WO2024060359A1 - 甘油磷脂类化合物在预防和治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖中的用途 - Google Patents

甘油磷脂类化合物在预防和治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖中的用途 Download PDF

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WO2024060359A1
WO2024060359A1 PCT/CN2022/128845 CN2022128845W WO2024060359A1 WO 2024060359 A1 WO2024060359 A1 WO 2024060359A1 CN 2022128845 W CN2022128845 W CN 2022128845W WO 2024060359 A1 WO2024060359 A1 WO 2024060359A1
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obesity
mice
dlpc
atherosclerosis
group
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PCT/CN2022/128845
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English (en)
French (fr)
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朱大海
张勇
李虎
陈梅红
胡晓娣
梁娜
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中国医学科学院基础医学研究所
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Publication of WO2024060359A1 publication Critical patent/WO2024060359A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the use of a glycerophospholipid compound in preparing drugs for preventing and/or treating hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity.
  • Non-communicable diseases including cardiovascular diseases, cancer and diabetes, account for 70% of early deaths worldwide and are the leading causes of death and premature disability [1] .
  • Obesity is a major risk factor for non-communicable diseases and is associated with reduced life expectancy, which is estimated to be reduced by 20 years depending on the severity of the condition and comorbid diseases.
  • the World Health Organization defines obesity as excessive fat accumulation that may harm health, with a BMI of 30kg/m2 at diagnosis [4] .
  • metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease
  • cardiovascular diseases such as atherosclerosis, hyperlipidemia, myocardial infarction and stroke
  • musculoskeletal diseases such as bone arthritis
  • Alzheimer's disease depression
  • cancer such as breast, ovarian, prostate, liver, kidney, and colon
  • the root cause of obesity is an energy imbalance between calories consumed and calories expended.
  • lifestyle and behavioral interventions aimed at reducing caloric intake and increasing energy expenditure have limited effectiveness because of complex and long-lasting hormonal, metabolic and neurochemical adaptations that resist weight loss and promote weight regain [6] .
  • most current obesity treatment drugs are appetite suppressants, aiming to suppress appetite by regulating neurotransmitters in the hypothalamus, or inhibitors of pancreatic and gastric lipase [7] .
  • this type of drugs has certain limitations, such as nutrient absorption impairment, dizziness, diarrhea, and high recurrence rate [8] .
  • maintaining a healthy lifestyle long-term without any changes is a huge challenge. People with obesity can easily become frustrated when faced with painful and endless lifestyle changes.
  • the object of the present invention is to provide a glycerophospholipid compound for use in preventing and/or treating hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity.
  • the present invention provides a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same for the prevention and/or treatment of hyperlipidemia, atherosclerosis, and non-alcoholic fatty liver disease. or use in obesity medications,
  • the present invention provides the use of a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same in the preparation of a medicament for preventing and/or treating obesity.
  • the present invention provides a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same in the preparation of medicaments for preventing and/or treating hyperlipidemia. use.
  • -OR is selected from -OH, choline group, L-serine group;
  • M + is selected from Na + , K + ;
  • the compound containing the structure represented by formula (I) is selected from the following compounds:
  • the use according to the present invention wherein the obesity is obesity induced by high-fat diet.
  • the hyperlipidemia is hyperlipidemia induced by a high-fat diet.
  • the use according to the invention wherein the atherosclerosis is high-fat diet induced atherosclerosis.
  • non-alcoholic fatty liver disease is non-alcoholic fatty liver disease induced by high-fat diet.
  • the use according to the present invention is characterized in that the compound reduces the volume of white adipocytes, preferably inducing browning of white adipose tissue, thereby preventing or treating the hyperlipidemia and atherosclerosis. cirrhosis, non-alcoholic fatty liver disease and obesity.
  • the use according to the present invention is characterized in that the compound reduces pathological symptoms and signs to prevent or treat hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity.
  • the use according to the present invention is characterized in that the compound prevents or treats hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity by slowing down the weight gain induced by a high-fat diet.
  • the use according to the present invention is characterized in that the compound prevents or treats the hyperlipidemia, atherosclerosis, and non-alcoholic fat by reducing the size of white adipocytes. Liver and obesity.
  • the use according to the present invention is characterized in that the compound prevents or treats the hyperlipidemia, atherosclerosis, non-alcoholic disease by reducing the deposition of lipids in serum. Fatty liver disease and obesity.
  • the use according to the present invention is characterized in that the compound prevents or treats hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity by enhancing the body's glucose tolerance and insulin sensitivity.
  • the use according to the present invention is characterized in that the compound prevents or treats the hyperlipidemia, atherosclerosis, and non-alcoholic fat by promoting the browning of white adipose tissue. Liver and obesity.
  • the pharmaceutical composition contains a therapeutically effective amount of a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable salt thereof.
  • Acceptable carrier or excipient a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable salt thereof.
  • the therapeutic method is preferably Chemotherapy, targeted therapy
  • the therapeutic agent is preferably another agent for the prevention and/or treatment of hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity.
  • the structural characteristic of the compound containing the structure shown in formula (I) of the present invention is that the fatty acyl side chain in the phospholipid is a double-chain octadecadienoic acid, and the phosphate group of the fatty acyl side chain binding part can be connected to different
  • the polar head group forms different glycerophospholipids, preferably 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine) (18 :2 PC, DLPC).
  • the choline base of the present invention is group; the L-serine group is group.
  • the "pharmaceutically acceptable salts" mentioned in the present invention refer to pharmaceutically non-toxic acid addition salts and base addition salts.
  • the acid addition salt is a salt formed by a compound and a suitable inorganic acid or organic acid, including hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalic acid Salt, malonate, valerate, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, malic acid Salt, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate and lauryl sulfonate, etc.
  • the base addition salt is a salt formed by a compound and a suitable inorganic base or organic base, including salts formed with alkali metal, amine or quaternary ammonium compounds, such as sodium salt, lithium salt, potassium salt, calcium salt , magnesium salts, amine salts, tetramethyl quaternary ammonium salts, tetraethyl quaternary ammonium salts, choline salts, especially sodium salts and choline salts; amine salts, including ammonia (NH 3 ), primary amines, secondary amines Or salts formed from tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, ethanolamine salts, serine salts, lysine salts and arginine salts, especially serine salts .
  • alkali metal, amine or quaternary ammonium compounds such as sodium salt, lithium salt,
  • compositions containing the active ingredients may be in forms suitable for oral administration, such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixir.
  • Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives, to provide medicinal preparations that are pleasing to the eye and palatable.
  • Tablets contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients suitable for the manufacture of tablets.
  • excipients may be inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium or sodium phosphate; granulating agents and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, corn starch or alginic acid; a binder such as starch, gelatin, polyvinylpyrrolidone or gum arabic; and a lubricant such as magnesium stearate, stearic acid or talc.
  • These tablets may be uncoated or may be coated by known techniques to mask the taste of the drug or to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained release over an extended period of time.
  • water-soluble taste masking substances such as hydroxypropyl methyl cellulose or hydroxypropyl cellulose, or time extending substances such as ethyl cellulose, cellulose acetate butyrate may be used.
  • Hard gelatin capsules may also be used in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin or olive oil. Oral formulation is available in soft gelatin capsules.
  • an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin
  • a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin or olive oil.
  • Oral formulation is available in soft gelatin capsules.
  • Aqueous suspensions contain the active substances and excipients suitable for the preparation of aqueous suspensions for mixing.
  • excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and gum arabic; dispersing or wetting agents, which may be natural
  • the resulting phospholipids such as lecithin, or the condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or the condensation products of ethylene oxide with long-chain fatty alcohols, such as heptadecanoethyleneoxycetylene Heptadeca ethyl eneoxy cetanol, or the condensation product of ethylene oxide with partial esters derived from fatty acids and hexitols, such as polyethylene oxide sorbitol monooleate, or ethylene oxide with Condensation products of partial esters derived from fatty acids and hexitol anhydrides,
  • Aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more colorants, one or more flavoring agents and one or more sweeteners.
  • preservatives such as ethylparaben or n-propylparaben
  • colorants such as ethylparaben or n-propylparaben
  • flavoring agents such as sucrose, saccharin or aspartame.
  • Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis, olive, sesame or coconut oil, or a mineral oil, such as liquid paraffin.
  • Oil suspensions may contain thickening agents such as beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents as described above may be added to provide a palatable preparation. These compositions can be preserved by adding antioxidants such as butylated hydroxyanisole or alpha-tocopherol.
  • Dispersible powders and granules suitable for the preparation of aqueous suspensions may provide the active ingredient and for mixing a dispersing or wetting agent, a suspending agent or one or more preservatives, by the addition of water. Suitable dispersing or wetting agents and suspending agents are as described above. Other excipients such as sweetening agents, flavoring agents and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid.
  • the pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oil phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin or mixtures thereof.
  • Suitable emulsifiers may be naturally occurring phospholipids, such as soy lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation of said partial esters with ethylene oxide. Products such as polyethylene oxide sorbitan monooleate.
  • Emulsions may also contain sweetening agents, flavoring agents, preservatives and antioxidants.
  • Syrups and elixirs may be formulated with sweeteners such as glycerin, propylene glycol, sorbitol, or sucrose. Such preparations may also contain demulcents, preservatives, coloring agents and antioxidants.
  • compositions of the invention may be in the form of sterile injectable aqueous solutions.
  • Acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution.
  • Sterile injectable preparations may be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in an oily phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerol and treated to form a microemulsion.
  • injectable solutions or microemulsions can be injected into the patient's bloodstream via localized mass injections.
  • solutions and microemulsions are preferably administered in a manner that maintains constant circulating concentrations of the compounds of the invention. To maintain this constant concentration, continuous intravenous drug delivery devices can be used.
  • the pharmaceutical composition of the present invention can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be prepared according to known techniques with the above-mentioned suitable dispersants or wetting agents and suspending agents.
  • the sterile injection preparation can also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol.
  • sterile fixed oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixed oils including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health, the patient's behavior, the patient's Diet, administration time, administration method, excretion rate, drug combination, etc.
  • the optimal treatment method such as the mode of treatment, the daily dosage of the general compound or the type of pharmaceutically acceptable salt can be verified according to the traditional treatment plan.
  • the present invention may include a compound containing the structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition, and prepare it into a clinically acceptable dosage form .
  • the compounds of the present invention can be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions and the like.
  • the compound of the present invention can be used as the only active ingredient or can be used in combination with other drugs.
  • Combination therapy is accomplished by administering the individual treatment components simultaneously, separately, or sequentially.
  • the "reducing pathological symptoms and signs" mentioned in the present invention mainly refers to the prevention and treatment of overweight and obesity caused by high-fat diet, the prevention and treatment of excessive lipid deposition in the blood, the prevention and treatment of non-alcoholic fatty liver disease, and the reduction of fatty liver disease in the liver. Lipid deposition and prevention of atherosclerosis.
  • the present invention proves that compounds containing glycerophospholipid structures can effectively induce the reduction of white adipocyte volume and browning of white adipose tissue, reduce lipid deposition in the blood, reduce lipid deposition in the liver, and inhibit atherosclerosis. It can prevent or treat hyperlipidemia, atherosclerosis, non-alcoholic fatty liver disease and obesity.
  • Figure 1 shows the body weight statistics of 18:2 PC (DLPC) and the solvent BSA after acting on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of the four groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the control group; ** indicates that the p value is ⁇ 0.01, indicating that the data in this group is significantly different from the control group. There is a significant statistical difference between the group data.
  • Figure 2 is a representative photo of the anatomy of adipose tissue in high-fat diet-induced obese mice (prevention) treated with 18:2 PC (DLPC) and solvent BSA.
  • iWAT is inguinal white fat
  • gWAT is epididymal white fat
  • BAT is brown fat.
  • Figure 3 is a statistical diagram of the anatomical weight of adipose tissue after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (prevention).
  • DLPC 18:2 PC
  • solvent BSA solvent BSA on high-fat diet-induced obese mice
  • Figure 4 is a statistical diagram of the anatomical weight of liver tissue after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that the data in this group are statistically different from the data in the control group; *** indicates that the p value is ⁇ 0.001, indicating that the data in this group There are extremely significant statistical differences compared with the control group data.
  • Figure 5 is a HE staining picture of adipose tissue and liver tissue after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention).
  • DLPC DLPC
  • solvent BSA acted on high-fat diet-induced obese mice (prevention).
  • iWAT is inguinal white fat
  • gWAT is epididymal white fat
  • BAT is brown fat.
  • Figure 6 is a statistical graph showing the detection of glucose tolerance after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of line graphs. * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group; **** indicates that the p value is ⁇ 0.0001, indicating that the group There are extremely significant statistical differences between the data and the control group data.
  • Figure 7 is a statistical graph showing the detection of insulin sensitivity in obese mice induced by high-fat diet (prevention) using 18:2 PC (DLPC) and solvent BSA. Perform a statistical t test on the values of several groups of line graphs. * indicates that the p value is ⁇ 0.05, which means that the data in this group are statistically different from the data in the control group; ** indicates that the p value is ⁇ 0.01, which means that the data in this group is significantly different from the data in the control group.
  • Figure 8 is a statistical graph showing the thermogenic ability of 18:2 PC (DLPC) and solvent BSA after acting on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of line graphs. ** indicates that the p value is ⁇ 0.01, indicating that there is a significant statistical difference between the data in this group and the data in the control group; *** indicates that the p value is ⁇ 0.001, indicating that There is an extremely significant statistical difference between the data in this group and the data in the control group; **** means that the p value is ⁇ 0.0001, which means that the data in this group have an extremely significant statistical difference compared with the data in the control group.
  • Figure 9 is a graph showing the detection of triglyceride (TG) content in serum after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group.
  • TG triglyceride
  • Figure 10 shows the detection of free fatty acid (NEFA) content in serum after 18:2 PC (DLPC) and solvent BSA were treated with high-fat diet-induced obese mice (prevention).
  • NEFA free fatty acid
  • Figure 11 shows the detection of free total cholesterol (CHO) content in the serum of 18:2 PC (DLPC) and the solvent BSA in high-fat diet-induced obese mice (prevention).
  • Figure 12 is a statistical diagram of UCP1 gene expression levels in iWAT after 18:2 PC (DLPC) and solvent BSA were treated with high-fat diet-induced obese mice (prevention).
  • Perform a statistical t test on the values of the four groups of histograms where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group; ** indicates that the p value is ⁇ 0.01, indicating that the data in this group is significantly different from the data in the control group.
  • *** indicates that the p value is ⁇ 0.001, which means there is an extremely significant statistical difference between the data in this group and the data in the control group.
  • Figure 13 is a statistical diagram of the body weight of 18:2 PC (DLPC) and the solvent BSA after high-fat diet-induced obese mice (treatment). Perform a statistical t test on the values of the four groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group.
  • Figure 14 is a representative photo of the anatomy of adipose tissue after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (treatment).
  • DLPC 18:2 PC
  • gWAT is epididymal white fat
  • BAT is brown fat.
  • Figure 15 shows the weight statistics of iWAT after dissection of 18:2 PC (DLPC) and solvent BSA in high-fat diet-induced obese mice (treatment). Perform a statistical t test on the values of several groups of histograms, where *** indicates that the p value is ⁇ 0.001, indicating that there is an extremely significant statistical difference between the data in this group and the data in the control group; ** indicates that the p value is ⁇ 0.01, indicating that There is a significant statistical difference between this group of data and the control group's data.
  • Figure 16 is a graph showing the weight statistics of gWAT after dissection of 18:2 PC (DLPC) and solvent BSA in high-fat diet-induced obese mice (treatment). Perform a statistical t test on the values of several groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group.
  • Figure 17 is a picture of HE staining of liver tissue after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (treatment).
  • DLPC 18:2 PC
  • solvent BSA solvent BSA on high-fat diet-induced obese mice
  • Figure 18 shows the detection of glucose tolerance after the action of 18:2 PC (DLPC) and solvent BSA on high-fat diet-induced obese mice (treatment). Perform a statistical t test on the values of several groups of line charts. ** indicates that the p value is ⁇ 0.01, which means that there is a significant statistical difference between the data in this group and the data in the control group; * indicates that the p value is ⁇ 0.05, which means that this group The data were statistically different from those of the control group.
  • Figure 19 is a test chart of insulin sensitivity after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (treatment).
  • the values of several groups of bar graphs were subjected to statistical t-tests, where * indicates p value ⁇ 0.05, indicating that the data in this group are statistically different from the data in the control group; ** indicates p value ⁇ 0.01, indicating that the data in this group are significantly statistically different from the data in the control group; *** indicates p value ⁇ 0.001, indicating that the data in this group are extremely significantly statistically different from the data in the control group; **** indicates p value ⁇ 0.0001, indicating that the data in this group are extremely significantly statistically different from the data in the control group.
  • Figure 20 is a test graph of the heat-producing capacity of 18:2 PC (DLPC) and solvent BSA after acting on high-fat diet-induced obese mice (treatment).
  • Figure 21 is a graph showing the detection of triglyceride (TG) content in serum after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention).
  • TG triglyceride
  • Figure 22 is a graph showing the detection of free fatty acid (NEFA) content in serum after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of histograms, where * indicates that the p value is ⁇ 0.05, indicating that there is a statistical difference between the data in this group and the data in the control group.
  • NEFA free fatty acid
  • Figure 23 is a graph showing the detection of free total cholesterol (CHO) content in serum after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of histograms. ** indicates that the p value is ⁇ 0.01, indicating that there is a significant statistical difference between the data in this group and the data in the control group; *** indicates that the p value is ⁇ 0.001, indicating that There is an extremely significant statistical difference between this group of data and the control group's data.
  • Figure 24 is a representative picture of oil red O staining of the aorta after 18:2 PC (DLPC) and solvent BSA were used to treat high-cholesterol diet-induced atherosclerosis in mice (prevention).
  • SD means standard diet
  • HCD means high cholesterol diet.
  • Figure 25 is a picture of the oil red O staining results of the aortas of all experimental individuals after 18:2 PC (DLPC) and solvent BSA were used to treat high-cholesterol diet-induced atherosclerosis in mice (prevention).
  • SD means standard diet
  • HCD means high cholesterol diet.
  • Figure 26 shows the statistical results of aortic plaque area after 18:2 PC (DLPC) and solvent BSA acted on mice with atherosclerosis induced by high cholesterol diet (prevention). *** indicates that the p value is ⁇ 0.001, which means that there is an extremely significant statistical difference between the data of this group and the data of the control group. Among them, SD means standard diet.
  • Figure 27 is a representative photo of the liver tissue anatomy of 18:2 PC (DLPC) and solvent BSA in 60% high-fat diet-induced obese mice (prevention).
  • SD means standard diet
  • HFD means high-fat diet.
  • Figure 28 is a statistical diagram of the anatomical weight of liver tissue after 18:2 PC (DLPC) and solvent BSA acted on obese mice induced by 60% high-fat diet (prevention). Perform a statistical t test on the values of several groups of histograms. ** indicates that the p value is ⁇ 0.01, which indicates that there is a significant statistical difference between the data in this group and the data in the control group; **** indicates that the p value is ⁇ 0.0001. It means that there is an extremely significant statistical difference between the data of this group and the data of the control group. Among them, SD means standard diet and HFD means high-fat diet.
  • Figure 29 is a picture of HE staining of liver tissue after 18:2 PC (DLPC) and solvent BSA acted on 60% high-fat diet-induced obese mice (prevention). Among them, SD represents standard diet and HFD represents high-fat diet.
  • Figure 30 is a picture of Oil Red O staining of liver tissue after 18:2 PC (DLPC) and solvent BSA were used in obese mice induced by 60% high-fat diet (prevention). Among them, SD means standard diet and HFD means high-fat diet.
  • Figure 31 is a graph showing the detection of triglyceride (TG) content in liver tissue after 18:2 PC (DLPC) and solvent BSA acted on 60% high-fat diet-induced obese mice (prevention). Perform a statistical t test on the values of several groups of histograms. ** indicates that the p value is ⁇ 0.01, indicating that there is a significant statistical difference between the data in this group and the data in the control group; *** indicates that the p value is ⁇ 0.001, indicating that There is an extremely significant statistical difference between this group of data and the control group's data. Among them, SD means standard diet and HFD means high-fat diet.
  • Figure 32 is a graph showing the detection of total cholesterol (CHO) content in liver tissue after 18:2 PC (DLPC) and solvent BSA acted on high-fat diet-induced obese mice (prevention).
  • Perform a statistical t test on the values of several groups of histograms where * indicates that the p value is ⁇ 0.05, indicating that the data in this group are statistically different from the data in the control group; ** indicates that the p value is ⁇ 0.01, indicating that the data in this group is significantly different from the data in the control group.
  • *** indicates that the p value is ⁇ 0.001, which means there is an extremely significant statistical difference between the data in this group and the data in the control group.
  • SD means standard diet
  • HFD means high-fat diet.
  • BSA bovine serum albumin
  • DLPC bovine serum albumin
  • mice were given a sufficient amount of high-fat diet (45kcal%, Jiangsu Medison Biopharmaceutical Co., Ltd.). At the same time, the mice were intraperitoneally injected with the solvent BSA (control group) at regular intervals every day. , 18:2 PC (DLPC) working solution of 50mg/kg, 100mg/kg, and 200mg/kg, and continue to process for 2 weeks. After 2 weeks, the body weight of the mice was counted.
  • high-fat diet 45kcal%, Jiangsu Medison Biopharmaceutical Co., Ltd.
  • Figure 1 shows the statistical results of body weight after 2 consecutive weeks of high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA.
  • Body weight statistics reflect the degree of obesity in mice. It can be seen from this figure that the body weight of the mice in the 18:2 PC (DLPC) group injected with 50 mg/kg, 100 mg/kg, and 200 mg/kg at the same time after being fed a high-fat diet for 2 weeks was significantly lower than that of the solvent BSA injection group, and the weight appeared There is a decreasing trend with the increase in the dosage of 18:2 PC (DLPC).
  • mice treated with 18:2 PC (DLPC) or BSA for 2 weeks were killed by cervical dislocation.
  • the subcutaneous fat and abdominal fat of the mice were dissected and separated. After weighing, they were placed on the test bench according to groups for morphological comparison. Use Excel software to analyze experimental results.
  • Figure 2 is a representative photo of the adipose tissue of mice fed with a high-fat diet for 2 consecutive weeks and injected with different dosages of 18:2 PC (DLPC) and the solvent BSA.
  • the weight of white adipose tissue of mice injected with different dosages of 18:2 PC (DLPC) was significantly lower than that of the solvent BSA group.
  • Representative images show that the volume of white fat in mice in the 18:2 PC (DLPC) group was significantly reduced.
  • Figure 3 shows the statistical results of adipose tissue weight after 2 weeks of continuous high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA. It shows that the weight of adipose tissue decreases with the increase in the dosage of 18:2 PC (DLPC).
  • Figure 4 shows the statistical results of liver tissue weight after high-fat diet feeding for 2 consecutive weeks combined with injection of 50 mg/kg, 100 mg/kg, and 200 mg/kg of 18:2 PC (DLPC) and solvent BSA. It was shown that the weight of liver tissue showed a decreasing trend as the dosage of 18:2 PC (DLPC) increased.
  • DLPC 18:2 PC
  • mice treated with 18:2 PC (DLPC) or BSA for 2 weeks were killed by cervical dislocation.
  • the subcutaneous fat and abdominal fat of the mice were dissected and separated.
  • a small piece of adipose tissue and liver tissue were cut out with a scalpel and placed in the Fix in 4% paraformaldehyde overnight.
  • paraffin sections were prepared and HE stained. The HE results were observed under an OLYMPUS optical microscope (model TH4-200).
  • Figure 5 shows the HE staining results of adipose tissue and liver tissue after 2 weeks of high-fat diet feeding combined with injection of 50 mg/kg, 100 mg/kg, and 200 mg/kg of 18:2 PC (DLPC) and solvent BSA.
  • DLPC 18:2 PC
  • the diameter and volume of adipocytes in iWAT and gWAT of mice treated with different doses of DLPC were significantly reduced, and lipid deposition in the liver was also significantly reduced.
  • mice in each group were first weighed and moved into a clean cage at 5 pm the day before the experiment and fasted for 16 hours until 9 am the next day. During the fasting period, the mice maintained normal drinking water; at 8 a.m. the next day, each mouse was injected with 50 mg/kg, 100 mg/kg, and 200 mg/kg of 18:2 PC (DLPC) or BSA. At 9 a.m., glucose tolerance was started. Subject to experimentation. Weigh the body weight of each mouse.
  • Determination of fasting basal blood sugar Take the mouse out of the cage, use scissors to cut off about 1-2mm from the end of the mouse's tail, gently squeeze the mouse's tail to allow the blood to condense into a drop, and use a blood glucose meter (Johnson & Johnson Wenhaobeiyou) type blood glucose meter) to measure fasting blood glucose, and the measured value is regarded as the blood glucose value at 0 min. After allowing the mice to adapt for 30 minutes, preparations were made for intraperitoneal injection of glucose. Mice were intraperitoneally injected with glucose solution (Sigma) using a 1 ml insulin needle (BD). The volume of glucose injected was determined according to the body weight of the mouse, with 5 ⁇ l injected per g of body weight.
  • the GTT test usually tests the body's ability to regulate blood sugar and whether there is abnormal glucose metabolism. As shown in Figure 6, compared with the BSA group, injection of different dosages of 18:2 PC (DLPC) can significantly improve the glucose tolerance of mice.
  • DLPC 18:2 PC
  • mice On the day of the experiment, the mice were moved into a clean cage at 9 a.m. and fasted for 4 hours until 1 p.m. During the fasting period, the mice maintained normal drinking water. Before fasting, each mouse was injected with 50mg/kg, 100mg/kg, 200mg/kg 18:2 PC (DLPC) or BSA. Weigh the weight of each mouse; measure fasting basal blood sugar: take the mouse out of the cage, use scissors to cut off about 1-2mm from the end of the mouse's tail, gently squeeze the mouse's tail, and let the blood concentrate into a drop.
  • DLPC fasting basal blood sugar
  • the ITT test usually tests the body's sensitivity to insulin, thereby regulating blood sugar homeostasis, and indicates whether there will be abnormal insulin resistance. As shown in Figure 7, compared with the BSA group, injection of different dosages of 18:2 PC (DLPC) can significantly improve the sensitivity of mice to insulin.
  • DLPC 18:2 PC
  • mice 6-8 mice were randomly selected from each group and treated with 50mg/kg, 100mg/kg, 200mg/kg 18:2 PC (DLPC) or BSA for 4 weeks.
  • the rectal temperature of each group of mice was measured using a rectal temperature detector before being placed in a 4°C cold room, and recorded as the rectal temperature value at 0h.
  • the mice were then placed in a 4°C cold room, and the rectal temperature was measured every 1 hour, for a total of 4 time points (4 times in total).
  • the experimental results were analyzed using Excel software.
  • Rectal temperature testing is usually used to characterize the body's ability to maintain core body temperature under cold stimulation, that is, the body's thermogenic metabolic capacity.
  • the rectal temperature detection results in Figure 8 show that 18:2 PC (DLPC) can significantly increase the body's ability to maintain body temperature, that is, the body's heat production, in mice under cold stimulation.
  • DLPC DLPC
  • mice Collect the whole blood of mice by taking blood from the eyeballs, store it at 4°C for 4 hours, and centrifuge at 8000 rpm for 15 minutes to collect the supernatant, which is serum.
  • a 96-well cell culture plate and follow the detection methods of the NEFA detection kit, TG detection kit, and CHO detection kit (all purchased from Nanjing Jiancheng Bioengineering Institute) to take appropriate amounts of serum from mice in different treatment groups for detection.
  • the multifunctional microplate reader FlexStation3 from Molecular Devices was used to detect the absorbance value at 546nm.
  • FIG-11 shows the detection results of triglycerides (TG), free fatty acids (NEFA), and free total cholesterol (CHO) in the serum of mice treated with different dosages of 18:2 PC (DLPC) and solvent BSA.
  • TG, NEFA, and CHO experiments were used to detect lipid deposition in serum. It can be seen from this figure that the contents of TG, NEFA, and CHO in the serum of mice treated with 18:2 PC (DLPC) at different dosages were significantly less than those in the control group, and the contents of various lipids in the serum increased with 18:2. decreased with increasing dosage of PC (DLPC).
  • RNA in iWAT was extracted by TRIzol method, and then cDNA was synthesized by reverse transcription.
  • the expression level of UCP1 gene was detected by real-time PCR.
  • the UCP1 gene is a marker gene for browning of adipose tissue.
  • the detection of UCP1 expression level shows that 18:2 PC (DLPC) promotes the browning of white fat by regulating the expression of the UCP1 gene, thereby achieving fat reduction.
  • Figure 12 shows the statistical results of UCP1 gene expression levels in iWAT. The expression of UCP1 gene in iWAT of mice treated with 18:2 PC (DLPC) at different dosages was significantly higher than that in the control group, indicating that 18:2 PC (DLPC) significantly caused the browning of white adipose tissue and had significant fat reducing effect.
  • Figure 13 shows the statistical results of body weight after 4 weeks of continuous high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA.
  • Body weight statistics reflect the degree of obesity in mice. It can be seen from this figure that after 4 weeks of high-fat diet feeding, the body weight of the mice in the 18:2 PC (DLPC) group injected with 50 mg/kg, 100 mg/kg, and 200 mg/kg was significantly lower than that in the solvent BSA group, and the body weight appeared There is a decreasing trend with the increase in the dosage of 18:2 PC (DLPC).
  • DLPC 18:2 PC
  • mice treated with 18:2 PC (DLPC) or BSA for 4 weeks were killed by cervical dislocation.
  • the subcutaneous fat and abdominal fat of the mice were dissected and separated. After weighing, they were placed on the test bench according to groups for morphological comparison. Use Excel software to analyze experimental results.
  • Figure 14 is a representative photo of the adipose tissue of mice fed with a high-fat diet for 4 consecutive weeks and injected with different dosages of 18:2 PC (DLPC) and the solvent BSA.
  • the weight of white adipose tissue of mice injected with different dosages of 18:2 PC (DLPC) was significantly lower than that of the solvent BSA group.
  • Representative images show that the volume of white fat in mice in the 18:2 PC (DLPC) group was significantly reduced.
  • Figures 15 and 16 show the weight statistical results of iWAT and gWAT after autopsy after 4 weeks of high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA. It was shown that the weight of adipose tissue decreased as the dosage of 18:2 PC (DLPC) increased.
  • mice treated with 18:2 PC (DLPC) or BSA for 4 weeks were killed by cervical dislocation.
  • the livers of the mice were dissected and separated. After weighing, a small piece of liver tissue was cut out with a scalpel and fixed in 4% paraformaldehyde. overnight. After the tissue was fixed, paraffin sections were prepared and HE stained. The HE results were observed under an OLYMPUS optical microscope (model TH4-200).
  • Figure 17 shows the HE staining results of liver tissue after 4 weeks of continuous high-fat diet feeding combined with different dosages of 18:2 PC (DLPC) and solvent BSA injection. Compared with mice in the BSA-injected group, lipid deposition in the livers of mice treated with different dosages of DLPC was significantly reduced.
  • DLPC 18:2 PC
  • mice in each group were first weighed and moved into a clean cage at 5 pm the day before the experiment and fasted for 16 hours until 9 am the next day. During the fasting period, the mice maintained normal drinking water. At 8 a.m. the next day, each mouse was injected with 50 mg/kg, 100 mg/kg, and 200 mg/kg of 18:2 PC (DLPC) or BSA. At 9 a.m., the glucose tolerance experiment was started. Weigh the body weight of each mouse.
  • Determination of fasting basal blood sugar Take the mouse out of the cage, use scissors to cut off about 1-2mm from the end of the mouse's tail, gently squeeze the mouse's tail to allow the blood to condense into a drop, and use a blood glucose meter (Johnson & Johnson Wenhaobeiyou) type blood glucose meter) to measure fasting blood glucose, and the measured value is regarded as the blood glucose value at 0 min. After allowing the mice to adapt for 30 minutes, preparations were made for intraperitoneal injection of glucose. Mice were injected intraperitoneally with glucose solution (Sigma) using a 1 ml insulin needle (BD). The volume of glucose injected is determined according to the body weight of the mouse, with 5 ⁇ l injected per g of body weight.
  • the GTT test usually tests the body's ability to regulate blood sugar and whether there is abnormal glucose metabolism. As can be seen from Figure 18, compared with the BSA group, injection of different dosages of 18:2 PC (DLPC) can significantly improve the glucose tolerance of mice.
  • DLPC 18:2 PC
  • mice On the day of the experiment, the mice were moved into a clean cage at 9 a.m. and fasted for 4 hours until 1 p.m. During the fasting period, the mice maintained normal drinking water. Before fasting, each mouse was injected with 50 mg/kg, 100 mg/kg, or 200 mg/kg of 18:2 PC (DLPC) or BSA. Weigh the body weight of each mouse.
  • DLPC 18:2 PC
  • Determination of fasting basal blood sugar Take the mouse out of the cage, use scissors to cut off about 1-2mm from the end of the mouse's tail, gently squeeze the mouse's tail to allow the blood to condense into a drop, and use a blood glucose meter (Johnson & Johnson Wenhaobeiyou) type blood glucose meter) to measure fasting blood glucose, and the measured value is regarded as the blood glucose value at 0 min. After allowing the mice to adapt for 30 minutes, preparations were made for intraperitoneal injection of insulin. The mice were intraperitoneally injected with insulin solution (Novo Nordisk (China) Pharmaceutical Co., Ltd.) using a 1 ml insulin needle (BD).
  • insulin solution Novo Nordisk (China) Pharmaceutical Co., Ltd.
  • the volume of insulin injected was determined according to the body weight of the mouse, with 5 ⁇ l injected per g of body weight. Start timing from the completion of injection, and measure the blood glucose value of each mouse at various time points at 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min after injection. After the experiment, the mice in each cage were supplemented with feed. Use Excel software to analyze experimental results.
  • the ITT test usually tests the body's sensitivity to insulin, thereby regulating blood sugar homeostasis, and indicates whether there will be abnormal insulin resistance. As shown in Figure 19, compared with the BSA group, injection of different dosages of 18:2 PC (DLPC) can significantly improve the sensitivity of mice to insulin.
  • DLPC DLPC
  • Each group randomly selects 6-8 mice treated with 50mg/kg, 100mg/kg, 200mg/kg 18:2 PC (DLPC) or BSA for 4 weeks, and uses a rectal temperature detector before placing them in a 4°C cold room.
  • the rectal temperature of mice in each group was recorded as the rectal temperature value at 0 h.
  • the mice were then placed in a 4°C cold room, and the rectal temperature was measured every 1 hour.
  • the rectal temperature values were measured at 4 time points in total (4 times in total). ).
  • Use Excel software to analyze experimental results.
  • Rectal temperature testing is usually used to characterize the body's ability to maintain core body temperature under cold stimulation, that is, the body's thermogenic metabolic capacity.
  • the rectal temperature detection results in Figure 20 show that 18:2 PC (DLPC) can significantly increase the body's ability to maintain body temperature, that is, the body's heat production, in mice under cold stimulation.
  • DLPC DLPC
  • mice Collect the whole blood of mice by taking blood from the eyeballs, store it at 4°C for 4 hours, and centrifuge at 8000 rpm for 15 minutes to collect the supernatant, which is serum.
  • a 96-well cell culture plate and follow the detection methods of the NEFA detection kit, TG detection kit, and CHO detection kit (all purchased from Nanjing Jiancheng Bioengineering Institute) to take appropriate amounts of serum from mice in different treatment groups for detection.
  • the multifunctional microplate reader FlexStation3 from Molecular Devices was used to detect the absorbance value at 546nm.
  • FIGS 21-23 show the detection results of triglycerides (TG), free fatty acids (NEFA), and free total cholesterol (CHO) in the serum of mice treated with different dosages of 18:2 PC (DLPC) and solvent BSA.
  • TG, NEFA, and CHO experiments were used to detect lipid deposition in serum. It can be seen from this figure that the contents of TG, NEFA, and CHO in the serum of mice treated with 18:2 PC (DLPC) at different dosages were significantly less than those in the control group, and the contents of various lipids in the serum increased with 18:2. decreased with increasing dosage of PC (DLPC).
  • mice Purchased 5-week-old ApoE-/- male mice (purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups (not less than 10 in each group) according to body weight to minimize the weight difference between each group. The mice were given a sufficient amount of high-cholesterol diet (21% fat, 0.15% cholesterol, Beijing Huafukang Company), and the mice were injected with solvent BSA (control group) and 50 mg/kg of 18:2 PC (DLPC) at regular intervals every day. The working solution (preparation method is the same as in Example 1) was continuously administered for 12 weeks, and atherosclerosis in mice was detected after 12 weeks.
  • high-cholesterol diet 21% fat, 0.15% cholesterol, Beijing Huafukang Company
  • mice treated with 18:2 PC (DLPC) or BSA for 12 weeks were killed by cervical dislocation. Cardiac perfusion was performed with PBS until all blood in the aorta was drained. The aorta of the mouse was dissected and isolated, and the surface connective tissue was peeled off and soaked in in PBS. After the aortas of all mice were collected, the aortas were fixed in 4% paraformaldehyde (Sigma) for about 15 minutes, washed with 60% isopropyl alcohol (Sinopharm) for 5 minutes, and stained with Oil Red O staining solution (Beijing Soc. Lebao Technology Co., Ltd.) Dye for 10-15 minutes, separate with 60% isopropyl alcohol, wash off excess Oil Red O staining solution, rinse with PBS for observation and photography.
  • Figures 24-25 are pictures of the results of Oil Red O staining of the aorta of mice with high-cholesterol diet-induced atherosclerosis using 18:2 PC (DLPC) and the solvent BSA.
  • the formation of aortic plaques in mice injected with 50 mg/kg of 18:2 PC (DLPC) was significantly improved compared with the solvent BSA group.
  • Figure 26 shows the statistical results of aortic plaque area after 50mg/kg 18:2 PC (DLPC) and solvent BSA acted on mice with atherosclerosis induced by high cholesterol diet (prevention).
  • the plaque area was counted using ImageJ software.
  • the area of aortic plaques in mice injected with 50 mg/kg of 18:2 PC (DLPC) was significantly reduced compared with the solvent BSA group.
  • mice Purchased 8-week-old C57BL/6 male mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into groups according to body weight (not less than 10 in each group) to minimize the weight difference between each group. Mice were given a sufficient amount of high-fat diet (60kcal%, Jiangsu Medison Biopharmaceutical Co., Ltd.) to construct a mouse model of fatty liver induced by high-fat diet. At the same time, the mice were injected with solvent BSA (control group) regularly every day, 50 mg/ kg, 100mg/kg, 200mg/kg of 18:2 PC (DLPC) (the preparation method is the same as in Example 1). Treatment continues for 14 weeks. After 14 weeks, the liver weight and lipid deposition degree of the mice were counted.
  • solvent BSA control group
  • DLPC DLPC
  • mice treated with 18:2 PC (DLPC) or BSA for 14 weeks were killed by cervical dislocation.
  • the subcutaneous fat and abdominal fat of the mice were dissected and separated. After weighing, they were placed on the test bench according to groups for morphological comparison. Use Excel software to analyze experimental results.
  • Figure 27 is a representative photo of the liver tissue of mice fed with a high-fat diet for 14 consecutive weeks and injected with different dosages of 18:2 PC (DLPC) and the solvent BSA.
  • the volume of liver tissue in mice injected with different dosages of 18:2 PC (DLPC) was significantly smaller than that in the BSA group.
  • Representative images show that the volume of white fat in mice in the 18:2 PC (DLPC) group was significantly reduced.
  • Figure 28 shows the weight statistical results of liver tissue dissection after 14 weeks of continuous high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA. It was shown that the weight of liver tissue showed a decreasing trend as the dosage of 18:2PC (DLPC) increased.
  • DLPC 18:2 PC
  • mice treated with 18:2 PC (DLPC) or BSA for 14 weeks were killed by cervical dislocation.
  • the livers of the mice were dissected and separated. After weighing, a small piece of liver tissue was cut out with a scalpel and fixed in 4% paraformaldehyde. overnight. After the tissue was fixed, paraffin sections were prepared and HE stained. The HE results were observed under an OLYMPUS optical microscope (model TH4-200).
  • Figure 29 shows the results of HE staining of liver tissue after 14 weeks of high-fat diet feeding combined with different doses of 18:2 PC (DLPC) and solvent BSA injection. Compared with the mice injected with BSA, the lipid deposition in the liver of mice treated with different doses of DLPC was significantly reduced.
  • DLPC 18:2 PC
  • mice treated with 18:2 PC (DLPC) or BSA for 14 weeks were killed by cervical dislocation, the liver was removed, a piece of liver tissue was cut out, and the scalpel was cut into a regular strip or square shape, and OCT embedding agent (SAKURA Sakura) was used. After embedding and quick freezing in liquid nitrogen, frozen sections of liver tissue were prepared. The sections were fixed in 4% paraformaldehyde (Sigma) for about 10 minutes, washed with 60% isopropyl alcohol (Sinopharm) for 5 minutes, and stained with Oil Red O staining solution (Beijing Solebao Technology Co., Ltd.) for 10-15 minutes.
  • Figure 30 shows the results of Oil Red O staining of liver tissue after 14 consecutive weeks of high-fat diet feeding combined with injection of different dosages of 18:2 PC (DLPC) and solvent BSA. Compared with mice in the BSA injection group, the lipid content in the livers of mice treated with different doses of DLPC was significantly reduced, and showed a dose-dependent trend.
  • DLPC 18:2 PC
  • liver tissue Take an appropriate volume of liver tissue and homogenize it in ice-cold PBS, and take out part of the homogenate for BCA protein quantification.
  • Molecular Devices' multifunctional microplate reader FlexStation3 was used to detect the corresponding absorbance value. The content of various lipids in the liver tissue was calculated based on the protein concentration of the tissue homogenate tested for each individual.
  • Figures 31-32 show the test results of triglyceride (TG) and free total cholesterol (CHO) in the liver tissue of mice treated with different doses of 18:2 PC (DLPC) and solvent BSA.
  • the TG and CHO experiments are to detect lipid deposition in liver tissue.
  • the TG and CHO contents in the liver tissue of mice treated with different doses of 18:2 PC (DLPC) were significantly lower than those in the control group, and the TG content decreased with the increase of the dose of 18:2 PC (DLPC).

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Abstract

本发明涉及甘油磷脂类化合物在预防和治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖中的用途。具体地，本发明涉及含有式(I)所示结构的甘油磷脂类化合物在制备用于预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝、或肥胖的药物中的用途。本发明所述的化合物可以通过诱导白色脂肪组织体积减小或白色脂肪组织发生棕色化，从而具有减脂、缓解动脉粥样硬化、缓解非酒精性脂肪肝和对抗肥胖的作用效果。

Description

甘油磷脂类化合物在预防和治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖中的用途 技术领域
本发明涉及一种甘油磷脂类化合物在制备预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖的药物中的用途。
背景技术
非传染性疾病，包括心血管疾病、癌症和糖尿病在内，占全世界早期死亡的70％，是死亡和过早残疾的主要原因 [1]。肥胖是非传染性疾病的主要危险因素，与预期寿命减少有关，根据病情和并发症疾病的严重程度，预期寿命估计减少20年。世界卫生组织将肥胖定义为可能损害健康的过度脂肪堆积，诊断时BMI为30kg/m 2[4]。肥胖大大增加了代谢疾病 [2-4](如2型糖尿病和非酒精性脂肪肝疾病)、心血管疾病(如动脉粥样硬化、高血脂、心肌梗死和中风)、肌肉骨骼疾病(如骨关节炎)、阿尔茨海默病、抑郁症和某些类型的癌症(如乳腺癌、卵巢癌、前列腺癌、肝、肾和结肠)的风险。世界肥胖联合会和包括美国和加拿大医学协会在内的其他组织已经宣布，肥胖是一种慢性进行性疾病，而不仅仅是导致其他疾病的风险因素 [5]。
肥胖的根本原因是摄入的卡路里和消耗的卡路里之间的能量不平衡。治疗肥胖的方法有很多，包括饮食控制、体育锻炼、改变生活方式和减肥药。首先，以减少卡路里摄入和增加能量消耗为目标的生活方式和行为干预措施效果有限，因为复杂而持久的激素、代谢和神经化学适应能够抵抗体重减轻并促进体重恢复 [6]。其次，目前大多数肥胖治疗药物都是食欲抑制剂，旨在通过调节下丘脑的神经递质来抑制食欲，或者是胰腺和胃脂肪酶的抑制剂 [7]。但此类药物存在一定的局限性，如营养吸收障碍、头晕、腹泻、复发率高 [8]。此外，在没有任何改变的情况下，长期保持健康的生活方式是一个巨大的挑战。肥胖患者在面对痛苦而又无穷无尽的生活方式改变时，很容易感到沮丧。
因此开发出健康、无毒、安全的小分子治疗药物是理想的减肥治疗以及相关代谢病发生预防的方法。
发明内容
本发明的目的在于提供一种甘油磷脂类化合物用于预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖的用途。
因此，本发明提供含有式(I)所示结构的化合物或其可药用盐或者含有其的药物组合物在制备用于预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝或肥胖的药物中的用途，
Figure PCTCN2022128845-appb-000001
在一个优选的实施方案中，本发明提供含有式(I)所示结构的化合物或其可药用盐或者含有其的药物组合物在制备用于预防和/或治疗肥胖的药物中的用途。
在另一个优选的实施方案中，本发明提供含有式(I)所示结构的化合物或其可药用盐或者含有其的药物组合物在制备用于预防和/或治疗高血脂的药物中的用途。
在一个具体的实施方案中，根据本发明所述的用途，其中含有式(I)所示结构的化合物如下式(II)所示：
Figure PCTCN2022128845-appb-000002
其中：
-OR选自-OH、胆碱基、L-丝氨酸基；
M +选自Na +、K +；
其中当-OR为胆碱基时，M +不存在。
在另一个具体的实施方案中，根据本发明所述的用途，其中含有式(I)所示结构的化合物选自以下化合物：
18:2 PC(DLPC)
Figure PCTCN2022128845-appb-000003
在一个优选的实施方案中，根据本发明所述的用途，其中所述肥胖为高脂饮食引发的肥胖。
在另一个优选的实施方案中，根据本发明所述的用途，其中所述高血脂为高脂饮食引发的高血脂。
在另一个优选的实施方案中，根据本发明所述的用途，其中所述动脉粥样硬化为高脂饮食引发的动脉粥样硬化。
在另一个优选的实施方案中，根据本发明所述的用途，其中所述非酒精性脂肪肝为高脂饮食引发的非酒精性脂肪肝。
在一个具体的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过白色脂肪细胞体积减小，优选诱导白色脂肪组织棕色化，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个具体的实施方案中，根据本发明所述的用途，其特征在于，所述化 合物减轻病理症状和体征预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在一个优选的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过减缓高脂饮食诱导的体重增加从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个优选的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过缩小白色脂肪细胞的体积，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个优选的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过减少血清中脂质的沉积，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个优选的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过增强机体的葡萄糖耐受和胰岛素敏感性，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个优选的实施方案中，根据本发明所述的用途，其特征在于，所述化合物通过促进白色脂肪组织棕色化，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
在另一个具体的实施方案中，根据本发明所述的用途，其中所述药物组合物含有治疗有效量的含有式(I)所示结构的化合物或其可药用盐作为活性成分和药学上可接受的载体或赋形剂。
在另一个具体的实施方案中，根据本发明所述的用途，其中所述含有式(I)所示结构的化合物与另一种或多种治疗方法或治疗剂组合使用，所述治疗方法优选化学疗法、靶向疗法，所述治疗剂优选另一种预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖的试剂。
本发明所述的含有式(I)所示结构的化合物，其结构特征在于：磷脂中脂肪酰侧链为双链十八碳二烯酸，脂肪酰侧链结合部的磷酸基团可以连接不同的极性头部，形成不同的甘油磷脂，优选是1,2-二亚油酰基-sn-甘油-3-磷脂酰胆碱(1,2-dilinoleoyl-sn-glycero-3-phosphocholine)(18:2 PC，DLPC)。
本发明所述的胆碱基为
Figure PCTCN2022128845-appb-000004
基团；所述的L-丝氨酸基为
Figure PCTCN2022128845-appb-000005
基团。
本发明所述的“可药用盐”是指在药学上无毒的酸加成盐和碱加成盐。所述的酸加成盐为化合物与合适的无机酸或者有机酸形成的盐，包括盐酸盐、磷酸盐、磷酸氢盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、丙二酸盐、戊酸盐、谷氨酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、对甲苯磺酸盐、 甲磺酸盐、苹果酸盐、酒石酸盐、苯甲酸盐、双羟萘酸盐、水杨酸盐、香草酸盐、扁桃酸盐、琥珀酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述的碱加成盐为化合物与合适的无机碱或者有机碱形成的盐，包括如与碱金属、胺类或季铵类化合物形成的盐，如钠盐、锂盐、钾盐、钙盐、镁盐、胺盐、四甲基季铵盐、四乙基季铵盐、胆碱盐，特别是钠盐和胆碱盐；胺盐，包括与氨(NH 3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐、乙醇胺盐、丝氨酸盐、赖氨酸盐和精氨酸盐，特别是丝氨酸盐。
含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂。可按照本领域任何已知制备药用组合物的方法制备口服组合物，此类组合物可含有一种或多种选自以下的成分：甜味剂、矫味剂、着色剂和防腐剂，以提供悦目和可口的药用制剂。片剂含有活性成分和用于混合的适宜制备片剂的无毒的可药用的赋形剂。这些赋形剂可以是惰性赋形剂，如碳酸钙、碳酸钠、乳糖、磷酸钙或磷酸钠；造粒剂和崩解剂，例如微晶纤维素、交联羧甲基纤维素钠、玉米淀粉或藻酸；粘合剂，例如淀粉、明胶、聚乙烯吡咯烷酮或阿拉伯胶；和润滑剂，例如硬脂酸镁、硬脂酸或滑石粉。这些片剂可以不包衣或可通过掩盖药物的味道或在胃肠道中延迟崩解和吸收，因而在较长时间内提供缓释作用的已知技术将其包衣。例如，可使用水溶性味道掩蔽物质，例如羟丙基甲基纤维素或羟丙基纤维素，或延长时间物质例如乙基纤维素、醋酸丁酸纤维素。
也可用其中活性成分与惰性固体稀释剂例如碳酸钙、磷酸钙或高岭土混合的硬明胶胶囊，或其中活性成分与水溶性载体例如聚乙二醇或油溶媒例如花生油、液体石蜡或橄榄油混合的软明胶胶囊提供口服制剂。
水混悬液含有活性物质和用于混合的适宜制备水混悬液的赋形剂。此类赋形剂是悬浮剂，例如羧基甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯吡咯烷酮和阿拉伯胶；分散剂或湿润剂，可以是天然产生的磷脂例如卵磷脂，或烯化氧与脂肪酸的缩合产物，例如聚氧乙烯硬脂酸酯，或环氧乙烷与长链脂肪醇的缩合产物，例如十七碳亚乙基氧基鲸蜡醇(heptadeca乙基ene氧基cetanol)，或环氧乙烷与由脂肪酸和己糖醇衍生的部分酯的缩合产物，例如聚环氧乙烷山梨醇单油酸酯，或环氧乙烷与由脂肪酸和己糖醇酐衍生的偏酯的缩合产物，例如聚环氧乙烷脱水山梨醇单油酸酯。水混悬液也可以含有一种或多种防腐剂例如尼泊金乙酯或尼泊金正丙酯、一种或多种着色剂、一种或多种矫味剂和一种或多种甜味剂，例如蔗糖、糖精或阿司帕坦。
油混悬液可通过使活性成分悬浮于植物油如花生油、橄榄油、芝麻油或椰子油，或矿物油例如液体石蜡中配制而成。油混悬液可含有增稠剂，例如蜂蜡、硬石蜡或鲸蜡醇。可加入上述的甜味剂和矫味剂，以提供可口的制剂。可通过加入抗氧化剂例如丁羟茴醚或α-生育酚保存这些组合物。
通过加入水，适用于制备水混悬液的可分散粉末和颗粒可以提供活性成分和用于混合的分散剂或湿润剂、悬浮剂或一种或多种防腐剂。适宜的分散剂或湿润剂和悬浮剂如上所述。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂例如抗坏血酸保存这些组合物。
本发明的药物组合物也可以是水包油乳剂的形式。油相可以是植物油例如橄榄油或花生油，或矿物油例如液体石蜡或其混合物。适宜的乳化剂可以是天然产生的磷脂，例如大豆卵磷脂，和由脂肪酸和己糖醇酐衍生的酯或偏酯，例如山梨坦单油酸酯，和所述偏酯和环氧乙烷的缩合产物，例如聚环氧乙烷山梨醇单油酸酯。乳剂也可以含有甜味剂、矫味剂、防腐剂和抗氧剂。可用甜味剂例如甘油、丙二醇、山梨醇或蔗糖配制的糖浆和酏剂。此类制剂也可含有缓和剂、防腐剂、着色剂和抗氧剂。
本发明的药物组合物可以是无菌注射水溶液形式。可以使用的可接受的溶媒和溶剂有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。
本发明的药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如在1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
本领域技术人员熟知，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用特定化合物的活性、病人的年龄、病人的体重、病人的健康状况、病人的行被、病人的饮食、给药时间、给药方式、排泄的速率、药物的组合等。另外，最佳的治疗方式如治疗的模式、通式化合物的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。
本发明可以包含含有式(I)所示结构的化合物或其可药用盐作为活性成分，与药学上可接受的载体或赋型剂混合制备成组合物，并制备成临床上可接受的剂型。
本发明的化合物可以与其他活性成分组合使用，只要它们不产生其他不利的作用，例如过敏反应等。本发明化合物可作为唯一的活性成分，也可以与其它药物联合使用。联合治疗通过将各个治疗组分同时、分开或相继给药来实现。
本发明所述的“减轻病理症状和体征”主要指预防和治疗高脂饮食引起的超重和肥胖、预防和治疗血液中过度的脂质沉积、预防和治疗非酒精性脂肪肝、减 少肝脏中的脂质沉积、预防动脉粥样硬化。
本发明通过具体实验证明了含有甘油磷脂结构的化合物能够有效诱导白色脂肪细胞体积的减小和诱导白色脂肪组织棕色化、减少血液中的脂质沉积、减少肝脏中的脂质沉积、抑制动脉粥样硬化斑块的形成从而达到预防或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖的作用。
附图说明
图1为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的体重统计图。对四组柱状图的数值进行统计学t检验，其中*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异。
图2为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)的脂肪组织解剖代表性照片。其中：iWAT为腹股沟白色脂肪，gWAT为附睾白色脂肪，BAT为棕色脂肪。
图3为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的脂肪组织解剖重量统计图。对几组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异；其中，iWAT为腹股沟白色脂肪，gWAT为附睾白色脂肪，BAT为棕色脂肪。
图4为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织解剖重量统计图。对几组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图5为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的脂肪组织和肝脏组织HE染色图片。其中，iWAT为腹股沟白色脂肪，gWAT为附睾白色脂肪，BAT为棕色脂肪。
图6为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的葡萄糖耐受的检测统计图。对几组折线图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；****表示p值<0.0001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图7为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)胰岛素敏感性的检测统计图。对几组折线图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异；****表示p值<0.0001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图8为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的产热能力的检测统计图。对几组折线图的数值进行统计学t检验，其中，**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异；****表示p值<0.0001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图9为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中甘油三酯(TG)含量检测图。对几组柱状图的数值进行统计学t检验，其中*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异。
图10为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中游离脂肪酸(NEFA)含量检测图。对几组柱状图的数值进行统计学t检验，其中*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异。
图11为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中游离总胆固醇(CHO)含量检测图。对几组柱状图的数值进行统计学t检验，其中*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异。
图12为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的iWAT中UCP1基因表达水平的统计图。对四组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图13为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的体重统计图。对四组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异。
图14为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的脂肪组织解剖代表性照片。其中，iWAT为腹股沟白色脂肪，gWAT为附睾白色脂肪，BAT为棕色脂肪。
图15为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的iWAT解剖后的重量统计图。对几组柱状图的数值进行统计学t检验，其中***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异。
图16为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的gWAT解剖后的重量统计图。对几组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异。
图17为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的肝脏组织HE染色图片。
图18为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的葡萄糖耐受的检测图。对几组折线图的数值进行统计学t检验，其中，**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异。
图19为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的胰岛素敏感性的检测图。对几组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异；****表示p值<0.0001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图20为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(治疗)后的产热能力的检测图。
图21为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中甘油三酯(TG)含量检测图。
图22为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中游离脂肪酸(NEFA)含量检测图。对几组柱状图的数值进行统计学t检验，其中*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异。
图23为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的血清中游离总胆固醇(CHO)含量检测图。对几组柱状图的数值进行统计学t检验，其中，**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。
图24为18:2 PC(DLPC)与溶剂BSA作用于高胆固醇饮食诱导的动脉粥样硬化小鼠(预防)后主动脉大体油红O染色代表性图片。其中，SD表示标准饮食；HCD表示高胆固醇饮食。
图25为18:2 PC(DLPC)与溶剂BSA作用于高胆固醇饮食诱导的动脉粥样硬化小鼠(预防)后所有实验个体主动脉大体油红O染色结果图片。其中，SD表示标准饮食；HCD表示高胆固醇饮食。
图26为18:2 PC(DLPC)与溶剂BSA作用于高胆固醇饮食诱导的动脉粥样硬化小鼠(预防)后主动脉斑块面积统计结果。***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。其中，SD表示标准饮食。
图27为18:2 PC(DLPC)与溶剂BSA作用于60％高脂饮食诱导的肥胖小鼠(预防)的肝脏组织解剖代表性照片。其中，SD表示标准饮食，HFD表示高脂饮食。
图28为18:2 PC(DLPC)与溶剂BSA作用于60％高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织解剖重量统计图。对几组柱状图的数值进行统计学t检验， 其中，**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；****表示p值<0.0001，代表该组数据与对照组数据相比有极其显著的统计学差异。其中，SD表示标准饮食，HFD表示高脂饮食。
图29为18:2 PC(DLPC)与溶剂BSA作用于60％高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织HE染色图片。其中，SD表示标准饮食，HFD表示高脂饮食。
图30为18:2 PC(DLPC)与溶剂BSA作用于60％高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织油红O染色图片。其中，SD表示标准饮食，HFD表示高脂饮食。
图31为18:2 PC(DLPC)与溶剂BSA作用于60％高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织中甘油三酯(TG)含量检测图。对几组柱状图的数值进行统计学t检验，其中，**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。其中，SD表示标准饮食，HFD表示高脂饮食。
图32为18:2 PC(DLPC)与溶剂BSA作用于高脂饮食诱导的肥胖小鼠(预防)后的肝脏组织中总胆固醇(CHO)含量检测图。对几组柱状图的数值进行统计学t检验，其中，*表示p值<0.05，代表该组数据与对照组数据相比有统计学差异；**表示p值<0.01，代表该组数据与对照组数据相比有显著的统计学差异；***表示p值<0.001，代表该组数据与对照组数据相比有极其显著的统计学差异。其中，SD表示标准饮食，HFD表示高脂饮食。
具体实施方式
以下通过具体实施例进一步描述本发明，但应理解这些实施例仅是为了阐明本发明，并不以任何形式限制本发明的范围。
实验材料
18:2 PC(DLPC)的结构如下所示：
18:2 PC(DLPC)
Figure PCTCN2022128845-appb-000006
18:2 PC(DLPC)购自北京世康合成医药科技有限公司。
实验所用细胞：
Figure PCTCN2022128845-appb-000007
实施例1：18:2 PC(DLPC)高脂饮食诱导的肥胖小鼠的预防作用
(1)体重检测实验
用天平称取适量18:2 PC(DLPC)，于超净台中溶于0.5％BSA中(BSA中文名为牛血清白蛋白，购自Biotopped公司)，分别配制成50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)工作液，-20℃保存。称量购买的每只8周龄的C57BL/6雄鼠(购自北京华阜康生物科技股份有限公司)的重量，再根据体重进行随机分组(每组不少于10只)，使每组间的体重差异最小。在小鼠适应动物房环境以后开始进行实验，给予小鼠足量的高脂饮食(45kcal％，江苏美迪森生物医药有限公司)，同时每天定时分别给小鼠腹腔注射溶剂BSA(对照组)，50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)工作液，持续处理2周。2周后，对小鼠的体重进行统计。
结果：
图1为连续2周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的体重统计结果。体重统计体现小鼠肥胖的程度。由该图可知在高脂饮食喂养2周，同时注射50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)组的小鼠的体重明显低于注射溶剂BSA组，并且体重出现了随18:2 PC(DLPC)给药量增加而减少的趋势。
(2)小鼠脂肪组织代表性图片及组织重量的统计
脱颈处死如上18:2 PC(DLPC)或BSA处理2周的小鼠，解剖分离出小鼠的皮下脂肪和腹腔脂肪，称重后按组别摆在试验台上进行形态学的比较。用Excel软件分析实验结果。
结果：
图2为连续2周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的小鼠脂肪组织的代表性照片。注射不同给药量的18:2 PC(DLPC)组小鼠白色脂肪组织的重量显著低于溶剂BSA组。代表性图片显示18:2 PC(DLPC)组小鼠白色脂肪的体积显著减小。
图3为连续2周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的脂肪组织重量统计结果。显示脂肪组织重量出现了随18:2 PC(DLPC)给药量增加而减少的趋势。
图4为连续2周高脂饮食喂养联合50mg/kg，、100mg/kg、200mg/kg的18:2 PC(DLPC)和溶剂BSA注射后的肝脏组织重量统计结果。显示肝脏组织重量出现了随18:2 PC(DLPC)给药量增加而减少的趋势。
(3)小鼠脂肪组织和肝脏组织HE染色
脱颈处死18:2 PC(DLPC)或BSA处理2周的小鼠，解剖分离出小鼠的皮下脂肪和腹腔脂肪，称重后用解剖刀切下一小块脂肪组织和肝脏组织，置于4％多聚甲醛中固定过夜。待组织固定好以后制备石蜡切片以及HE染色。HE结果在OLYMPUS光学显微镜(型号TH4-200)下进行观察。
结果：
图5为连续2周高脂饮食喂养联合50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)和溶剂BSA注射后的脂肪组织和肝脏组织HE染色结果。与注射BSA组小鼠相比,不同的给药量的DLPC处理组小鼠的iWAT和gWAT中脂肪细胞的直径及体积明显减小，同时肝脏中的脂质沉积也显著减少。
(4)小鼠葡萄糖耐受实验(Glucose tolerance test,GTT)
实验前首先对各组小鼠进行体重的称量并且于实验前一天下午5点将小鼠换入干净的笼子禁食，禁食16小时，至次日上午9点。禁食期间，小鼠保持正常的饮水；次日上午8点每只小鼠注射50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)或BSA，上午9点，开始葡萄糖耐受实验。称取每只小鼠的体重。空腹基础血糖的测定：将小鼠从笼子中取出，用剪刀剪去小鼠尾巴末端约1-2mm，轻轻挤压小鼠尾巴，让血液富集成一滴，用血糖仪(强生稳豪倍优型血糖仪器)测定空腹血糖，测定值认定为0min的血糖值。让小鼠适应30分钟之后，开始准备腹腔注射葡萄糖。用1ml胰岛素针(BD)给小鼠腹腔注射葡萄糖溶液(Sigma)。注射葡萄糖的体积根据小鼠的体重决定，每g体重注射5μl。从注射完毕一刻起，开始计时，在注射后15min、30min、60min、90min、120min测定每只小鼠各个时间点的血糖值。实验完毕后，将每笼小鼠补充上饲料。用Excel软件分析实验结果。
结果：GTT实验通常检测的是机体对血糖的调节能力，是否存在糖代谢异常的情况。由图6可知，与BSA组相比，注射不同给药量的18:2 PC(DLPC)可显著改善小鼠的葡萄糖耐受能力。
(5)小鼠胰岛素耐受实验(Insulin tolerance test,ITT)
实验当天的上午9点将小鼠换入干净的笼子禁食，禁食4小时，至下午1点。禁食期间，小鼠保持正常的饮水。禁食前每只小鼠注射50mg/kg、100mg/kg、200mg/kg 18:2 PC(DLPC)或BSA。称取每只小鼠的体重；空腹基础血糖的测定：将小鼠从笼子中取出，用剪刀剪去小鼠尾巴末端约1-2mm，轻轻挤压小鼠尾巴，让血液富集成一滴，用血糖仪(强生稳豪倍优型血糖仪器)测定空腹血糖，测定值认定为0min的血糖值。让小鼠适应30分钟之后，开始准备腹腔注射胰岛素。用1ml胰岛素针(BD)给小鼠腹腔注射胰岛素溶液(诺和诺德(中国)制药有限公司)。注射胰岛素的体积根据小鼠的体重决定，每g体重注射5μl。从注射完毕一刻起，开始计时；在注射后15min、30min、45min、60min、90min、120min测定每只小鼠各个时间点的血糖值。实验完毕后，将每笼小鼠补充上饲料。用Excel软件分析实验结果。
结果：ITT实验通常检测的是机体对胰岛素的敏感性，进而调控血糖稳态的能力，指征是否会出现胰岛素抵抗的异常的情况。由图7可知，与BSA组相比，注射不同给药量的18:2 PC(DLPC)可显著改善小鼠对胰岛素的敏感性。
(6)小鼠肛温的检测
每组随机挑选6-8只50mg/kg、100mg/kg、200mg/kg 18:2 PC(DLPC)或BSA处理4周的小鼠，在放入4℃冷室前使用肛温检测仪检测每组小鼠的肛温，记为0h的肛温值。随后将小鼠放入4℃冷室，每隔1小时检测一次肛温，一共检测4个时间点的肛温值(共4次)。用Excel软件分析实验结果。
结果：
肛温检测通常是为了表征在冷刺激下，机体对核心体温的维持能力即全身的产热代谢能力。图8的肛温检测结果显示，18:2 PC(DLPC)可显著增加小鼠在冷刺激下维持体温即机体产热能力。
(7)血清中脂质的检测
通过眼球取血的方式收集小鼠的全血，置于4℃保存4h后，8000转离心15分钟收集上清即血清。使用96孔细胞培养板，按照NEFA检测试剂盒、TG检测试剂盒、CHO检测试剂盒(均购自南京建成生物工程研究所)说明书的检测方法，取适量不同处理组小鼠的血清进行检测。最后使用Molecular Devices公司的多功能酶标仪FlexStation3检测546nm处吸光度值。
结果：
图9-11为不同给药量的18:2 PC(DLPC)与溶剂BSA处理小鼠的血清中甘油三酯(TG)、游离脂肪酸(NEFA)、游离总胆固醇(CHO)的检测结果图。TG、NEFA、CHO实验检测血清中的脂质沉积情况。由该图可知，不同给药量的18:2 PC(DLPC)处理组小鼠血清中的TG、NEFA、CHO的含量均显著少于对照组，血清中各种脂质的含量随18:2 PC(DLPC)给药量的增加而减少。
(8)iWAT中UCP1基因表达水平的检测
TRIzol法提取iWAT中的RNA，随后以逆转录的方式合成cDNA，通过实时PCR的方式检测UCP1基因的表达水平。
结果：
UCP1基因是脂肪组织棕色化的标志基因，UCP1表达水平的检测为了说明18:2 PC(DLPC)是通过调节UCP1基因的表达，促进白色脂肪棕色化，进而达到减脂作用。图12为iWAT中UCP1基因表达水平的统计结果。不同给药量的18:2 PC(DLPC)处理组小鼠iWAT中UCP1基因的表达量显著高于对照组，说明18:2 PC(DLPC)显著的引起了白色脂肪组织的棕色化，具有显著的减脂作用。
实施例2：18:2 PC(DLPC)对高脂饮食诱导的肥胖小鼠的治疗作用
(1)体重检测实验
将购买的8周龄C57BL/6雄鼠(购自北京华阜康生物科技股份有限公司)根据体重进行随机分组(每组不少于10只)，使每组间的体重差异最小。连续给予8周高脂饮食(45kcal％，江苏美迪森生物医药有限公司)后，再根据体重进行随机分组(每组不少于10只)，开始进行实验。给予小鼠足量高脂饮食同时每天定时 分别给小鼠注射溶剂BSA(对照组)，50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)(配制方法与实施例1中相同)。持续处理4周。4周后对小鼠的体重进行统计。
结果：
图13为连续4周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的体重统计结果。体重统计体现小鼠肥胖的程度。由该图可知在高脂饮食喂养4周后，注射50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)组的小鼠的体重明显低于注射溶剂BSA组，并且体重出现了随18:2 PC(DLPC)给药量增加而减少的趋势。
(2)小鼠脂肪组织代表性图片及组织重量的统计
脱颈处死如上18:2 PC(DLPC)或BSA处理4周的小鼠，解剖分离出小鼠的皮下脂肪和腹腔脂肪，称重后按组别摆在试验台上进行形态学的比较。用Excel软件分析实验结果。
结果：
图14为连续4周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的小鼠脂肪组织的代表性照片。注射不同给药量的18:2 PC(DLPC)组小鼠白色脂肪组织的重量显著低于溶剂BSA组。代表性图片显示18:2 PC(DLPC)组小鼠白色脂肪的体积显著减小。
图15和图16为连续4周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后，iWAT和gWAT解剖后的重量统计结果。显示脂肪组织重量出现了随18:2 PC(DLPC)给药量增加而减少的趋势。
(3)小鼠脂肝脏组织HE染色
脱颈处死18:2 PC(DLPC)或BSA处理4周的小鼠，解剖分离出小鼠的肝脏，称重后用解剖刀切下一小块肝脏组织，置于4％多聚甲醛中固定过夜。待组织固定好以后制备石蜡切片以及HE染色。HE结果在OLYMPUS光学显微镜(型号TH4-200)下进行观察。
结果：
图17为连续4周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射4周后的肝脏组织HE染色结果。与注射BSA组小鼠相比，不同的给药量的DLPC处理组小鼠肝脏中的脂质沉积显著减少。
(4)小鼠葡萄糖耐受实验(Glucose tolerance test,GTT)
实验前首先对各组小鼠进行体重的称量并且于实验前一天下午5点将小鼠换入干净的笼子禁食，禁食16小时，至次日上午9点。禁食期间，小鼠保持正常的饮水。次日上午8点每只小鼠注射50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)或BSA，上午9点，开始葡萄糖耐受实验。称取每只小鼠的体重。空腹基础血糖的测定：将小鼠从笼子中取出，用剪刀剪去小鼠尾巴末端约1-2mm，轻轻挤压小 鼠尾巴，让血液富集成一滴，用血糖仪(强生稳豪倍优型血糖仪器)测定空腹血糖，测定值认定为0min的血糖值。让小鼠适应30分钟之后，开始准备腹腔注射葡萄糖。用1ml胰岛素针(BD)给小鼠腹腔注射葡萄糖溶液(Sigma)。注射葡萄糖的体积根据小鼠的体重决定，每g体重注射5μl。从注射完毕起开始计时，在注射后15min、30min、60min、90min、120min测定每只小鼠各个时间点的血糖值。实验完毕后，将每笼小鼠补充上饲料。用Excel软件分析实验结果。
结果：
GTT实验通常检测的是机体对血糖的调节能力，是否存在糖代谢异常的情况。由图18可知，与BSA组相比，注射不同给药量的18:2 PC(DLPC)可显著改善小鼠的葡萄糖耐受能力。
(5)小鼠胰岛素耐受实验(Glucose tolerance test,GTT)
实验当天的上午9点将小鼠换入干净的笼子禁食，禁食4小时，至下午1点。禁食期间，小鼠保持正常的饮水。禁食前每只小鼠注射50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)或BSA。称取每只小鼠的体重。空腹基础血糖的测定：将小鼠从笼子中取出，用剪刀剪去小鼠尾巴末端约1-2mm，轻轻挤压小鼠尾巴，让血液富集成一滴，用血糖仪(强生稳豪倍优型血糖仪器)测定空腹血糖，测定值认定为0min的血糖值。让小鼠适应30分钟之后，开始准备腹腔注射胰岛素。用1ml胰岛素针(BD)给小鼠腹腔注射胰岛素溶液(诺和诺德(中国)制药有限公司)。注射胰岛素的体积根据小鼠的体重决定，每g体重注射5μl。从注射完毕起开始计时，在注射后15min、30min、45min、60min、90min、120min测定每只小鼠各个时间点的血糖值。实验完毕后，将每笼小鼠补充上饲料。用Excel软件分析实验结果。
结果：ITT实验通常检测的是机体对胰岛素的敏感性，进而调控血糖稳态的能力，指征是否会出现胰岛素抵抗的异常的情况。由图19可知，与BSA组相比，注射不同给药量的18:2 PC(DLPC)可显著改善小鼠对胰岛素的敏感性。
(6)小鼠肛温的检测
每组随机挑选6-8只50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)或BSA处理4周的小鼠，在放入4℃冷室前使用肛温检测仪检测每组小鼠的肛温，记为0h的肛温值，随后将小鼠放入4℃冷室，每隔1小时检测一次肛温，一共检测4个时间点的肛温值(共4次)。用Excel软件分析实验结果。
结果：
肛温检测通常是为了表征在冷刺激下，机体对核心体温的维持能力即全身的产热代谢能力。图20的肛温检测结果显示，18:2 PC(DLPC)可显著增加小鼠在冷刺激下维持体温即机体产热能力。
(7)血清中脂质的检测
通过眼球取血的方式收集小鼠的全血，置于4℃保存4h后，8000转离心15 分钟收集上清即血清。使用96孔细胞培养板，按照NEFA检测试剂盒、TG检测试剂盒、CHO检测试剂盒(均购自南京建成生物工程研究所)说明书的检测方法，取适量不同处理组小鼠的血清进行检测。最后使用Molecular Devices公司的多功能酶标仪FlexStation3检测546nm处吸光度值。
结果：
图21-23为不同给药量的18:2 PC(DLPC)与溶剂BSA处理小鼠的血清中甘油三酯(TG)、游离脂肪酸(NEFA)、游离总胆固醇(CHO)的检测结果图。TG、NEFA、CHO实验检测血清中的脂质沉积情况。由该图可知，不同给药量的18:2 PC(DLPC)处理组小鼠血清中的TG、NEFA、CHO的含量均显著少于对照组，血清中各种脂质的含量随18:2 PC(DLPC)给药量的增加而减少。
实施例3：18:2 PC(DLPC)高胆固醇饮食诱导的动脉粥样硬化小鼠的预防作用
将购买的5周龄ApoE-/-雄鼠(购自北京维通利华实验动物技术有限公司)根据体重进行随机分组(每组不少于10只)，使每组间的体重差异最小。给予小鼠足量高胆固醇饮食(21％脂肪，0.15％胆固醇，北京华阜康公司)，同时每天定时分别给小鼠注射溶剂BSA(对照组)、50mg/kg的18:2 PC(DLPC)工作液(配制方法与实施例1中相同)，持续给药12周，12周后对小鼠动脉粥样硬化进行检测。
(1)小鼠主动脉油红O染色
脱颈处死如上18:2 PC(DLPC)或BSA处理12周的小鼠，用PBS进行心脏灌流直至主动脉中血液全部排出，解剖分离出小鼠的主动脉，剥离表面的结缔组织，浸泡在PBS中。待全部小鼠的主动脉取材完毕之后，将主动脉固定于4％多聚甲醛(Sigma)约15分钟，使用60％异丙醇(国药)洗5分钟，使用油红O染色液(北京索莱宝科技有限公司)染色10-15分钟，60％异丙醇分色，洗掉多余的油红O染色液，用PBS冲洗即可进行观察和拍照。
结果：
图24-25为18:2 PC(DLPC)与溶剂BSA作用于高胆固醇饮食诱导的动脉粥样硬化小鼠主动脉大体油红O染色结果图片。注射50mg/kg的18:2 PC(DLPC)组小鼠主动脉斑块的形成与溶剂BSA组相比显著改善。
(2)小鼠主动脉斑块面积统计
结果：
图26为50mg/kg 18:2 PC(DLPC)与溶剂BSA作用于高胆固醇饮食诱导的动脉粥样硬化小鼠(预防)后主动脉斑块面积统计结果。使用ImageJ软件对斑块面积进行统计。注射50mg/kg的18:2 PC(DLPC)组小鼠主动脉斑块的面积与溶剂BSA组相比显著减少。
实施例4：18:2 PC(DLPC)高脂饮食诱导的脂肪肝小鼠的预防作用
将购买的8周龄C57BL/6雄鼠(购自北京华阜康生物科技股份有限公司)根据体重进行随机分组(每组不少于10只)，使每组间的体重差异最小。给予小鼠足量高脂饮食(60kcal％，江苏美迪森生物医药有限公司)构建高脂饮食诱导的脂肪肝小鼠模型，同时每天定时分别给小鼠注射溶剂BSA(对照组)，50mg/kg、100mg/kg、200mg/kg的18:2 PC(DLPC)(配制方法与实施例1中相同)。持续处理14周。14周后对小鼠的肝脏重量和脂质沉积程度进行统计。
(1)小鼠肝脏组织代表性图片及组织重量的统计
脱颈处死如上18:2 PC(DLPC)或BSA处理14周的小鼠，解剖分离出小鼠的皮下脂肪和腹腔脂肪，称重后按组别摆在试验台上进行形态学的比较。用Excel软件分析实验结果。
结果：
图27为连续14周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的小鼠肝脏组织的代表性照片。注射不同给药量的18:2 PC(DLPC)组小鼠肝脏组织的体积显著小于BSA组。代表性图片显示18:2 PC(DLPC)组小鼠白色脂肪的体积显著减小。
图28为连续14周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后，肝脏组织解剖后的重量统计结果。显示肝脏组织重量出现了随18:2PC(DLPC)给药量增加而减少的趋势。
(2)小鼠肝脏组织HE染色
脱颈处死18:2 PC(DLPC)或BSA处理14周的小鼠，解剖分离出小鼠的肝脏，称重后用解剖刀切下一小块肝脏组织，置于4％多聚甲醛中固定过夜。待组织固定好以后制备石蜡切片以及HE染色。HE结果在OLYMPUS光学显微镜(型号TH4-200)下进行观察。
结果：
图29为连续14周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的肝脏组织HE染色结果。与注射BSA组小鼠相比，不同的给药量的DLPC处理组小鼠肝脏中的脂质沉积显著减少。
(3)小鼠肝脏组织油红O染色
脱颈处死如上18:2 PC(DLPC)或BSA处理14周的小鼠，取出肝脏，切下一块肝脏组织，解剖刀修成规则的长条或方块状，使用OCT包埋剂(SAKURA樱花)包埋，快速置于液氮中速冻后制备肝脏组织的冰冻切片。将切片固定于4％多聚甲醛(Sigma)约10分钟，使用60％异丙醇(国药)洗5分钟，使用油红O染色液(北京索莱宝科技有限公司)染色10-15分钟，60％异丙醇分色，洗掉多余的油红O染色液，PBS冲洗后使用苏木素染细胞核，流水下冲洗1分钟，使用甘油明胶(北京索莱宝科技有限公司)封片后即可进行观察和拍照。
结果：
图30为连续14周高脂饮食喂养联合不同给药量的18:2 PC(DLPC)和溶剂BSA注射后的肝脏组织油红O染色结果。与注射BSA组小鼠相比，不同的给药量的DLPC处理组小鼠肝脏中的脂质含量显著减少，且呈现剂量依赖趋势。
(4)肝脏中生化指标的检测
取适当体积的肝脏组织于冰冷的PBS中匀浆，取出部分匀浆液用于BCA蛋白定量。使用96孔细胞培养板，按照TG检测试剂盒、CHO检测试剂盒(均购自南京建成生物工程研究所)说明书的检测方法，取适量不同处理组小鼠肝脏组织匀浆液进行检测。最后使用Molecular Devices公司的多功能酶标仪FlexStation3检测对应吸光度值。以检测的每只个体的组织匀浆液蛋白浓度计算肝脏组织中各类脂质的含量。
图31-32为不同给药量的18:2 PC(DLPC)与溶剂BSA处理小鼠的肝脏组织中甘油三酯(TG)、游离总胆固醇(CHO)的检测结果图。TG、CHO实验为了检测肝脏组织中的脂质沉积情况。由该图可知，不同给药量的18:2 PC(DLPC)处理组小鼠肝脏组织的TG、CHO的含量均显著少于对照组，且TG的含量随18:2 PC(DLPC)给药量的增加而减少。
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Claims (8)

  1. 含有式(I)所示结构的化合物或其可药用盐或者含有其的药物组合物在制备用于预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝或肥胖的药物中的用途，
    Figure PCTCN2022128845-appb-100001
  2. 根据权利要求1所述的用途，其中含有式(I)所示结构的化合物如下式(II)所示：
    Figure PCTCN2022128845-appb-100002
    其中：
    -OR选自-OH、胆碱基、L-丝氨酸基团；
    M +选自Na +、K +；
    其中当-OR为胆碱基时，M +不存在。
  3. 根据权利要求1或2所述的用途，其中含有式(I)所示结构的化合物选自以下化合物：
    Figure PCTCN2022128845-appb-100003
  4. 根据权利要求1至3中任一项所述的用途，其中所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖为高脂饮食引发的高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
  5. 根据权利要求1至4中任一项所述的用途，其特征在于，所述化合物通过诱导白色脂肪细胞体积减小，优选诱导白色脂肪组织棕色化，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
  6. 根据权利要求1至5中任一项所述的用途，其特征在于，所述化合物通过减轻病理症状和体征预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖；优选通过减缓高脂饮食诱导的体重增加，和/或缩小白色脂肪细胞的体积，和/或减少血清中脂质的沉积，和/或增强机体的葡萄糖耐受和胰岛素敏感性，和/或促进白色脂肪组织棕色化，从而预防或治疗所述高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖。
  7. 根据权利要求1至6中任一项所述的用途，其中所述药物组合物含有治疗有效量的含有式(I)所示结构的化合物或其可药用盐作为活性成分和药学上可接受的载体或赋形剂。
  8. 根据权利要求1至7中任一项所述的用途，其中所述含有式(I)所示结构的化合物或其可药用盐与另一种或多种治疗方法或治疗剂组合使用，所述治疗方法优选化学疗法、靶向疗法，所述治疗剂优选另一种预防和/或治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖的试剂。
PCT/CN2022/128845 2022-09-21 2022-11-01 甘油磷脂类化合物在预防和治疗高血脂、动脉粥样硬化、非酒精性脂肪肝和肥胖中的用途 WO2024060359A1 (zh)

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