit the synthesis of lipids in the liver of mice and reduce lipid neogenesis. This is consistent with the reduction of lipid droplets in the liver of mice after ethyl lactate treatment in the fed state (Fig. 4B), the reduction of plasma triglyceride levels, and the increase of FGF21 expression and secretion in the liver.

[0218] In summary, ethyl lactate can also inhibit steatosis by increasing the expression and secretion of FGF21 in the liver of mice under physiological conditions.

[0219] Example 5 Ethyl lactate improves hangover induced by acute alcohol gavage in mice

[0220] 5.1 Method

[0221] Mice were induced to be drunk by gavage with 2.5 g / kg body weight of ethanol, and 1 h later, the open field and rotarod tests were used to determine the drunkenness of mice. Mice were induced to be hungover by gavage with 5 g / kg body weight of ethanol, and 6 h later, the open field and rotarod tests were used to determine the hangover of mice. Each mouse only performed one behavioral test after gavage. Ethyl lactate was added to the alcohol (10 g / L ethyl lactate in 52 vol% alcohol) to explore the effect of ethyl lactate on drunkenness and hangover.

[0222] 5.2 Results

[0223] As shown in Fig. 5, in the drunkenness state (2.5 g / kg ethanol gavage, 1 h), ethyl lactate did not affect the movement distance in the open field and the time on the rod of mice.

[0224] However, in the hangover state (5 g / kg ethanol gavage, 6 h), ethyl lactate significantly increased the open field movement distance and the rotarod time on the stick, indicating that ethyl lactate can improve the hangover state of mice. At the same time, ethyl lactate needs a certain time to induce FGF21 in vivo to resist the hangover state induced by alcohol.

[0225] Example 6 Lactic acid does not affect alcohol-induced liver steatosis

[0226] 6.1 Method

[0227] Since ethyl lactate can be hydrolyzed to lactic acid in vivo, to explore whether the improvement of ethyl lactate depends on its hydrolysis product lactic acid, we treated mice with lactic acid.

[0228] The modeling and experimental methods in this example are as described in the "Methods" section of the present application, wherein the ethyl lactate treatment of mice is replaced by lactic acid (52 vol% alcohol containing 1 g / L lactic acid) treatment.

[0229] 6.2 Results

[0230] As shown in Figure 6, after alcohol feeding, the liver showed significant lipid accumulation, and lactic acid treatment had no significant effect on alcohol-induced liver steatosis. This indicates that lactic acid does not play a role in the improvement of alcohol-related liver disease by ethyl lactate, but rather directly acts on hepatocytes to function.

[0231] Discussion

[0232] The present application discovers a food-derived natural small molecule compound, ethyl lactate, which has the effect and mechanism of improving liver damage and / or alcohol-related liver disease. Using a mouse ALD model induced by slow-acute alcohol feeding, it was found that ethyl lactate dose-dependently improved alcohol-induced liver steatosis, damage, oxidative stress and inflammatory response. At the same time, the expression level of key proteins for liver lipid synthesis was significantly decreased after ethyl lactate treatment, and in vitro cell experiments found that ethyl lactate inhibited alcohol-induced lipid synthesis by directly acting on hepatocytes. Further, mechanism studies found that ethyl lactate regulates liver metabolism by increasing the transcriptional activity of SIRT1, promoting the expression and secretion of liver FGF21 to improve alcoholic steatohepatitis.

[0233] The present application firstly discovers that ethyl lactate has in-vivo biological activity, participates in regulating the occurrence and development of ALD, and has a good improvement and treatment effect on mouse liver injury and / or alcohol-related liver disease. This provides a new idea and method for improving liver damage caused by excessive drinking or treating alcoholic steatohepatitis. At the same time, the downstream target of ethyl lactate, liver FGF21, its analog has shown good weight loss, blood lipid reduction, liver lipid reduction, improvement of fatty liver and fibrosis in clinical experiments. As a high-efficiency in-vivo inducer of liver FGF21, ethyl lactate is expected to also play an improvement role in other metabolic-related diseases.

[0234] All documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various alterations and modifications to this application will become apparent to those of ordinary skill in the art, and it is intended that the application encompass all such alterations and modifications within the scope of the appended claims.

Claims

1. Use of ethyl lactate or a pharmaceutically acceptable salt thereof, characterized in that, for the manufacture of a medicament or a pharmaceutical composition for one or more uses selected from the group consisting of: (a1) preventing or treating liver injury; (a2) preventing or treating alcohol-related liver disease; (a2) an agonist of SIRT1; (a3) an agonist of FGF21; (s4) improving hangover.

2. Use according to claim 1, characterized in that, The alcohol-related liver disease includes chronic alcohol-related liver disease, acute alcohol-related liver disease, or acute-on-chronic alcohol-related liver disease.

3. Use according to claim 1, characterized in that, The liver injury and alcohol-related liver disease are selected from the group consisting of liver steatosis, alcoholic steatohepatitis, liver oxidative stress, alcoholic fatty liver, or a combination thereof.

4. The use according to claim 1, characterized in that, The medicament or pharmaceutical composition is also for a use selected from the group consisting of: (a1) improving liver injury (a2) improving liver fat deposition; (a3) an inhibitor of alcohol-related liver disease inflammatory factor; (a4) inhibiting alcohol-induced lipid synthesis; (a5) an inhibitor of lipid synthesis-related factor.

5. The use according to claim 1, characterized in that, The medicament or pharmaceutical composition is administered to a subject selected from the group consisting of a mammal or a rodent.

6. Use according to claim 5, characterized in that, The subject has one or more characteristics selected from the group consisting of: (c1) liver injury (c2) long-term drinking of alcohol; (c3) suffering from alcohol-related liver disease; (c4) being drunk or having a hangover; (c5) high expression of alcohol-related liver disease inflammatory factor; (c6) high expression of lipid synthesis-related factor; (c7) high content of fatty acid synthase.

7. A beverage product for the prevention of liver damage and alcohol-related liver disease and / or the improvement of hangover, characterized in that, The alcoholic product comprises ethyl lactate.

8. The alcoholic product of claim 7, wherein, The content of ethyl lactate in the alcoholic product is 0.01-15 g / L.

9. The alcoholic product of claim 6, wherein, The content of ethyl lactate in the alcoholic product is 0.1-10 g / L.

10. The alcoholic product of claim 6, wherein, The content of ethyl lactate in the alcoholic product is 0.2-5 g / L.

11. A method of inhibiting hepatic lipid synthesis, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-10. The method comprises the step of: contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby inhibiting lipid synthesis in hepatocytes.

12. The method of claim 11, wherein, The concentration of ethyl lactate is 0.01-15 g / L.

13. A method of preventing and / or treating liver damage and / or alcohol-related liver disease, characterized in that, The method comprises the step of: administering a prophylactically or therapeutically effective amount of ethyl lactate or a pharmaceutically acceptable salt thereof to a subject in need, thereby preventing or treating liver injury and / or alcohol-related liver disease.

14. The method of claim 13, wherein, The alcohol-related liver disease includes chronic alcohol-related liver disease, acute alcohol-related liver disease, or acute-on-chronic alcohol-related liver disease.

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