Application of ethyl lactate in alcoholic related liver diseases

Abstract

The invention provides an application of ethyl lactate in prevention and treatment of alcoholic related liver diseases. Specifically, animal experiments are carried out by establishing an alcoholic related liver disease mouse model, and in-vivo and in-vitro experiments are carried out to verify that the ethyl lactate dose-dependently improves the alcoholic related liver disease. Experiments show that ethyl lactate inhibits alcohol-induced lipid synthesis, and the liver metabolism level is regulated by up-regulating expression of SIRTI and FGF21, so that the alcoholic related liver diseases are improved. Based on the invention, the ethyl lactate or the pharmaceutically acceptable salt thereof can be used for preparing the medicine for preventing and treating the alcoholic related liver diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the use of ethyl lactate in 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 manufacture of a medicament or a pharmaceutical composition for one or more uses selected from the group consisting of:

[0006] (a1) preventing or treating alcohol-associated liver disease;

[0007] (a2) an agonist of SIRT1;

[0008] (a3) an agonist of FGF21;

[0009] (s4) improving hangover.

[0010] In another preferred embodiment, the alcohol-associated liver disease comprises chronic alcohol-associated liver disease, acute alcohol-associated liver disease, or mixed acute-on-chronic alcohol-associated liver disease.

[0011] In another preferred embodiment, the alcohol-associated liver disease is selected from the group consisting of liver steatosis, alcoholic steatohepatitis, liver oxidative stress, alcoholic fatty liver, or a combination thereof.

[0012] In another preferred embodiment, the agonist of FGF21 promotes the upregulation of FGF21 in hepatocytes.

[0013] In another preferred embodiment, the agonist of SIRT1 promotes the upregulation of SIRT1 in hepatocytes.

[0014] In another preferred embodiment, the medicament or pharmaceutical composition is further used for a purpose selected from the group consisting of:

[0015] (a1) improving liver fat deposition;

[0016] (a2) an inhibitor of alcoholic liver disease-associated inflammatory factor;

[0017] (a3) inhibiting alcohol-induced lipid synthesis;

[0018] (a4) an inhibitor of lipid synthesis-associated factor.

[0019] In another preferred embodiment, the alcoholic liver disease-associated inflammatory factor is selected from the group consisting of IL-1β, IL-6, MCP1, ICAM1, CD11b, or a combination thereof.

[0020] In another preferred embodiment, the lipid synthesis-associated factor is selected from the group consisting of SREBP-1c, ACC1, FAS, SCD1, DGAT1, ASCL4, or a combination thereof.

[0021] 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.

[0022] In another preferred embodiment, the subject has one or more characteristics selected from the group consisting of:

[0023] (c1) long-term drinking of alcohol;

[0024] (c2) suffering from alcoholic liver disease;

[0025] (c3) being drunk or suffering from hangover;

[0026] (c4) high expression of alcoholic liver disease-associated inflammatory factor;

[0027] (c5) high expression of lipid synthesis-associated factor;

[0028] (c6) high content of fatty acid synthase.

[0029] In another preferred embodiment, the high expression means that the relative expression level of mRNA of the alcoholic liver disease-associated inflammatory factor or lipid synthesis-associated factor (Z1) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0, compared to a reference value (Z0), and the ratio of the two (Z1 / Z0) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0.

[0030] In another preferred embodiment, the high content of fatty acid synthase means that the content of fatty acid synthase (C1) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0, compared to a reference value (C0), and the ratio of the two (C1 / C0) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0. 1) In another preferred embodiment, the high content of fatty acid synthase means that the content of fatty acid synthase (C1) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0, compared to a reference value (C0), and the ratio of the two (C1 / C0) is greater than or equal to 1.2, preferably greater than or equal to 1.5, more preferably greater than or equal to 2.0.

[0031] In another preferred embodiment, the reference value refers to the mRNA relative expression level of an inflammation-related factor or a lipid synthesis-related factor or the fatty acid synthase content in a healthy person.

[0032] In another preferred embodiment, the pharmaceutical or pharmaceutical composition can be used alone or in combination in the prevention and treatment of alcoholic-related liver disease.

[0033] In another preferred embodiment, the combination use includes use in combination with other drugs for preventing and treating alcoholic-related liver disease.

[0034] In another preferred embodiment, the pharmaceutical composition further comprises an additional drug for treating alcoholic-related liver disease.

[0035] In another preferred embodiment, the additional drug for treating alcoholic-related 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.

[0036] In another preferred embodiment, the pharmaceutical composition is a solid or liquid preparation.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, preferably a tablet, a capsule, or a granule.

[0041] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the group consisting of subcutaneous, intravenous, and anorectal.

[0042] 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.

[0043] In another preferred embodiment, the pharmaceutical or pharmaceutical composition improves alcoholic-related liver disease by inhibiting liver lipid synthesis.

[0044] In another preferred embodiment, the medicament or pharmaceutical composition improves alcoholic-related liver disease by inhibiting fatty acid synthase.

[0045] In a second aspect of the present application, there is provided a product for preventing and / or treating alcoholic-related liver disease and / or hangover, the product comprising ethyl lactate.

[0046] In another preferred embodiment, the product comprises ethyl lactate at a concentration of 0.01 to 15 g / L, preferably 0.1 to 10 g / L, more preferably 0.2 to 5 g / L.

[0047] In a third aspect of the present application, there is provided a method for promoting SIRT1 expression in hepatocytes, the method comprising the steps of:

[0048] contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby promoting SIRT1 expression in hepatocytes.

[0049] In another preferred embodiment, the method is in vitro.

[0050] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0051] In another preferred embodiment, the ethyl lactate is at a concentration of 0.01 to 15 g / L, preferably 0.1 to 10 g / L, more preferably 0.2 to 5 g / L.

[0052] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0053] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0054] In another preferred embodiment, the non-human mammal includes rodents (e.g. rats, mice), primates (e.g. monkeys).

[0055] In a fourth aspect of the present application, there is provided a method for promoting FGF21 expression in hepatocytes, the method comprising the steps of:

[0056] contacting ethyl lactate or a pharmaceutically acceptable salt thereof with hepatocytes, thereby promoting FGF21 expression in hepatocytes.

[0057] In another preferred embodiment, the method is in vitro.

[0058] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0059] In another preferred embodiment, the ethyl lactate is at a concentration of 0.01 to 15 g / L, preferably 0.1 to 10 g / L, more preferably 0.2 to 5 g / L.

[0060] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0061] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0062] In another preferred embodiment, the non-human mammal includes: rodents (e.g., rats, mice), primates (e.g., monkeys).

[0063] In a fifth aspect of the present application, there is provided a method of inhibiting lipid synthesis in hepatocytes, comprising the step of:

[0064] contacting the hepatocytes with ethyl lactate or a pharmaceutically acceptable salt thereof, thereby inhibiting lipid synthesis in the hepatocytes.

[0065] In another preferred embodiment, the ethyl lactate or pharmaceutically acceptable salt thereof inhibits lipid synthesis by inhibiting fatty acid synthase.

[0066] In another preferred embodiment, the method is in vitro.

[0067] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0068] In another preferred embodiment, the ethyl lactate is present at a concentration of 0.01 to 15 g / L, preferably 0.1 to 10 g / L, more preferably 0.2 to 5 g / L.

[0069] In another preferred embodiment, the hepatocytes are primary hepatocytes.

[0070] In another preferred embodiment, the hepatocytes are derived from a mammal.

[0071] In another preferred embodiment, the hepatocytes are derived from a human or a non-human mammal.

[0072] In another preferred embodiment, the non-human mammal includes: rodents (e.g., rats, mice), primates (e.g., monkeys).

[0073] In a sixth aspect of the present application, there is provided a method of ameliorating alcoholic-related liver disease, comprising the step of:

[0074] administering to a subject in need thereof a prophylactically or therapeutically effective amount of ethyl lactate or a pharmaceutically acceptable salt thereof, thereby ameliorating alcoholic-related liver disease.

[0075] In another preferred embodiment, the alcoholic-related liver disease includes chronic alcoholic-related liver disease, acute alcoholic-related liver disease, or acute-on-chronic alcoholic-related liver disease.

[0076] In another preferred embodiment, the alcoholic-related liver disease is selected from the group consisting of: liver steatosis, alcoholic steatohepatitis, liver oxidative stress, alcoholic fatty liver, or a combination thereof.

[0077] It should be understood that, within 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

[0078] Figure 1 Ethyl lactate dose-dependently ameliorated alcoholic-associated 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.

[0079] Figure 2 Ethyl lactate directly acted 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.

[0080] Figure 3 Ethyl lactate increased the expression of SIRT1 and FGF21 in hepatocytes. (A) Mouse liver FGF21 expression levels and plasma FGF21 content. (B) Mouse liver SIRT1 expression levels. *p < 0.05 compared with the EtOH + PBS group. C) Mouse liver SIRT1 protein levels, n = 4. (D) Correlation analysis of mouse liver SIRT1 expression levels with FGF21 expression levels, plasma FGF21 content, and plasma triglyceride content. (E) FGF21 gene expression. (F) SIRT1 gene expression. *p < 0.05 compared with the control group, #p < 0.05 compared with 100 mM EtOH. (G) SIRT1 protein levels.

[0081] Figure 4 Ethyl lactate induced liver FGF21 to inhibit steatosis under starvation conditions.

[0082] Figure 5 Ethyl lactate improved mouse acute alcohol gavage-induced hangover.

[0083] Figure 6 It is shown that lactic acid does not affect alcohol-induced liver steatosis. DETAILED DESCRIPTION

[0084] The present inventors, through extensive and in-depth research, through a large number of experiments and screening, first accidentally found that ethyl lactate can effectively improve alcoholic-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 by up-regulating the expression of SIRT1, promotes the expression and secretion of liver FGF21 to improve alcoholic steatohepatitis. On this basis, the present application is completed.

[0085] TERMS

[0086] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application to which the present application pertains. Before the present application is described, it is to be understood that this 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.

[0087] As used herein, the term "comprises" or variations such as "comprising" or "comprises" is understood to mean including, but not excluding the presence of other elements or additional components.

[0088] The term "about" can refer to a value or a composition that is within an acceptable error range for the specific value or composition determined by one of ordinary skill in the art that is recited. By way of example, the expression "about 100" includes values such as 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0089] 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 and fraction thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0090] As used herein, the term "and / or" refers to any and all possible combinations of one or more of the associated listed items.

[0091] Ethyl lactate

[0092] As used herein, the terms "active ingredient of the invention", "ethyl lactate of the invention" are used interchangeably to refer to an active ingredient capable of preventing and treating alcoholic-related liver disease.

[0093] Ethyl lactate (C5H 10 O3) is a natural ester flavoring substance, mainly synthesized from 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, which has the characteristics of non-toxic, good solubility, not easy to volatilize, biodegradability, etc. However, there is no report on whether ethyl lactate has in vivo biological activity and regulates biological processes.

[0094] Fibroblast growth factor 21

[0095] Fibroblast growth factor 21 (FGF21) is a stress-induced hormone, which 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 large number of long-acting FGF21 analogs and agonistic monoclonal antibodies of FGFR1-β-klotho receptor complex have been developed and entered clinical trials. In these trials, substantial improvements in blood lipid abnormalities, liver fat content and serum markers of liver fibrosis in patients with non-alcoholic steatohepatitis (NASH) were observed. Therefore, drugs targeting liver FGF21 have great application potential in metabolic-related diseases.

[0096] SIRT1

[0097] Silent information regulator 1 (SIRT1) is a ubiquitously expressed protein that plays a complex role in the pathology, progression and treatment of various 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 various diseases such as aging, obesity, fatty liver, etc. In the fasting state, SIRT1 also mediates the expression and secretion of liver FGF21.

[0098] Fatty acid synthase

[0099] The biosynthesis of fatty acids affects a variety of cellular functions, and its dysregulation is associated with diseases such as cancer, obesity, and nonalcoholic fatty liver disease. Cellular fatty acid biosynthesis 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 associated with the development and progression of a variety of diseases.

[0100] Intoxication and hangover

[0101] Intoxication refers to the symptoms of ataxia, coma, etc. that occur in the body shortly after drinking, which is positively correlated with the blood ethanol concentration, and is alleviated as ethanol is metabolized and eliminated.

[0102] 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 eliminated.

[0103] Pharmaceutical composition and method of administration

[0104] 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 disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. Generally, a satisfactory result is obtained when the active ingredient of the present application is administered to an animal at a dosage of from about 0.00001 mg to 1 g per kg of animal body weight per day (preferably from 0.0001 mg to 0.5 g per kg of animal body weight per day) for experimental mice, 25 mg / kg. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. 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).

[0105] The pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to such pharmaceutical agents carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition, and that have no excessive toxicity after administration. These carriers are well known to those of ordinary skill in the art. A full 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, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0106] The pharmaceutically acceptable carrier in the therapeutic composition can contain a liquid such as water, saline, glycerol and ethanol. In addition, there can be present auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like.

[0107] Generally, the therapeutic composition can be prepared into an injectable agent such as a liquid solution or suspension, or into a solid form of a liquid carrier which is suitable for being made into a solution or suspension before injection.

[0108] Once the composition of the present application is prepared, it can be administered by a conventional route, including but not limited to intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal; particularly, a human.

[0109] When the pharmaceutical composition of the present application is used for actual treatment, various pharmaceutical compositions in different dosage forms can be used depending on the use. Preferably, an intravenous preparation is used.

[0110] The pharmaceutical composition can be prepared by mixing, diluting or dissolving according to a conventional method, and occasionally adding a suitable pharmaceutical additive such as an excipient, a disintegrant, a binder, a lubricant, a diluent, a buffer, an isotonicity, a preservative, a wetting agent, an emulsifying agent, a dispersing agent, a stabilizer and a solubilizer, and the preparation process can be performed in a conventional manner according to the dosage form.

[0111] For example, the preparation of an eye drop can be performed by dissolving the ethyl lactate or a pharmaceutically acceptable salt thereof of the present application in sterile water (in which a surfactant is dissolved) together with a base material, adjusting the osmotic pressure and the pH to a physiological state, and optionally adding a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonicity, an antioxidant and a viscosity increasing agent, and then completely dissolving it.

[0112] The pharmaceutical composition of the present application can also be administered in a sustained release agent form. 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, there can be exemplified ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, and the like, and preferably, biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be exemplified.

[0113] When the pharmaceutical composition of the present application is used for actual treatment, the dose of the ethyl lactate of the present application or the pharmaceutically acceptable salt thereof as an active ingredient can be reasonably determined depending on the body weight, age, sex, and degree of symptoms of each patient to be treated.

[0114] The main advantages of the present application include:

[0115] (1) Ethyl lactate dose-dependently improves alcoholic-related liver disease, with great potential for drug development.

[0116] (2) Ethyl lactate, as a food-derived small molecule with metabolic regulation function, can be applied to the development of food field to regulate metabolic homeostasis of the body.

[0117] (3) Ethyl lactate dose-dependently up-regulates the expression of SIRT1.

[0118] (4) Ethyl lactate can be used as an effective FGF21 inducer.

[0119] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0120] Materials

[0121] All wild-type C57BL / 6J mice were purchased from Shanghai Slac Laboratory Animal Co., Ltd. relying on the animal platform of the Institute of Nutritional and Health, Chinese Academy of Sciences.

[0122] Methods

[0123] (1) ALD modeling in mice

[0124] ALD in mice was induced by slow-acute alcohol feeding. Specifically, 12-14 week-old male or female mice were fed with 5-day liquid alcohol feed to adapt, and the alcohol volume content was gradually increased from 0% to 4%, and then fed with 5% volume alcohol content liquid alcohol feed for 10 days. 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 blood samples and liver samples were quickly collected. The control mice were fed with isocaloric malt dextrin instead of ethanol. During alcohol feeding and gavage, ethyl lactate (52 vol% alcohol containing ethyl lactate 1g, 3g, 10g / L) was added to the alcohol, and PBS was used for the control mice.

[0125] (2) Mouse liver tissue and cell total RNA extraction

[0126] Put 20-50 mg of mouse liver into a 1.5 ml centrifuge tube containing 2 steel balls and 1 ml of Trizol, then put it into a pre-cooled grinder for 120 s, 60 Hz grinding to fully lyse the tissue. After centrifugation 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 for 15 s, then stand at room temperature for 10 min, centrifuge at 9,000 rpm for 10 min at 4°C, and a white precipitate appears at the bottom, which is 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, then add 100-200 μl of DEPC water to dissolve the RNA. The fully dissolved RNA is detected for concentration.

[0127] (3) RNA reverse transcription

[0128] Dilute 12 μl of 3000 ng total RNA with DEPC water. Use the HiScript IIIRT SuperMix for qPCR (+gDNA wiper) kit to reverse transcribe the previously extracted RNA:

[0129] • Genomic DNA removal

[0130] RNA 12 μl

[0131] 4×gDNA wiper Mix 0.2 μl

[0132] 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.

[0133] • RNA reverse transcription

[0134] Add 5×HiScript III qRTSuperMix directly to the reaction tube of the previous step to configure a 20 μl reverse mixing system:

[0135] 5×HiScript III qRTSuperMix 4 μl

[0136] 16 μl of the previous reaction system

[0137] After vortexing and centrifuging to remove air bubbles from the PCR tube, incubate in a PCR instrument at 37°C for 15 min, then at 85°C for 5 s to complete RNA transcription and obtain cDNA.

[0138] ●cDNA dilution

[0139] Add 180 μl of dd water to the reaction tube from the previous step to dilute it 10 times, and set aside.

[0140] (4) Real-time quantitative PCR (qPCR)

[0141] ●qPCR reaction system

[0142] SYBR MIX 5μl

[0143] Forward primer (10 μM) 0.5 μl

[0144] Reverse primer (10 μM) 0.5 μl

[0145] After centrifuging the prepared mixture with vortexing, transfer it to a 384-well qPCR plate. Add 4 μl of diluted cDNA to each well of the plate and seal with a qPCR membrane. Centrifuge at 2000 rpm for 2 min at room temperature. Place the plate in a qPCR instrument and perform amplification and detection according to the following reaction conditions.

[0146] ●Reaction conditions

[0147]

[0148] (5) Isolation of mouse primary hepatocytes

[0149] Normally fed C57BL / 6 mice were anesthetized with isoflurane, and a 24G indwelling intravenous catheter was inserted into their portal vein. The mouse livers were perfused with 50 ml of PBS (calcium and magnesium-free) containing 0.5 mM EDTA. The livers were then perfused with collagenase buffer (66.7 mM NaCl, 6.7 mM KCl, 6.3 mM CaCl2, 0.05% collagenase 2, 0.226 mM BSA, 100 mM HEPES, pH 7.4) for 3-5 min to digest the livers. After complete digestion, the livers were transferred to 10 cm culture dishes to release hepatocytes, followed by filtration through a 40 μm filter. The hepatocytes were washed with PBS and centrifuged at 500 rpm for 5 min at 4 °C. After centrifugation, the cells were resuspended in low-glucose medium containing 10% FBS. Cells were seeded into 6-well plates according to experimental requirements and cultured in a 5% CO2 incubator at 37 °C.

[0150] (6) BODIPY staining of mouse primary hepatocytes

[0151] Freshly isolated mouse primary hepatocytes were evenly spread in 6-well plates with cover glass at the bottom and cultured in low-sugar medium overnight. Serum-free low-sugar medium containing 100 mM ethanol was prepared and used to treat the cells with ethanol and ethyl lactate. Fresh medium containing ethanol and ethyl lactate was replaced every 24 hours for a total of 48 hours. A 5 mM BODIPY dye working solution was prepared by diluting the stock solution 2500-fold. 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 three times, 3 mL per well. Each well was stained with 3 mL and incubated at 37 °C for 15 minutes. From this point on, light was avoided as much as possible. After staining, the cells were washed with 3 mL PBS twice. The cells were fixed with 3 mL 4% paraformaldehyde solution at room temperature for 30 minutes. The fixing solution was removed and the cells were washed with 3 mL PBS three times, 5 minutes each time. The cells were incubated with 2 mL 2 ug / mL DIPY solution for 10 minutes and then washed with PBS three times. After mounting with glycerol gelatin, the cells were stored at 4 °C and photographed.

[0152] (7) Determination of ALT and AST activities in mouse plasma

[0153] The mouse plasma ALT and AST activity assay kit was used according to the instructions. First, 7.5 μL of plasma and 150 μL of reagent one were added to a 96-well plate, which was then incubated at 37 °C for 10 minutes. Then, 50 μL of reagent two was added, and the absorbance was measured at 340 nm for a total of 15 cycles.

[0154] The absorbance values of the fourth and fourteenth cycles were selected for calculation. The ALT and AST activities were calculated using the following formula: ALT / AST (U / L) = ΔOD 340 / ΔT s x 60 x 207.5 / 6.22 / 7.5 / 0.6 x 1000.

[0155] (8) Determination of TG and TC contents in mouse plasma

[0156] The mouse plasma TG and TC content assay kit was used according to the instructions. After adding 2.5 μL of plasma or standard solution to a 96-well plate, 250 μL of working solution was added, and the plate was incubated at 37 °C for 10 minutes. The wavelength was 500 nm, and the enzyme label was used to determine the wavelength of each well with distilled water as a blank control. The concentration of the standard was used to calculate the content of the sample.

[0157] (9) Data statistics and analysis

[0158] The experimental data are expressed as mean ± standard error. The two-tailed unpaired t-test method was used in Excel to compare the differences between the two groups. A statistically significant difference was considered to be P less than or equal to 0.05.

[0159] Example 1: Ethyl lactate dose-dependently improves alcoholic liver disease.

[0160] 1.1 Method

[0161] This study investigated the effect of ethyl lactate on ALD in mice using a slow-on-acute alcohol feeding model (NIAAA or Gao-Binge model). This model simulates the human pattern of long-term heavy drinking followed by recent binge drinking and reproduces the pathological characteristics of human ALD. To investigate the ameliorative effect of ethyl lactate on ALD in mice, the Gao-Binge model was used. During the slow-on-acute alcohol feeding process, alcohol was added to the mice at dose gradients of 1 g / L, 3 g / L, and 10 g / L (groups), and the dose-response relationship of ethyl lactate in improving ALD was verified.

[0162] Modeling and related experiments are as described in the "Methods" section of this application.

[0163] 1.2 Results

[0164] like Figure 1 As shown in Figure A, slow-on-acute alcohol feeding resulted in hepatic steatosis in mice (see white circular vacuoles in H&E staining and red positive areas in Oil Red O staining), while ethyl lactate treatment dose-dependently improved hepatic steatosis in ALD mice. At 1 g / L, ethyl lactate treatment already showed a relatively significant improvement in lipid deposition in mouse livers, and when the dose was increased to 3 g / L and 10 g / L, the effect of ethyl lactate in improving hepatic steatosis in ALD mice became highly significant.

[0165] like Figure 1 As shown in Figure B, ethyl lactate tends to improve liver damage in ALD mice and exhibits a certain dose-response effect.

[0166] like Figure 1 As shown in Figure C, ethyl lactate can significantly reduce hyperlipidemia in mice induced by long-term alcohol feeding, and the effect is more significant with increasing dose of ethyl lactate.

[0167] Oxidative stress and inflammation are considered two key characteristics of ALD (Alcoholic Diet). Prolonged alcohol consumption leads to a compensatory increase in the hepatic microsomal alcohol metabolism pathway. While ethanol is metabolized via CYP2E1, a large amount of reactive oxygen species (ROS) are generated, causing oxidative stress in hepatocytes. These ROS directly lead to lipid peroxidation, increasing the levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) in hepatocytes. These MDA products then form adducts with intracellular proteins, producing cytotoxicity and exacerbating oxidative stress. Therefore, immunohistochemical staining of liver sections for 4-HNE and MDA can be used to determine hepatic ROS production and oxidative stress levels.

[0168] like Figure 1 As shown in Figure D, ethyl lactate dose-dependently reduced the formation of 4-HNE and MDA protein adducts in the liver of ALD mice, thereby improving hepatic ROS production and oxidative stress.

[0169] Neutrophils and macrophages are the most prevalent infiltrating immune cells in the liver of patients with atrial fibrillation (ALD), responding to the inflammatory immune response in the liver. Myeloperoxidase (MPO) is highly expressed in neutrophils and is a common marker protein for neutrophil detection. F4 / 80, on the other hand, is a specific marker protein for macrophages. A certain number of resident macrophages (Kupffer cells) exist within hepatocytes, while activated macrophages aggregate in clusters.

[0170] like Figure 1 As shown in Figure E, ethyl lactate reduced neutrophil and macrophage infiltration in the liver of ALD mice in a dose-dependent manner, thus improving liver inflammation.

[0171] Inflammatory cells and pro-inflammatory factors are associated with genes such as pro-inflammatory cytokines IL-1β (Interleukin-1β) and IL-6 (Interleukin-6), chemokines MCP1 (Monocyte Chemoattractant Protein-1, MCP1) (also known as CCL2, (Chemokine(CC-motif)ligand 2)), and adhesion molecules required for immune cells to function, such as ICAM1 (Intercellular Adhesion Molecule 1) and CD11b.

[0172] like Figure 1As shown in FIG. 6, ethyl lactate down-regulated the expression of inflammation-related genes in the liver of ALD mice in a dose-dependent manner. At a dose of 1 g / L, ethyl lactate significantly improved liver inflammation.

[0173] The inhibition of lipid synthesis in hepatocytes by ethyl lactate reduced the lipid toxicity caused by excessive lipid accumulation, thereby reducing the damage and death of hepatocytes. Further, the release of Damage-Associated Molecular Patterns (DAMPs) by dead hepatocytes was reduced, which to some extent reduced the immune response of the liver, thereby improving the inflammation of the liver of ALD mice.

[0174] The above results show that ethyl lactate has a good effect on improving alcoholic 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 3 g / L and 10 g / L, ALD can be significantly improved, which has the potential to treat alcoholic liver disease.

[0175] Example 2 Ethyl lactate directly acts on hepatocytes to inhibit alcohol-induced lipid synthesis

[0176] 2.1 Method

[0177] Simple lipid deposition is an early stage of ALD development, and excessive lipid accumulation can lead to liver steatosis, which develops into alcoholic fatty liver (AFL). With continuous drinking, liver cell death and inflammation occur, 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.

[0178] Modeling and related experiments are described in the "Methods" section of this application.

[0179] 2.1 Results

[0180] As shown in Figure 2 Under non-alcoholic feeding, the FAS level in the mouse liver was very low and almost undetectable. After slow and acute alcohol feeding, the FAS content in the mouse liver increased significantly and showed spatial distribution.

[0181] The expression of FAS gradually decreased from the portal vein (PV) to the central vein (CV) of the liver, and FAS was most highly expressed in the PV region, which is rich in nutrients. Because alcohol is mostly absorbed in the small intestine and then enters the liver through the PV for catabolism.

[0182] After treatment with ethyl lactate, the FAS level in the liver of mice was significantly reduced, and its spatial distribution was 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.

[0183] As shown in Figure 2 As shown in B, ethyl lactate significantly down-regulated the expression of the above-mentioned lipid synthesis-related genes, and had a dose effect, indicating that ethyl lactate reduced liver lipid deposition by inhibiting alcohol metabolism-induced de novo synthesis of fatty acids, thereby improving acute-on-chronic induced mice ALD.

[0184] In order to prove that ethyl lactate exerts its inhibitory effect on lipid synthesis by directly interacting with hepatocytes, the applicant isolated normal feeding mouse liver primary cells, and verified the improvement effect of ethyl lactate by in vitro cell experiments.

[0185] The liver primary cells were cultured in serum-free low-glucose DMEM medium added with 100 mM ethanol for 48 hours, and treated with 1 mM and 10 mM ethyl lactate. Then BODIPY 493 / 503 staining was performed to determine the lipid synthesis.

[0186] As shown in Figure 2 As shown in C, after 100 mM ethanol incubation for 48 hours, a large number of lipid droplets of different sizes and distributed around the nucleus appeared in the liver cells, indicating that the liver cells synthesized a large amount of lipids after ethanol exposure.

[0187] When treated with a gradient concentration of ethyl lactate, the lipid droplets around the liver cells were significantly reduced Figure 2 D), and still had a dose effect.

[0188] The above results show that ethyl lactate inhibits alcohol-induced de novo synthesis of lipids by directly acting on liver cells.

[0189] In summary, the in vivo and in vitro experimental data prove that ethyl lactate inhibits alcohol-induced lipid synthesis by reducing the level of lipid synthesis-related proteins in liver cells, thereby improving liver steatosis in ALD mice.

[0190] Example 3 Ethyl lactate increases the expression of SIRT1 and FGF21 in liver cells

[0191] Fibroblast growth factor 21 (FGF21) is a hepatic secretory factor primarily secreted by the liver with metabolic regulatory functions, acting on various metabolic organs throughout the body, such as adipose tissue and the liver. Following alcohol consumption, FGF21 is significantly induced within a short period, regulating the metabolic stress induced by excessive alcohol intake. Therefore, the applicant hypothesizes whether FGF21 is a potential downstream target for ethyl lactate to improve ALD.

[0192] The experimental method in this embodiment is as described in the "Methods" section of this application.

[0193] 3.1 In vivo experiments

[0194] like Figure 3 As shown in Figure A, slow-on-acute alcohol feeding induced an increase in FGF21 expression and secretion (plasma FGF21 level) in the mouse liver. Ethyl lactate treatment further significantly increased FGF21 expression and secretion in the mouse liver, exhibiting a dose-response effect.

[0195] Under starvation, FGF21 transcription is regulated by SIRT1. Liver-specific knockout of SIRT1 reduces FGF21 expression and exacerbates starvation-induced physiological steatosis of the liver.

[0196] The applicant detected SIRT1 expression in mouse liver using RT-qPCR, and the results are as follows: Figure 3 As shown in Figure B, SIRT1 expression in mouse liver increased in a dose-dependent manner after treatment with ethyl lactate.

[0197] At the same time, the protein level of SIRT1 in the mouse liver was also significantly increased. Figure 3 (C) This suggests that increased SIRT1 may promote FGF21 transcription.

[0198] Correlation analysis showed that after ethyl lactate treatment, the expression of SIRT1 in mouse liver was significantly positively correlated with the expression and plasma FGF21 levels, while it was significantly negatively correlated with the plasma TG levels in mice. Figure 3 (D).

[0199] Further evidence indicates that ethyl lactate improves ALD in mice by dose-dependently increasing the expression of SIRT1 and FGF21 in the liver, and the increased FGF21 then acts on the liver itself to regulate metabolism and energy homeostasis.

[0200] 3.2 In vitro experiments

[0201] Using the normal mouse hepatocyte line AML12, the cells were cultured in a medium containing 100 mM ethanol for 48 hours to simulate the ethanol exposure of hepatocytes in vitro, while simultaneously being treated with ethyl lactate (1 mM or 10 mM) for 48 hours.

[0202] As shown in FIG. 13A, ethanol exposure increased the expression of FGF21 in hepatocytes, and ethyl lactate treatment further increased the expression of FGF21 in a dose-dependent manner. Figure 3

[0203] Ethyl lactate treatment increased the gene expression and protein level of SIRT1 down-regulated after alcohol exposure (FIGS. 13F and 13G). Figure 3

[0204] In vitro experimental data again confirmed that ethyl lactate has the effect of inducing the expression of FGF21 and SIRT1 in hepatocytes.

[0205] 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 liver lipid synthesis-related genes, thereby reducing liver lipid synthesis and reducing liver lipid deposition, thereby improving liver damage and inflammation.

[0206] Example 4 Ethyl lactate induces liver FGF21 to inhibit lipid degeneration under starvation

[0207] 4.1 Method

[0208] FGF21 is an energy homeostasis regulating hormone in response to metabolic stress, and the inducing factor is 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. The liver will absorb and utilize these fatty acids, so that the liver also appears short-term physiological fat accumulation. Whether ethyl lactate can also induce FGF21 in the liver of mice under physiological conditions was explored by using a 24-hour fasting mouse model.

[0209] Wild-type mice were fed with normal standard feed, and at the same time, they were allowed to drink water containing 3 g / L ethyl lactate freely for one month. In the last week, the mice were additionally injected with 30 mg / kg ethyl lactate intraperitoneally every day. Before being sacrificed, the mice were subjected to 24-hour fasting with free water. The liver tissues of the mice were collected, and H&E and oil red O staining were performed to determine the liver steatosis.

[0210] 4.2 Results

[0211] As shown in FIG. 14A, compared with the liver of mice fed normally, a large number of small lipid droplets appeared in the hepatocytes of mice after 24 hours of fasting, indicating that physiological steatosis appeared in the liver after long-term starvation. Figure 4

[0212] ​​​While the liver lipid droplets of the mice treated with ethyl lactate were significantly reduced after 24 hours of fasting, the lipid droplets were reduced in H&E staining and the ORO red positive staining area was reduced. The ORO staining area quantification results are shown in Figure 4 A.

[0213] Meanwhile, the liver weight index of the fasting mice treated with ethyl lactate was significantly reduced compared with the control group Figure 4 B). This indicates that most of the absorbed free fatty acids in the liver of the 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.

[0214] After fasting, the TG in the plasma was significantly increased due to the lipolysis of adipose tissue, while the TG in the plasma was significantly reduced after treatment with ethyl lactate Figure 4 D). This indicates that more fatty acids are absorbed by the liver after treatment with ethyl lactate, 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.

[0215] As shown in Figure 4 E and F, the expression and secretion of FGF21 in the liver of the mice were significantly increased after fasting, and the expression and secretion of FGF21 in the liver were further increased after treatment with ethyl lactate. And ethyl lactate also significantly increased the expression of FGF21 in the liver of the mice in the fed state, which indicates that ethyl lactate can effectively induce FGF21 in the liver, thereby playing a metabolic improvement role.

[0216] The expression of SIRT1 in the liver was significantly increased after fasting, thereby increasing the transcription of FGF21. The expression of SIRT1 in the liver was further significantly increased after treatment with ethyl lactate, corresponding to the further increased expression of FGF21. However, there was no significant difference in the expression of SIRT1 in the liver of the mice in the fed state.

[0217] In order to determine the functional results of the increased FGF21 in the liver after treatment with ethyl lactate, the 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 such as FAS and SCD1.

[0218] As shown in Figure 4 G, the fatty acid oxidation related genes in the liver of the mice were significantly increased by more than 2.5 times after fasting, thereby increasing the oxidative decomposition of the fatty acids absorbed by the liver from the lipolysis of adipose tissue, thereby providing important energy support for maintaining normal metabolism of the liver.

[0219] After treatment with ethyl lactate, the fatty acid oxidation gene was significantly increased, which corresponds to the increased expression of FGF21. This further indicates that ethyl lactate inhibits starvation-induced steatosis in mouse liver by increasing the expression and secretion of FGF21 in the liver, thereby increasing downstream fatty acid oxidation, so that the free fatty acids absorbed by the liver are fully utilized, and thus reducing the accumulation of lipid droplets in hepatocytes.

[0220] like Figure 4 As shown in Figure H, fasting significantly downregulated the expression of hepatic lipid synthesis genes, thereby reducing energy expenditure under energy deficiency. Furthermore, ethyl lactate treatment further significantly downregulated the expression of FAS and SCD1 genes in the liver of fasted mice, thereby further reducing hepatic lipid synthesis.

[0221] Even in a fed state, ethyl lactate treatment significantly downregulated the expression of FAS and SCD1 in the mouse liver. This indicates that even in a fed state, ethyl lactate treatment can inhibit lipid synthesis and reduce lipidogenesis in the mouse liver. This is consistent with the reduction in lipid droplets in the mouse liver after ethyl lactate treatment in a fed state. Figure 4 The decrease in plasma triglyceride levels (B) is consistent with the increase in hepatic FGF21 expression and secretion.

[0222] In conclusion, ethyl lactate can inhibit steatosis in mouse liver under physiological conditions by increasing the expression and secretion of FGF21.

[0223] Example 5: Ethyl lactate improves acute alcohol gavage-induced hangover in mice.

[0224] 5.1 Method

[0225] Mice were induced to become intoxicated by gavage with 2.5 g / kg body weight of ethanol. The degree of intoxication was assessed 1 hour later using open field and rotarod tests. Mice were then induced to experience hangovers by gavage with 5 g / kg body weight of ethanol. The hangover status was assessed 6 hours later using open field and rotarod tests. Each mouse underwent only one behavioral test after gavage. The effects of ethyl lactate (10 g / L of ethyl lactate in 52 vol% ethanol) on intoxication and hangovers were investigated by simultaneously adding ethyl lactate to the alcohol.

[0226] 5.2 Results

[0227] like Figure 5 As shown, under intoxication (2.5 g / kg ethanol administered by gavage for 1 h), ethyl lactate did not affect the distance mice traveled in the open field or the time spent on the rotarod.

[0228] 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, 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.

[0229] Example 6 Lactic acid does not affect alcohol-induced liver steatosis

[0230] 6.1 Method

[0231] 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.

[0232] The modeling and experimental methods in this example are as described in the "Methods" section of the present application, in which the ethyl lactate treatment of mice is replaced by lactic acid (52 vol% alcohol containing 1 g / L lactic acid) treatment.

[0233] 6.2 Results

[0234] 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 improving alcoholic liver disease, but rather directly acts on hepatocytes to function.

[0235] Discussion

[0236] This patent discovers a food-derived natural small molecule compound, ethyl lactate, which has the effect and mechanism of improving alcoholic 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 up-regulated the expression of SIRT1, promoted the expression and secretion of liver FGF21 to regulate liver metabolism, and improved alcoholic steatohepatitis.

[0237] The patent first discovers that ethyl lactate has in vivo biological activity, participates in the regulation of ALD occurrence and development, and has a good improvement and treatment effect on alcoholic liver disease in mice. 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.

[0238] All documents mentioned in the present application are incorporated herein by reference as if each individual document were specifically and individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits depending on the precise placement of the elements, or practices, of the present application or depending on the exact composition of the compositions or methods, in some cases, features of the application can be used to advantage without a corresponding use of other features.

Claims

1. The use of ethyl lactate or a pharmaceutically acceptable salt thereof, characterized in that, For use in the preparation of a pharmaceutical or pharmaceutical composition, said pharmaceutical or pharmaceutical composition for one or more uses selected from the group consisting of: (a1) Prevention and treatment of alcoholic liver disease; (a2) SIRT1 agonists; (a3) An agonist of FGF21; (s4) Improves hangovers.

2. The use as described in claim 1, characterized in that, The alcoholic liver disease includes chronic alcoholic liver disease, acute alcoholic liver disease, or a combination of chronic-onset and acute alcoholic liver disease.

3. The use as described in claim 1, characterized in that, The alcoholic liver disease is selected from the following group: hepatic steatosis, alcoholic steatohepatitis, hepatic oxidative stress, alcoholic fatty liver, or a combination thereof.

4. The use as described in claim 1, characterized in that, The drug or drug composition is also used for purposes selected from the group consisting of: (a1) Improves liver fat deposition; (a2) Inhibitors of inflammatory factors in alcoholic liver disease; (a3) Inhibits alcohol-induced lipid synthesis; (a4) Inhibitors of lipid synthesis-related factors.

5. The use as described in claim 1, characterized in that, The drug or drug composition is administered to animals selected from the group consisting of mammals or rodents.

6. An alcoholic beverage product for preventing and treating alcohol-related liver disease and / or improving hangovers, characterized in that, The alcoholic beverages contain ethyl lactate.

7. The product as described in claim 6, characterized in that, The content of ethyl lactate in the alcoholic beverage is 0.01–15 g / L, preferably 0.1–10 g / L, and more preferably 0.2–5 g / L.

8. A method for promoting SIRT1 expression in hepatocytes, characterized in that, Including the following steps: Contacting hepatocytes with ethyl lactate or its pharmaceutically acceptable salts promotes SIRT1 expression in hepatocytes.

9. A method for promoting FGF21 expression in hepatocytes, characterized in that, Including the following steps: Contacting hepatocytes with ethyl lactate or its pharmaceutically acceptable salts promotes the expression of FGF21 in hepatocytes.

10. A method for inhibiting lipid synthesis in hepatocytes, characterized in that, Including the following steps: Ethyl lactate or its pharmaceutically acceptable salts are brought into contact with hepatocytes, thereby inhibiting lipid synthesis in hepatocytes.

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