Application of acetylcholine ACh in preparation of medicine for regulating ACSL4 ubiquitination degradation

Abstract

The invention discloses application of acetylcholine ACh in preparation of medicine for regulating and controlling ACSL4 ubiquitination degradation, and belongs to the field of biomedicine.The ubiquitination degradation of ACSL4 protein can be regulated by using acetylcholine or pharmaceutically acceptable salt of acetylcholine, and the invention particularly discloses a related pharmaceutical composition and application of the pharmaceutical composition in preparation. The invention also relates to a method for adjusting the ubiquitination degree of ACSL4 in in-vitro cells or tissues for a non-therapeutic purpose and a method for constructing a cell model with low expression of ACSL4 and / or high ubiquitination of ACSL4 protein.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to the application of acetylcholine (ACh) in the preparation of drugs for regulating the ubiquitination degradation of ACSL4. Background Art

[0002] Acetylcholine (ACh) is an important neurotransmitter responsible for transmitting signals between neurons. It is involved in the regulation of muscle contraction, memory formation, attention, and arousal. At the neuromuscular junction, acetylcholine activates muscle contraction and is essential for movement and coordination. The roles of acetylcholine in the autonomic nervous system include regulation of heart rate, vasodilation, and glandular secretion.

[0003] ACSL4 (long-chain acyl-CoA synthetase family member 4) can catalyze the binding of long-chain fatty acids to coenzyme A to form acyl-CoA, which is a key step in the process of fatty acids participating in cell membrane phospholipid synthesis, energy metabolism, etc. Ubiquitination is an important post-translational modification process of proteins, which regulates the function, localization and stability of proteins by covalently linking ubiquitin molecules to target proteins. The ubiquitination process involves a series of enzymatic reactions, mainly including the participation of ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides the use of acetylcholine or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for regulating ubiquitination of ACSL4.

[0006] Furthermore, the pharmaceutical composition is administered orally, via suppository, around a lesion, on a lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

[0007] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable adjuvant.

[0008] Furthermore, the pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0009] Further, the acid addition salt includes hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, argininate, fumarate, nicotinate, phthalate or oxalate of acetylcholine.

[0010] Furthermore, the base addition salts include lithium salts, sodium salts, potassium salts, barium salts, calcium salts, magnesium salts, aluminum salts, iron salts, ferrous salts, copper salts, and zinc salts of acetylcholine.

[0011] Furthermore, the ubiquitination of ACSL4 includes total ubiquitination of ACSL4 protein and / or K48-linked ubiquitination.

[0012] In some embodiments, the compounds used in the pharmaceutical compositions of the present invention can be used in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts are conventional non-toxic salts or quaternary ammonium salts, for example, from non-toxic organic or inorganic acids. These salts can be prepared in situ in the administration adjuvant or during the preparation of the dosage form, or by reacting the compounds described herein in their free base or acid form with a suitable organic or inorganic acid or base, respectively, and separating the salts formed in the subsequent purification process.

[0013] In some embodiments, suitable acids capable of forming salts with the compounds disclosed herein include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, etc., and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, p-aminobenzenesulfonic acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Suitable bases capable of forming salts with the compounds disclosed herein include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc., and organic bases such as monoalkylamines, dialkylamines and trialkylamines and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, pyridine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc.) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine, triethanolamine, etc.).

[0014] The term "pharmaceutically acceptable" as used herein designates a carrier that is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof. Pharmaceutically acceptable adjuvants used in the vaccine compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection or Ringer's dextrose and lactate injection), non-aqueous vehicles (e.g., non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffer), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween 80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0015] The present invention provides a pharmaceutical composition for regulating ubiquitination of ACSL4, wherein the pharmaceutical composition comprises acetylcholine or a pharmaceutically acceptable salt thereof.

[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0017] Further, the adjuvant includes an emulsifier, a glidant, a sweetener, a surfactant, a preservative, a dye / colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant, a suspending agent, a stabilizer, an isotonic agent, a pH adjusting and / or buffering agent, a solvent, a diluent or an excipient.

[0018] In some embodiments, the pharmaceutical composition also includes other drugs for assisting in regulating ubiquitination, including proteasome inhibitors and deubiquitinase (DUB) inhibitors, wherein proteasome inhibitors inhibit the activity of proteasomes and prevent the degradation of ubiquitinated proteins, thereby affecting the homeostasis of intracellular proteins. Common proteasome inhibitors include Bortezomib, Carfilzomib, and MG-132. Deubiquitinase (DUB) inhibitors inhibit the activity of deubiquitinases, affecting the degradation and stability of ubiquitinated proteins, thereby regulating the function of intracellular proteins. PR-619 is a non-selective, reversible inhibitor of ubiquitin and ubiquitin-like isopeptidase, which is cell permeable and can be used to study the regulation of intracellular ubiquitination processes.

[0019] For administration to a subject, the compounds described herein may be provided in the form of a pharmaceutically acceptable (e.g., sterile) composition. These pharmaceutically acceptable compositions comprise an effective amount of one or more compounds described herein, formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical compositions of the invention may be administered orally, for example, in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example, in the form of suppositories, or transdermally or topically, for example, in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or by inhalation, for example, in the form of nasal sprays or aerosol mixtures, or, for example, in the form of microcapsules, implants or rods.

[0020] The present invention provides a method for regulating the ubiquitination degree of ACSL4 in cells or tissues in vitro for non-therapeutic purposes, which comprises using acetylcholine or a pharmaceutically acceptable salt thereof.

[0021] Furthermore, the cells or tissues are selected from lung cells, microglia, tumor cells, and chondrocytes.

[0022] Furthermore, the cells or tissues are lung epithelial cells, MLE12 cells, or A549 cells.

[0023] The present invention provides a method for constructing a cell model of low expression of ACSL4 / or high ubiquitination of ACSL4 protein, the method comprising the step of treating cells in vitro with acetylcholine.

[0024] The present invention provides the use of acetylcholine or a pharmaceutically acceptable salt thereof in preparing a pharmaceutical composition for inhibiting ACSL4 expression.

[0025] Furthermore, the cells that inhibit ACSL4 expression are human or non-human mammalian cells.

[0026] Furthermore, the cells include lung cells, microglia, tumor cells, and chondrocytes.

[0027] Furthermore, the cells that inhibit ACSL4 expression are lung epithelial cells, MLE12 cells, and A549 cells.

[0028] The present invention provides the use of a reagent for detecting the level of acetylcholine or a pharmaceutically acceptable salt thereof in the preparation of a product for detecting the ubiquitination level of ACSL4.

[0029] The present invention provides a product comprising a reagent for detecting the level of acetylcholine or a pharmaceutically acceptable salt thereof.

[0030] Furthermore, the sample of the product is derived from a human or non-human mammal.

[0031] Furthermore, the non-human mammals include mice, cows, sheep, horses, rabbits, dogs, camels, and pigs.

[0032] Furthermore, the sample includes blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, tissue biopsy or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid or the subject's exhaled breath.

[0033] In some embodiments, the sample is taken directly from the subject without further processing, for example, blood can be obtained from the subject's peripheral circulatory system (e.g., via a blood collection needle). The sample may include, for example, blood, urine, feces, saliva, cerebrospinal fluid, and sweat. Non-limiting examples of such samples include blood (or components of blood - e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location of a subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, tissue fluid from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, ocular fluids, sweat, mucus, earwax, oils, glandular secretions, cerebrospinal fluid, hair, nails, skin cells, plasma, nasal swab or nasopharyngeal wash, cerebrospinal fluid, umbilical cord blood, lymphatic fluid, and / or other excreta or body tissues.

[0034] The present invention uses the term "level of expression" or "expression level" to generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetic coding and / or epigenetic information) is converted into structures that exist and function in a cell. Therefore, as used herein, "expression" can refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., by proteolysis). "Expressed genes" include genes that are transcribed into polynucleotides (e.g., mRNA) and then translated into polypeptides, as well as genes that are transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of ACSL4 protein expression in cells induced by S. a being regulated by ACh are shown in Figure 1. A is the result of lung epithelial cells, B is the result of MLE12 cells, and C is the result of A549 cells.

[0036] Figure 2 This is a result diagram that proves that the addition of ACh to cells can regulate the post-translational modification of ACSL4 protein. Among them, A shows that under the premise of adding ribosomal inhibitor (CHX), ACh significantly reduces the level of ACSL4 protein in MLE12 cells, and B shows that under the premise of adding ribosomal inhibitor (CHX) and proteasome inhibitor (MG132) at the same time, the phenomenon of ACh reducing the level of ACSL4 protein is inhibited.

[0037] Figure 3 The total ubiquitination modification and K48-linked ubiquitination modification result diagram of ACSL4 protein after ACh was used in lung tissue epithelial cells. Among them, A is the total ubiquitination modification result diagram, and B is the K48-linked ubiquitination modification result diagram.

[0038] Figure 4 The following are the results of ubiquitination and degradation of ACSL4 protein in MLE12 cells and A549 cells after ACh treatment. A is the result of total ubiquitination modification in MLE12 cells, B is the result of K48-linked ubiquitination modification in MLE12 cells, C is the result of total ubiquitination modification in A549 cells, and D is the result of K48-linked ubiquitination modification in A549 cells.

[0039] Figure 5 This is a diagram showing the therapeutic effect of ACh on acute lung injury in mice infected with S. a. A is the result of lung pathological injury, B is the result of survival rate, C is the result of lung injury score, D is the result of lung dry-wet ratio, E is the result of total cell count in bronchoalveolar lavage fluid, and F is the result of alveolar epithelial cell permeability. DETAILED DESCRIPTION

[0040] Unless otherwise indicated, the term "and / or" is used in this disclosure to mean "and" or "or". Unless explicitly indicated, the term "or" is used to mean "and / or" to mean the only alternative or that the alternatives are mutually exclusive, although this disclosure supports the only alternative and the definition of "and / or".

[0041] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" etc. will be understood to include the stated elements or components without excluding other elements or other components. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0042] In this article, the disclosed pharmaceutically acceptable salts of acetylcholine are encompassed in the present invention. Representative "pharmaceutically acceptable salts" include, for example, water-soluble and water-insoluble salts, such as acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium salt, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycollylarsanilate e), hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate, lactate, lactobionate, laurate, magnesium salt, malate, maleate, mandelate, methanesulfonate, methyl bromide, methylnitrate, methylsulfate, mucate, naphthylsulfonate, nitrate, N-glucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1 , 1-methylene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, toluenesulfonate, triethyl iodide, and valerate.

[0043] The term "treating" with respect to a subject refers to ameliorating at least one symptom of a disorder in the subject. Treatment includes curing, ameliorating or at least partially alleviating a disorder.

[0044] With respect to a subject, the term "prevent" or "preventing" refers to preventing a disease or condition from causing suffering to the subject. Prevention includes prophylactic treatment. For example, prevention may include administering a compound disclosed herein to a subject before the subject suffers from the disease, and the administration will prevent the subject from suffering from the disease.

[0045] In the present invention, treatment / prevention may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. In the present invention, treatment / prevention may refer to both treatment and prevention. Beneficial or desired clinical results include, but are not limited to, relief of symptoms; alleviation of the extent of a disorder, disorder or disease; stabilization (i.e., not worsening) of the state of a disorder, disorder or disease; delaying the occurrence of a disorder, disorder or disease or slowing its progression; improving a disorder, disorder or disease state; and alleviation (whether partial or complete) (whether detectable or undetectable) or improvement or improvement of a disorder, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a prolonged survival period compared to the expected survival period when not receiving treatment.

[0046] The terms "inhibit" and "mitigate" or any variation of these terms include any measurable or complete inhibition that achieves the desired result. For example, a decrease in activity (e.g., ACSL4 protein binding activity) may be about, at most about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to normal, or any range derivable therein.

[0047] As used in this disclosure, the term "administering" refers to directly administering a disclosed compound or a pharmaceutically acceptable salt or composition of a disclosed compound to a subject, or administering a prodrug derivative or analog or composition of the compound or a pharmaceutically acceptable salt of the compound to a subject, thereby forming an equivalent amount of the active compound in the subject.

[0048] In embodiments of the present invention, patients and subjects of humans or other mammals falling within the scope of the present invention, and including any individual desired to be examined or treated using the methods of the present invention. Suitable mammals include, but are not limited to, primates, livestock animals (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captured wild animals (e.g., foxes, deer, dingoes).

[0049] The content of the present invention is described in detail below in conjunction with specific embodiments and drawings. In the following, only certain exemplary embodiments are briefly described. As can be appreciated by those skilled in the art, the described embodiments can be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be illustrative and non-restrictive in nature. The acquisition methods of the various biological materials described in the embodiments are only to provide a way of experimental acquisition to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0050] Example 1

[0051] 1. Experimental instruments High-speed cryogenic centrifuge and high-speed cryogenic tissue grinder were purchased from Wuhan Sewell Biotechnology Co., Ltd.; animal lung quantitative atomization syringe, mouse laryngoscope, and mouse airway intubation auxiliary fixing table.

[0052] 2. Experimental reagents Cycloheximide (CHX) and MG132 were purchased from SELLECT; Acetylcholine chloride (ACh) was purchased from MCE, USA.

[0053] 3. Experimental strains USA300 methicillin-resistant Staphylococcus aureus (MRSA) was frozen and stored in our laboratory. 1 mL of glycerol was taken out from the -80℃ freezer and added to the sterilized and cooled 100 mL BHI agar medium. The culture was carried out at 37℃ and 200 rpm for 16 hours (the OD value at 600 nm was about 0.5-1.0 during the logarithmic growth phase). The colony count was used to determine the number of colonies (about 2.5×10 9 CFU / mL). After the bacterial solution is shaken, 1 mL is pipetted into a sterile enzyme-free centrifuge tube, centrifuged at 3000 r / 10 min, the supernatant is discarded, and the colonies are resuspended with sterile enzyme-free PBS to adjust the concentration to 1×10 9 CFU / mL, and place in a 4°C refrigerator for later use.

[0054] 4. Experimental mice 6-8 week old male C57BL / 6J mice were purchased from Beijing SpEffect Biotechnology Co., Ltd.

[0055] 5. Experimental cells The human or mouse normal alveolar epithelial cell line A549 / MLE12 cell line was purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0056] 6. Experimental methods 6.1 Construction of mouse acute lung injury model 1) Anesthesia: Inject 1% sodium pentobarbital (10 μL / g) intraperitoneally into C57BL / 6J mice. Pinch the mouse's tail with your fingers. If there is no reaction, it means that the anesthesia is successful. The anesthesia lasts for about 10 minutes.

[0057] 2) Fixation: After successful anesthesia, place the mouse on an airway intubation-assisted fixation table, fix it in a supine position, hang a rubber band on the upper incisors, and lay the mouse straight and flat.

[0058] 3) Intubation: Place the lighting lamp close to the front of the mouse's neck, lift the lower jaw with your left hand, fix the pulled-out tongue, insert curved tweezers into the oral cavity, hold the mouse's upper jaw, press the root of the mouse's tongue, expose the field of vision as much as possible, and observe the position of the vocal cords with the help of a cold light source. Note that the mouse's trachea is relatively close to the root of the tongue, and is on the back of the neck of the esophagus. However, the opening of the esophagus is not visible during the operation, so you should pay close attention to changes in the vocal cords. Under the illumination of a cold light source, the opening of the mouse's trachea is a bright spot, and the syringe is gently inserted into the trachea.

[0059] 4) Injection: Push the syringe slowly to inject bacterial solution or an equal amount of PBS. After the injection is completed, slowly pull out the syringe needle to prevent the injection solution from flowing back.

[0060] 6.2 Experimental design The experimental mice were divided into 3 groups. The control group: an equal amount of PBS was injected into the airways of the mice; the bacterial infection group: 1×10 8 CFU Staphylococcus aureus; bacterial infection plus ACh treatment group: 1×10 8 CFU of Staphylococcus aureus and treated with intraperitoneal injection of ACh (20 mg / kg).

[0061] 6.3 Observation of mouse survival curve In order to determine the effect of ACh on the survival time of Sa-infected mice, C57BL / 6 mice were grouped according to the above method. After completing the corresponding treatment, the survival of the mice was recorded. After 48 hours, the surviving mice in all groups were euthanized and the survival rate of the mice was calculated.

[0062] 6.4 HE staining of mouse lung tissue After the mice were anesthetized and cardiac perfusion was performed, the lung tissue of the mice was carefully peeled off. The lung tissue of the mice was fixed with 4% paraformaldehyde solution for 12 hours. The lung tissue of the mice was dehydrated, embedded, frozen sectioned, hematoxylin stained, differentiated, bluing, eosin stained, dehydrated, and sealed, and then observed under an optical microscope and photographed for preservation.

[0063] 6.5 Mouse lung pathology scoring After HE staining, the morphology of mouse lung tissue was observed under a microscope. The Smith scoring method was used to perform semi-quantitative analysis of pulmonary edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, atelectasis, and hyaline membrane formation, with a score of 0-4 (no damage 0 point, lesion range <25% = 1 point, lesion range 25%-50% = 2 points, lesion range 50%-75% = 3 points, lesion full field of view = 4 points). Three high-power fields were analyzed for each specimen, and the average value was taken as the lung injury score.

[0064] 6.6 Wet to dry ratio (W / D) of mouse lung tissue The left lower lung of the mouse was weighed, placed on tin foil, baked in an oven at 60°C for 72 h until constant weight was reached, and then weighed again to calculate the W / D of the lung tissue.

[0065] 6.7 Detection of cell number in mouse bronchoalveolar lavage fluid (BALF) After anesthetizing the mice, BALF was obtained through tracheal intubation. The lungs were lavaged with 1 mL PBS for 3 times. The obtained BALF was centrifuged and resuspended with 1 ml PBS and counted on a cell counter.

[0066] 6.8 Lung tissue permeability test Evans blue dye was injected through the tail vein, and the lung tissue was removed by thoracotomy 1 hour later, and the coloration of the lung tissue was observed by photographing.

[0067] 7. Experimental results like Figure 1 As shown, ACh can inhibit the expression of ACSL4 protein in lung epithelial cells induced by Staphylococcus aureus infection (S. a). Figure 1 A indicates that ACh can inhibit the expression of ACSL4 protein in lung epithelial cells infected with S. a. Figure 1 B shows that ACh can inhibit the expression of ACSL4 protein in MLE12 cells infected with S. a. Figure 1 C shows that ACh can inhibit the expression of ACSL4 protein in A549 cells infected with S. a.

[0068] like Figure 2 As shown, ACh regulates the post-translational modification of ACSL4 protein. Figure 2 A When ribosomal inhibitor (CHX) inhibited protein synthesis, ACh significantly reduced the ACSL4 protein level in MLE12 cells, suggesting the existence of post-translational modification; Figure 2B shows that in the presence of a proteasome inhibitor (MG132), the phenomenon of ACh reducing ACSL4 protein levels was inhibited, suggesting that ACh reduces ACSL4 protein levels through protein degradation.

[0069] like Figure 3 As shown, ACh promotes the ubiquitination and degradation of ACSL4 protein in lung epithelial cells. Figure 3 A represents the total ubiquitination modification of ACSL4 protein in lung epithelial cells promoted by ACh; Figure 3 B: ACh promotes K48-linked ubiquitination modification of ACSL4 protein in lung epithelial cells.

[0070] like Figure 4 As shown in the figure, ACh promotes the ubiquitination and degradation of ACSL4 protein in MLE12 cells and A549 cells. Figure 4 A and Figure 4 B shows that ACh promotes the total ubiquitination and K48-linked ubiquitination modification of ACSL4 protein in MLE12 cells; Figure 4 C and Figure 4 D shows that ACh promotes the total ubiquitination and K48-linked ubiquitination modification of ACSL4 protein in A549 cells.

[0071] like Figure 5 As shown, ACh significantly alleviated the acute lung injury in mice infected with S. a. Figure 5 A is that ACh reduces the pathological damage of the lung caused by S. a infection; Figure 5 B is that ACh significantly improves the survival rate of mice infected with S. a; Figure 5 C: ACh reduces the lung injury score of mice infected with S. a; Figure 5 D: ACh can reduce the lung dry-wet ratio of S. a-infected mice; Figure 5 E: ACh can reduce the total number of cells in the bronchoalveolar lavage fluid of mice infected with S. a; Figure 5 F: ACh can reduce the permeability of alveolar epithelial cells in mice infected with S. a.

[0072] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of acetylcholine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for regulating ubiquitination of ACSL4.

2. The use according to claim 1, wherein the pharmaceutical composition is administered orally, by suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable adjuvant.

3. The use according to claim 1, wherein the pharmaceutically acceptable salts include acid addition salts and base addition salts; Preferably, the acid addition salt comprises a hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, argininate, fumarate, nicotinate, phthalate or oxalate of acetylcholine; Preferably, the base addition salt comprises lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt of acetylcholine; Preferably, the ubiquitination of ACSL4 includes total ubiquitination and / or K48-linked ubiquitination of ACSL4 protein.

4. A pharmaceutical composition for regulating ubiquitination of ACSL4, comprising acetylcholine or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable adjuvant; Preferably, the adjuvant comprises an emulsifier, a glidant, a sweetener, a surfactant, a preservative, a dye / colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant, a suspending agent, a stabilizer, an isotonic agent, a pH adjusting and / or buffering agent, a solvent, a diluent or an excipient.

6. A method for regulating the ubiquitination degree of ACSL4 in cells or tissues in vitro for non-therapeutic purposes, the method comprising using acetylcholine or a pharmaceutically acceptable salt thereof; Preferably, the cells or tissues are selected from lung cells, microglia, tumor cells, chondrocytes.

7. A method for constructing a cell model of low ACSL4 expression / or high ubiquitination of ACSL4 protein, the method comprising the step of treating cells in vitro with acetylcholine.

8. Use of acetylcholine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for inhibiting ACSL4 expression.

9. Use of a reagent for detecting the level of acetylcholine or a pharmaceutically acceptable salt thereof in the preparation of a product for detecting the ubiquitination level of ACSL4.

10. A product comprising a reagent for detecting the level of acetylcholine or a pharmaceutically acceptable salt thereof; Preferably, the sample of the product is derived from a human or non-human mammal; Preferably, the non-human mammals include mice, cows, sheep, horses, rabbits, dogs, camels, and pigs.

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