Effects of cell-free fat extract on macrophage polarization regulation and disease treatment

Abstract

The present invention relates to the effects of acellular fat extract on macrophage polarization regulation and disease treatment. Specifically, the present invention provides a use of acellular fat extract for (i) promoting macrophage transformation from the M1 to the M2 subtype; (ii) preventing and / or treating diabetes and its complications; (iii) preventing and / or treating inflammation; and / or (iv) improving insulin resistance. The cellular fat extract of the present invention has excellent effects in promoting macrophage transformation from the M1 to the M2 subtype, preventing and / or treating diabetes and its complications, inflammation, and improving insulin resistance.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the effects of acellular fat extract on macrophage polarization regulation and disease treatment. Background Art

[0002] Immune inflammatory responses play a crucial role in the development and progression of a variety of pathological processes, including inflammatory diseases, metabolic diseases, infectious diseases, autoimmune diseases, and tissue damage and repair. As key players in innate immunity, macrophages influence and regulate the course of immune inflammatory responses through cytokine secretion and antigen presentation. In recent years, a growing number of studies have demonstrated that imbalances in macrophage number, distribution, function, and polarization balance play a key or dominant role in numerous disease processes.

[0003] Macrophages in tissues originate from monocytes in the blood and can migrate from peripheral blood to nearly all tissues to participate in the regulation of tissue homeostasis and inflammatory responses. They possess functional plasticity and diversity, and their functions undergo significant changes in response to environmental stimuli, a process known as macrophage polarization. This can produce macrophage subsets with distinct phenotypes and functions. Based on their function after activation, macrophages can be broadly divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). The M1 macrophage secretes a large number of proinflammatory cytokines, such as TNF-a, IL-1β, IL-6, and NO, as well as ROS / NOS products and Th1 chemokines. These macrophages play a crucial role in the initial stages of the inflammatory response, inducing cell apoptosis, tissue damage, and promoting foreign body clearance. M2 macrophages, on the other hand, play a distinct role. In inflammatory diseases, they suppress inflammatory responses and promote tissue repair. They secrete large amounts of anti-inflammatory cytokines such as IL-10 and TGF-β, inhibiting M1 macrophage-mediated inflammatory responses, promoting angiogenesis, tissue repair, wound healing, and boosting Th2 immunity. The polarized M1 or M2 macrophages undergo dynamic changes and mutual transformations based on different environmental signals. These polarized subtypes can promote tissue regeneration and repair, or stimulate inflammation and exacerbate tissue damage. Differences in polarization subtypes directly determine the outcome of inflammatory responses. Therefore, abnormal macrophage polarization regulation and balance are key factors in the development, progression, and outcome of many inflammatory diseases.

[0004] Therefore, there is a need in the art to develop a drug for preventing and treating abnormal macrophage polarization regulation / balance and related diseases. Summary of the Invention

[0005] The present invention aims to provide a use of a cell-free fat extract in promoting the transformation of macrophages from M1 to M2 subtypes and in preventing and / or treating diabetes and its complications, inflammation and improving insulin resistance.

[0006] In a first aspect, the present invention provides a use of a cell-free fat extract for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (i) promoting the transformation of macrophages from M1 to M2 subtype; (ii) preventing and / or treating diabetes and its complications; (iii) preventing and / or treating inflammation; and / or (iv) improving insulin resistance.

[0007] In another preferred embodiment, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, or a combination thereof.

[0008] In another preferred embodiment, the diabetes includes diabetes caused by insulin resistance.

[0009] In another preferred embodiment, the diabetes mellitus includes obese type diabetes mellitus.

[0010] In another preferred embodiment, the diabetes includes diabetes caused by a high-fat diet.

[0011] In another preferred embodiment, the diabetic complications are selected from the group consisting of diabetic retinopathy, diabetes-related uveitis, diabetic cataracts, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, diabetic neuropathy, or a combination thereof.

[0012] In another preferred embodiment, the prevention and / or treatment of diabetes and its complications includes one or more selected from the following groups for prevention and / or treatment:

[0013] (ii-1) lowering blood sugar levels;

[0014] (ii-2) Improve insulin resistance

[0015] (ii-3) Reduce macrophage infiltration in peripheral tissues.

[0016] In another preferred embodiment, the insulin resistance includes insulin resistance caused by obesity.

[0017] In another preferred embodiment, the insulin resistance includes insulin resistance caused by a high-fat diet.

[0018] In another preferred embodiment, the diabetes mellitus includes insulin resistance caused by inflammation of peripheral tissues and organs and / or macrophage infiltration of peripheral tissues and organs.

[0019] In another preferred embodiment, the insulin resistance includes insulin resistance caused by inflammation of peripheral tissues and organs and / or macrophage infiltration of peripheral tissues and organs.

[0020] In another preferred embodiment, the prevention and / or treatment of inflammation includes reducing the level of inflammatory factors.

[0021] In another preferred embodiment, the inflammation is obesity-related inflammation.

[0022] In another preferred embodiment, the inflammatory factor is selected from the following group: IL-1b, IL-6, TNF-α, F4 / 80, or a combination thereof.

[0023] In another preferred embodiment, the inflammation includes inflammation of peripheral tissues and organs.

[0024] In another preferred embodiment, the inflammation includes inflammation caused by obesity.

[0025] In another preferred embodiment, the inflammation includes inflammation caused by a high-fat diet.

[0026] In another preferred embodiment, the improvement of insulin resistance comprises one or more selected from the following groups:

[0027] (iv-1) Improve inflammation in peripheral tissues and organs;

[0028] (iv-2) Improve macrophage infiltration in peripheral tissues and organs.

[0029] In another preferred embodiment, the peripheral tissue is selected from the group consisting of adipose tissue, skeletal muscle tissue, or a combination thereof.

[0030] In another preferred embodiment, the adipose tissue includes inguinal adipose tissue.

[0031] In another preferred embodiment, the skeletal muscle tissue includes gastrocnemius muscle tissue.

[0032] In another preferred embodiment, the organ includes the liver.

[0033] In another preferred embodiment, the macrophages include macrophages expressing CD68.

[0034] In another preferred embodiment, the acellular fat extract is an acellular fat extract extracted from fat of human or non-human mammals.

[0035] In another preferred embodiment, the non-human mammal is a monkey, an ape, a cow, a pig, a dog, a sheep, a mouse or a rabbit.

[0036] In another preferred embodiment, the composition or preparation includes a pharmaceutical composition.

[0037] In another preferred embodiment, the composition or preparation further comprises a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the composition or preparation is in the form of an oral preparation, an external preparation or an injection preparation.

[0039] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0040] In another preferred embodiment, the composition or preparation is administered externally, topically, or by subcutaneous injection.

[0041] In another preferred embodiment, the cell-free fat extract does not contain cells and does not contain lipid droplets.

[0042] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0043] In another preferred embodiment, the “free from fat droplets” means that in the cell-free fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0044] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0045] In another preferred embodiment, the “cell-free” means that the average number of cells in 1 ml of cell-free fat extract is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0046] In another preferred embodiment, the cell-free fat extract is a naturally obtained nano fat extract without any added ingredients.

[0047] In another preferred embodiment, the “additive-free” means that except for the rinsing step, no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation of the fat extract.

[0048] In another preferred embodiment, the fat extract is prepared by emulsifying adipose tissue and then centrifuging it.

[0049] In another preferred embodiment, the fat extract contains but is not limited to one or more components selected from the following group: growth factors IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

[0050] In another preferred embodiment, the cell-free fat extract is prepared by the following method:

[0051] (1) providing a fat tissue raw material, chopping the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0052] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0053] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing adipocytes);

[0054] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0055] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0056] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0057] In a second aspect, the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:

[0058] (1) providing a fat tissue raw material, chopping the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0059] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0060] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing adipocytes);

[0061] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0062] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0063] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0064] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0065] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0066] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0067] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0068] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way pipe and push and blow repeatedly at a uniform speed.

[0069] In another preferred embodiment, in the step (4), the emulsification is performed by crushing with a tissue homogenizer.

[0070] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0071] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0072] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0073] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0074] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0075] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is divided into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0076] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0077] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0078] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0079] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0080] In another preferred embodiment, in step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0081] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0082] In another preferred embodiment, the filter is a microporous membrane filter.

[0083] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0084] In another preferred embodiment, in step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0085] In another preferred embodiment, step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for future use; can be thawed at low temperature (e.g., -4°C) or room temperature and used directly, or can be thawed and stored at low temperature (e.g., 4°C) for a period of time before use).

[0086] The third aspect of the present invention provides a cell-free fat extract, which is prepared by the method described in the second aspect of the present invention.

[0087] In a fourth aspect, the present invention provides a composition or preparation, comprising (a) the cell-free fat extract according to the third aspect of the present invention; and (b) a pharmaceutically acceptable carrier or excipient.

[0088] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0089] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0090] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semisolid dosage form, or a liquid dosage form, such as a solution, a gel, a cream, an emulsion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0091] In another preferred embodiment, in the composition or preparation, the mass percentage of the acellular fat extract is 5 wt %, preferably 1-20 wt %, based on the total weight of the cosmetic composition.

[0092] In a fifth aspect, the present invention provides a method for preparing the composition or preparation as described in the fourth aspect of the present invention, the method comprising the steps of: mixing the cell-free fat extract as described in the third aspect of the present invention with a pharmaceutically acceptable carrier or excipient to form a composition or preparation.

[0093] In a sixth aspect, the present invention provides a method for (i) promoting the transformation of macrophages from M1 to M2 subtype; (ii) preventing and / or treating diabetes and its complications; (iii) preventing and / or treating inflammation; and / or (iv) improving insulin resistance, comprising administering the cell-free fat extract as described in the third aspect of the present invention to a subject in need.

[0094] In another preferred embodiment, the subject is a human or non-human mammal.

[0095] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0096] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 Flow cytometry was used to detect the number and percentage of positive cells for the surface markers CD86 on M1 subtype macrophages and CD206 on M2 subtype macrophages.

[0098] Figure 2 This is the result of a glucose tolerance test.

[0099] Figure 3 Insulin tolerance test results.

[0100] Figure 4 The relative expression of inflammatory factor genes in peripheral tissues and organs was detected by RT-PCR.

[0101] Figure 5 The results of staining for the macrophage marker CD68 in liver, adipose and skeletal muscle tissues. DETAILED DESCRIPTION

[0102] After extensive and in-depth research, the inventors have developed for the first time a cell-free fat extract that can effectively promote the transformation of macrophages from the M1 to M2 subtype, demonstrating excellent ameliorative effects on diabetes, inflammation, and insulin resistance. This invention was completed on this basis.

[0103] the term

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0105] As used herein, the terms "include," "comprise," and "contain" are used interchangeably to encompass not only open definitions but also semi-closed and closed definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0106] As used herein, "diabetes" refers to a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by impaired insulin secretion, impaired insulin action, or both. Long-term high blood sugar levels in diabetes lead to chronic damage and dysfunction of various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves. Typically, hyperglycemia includes both type 1 diabetes and type 2 diabetes.

[0107] As used herein, "type 1 diabetes," also known as insulin-dependent diabetes, is caused by an absolute insulin deficiency. It most commonly occurs in children and adolescents, but can occur in people of all ages. The onset is rapid, and absolute insulin deficiency can easily lead to ketoacidosis. Insulin therapy is essential for achieving satisfactory results; otherwise, it can be life-threatening.

[0108] As used herein, "type 2 diabetes" refers to a patient whose body's ability to produce insulin is not completely lost. Some patients even produce too much insulin, but the effect of the insulin is poor.

[0109] As used herein, "insulin resistance" is an abnormal physiological state in which the body's response to either endogenous secretion or exogenous injection of insulin is reduced. Insulin resistance occurs when, due to various factors, the efficiency of insulin in promoting glucose uptake and utilization decreases, and the body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain blood sugar stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes. In the 1950s, Yallow et al. used radioimmunoassay to measure plasma insulin concentrations and found that patients with lower plasma insulin levels had higher insulin sensitivity, while those with higher plasma insulin levels were less sensitive to insulin. This led to the concept of insulin resistance.

[0110] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. As used herein, "prevention" also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0111] The "treatment" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention reduces, inhibits and / or reverses diabetes, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the levels observed in the absence of the composition, kit, or active ingredient combination described in the present invention.

[0112] As used herein, "ameliorate" includes preventing, treating, alleviating, reversing, and relieving, among others.

[0113] As used herein, "IL-1b" refers to interleukin-1b.

[0114] As used herein, "IL-6" refers to interleukin-6.

[0115] As used herein, "TNF-α" refers to tumor necrosis factor alpha.

[0116] Cell free fat extract (CEFFE) and preparation method thereof

[0117] As used herein, the terms "acellular adipose extract of the present invention," "extract of the present invention," "fat extract of the present invention," and the like are used interchangeably to refer to an extract (or extractant) derived from adipose tissue that is prepared without the addition of any solutions, solvents, small molecules, chemicals, or biological additives during the fat extract preparation process (except for the rinsing step). A typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention. Furthermore, it should be understood that while it is not necessary to add any additives (or added ingredients) to the extract of the present invention during its preparation, some or a small amount of safe substances (e.g., a small amount of water) that do not negatively or adversely affect the activity of the extract of the present invention may be added.

[0118] Typically, the cell-free fat extract of the present invention is prepared by the following method:

[0119] (1) providing a fat tissue raw material, chopping the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0120] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0121] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing adipocytes);

[0122] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0123] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0124] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0125] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0126] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0127] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0128] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0129] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way pipe and push and blow repeatedly at a uniform speed.

[0130] In another preferred embodiment, in the step (4), the emulsification is performed by crushing with a tissue homogenizer.

[0131] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0132] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0133] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0134] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0135] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0136] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is divided into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0137] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0138] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0139] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0140] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0141] In another preferred embodiment, in step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0142] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0143] In another preferred embodiment, the filter is a microporous membrane filter.

[0144] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0145] In another preferred embodiment, in step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0146] In another preferred embodiment, step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for future use; can be thawed at low temperature (e.g., -4°C) or room temperature and used directly, or can be thawed and stored at low temperature (e.g., 4°C) for a period of time before use).

[0147] use

[0148] The cell-free fat extract of the present invention can effectively promote the transformation of macrophages from M1 to M2 subtypes, and has excellent improving effects on diabetes, inflammation and insulin resistance.

[0149] Typically, the cell-free fat extract of the present invention includes one or more uses selected from the following groups: (i) promoting the transformation of macrophages from M1 to M2 subtype; (ii) preventing and / or treating diabetes and its complications; (iii) preventing and / or treating inflammation; and / or (iv) improving insulin resistance.

[0150] In a preferred embodiment, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, or a combination thereof.

[0151] In another preferred embodiment, the diabetes includes diabetes caused by insulin resistance.

[0152] In another preferred embodiment, the diabetes mellitus includes obese type diabetes mellitus.

[0153] In another preferred embodiment, the diabetes includes diabetes caused by a high-fat diet.

[0154] Typically, the diabetic complication is selected from the group consisting of diabetic retinopathy, diabetes-related uveitis, diabetic cataract, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, diabetic neuropathy, or a combination thereof.

[0155] In another preferred embodiment, the prevention and / or treatment of diabetes and its complications includes one or more selected from the following groups for prevention and / or treatment:

[0156] (ii-1) lowering blood sugar levels;

[0157] (ii-2) Improve insulin resistance

[0158] (ii-3) Reduce macrophage infiltration in peripheral tissues.

[0159] In another preferred embodiment, the insulin resistance includes insulin resistance caused by obesity.

[0160] In another preferred embodiment, the insulin resistance includes insulin resistance caused by a high-fat diet.

[0161] In another preferred embodiment, the diabetes mellitus includes insulin resistance caused by inflammation of peripheral tissues and organs and / or macrophage infiltration of peripheral tissues and organs.

[0162] In another preferred embodiment, the insulin resistance includes insulin resistance caused by inflammation of peripheral tissues and organs and / or macrophage infiltration of peripheral tissues and organs.

[0163] In another preferred embodiment, the prevention and / or treatment of inflammation includes reducing the level of inflammatory factors.

[0164] In another preferred embodiment, the inflammatory factor is selected from the following group: IL-1b, IL-6, TNF-α, F4 / 80, or a combination thereof.

[0165] In another preferred embodiment, the inflammation includes inflammation of peripheral tissues and organs.

[0166] In another preferred embodiment, the inflammation includes inflammation caused by obesity.

[0167] In another preferred embodiment, the inflammation includes inflammation caused by a high-fat diet.

[0168] In another preferred embodiment, the improvement of insulin resistance comprises one or more selected from the following groups:

[0169] (iv-1) Improve inflammation in peripheral tissues and organs;

[0170] (iv-2) Improve macrophage infiltration in peripheral tissues and organs.

[0171] In another preferred embodiment, the peripheral tissue is selected from the group consisting of adipose tissue, skeletal muscle tissue, or a combination thereof.

[0172] In another preferred embodiment, the adipose tissue includes inguinal adipose tissue.

[0173] In another preferred embodiment, the skeletal muscle tissue includes gastrocnemius muscle tissue.

[0174] In another preferred embodiment, the organ includes the liver.

[0175] In another preferred embodiment, the macrophages include macrophages expressing CD68.

[0176] The present invention also provides a method for (i) promoting the transformation of macrophages from M1 to M2 subtype; (ii) preventing and / or treating diabetes and its complications; (iii) preventing and / or treating inflammation; and / or (iv) improving insulin resistance, comprising the steps of administering the cell-free fat extract of the present invention to a subject in need.

[0177] In another preferred embodiment, the subject is a human or non-human mammal.

[0178] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0179] Composition and administration

[0180] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, etc.

[0181] Typically, the cell-free fat extract of the present invention can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, microgranules, solutions, lozenges, jellies, creams, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the medicine.

[0182] The composition of the present invention may also include a pharmaceutically, food, health product or dietary acceptable carrier. "Pharmaceutically, food, health product or dietary acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically, food, health product or dietary acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0183] There is no particular limitation on the administration of the composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration), and topical administration. The preferred administration methods are oral administration and injection administration.

[0184] Solid dosage forms for oral administration or delivery include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0185] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

[0186] Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures thereof.

[0187] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.

[0188] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0189] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0190] Dosage forms for topical or topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0191] The cell-free fat extract of the present invention can be administered alone or in combination with other drugs for preventing and / or treating fatty liver and / or its complications.

[0192] When administering the composition, a safe and effective amount of the acellular fat extract of the present invention is applied to a human or non-human animal (such as rats, mice, dogs, cats, cattle, chickens, ducks, etc.) in need of treatment, wherein the dosage during administration is an effective dosage that is acceptable for pharmaceutical use, food use, or health supplements. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "safe and effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs. For example, for a 60 kg person, the daily dosage is generally 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0193] The main advantages of the present invention include:

[0194] This study discovered for the first time that cell-free fat extract can promote the transformation of macrophages from M1 to M2 subtypes. Using a high-fat diet-induced type II diabetes model mouse model, it was confirmed that CEFFE treatment can regulate peripheral tissue macrophage polarization, reduce macrophage recruitment, improve obesity-related chronic inflammation, and enhance insulin sensitivity. This suggests the therapeutic potential of CEFFE in obesity-related metabolic diseases, and also suggests the therapeutic value of CEFFE in diseases related to immune inflammatory responses in which macrophage polarization is involved.

[0195] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0196] Example 1

[0197] 1. Preparation of Cell Free Fat Extract (CEFFE)

[0198] Fat was obtained from volunteers with informed consent. The preparation method of acellular adipose tissue extract is as follows:

[0199] (1) Take the fat obtained by suction or surgical resection, cut it into small pieces, and rinse it with saline three times.

[0200] (2) The washed adipose tissue was placed in a centrifuge tube, and centrifuged at 1200 g for 3 minutes to obtain a layered mixture.

[0201] (3) For the stratified mixture, the upper oil layer and the lower water layer are removed, and the middle layer (i.e., the fat layer containing fat cells) is collected.

[0202] (4) The intermediate layer was mechanically emulsified by repeatedly pushing and hitting the intermediate layer 30 times at a constant speed using two 10 ml syringes connected to a three-way tube, and a mechanically emulsified fat mixture (also called nano fat) was obtained.

[0203] (5) The mechanically emulsified fat mixture is placed in a -80°C freezer and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture is centrifuged at 1200g for 5 minutes to obtain a layered mixture. The layered mixture is divided into 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer, and the fourth layer is a cell / tissue fragment precipitation layer. The oil layer and the residual fat tissue layer are removed, and the liquid layer is aspirated. During the aspiration process, contamination of the cell / tissue fragment precipitation layer is avoided, thereby obtaining a primary fat extract.

[0204] (6) The obtained primary fat extract was sterilized by filtering through a 0.22 μm filter to sterilize and remove any living cells that may be mixed in, thereby obtaining a cell-free fat extract, which was then aliquoted and frozen at -20°C and thawed at 4°C before use.

[0205] 2. Cell Culture:

[0206] The mouse mononuclear macrophage cell line RAW264.7 (M0 macrophages) was purchased from the Chinese Academy of Sciences Cell Bank. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were purchased from Sigma. RAW264.7 (M0 macrophages) were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The medium was changed every other day and the cells were passaged at 90% cell density to obtain M0 macrophage cultures.

[0207] 100 ng / mL LPS and 30 ng / mL IFN were added to the macrophage culture medium to establish an in vitro inflammation model.

[0208] In the CEFFE-treated group, 10% CEFFE (v / v) was added into the cell culture medium.

[0209] 3. Flow Cytometry

[0210] The M0 macrophages cultured in vitro in step 2 were divided into a blank group (Control group), a CEFFE group, an LPS+IFN-γ group, and an LPS+IFN-γ+CEFFE group. In the CEFFE group, CEFFE was added to the M0 macrophage culture medium, and the concentration of CEFFE was 10% (v / v) of the cell culture medium; in the LPS+IFN-γ group, LPS (100 ng / mL) and IFN-γ (30 ng / mL) were added to the M0 macrophage culture medium; in the LPS+IFN-γ+CEFFE group, LPS (100 ng / mL), IFN-γ (30 ng / mL), and CEFFE (at a concentration of 10% (v / v) in the cell culture medium) were added to the M0 macrophage culture medium; no drug treatment was added to the Control group. After culturing M0 macrophages in vitro for 24 h, the cells in different groups were digested and collected, and incubated with fluorescently labeled CD86 and CD206 antibodies at 4°C for 30 min, washed, resuspended, and the cell fluorescence expression was detected by flow cytometry.

[0211] LPS and IFN can stimulate the polarization of M0 macrophages to the M1 subtype, thereby establishing an in vitro inflammatory model. CD86 is a surface marker of M1 subtype macrophages, and CD206 is a surface marker of M2 subtype macrophages. After 24 hours of culture, the number and percentage of positive cells for the surface markers CD86 and CD206 of M1 subtype macrophages and M2 subtype macrophages were detected by flow cytometry. Figure 1 shown.

[0212] from Figure 1 Compared with the control group, CEFFE culture in the CEFFE group failed to induce a significant polarization of M0 macrophages. Although the percentage of CD86+ cells increased slightly to 3.73%, it was still much lower than that in the LPS+IFN-γ group (35.96%). Compared with the conventional M1-induced LPS+IFN-γ group, the addition of CEFFE to the LPS+IFN-γ+CEFFE group significantly reduced the percentage of CD86+ cells and increased the proportion of CD206+ cells. The decrease and increase in the proportion of cells of the two phenotypes were basically the same (approximately 12%), indicating that CEFFE treatment promoted the transformation of macrophages from the M1 to the M2 subtype.

[0213] 4. Establishment of a high-fat-induced insulin resistance mouse model and pharmacodynamic study

[0214] 4.1 Establishment, grouping, and drug treatment of high-fat-induced insulin resistance mouse model

[0215] Six-week-old C57 mice were purchased from the Shanghai Experimental Animal Center. The mouse insulin resistance model was induced by a high-fat diet for 15 consecutive weeks. After 15 weeks of feeding, the mice underwent fasting glucose tolerance and insulin tolerance tests. Subsequently, the mice were divided into three groups: a blank control group (normal diet, Chow group), a negative control group (high-fat diet + PBS injection, PBS group), and a CEFFE group (high-fat diet + CEFFE injection, CEFFE group). The mice were injected into the tail vein once every 4 days, for a total of 7 injections, with a treatment cycle of 30 days. Each CEFFE injection dose was 250 μl. PBS injection was used as a negative control, and Chow was used as a blank control. Random blood glucose and body weight were monitored regularly during treatment.

[0216] After 30 days of treatment, fasting glucose tolerance test and insulin tolerance test were conducted again, and blood was drawn from the mice for hematological examination. The liver, inguinal fat and gastrocnemius muscle tissues of the model mice were collected for RT-PCR detection of inflammatory factor expression in peripheral tissues and immunostaining. The results are as follows:

[0217] 4.2 Pharmacodynamic studies in a high-fat-induced insulin resistance mouse model

[0218] Statistical analysis: The data were analyzed using one-way ANOVA test using SPSS software, and the results were expressed as mean ± standard deviation.

[0219] 4.2.1 Glucose tolerance and insulin tolerance test:

[0220] Glucose tolerance test: After fasting for 12 hours, glucose was injected into the abdominal cavity of the mice and the blood glucose level in the tail vein was measured at 0, 15, 30, 60, 90 and 120 minutes. Figure 2 shown.

[0221] Insulin tolerance test: After fasting for 6 hours, mice were injected with insulin intraperitoneally, and the blood glucose levels in the tail vein of mice were measured at 0, 15, 30, 60, 90 and 120 minutes. Figure 3 shown.

[0222] from Figure 2 and Figure 3 As can be seen, compared with the blank control group, the high-fat diet negative control group mice had significantly higher blood glucose levels at 0, 15, 30, 60, 90, and 120 minutes, and significantly increased the area under the glucose tolerance and insulin tolerance curves (AUCs). Compared with the negative control group, the blood glucose levels and AUCs of mice in the CEFFE-treated group were significantly reduced, indicating that CEFFE treatment can improve insulin sensitivity and ameliorate insulin resistance. These results suggest that CEFFE treatment can improve insulin resistance by regulating the polarization of peripheral tissue macrophages.

[0223] 4.2.2 RT-PCR detection of inflammatory cytokine expression in peripheral tissues

[0224] The liver, inguinal fat and gastrocnemius muscle tissues of model mice were collected, and total RNA was extracted with Trizol. The RNA concentration was calculated by spectrophotometry at a wavelength of 260 / 280 nm. After reverse transcription with the EZbioscience kit, the expression levels of IL-1b, IL-6, TNF-a, and F4 / 80 were detected by RT-PCR fluorescence. The total reaction system was 20ul, and the amplification conditions were: initial denaturation at 95℃ for 10min; 95℃ for 15s, 62℃ for 60s, and 40 cycles. After the reaction, the RT-PCR results were subjected to relative quantitative statistics. The relative expression of inflammatory factor genes in peripheral tissues and organs was detected by RT-PCR. Figure 4 As shown:

[0225] from Figure 4 It can be seen that compared with the blank control group, the expression of inflammatory factors in the liver, adipose and skeletal muscle tissues of mice in the high-fat fed negative control group increased, while it was significantly decreased after CEFFE treatment, indicating that CEFFE can effectively reduce inflammation in peripheral tissues and organs of diabetic mice.

[0226] 4.2.3 Immunostaining

[0227] The liver, inguinal fat, and gastrocnemius muscle tissues of the model mice were collected and immersed in 4% paraformaldehyde. After fixation for 24 hours, they were embedded in paraffin and routinely sectioned, dewaxed, hydrated, and then high-pressure fixed. After blocking with 5% BSA, they were incubated with 1:100 CD68 at 4°C overnight. The next day, after thorough washing, HRP-secondary antibody was added, and the tissues were incubated at 37°C for 30 minutes before DAB staining. Optical microscopy was used for photography and ImageJ was used for counting and analysis. The macrophage marker CD68 staining in liver, fat, and skeletal muscle tissues was as follows: Figure 5 shown.

[0228] from Figure 5 It can be seen that compared with the negative control group, the number of CD68+ macrophages in the CEFFE treatment group was significantly decreased, indicating that CEFFE treatment can effectively reduce the infiltration of macrophages in peripheral tissues.

[0229] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of a cell-free fat extract, characterized in that: For preparing a pharmaceutical composition or pharmaceutical preparation, the pharmaceutical composition or pharmaceutical preparation is used for one or more uses selected from the group consisting of: (i) preventing and / or treating type 2 diabetes; (ii) preventing and / or treating inflammation; The cell-free fat extract is prepared by a method comprising the following steps: (1) providing a fat tissue raw material, chopping the fat tissue raw material, and rinsing the fat tissue raw material to obtain rinsed fat tissue; (2) centrifuging the rinsed adipose tissue to obtain a layered mixture; (3) removing the upper oil layer and the lower water layer from the stratified mixture and collecting the middle layer, i.e., the fat layer containing fat cells; (4) emulsifying the intermediate layer to obtain an emulsified fat mixture, also known as nanofat; (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and (6) filtering and sterilizing the primary fat extract to obtain the cell-free fat extract; The cell-free fat extract contains no cells and no fat droplets, wherein "containing no fat droplets" means that the volume of oil droplets in the cell-free fat extract accounts for less than 1% of the total liquid; and "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤1.

2. The use according to claim 1, characterized in that The type 2 diabetes is diabetes caused by insulin resistance.

3. The use according to claim 1, characterized in that The type 2 diabetes is diabetes caused by a high-fat diet.

4. The use according to claim 1, wherein The term "free of lipid droplets" means that the volume of lipid droplets in the cell-free fat extract accounts for less than 0.5% of the total liquid; and the term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤0.

5.

5. The use according to claim 1, characterized in that The cell-free fat extract is a naturally obtained nano fat extract without any added ingredients; The term "additive-free" means that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation of the fat extract.

6. The use according to claim 1, wherein In the step (4), the emulsification is performed by crushing with a tissue homogenizer.

7. The use according to claim 1, characterized in that The pharmaceutical composition or pharmaceutical preparation is administered externally, topically, or by subcutaneous injection.

8. The use according to claim 1, characterized in that The fat extract contains the following components: growth factors IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH and G-CSF.

Images